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Steroids journal homepage: www.elsevier.com/locate/steroids 6 7
TXA9, a cardiac glycoside from Streptocaulon juventas, exerts a potent anti-tumor activity against human non-small cell lung cancer cells in vitro and in vivo
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a Development and Utilization Key Laboratory of Northeast Plant Materials, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China b School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China c The Animal Experimental Center, Shenyang Pharmaceutical University, Shenyang 110016, China d The People’s Liberation Army 463 Hospital, Shenyang 110042, China e Department of Bioscience, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama, Japan
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Rui Xue a, Na Han a, Mingyu Xia b, Chun Ye a, Zhihui Hao c, Lihui Wang b, Yu Wang d, Jingyu Yang b, Ikuo Saiki e, Jun Yin a,⇑
a r t i c l e Q2
i n f o
Article history: Received 6 May 2014 Received in revised form 2 October 2014 Accepted 18 December 2014 Available online xxxx Keywords: Acovenosigenin A 3-O-b-D-glucoside Anti-tumor NSCLC Streptocaulon juventas Cardiac glycosides Apoptosis
a b s t r a c t Non-small cell lung cancer is the most common type of lung cancer and the most common cause of cancer-related death in humans. TXA9, which is a natural product separated from an anti-tumor-active fraction of the roots of Streptocaulon juventas, may possess potent anti-proliferative activity according to the present study. In this study, the anti-tumor effects and toxicity of TXA9 were tested against human nonsmall cell lung cancer cell lines (A549, NCI-H1299, Ltep-a2, PC-9, and Lu99) and a normal human lung embryonic fibroblast cell (HE-lung) in vitro, and then toward A549 cells in vivo in a murine xenograft model. The results show that TXA9 exhibits potent cytotoxic activities against non-small lung cancer cells and has no toxic effect on the normal human lung embryonic fibroblast cells. The mechanistic studies demonstrate that TXA9 can induce the apoptosis of A549 cells through the extrinsic pathway. The in vivo study results reveal that the intravenous administration of TXA9 at high-dose (15 mg kg 1) induces significant tumor growth inhibition of non-small cell lung cancer xenografts with tumor inhibition rate up to 64.2%, compared with mice in the control group. The inhibitory effect was similar to that of taxol (62.5%). In particular, no significantly adverse effects were exerted by TXA9, which suggests that it is well tolerated. This promising natural product may be useful as a potential novel anti-tumor candidate. Ó 2014 Published by Elsevier Inc.
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1. Introduction Non-small cell lung cancer (NSCLC), accounts for approximately 85% of all lung cancers, and almost 65–70% of NSCLC patients preAbbreviations: NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; CGs, cardiac glycosides; SJ, Streptocaulon juventas; MTT, 3-(4,5-dimethylibiazol-2yl)-2,5-diphenyl-tetrazolium bromide; AO, acridine orange; PI, propidium iodide; i.v., intravenous; LTXA9, TXA9 at a dose of 5 mg kg 1; MTXA9, TXA9 at a dose of 10 mg kg 1; HTXA9, TXA9 at a dose of 15 mg kg 1; RBC, red blood cell; WBC, white blood cell; PLT, platelets; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; LDH, lactic dehydrogenase; BUN, blood urea nitrogen; TBIL, total bilirubin; Crea, creatinine; TP, total protein; FADD, Fasassociated death domain. ⇑ Corresponding author at: School of Traditional Chinese Materia Medica 48#, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China. Tel./fax: +86 24 23986491. E-mail address: [email protected] (J. Yin).
sent with advanced disease at the time of diagnosis [1]. The prognosis for NSCLC patients is very poor with a five-year survival rate of only 15%, and this malignancy remains difficult to treat [2]. Due to the limited therapeutic effect of standard chemotherapy, radiotherapy and surgery, no effective treatment has been developed to date. For many years, natural products have played a very important role in anticancer drug discovery and development because they offer a large structural diversity [3]. As a result, both terrestrial and marine organisms are presently considered important sources of novel lead compounds. Well known for their positive inotropic effect, cardiac glycosides (CGs) have been used clinically for many years for the treatment of heart failure and atrial arrhythmias. Since the middle of the 1960s, initial observations from epidemiological studies have shown that cancer patients receiving cardiac glycosides exhibit significantly lower mortality rates than patients without treatment
http://dx.doi.org/10.1016/j.steroids.2014.12.015 0039-128X/Ó 2014 Published by Elsevier Inc.
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with such drugs. Consequently, it is generally accepted that CGs have a putative anti-cancer effect, and the anti-proliferative activity of CGs, such as digitoxin on human cancer cells has been widely reported [4–8]. However, it is well known that the most significant adverse effect of digitoxin, which is frequently encountered in clinical treatment, is cardiotoxicity [9–10]. The narrow therapeutic window dramatically limits the application of digitoxin, because its toxicity is a major concern whenever it is considered for therapy [11]. Therefore, it is necessary to discover new candidate drugs that exhibit a significant anti-tumor effect but low toxicity. Streptocaulon juventas (SJ) (Asclepiadaceae), which is mainly distributed in Southeast Asia, is used in folk medicine to stimulate the spleen, tonify the body, and strengthen the kidney [12]. We previously reported that the 75% ethanol extract of SJ exhibits potent inhibitory activity in vivo on the growth of the human lung A549 adenocarcinoma cell line with only mild adverse effects [13]. A subsequent phytochemical study of the active extract confirmed that cardiac glycosides contribute to the observed anti-tumor activity [14–15]. Among the numerous cardiac glycosides obtained, TXA9 was chosen as the lead compound for further research due to its potent in vitro anti-proliferative activity and relatively simple structure. In the present study, the potential anti-proliferative activities of TXA9 against NSCLC in vitro and in vivo were evaluated, and any possible treatment-related toxic effects were assessed through a series of hematology and serum biochemistry tests, investigations of the visceral organs, and monitoring of body weight. Additionally, the apoptosis mechanism of TXA9 on A549 cells was investigated.
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2. Experimental
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2.1. Ethics statement
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All of the animal care and experimental procedures were approved by the Animal Ethical Committee of Shenyang Pharmaceutical University. All tumor implantations, blood collections, and sacrifices were performed under anesthesia and all efforts were made to minimize suffering.
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2.2. Plant material
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2.4. Cell culture
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The human lung A549 adenocarcinoma cell line, human nonsmall cell lung carcinoma NCI-H1299 cell line, human lung PC-9 adenocarcinoma cell line, and lung giant cell Lu99 carcinoma cell line were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). The human lung Ltep-a2 adenocarcinoma cell line was ordered from Typical Culture Preservation Commission Cell Bank (Chinese Academy of Sciences, Shanghai, China). The HE-lung embryonic fibroblast cell line was provided by Prof. Ikuo Saiki (Department of Bioscience, Institute of Natural Medicine, University of Toyama, Sugitani, Toyama, Japan) as a gift. All of the cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in an atmosphere of 5% CO2 and 95% air.
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2.5. Cell proliferation
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The effect of TXA9 on cell proliferation was assessed using the 3-(4,5-dimethylibiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, 96-well microtitre plates were seeded with 5 103 cells per well and incubated for 24 h. Culture media containing different concentrations of the samples were then added. After incubation for 12 h, 24 h, and 48 h, 100 lL of MTT from a stock solution (0.5 mg/mL) was added to each well, and the plate was incubated for 4 h. The absorbance of the resulting formazan product was measured at 492 nm using a microplate reader (Tecan, Austria).
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2.6. Observation of cell morphological changes and acridine orange staining analysis
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The morphological changes were observed as described previously [16]. Briefly, A549 cells were cultured at a density of 8 105 cells/flask in 25-mm2 cell culture flasks. After 24 h of incubation, the cells were treated with 600 nM TXA9 and incubated for an additional 24 h. At the end of the incubation period, the morphological changes in the cells were observed through photomicrography using a phase-contrast microscope. Acridine orange (AO) staining was used as an indicator of cell apoptosis. A549 cells were plated at 3 105 cells/well in six-well plates. After 24 h of incubation, the cells were treated with TXA9 at a concentration of 600 nM and incubated for 24 h. The prepared cells were washed once with PBS. After 10 mg/L AO was added to the cells, the cells were incubated for 30 min at 37 °C and then observed under a fluorescence microscope at an excitation wavelength of 488 nm and an emission wavelength of 515 nm.
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Roots of SJ were collected in Simao, Yunnan Province in 2009. No specific permissions were required for the collections because the crude drug is wild and is not an endangered or protected species. The roots were identified by Prof. Jun Yin (Division of Pharmacognosy, Shenyang Pharmaceutical University), and voucher samples (SPU 2162-2165) were deposited at the herbarium of Shenyang Pharmaceutical University, Shenyang, China. 2.3. Preparation of TXA9
2.7. Flow cytometry analysis by annexin V and propidium iodide staining
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TXA9 was isolated from the roots of SJ collected in Yunnan Province of China in 2009. The air-dried roots were extracted with EtOH–H2O (75:25, v/v) for 1.5 h, and this process was repeated three times. The combined solution was concentrated under reduced pressure until there was no trace of alcohol and then extracted with n-butanol four times. The concentrated extract was then subjected to silica-gel column chromatography with CH2Cl2–MeOH (25:1, 12:1, 1:1, v/v) to yield three fractions (A, B and C). Fraction B was applied to a silica-gel column, eluted with EtOAc–Me2CO–H2O (10:1:0.1, v/v/v), subjected to ODS silica-gel column chromatography with MeOH–H2O (50:50, v/v), and recrystallized using MeOH to obtain TXA9 (purity: 99%).
Annexin V-FITC/PI double staining was employed for the quantitative determination of the percentage of apoptotic cells. A549 cell at a density of 3 106 cells in each dish (ID: 6 cm) were incubated overnight, and then treated with sample or vehicle. After incubation for 6, 12, or 24 h with TXA9, the cells were digested with trypsin and suspended with PBS. After centrifugation, the PBS was removed, and the cells were stained with FITC-conjugated annexin V and propidium iodide (PI) for 15 min at 20 °C in a Ca2+enriched binding buffer (apoptosis detection kit, R&D Systems, Abingdon, UK; 20). The cells were immediately analyzed with a flow cytometer in their staining solution (Becton Dickinson FACScan, Franklin Lakes, NJ, USA).
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2.8. Cell cycle analysis by flow cytometry
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The degree of cell apoptosis was determined through forward/ side scatter analysis and PI staining using a FACScan flow cytometer (Becton Dickinson) equipped with an argon-ion laser as described previously [17]. Briefly, A549 cells were cultured in cell culture flasks and treated with 600 nM TXA9 for 12, 24, 36, and 48 h. The prepared cells were washed once with PBS, fixed in 70% ethanol overnight, washed once with PBS, and stained with PI solution (50 lg/mL PI and 50 lg/mL RNase A in PBS) for 30 min in the dark. The samples were then analyzed by flow cytometry.
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2.9. Western blotting analysis
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A549 cells were treated with 600 nM TXA9 for different time periods. Both adherent and floating cells were collected, and a Western blotting analysis was then performed as previously described [17]. Equal amounts of total proteins were separated by 12% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk in NaCl/Tris-T (10 mM Tris/HCl, pH 8.0, 150 mM NaCl and 0.05% Tween 20) buffer for 1 h at room temperature and then incubated at 4 °C overnight with primary antibodies against caspase 3, caspase 8, Fas, and FADD (Santa Cruz, CA, USA). After several washes, the membranes were then incubated with the corresponding HRP-conjugated secondary antibodies at room temperature for 1 h and visualized with the enhanced chemiluminescence detection system (GE Healthcare, USA).
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of treatment, all of the mice were killed, and their tumors and organs were removed, weighed, and then fixed in 10% formalin, embedded in paraplast, cut into 4-lm-thick sections, and stained with hematoxylin and eosin (H&E) for histopathological examination. The slices were investigated under the microscope.
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2.12. Efficacy and toxicity of TXA9
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The evaluation of efficacy and toxicity of TXA9 in nude mice was based on the analyses of tumor growth curves, tumor weight, organ indices as described previously [13]. Blood of each mouse was collected, then the hematological parameters were determined using a Y-159 blood analyzer (Nihon Kehden, Japan), and the serum was used to measure various biochemical indexes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactic dehydrogenase (LDH), blood urea nitrogen (BUN), total bilirubin (TBIL), creatinine (Crea), and total protein (TP), using the appropriate reagent kits and an ELISA reader (Tecan, Australia).
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2.13. Statistical analyses
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The in vitro experiments were repeated at least three times. The results are expressed as the means ± SD or means ± SEM. The statistical analyses were performed using one-way analysis of variance followed by the LSD test under the condition of variance homogeneity and Dunnett’s T3 test under the condition of variance heterogeneity. Statistical significance was considered to be obtained at P < 0.05.
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2.10. Animal and tumor xenograft model
3. Results
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3.1. TXA9 shows potent anti-proliferative activity against human nonsmall cell lung cancer cell lines
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2.11. Treatment
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When the average tumor size reached 100 mm3, the animals were divided into five groups (ten animals per group) and administered the following treatments: (A) control [vehicle (5% propylene glycol and 5% ethanol/saline), i.v.]; (B) taxol (5 mg kg 1, i.v.); (C) low dose of TXA9 (LTXA9, 5 mg kg 1, i.v.); (D) medial dose of TXA9 (MTXA9, 10 mg kg 1, i.v.) and (E) high dose of TXA9 (HTXA9, 15 mg kg 1, i.v.). Taxol and TXA9 were dissolved in 5% propylene glycol and 5% ethanol/saline. All of the compounds were injected three times a week for four consecutive weeks, starting on the 14th day after the tumor graft. Three times a week, each mouse was weighed, and the tumor volume was measured. After 28 days
On the basis of spectroscopic studies and comparison with a reference [18], the structure of TXA9 was elucidated as acovenosigenin A 3-O-b-D-glucoside (Fig. 1A). In the initial study, the antiproliferative activity of TXA9 against five types of human nonsmall cell lung cancer cell lines and one normal human lung cell line was first evaluated through the MTT assay, using taxol as a positive control. TXA9 exhibited inhibitory effects against A549 (IC50: 0.11 lM), Ltep-a2 (IC50: 0.03 lM), and PC-9 (IC50: 0.44 lM) cell lines similar to those of taxol (IC50: A549, 0.16 lM; Ltep-a2, 0.01 lM; and PC-9, 1.25 lM). However, the test compound showed more potent growth inhibition activities than taxol on the NCIH1299 (TXA9: 0.006 lM and taxol: 0.48 lM) and Lu99 (TXA9: 0.32 lM and taxol: 25.6 lM) cell lines. This promising result demonstrates that TXA9 can selectively inhibit the growth of tumor cell lines but has no toxic effect against normal cells (IC50 > 100 lM) (Table 1). Moreover, this finding also illustrates that TXA9 exhibits potent, dose- and time-dependent anti-proliferative activity at 12 h, 24 h, and 48 h (Fig. 1B). Based on these results, 0.53 lM was calculated as the IC50 value for TXA9 at 24 h and 600 nM was chosen for the subsequent mechanism studies. The analysis of the morphological changes in A549 cells [Fig. 2(I)] showed that shrinking or multi-blebbing cell shapes and the presence of apoptotic bodies are clearly observed in the cells treated with TXA9 at a concentration of 600 nM (Fig. 2B), which were quite different to those of the control (Fig. 2A). These morphological changes are typical of apoptosis [19]. Furthermore, the morphological changes in the cell nuclei were monitored by AO staining. The cell nuclei exposed to TXA9 at a concentration of 600 nM displayed distinct karyopyknosis and dense fluorescence, and the crescent bodies could be easily observed at the edge of
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Six-week-old female BALB/c nu/nu mice (weight: 18–20 g) purchased from the Animal Experiment Center of Shenyang Pharmaceutical University were maintained under pathogen-free conditions and fed irradiated chow. After an acclimation period of approximately 7 days, tumor inoculation was performed. A549 cells were harvested and resuspended at a final concentration of 3 107 cells/mL in PBS and Matrigel™ Matrix (BD Biosciences, Two Oak Park, Bedford, MA, USA) at a 1:1 ratio. The mice were inoculated s.c. into the right flank with 3 106 cells (0.1 mL). The tumors were measured with a vernier caliper every 3 days, and their volumes were calculated as described previously [13]. Before administration, TXA9 was subjected to an acute toxicity study to determine the optimal dose. This dose was determined through the intravenous (i.v.) administration of a single dose to healthy mice that had not received a tumor graft. The survival of the animals was monitored up to 7 days. Four different doses of TXA9 (i.e., 4.2, 10, 15 and 25 mg kg 1) were used to determine the index. The maximum administration dosage of TXA9 via the i.v. route was found to be 25 mg kg 1.
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Fig. 1. Chemical structure of TXA9 (A) and its in vitro growth-inhibitory activities against A549 cells (B). TXA9 was administered at six concentrations ranging from 3 to 480 nM, and each concentration was analyzed three times. The cells were cultured for 12 h, 24 h, and 48 h. The results are presented as the growth inhibitory rate of the cells. All of the experiments were performed three times to validate the results.
Table 1 The cytotoxic activities of TXA9 and taxol in vitro. Compounds
TXA9 Taxol
IC50 (lM) A549
NCI-H1299
Ltep-a2
PC-9
Lu99
HE-lung
0.11 0.16
0.006 0.48
0.03 0.01
0.44 1.25
0.32 25.6
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All of the IC50 values were acquired from a 48-h MTT assay with at least three independent experiments. A549: human lung adenocarcinoma cell; NCI-H1299: human non-small cell lung carcinoma cell; Ltep-a2: human lung adenocarcinoma cell; PC-9: human lung adenocarcinoma cell; Lu99: lung giant cell carcinoma cell; HE-lung: HE-lung embryonic fibroblast cell. Taxol was used as a positive control.
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the nuclear membrane (Fig. 2D), whereas the cell nuclei without TXA9 treatment exhibited a smooth surface and homogeneous fluorescence (Fig. 2C). All of these changes indicate that TXA9 may induce apoptosis in A549 cells. 3.2. TXA9 can induce apoptosis as determined by flow cytometry analysis
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To confirm the cell death type, the cells were stained with Annexin V and PI [Fig. 2(II)], and the cell death at 6 h, 12 h, and 24 h was analyzed by flow cytometry (Fig. 2E). As a result, the percentages of cells in the lower-right quadrant were 3.60%, 4.59%, and 25.99% after 6 h, 12 h, and 24 h respectively of treatment, respectively, showing that TXA9 can induce a significant increase in the percentage of apoptotic cells.
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3.3. TXA9 can induce cell cycle arrest in the SubG0/1 and G2/M periods
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The flow cytometric analysis of the cell cycle distribution displayed that the percentages of control cells in the SubG0/G1 phase was only 1.68%, and this percentage was obviously increased to
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3.51%, 13.29%, 25.33%, and 36.33% after treatment with 600 nM TXA9 for 12, 24, 36, and 48 h, respectively (Fig. 3). This finding further confirms that TXA9 can induce time-dependent apoptosis in A549 cells. In addition, compared with the percentage of control cells in the G2/M phase (12.78%), treatment with TXA9 for 12 and 24 h significantly increased the percentages of cells in this phase to 28.08% and 28.50%, respectively. This phenomenon indicates that TXA9 can cause a marked lever of G2/M cycle arrest in A549 cells. In association with the increased apoptosis rates, the G2/M phase percentage was decreased by treatment with TXA9 for 36 and 48 h. These results showed that TXA9 can obviously induce A549 cell cycle arrest at the G2/M periods within 24 h.
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3.4. TXA9 can induce apoptosis in A549 cells through the death receptor pathway
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Analyses of the cell lysates by Western blotting were performed to evaluate the protein expression of A549 cells after treatment with TXA9. After treatment with 600 nM TXA9 for 12, 24, 36, and 48 h, the protein levels of Fas and Fas-associated death domain (FADD), which can activated the cascade reaction of apoptosis via the extrinsic pathway, were up-regulated in a time-dependent manner. Additionally, TXA9 was found to time-dependently cleave procaspase-8 and procaspase-3 to the active forms, namely caspase-8 and caspase-3, respectively, as well as its death substrates (ICAD) (Fig. 4A and B). All of these lines of evidence prove that TXA9 activates the extrinsic apoptotic pathway. Such an obvious in vitro anti-proliferative effect encouraged us to conduct an in vivo study on nude mice inoculated with A549 cells.
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3.5. TXA9 inhibits the growth of lung carcinoma xenografts
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The potential in vivo anti-tumor efficacy of TXA9 was investigated in s.c. xenografts of human lung A549 adenocarcinoma cells in nude mice. When the average tumors reached 100 mm3, the mice were treated with different doses of TXA9 thrice a week for four consecutive weeks, and taxol was used as a positive control. The tumor volume and body weight of each mouse were measured every 3 days, and a tumor growth curve was constructed for each group. The tumor volumes of the mice in the control group exhibited a rapid growth, with the mean value increasing from 142.2 ± 30.4 mm3 on Day 1 to 1011.1 ± 108.9 mm3 on Day 28. Compared with the control group, taxol (the positive control) produced a markedly slow growth, with the mean value increasing from 170.7 ± 25.0 mm3 on Day 1 to 424.2 ± 43.4 mm3 on Day 28. Similar results were obtained for the mice treated with HTXA9 (15 mg kg 1 of TXA9): the mean tumor volume increased from 152.3 ± 18.7 mm3 to 343.9 ± 98.0 mm3. In addition, the tumor volume of the mice in the HTXA9 group was significantly different compared with that of the control on Day 13 and from Days 19 to 28, whereas the tumor volume in the positive control group displayed a significant difference from Days 19 to 28. These results confirm the time- and dose-dependent inhibitory effect of TXA9 on tumor volume over the intervention period of 28 days. According to the curve (Fig. 5A), HTXA9 exhibited slightly better anti-proliferation activity compared with taxol, although the difference was not significant. Although the volumes in the LTXA9 (5 mg kg 1 of TXA9) and MTXA9 (10 mg kg 1 of TXA9) groups exhibited slow proliferation, with the mean volumes increasing from 146.7 ± 26.4 mm3 to 503.0 ± 124.1 mm3 and from 3 156.7 ± 21.5 mm to 467.7 ± 67.5 mm3, respectively, there was no significant difference compared with the control. This finding indicates that the i.v. administration of HTXA9 was effective.
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Fig. 2. The morphological changes (I) and flow cytometry analysis by annexin V and propidium iodide staining of A549 cells treated with TXA9 (II). The cells were incubated in the medium alone for 24 h (A), and in medium containing 600 nM TXA9 for 24 h (B) (magnification = 200). An arrow indicates the multi-blebbing cells and apoptotic bodies. The scale bar represents 100 lm. A549 cells incubated in the medium alone for 24 h (C) (magnification = 200) and in medium containing 600 nM TXA9 for 24 h (D) (magnification = 200) were subjected to AO staining. The scale bar represents 200 lm. Two white arrows note the distinct karyopyknosis and dense fluorescence of the cell nuclei, and crescent bodies are observed at the edge of the nuclear membrane. Annexin V and propidium iodide double assay for A549 cells treated with TXA9 for 0 h, 6 h, 12 h, and 24 h (E). The X-axis indicates the fluorescence intensity of the tumor cells bound with FITC-conjugated Annexin V, and the Y-axis indicates the fluorescence intensity of tumor cells bound with PI. UL: upper-left quadrant; UR: upper-right quadrant; LL: lower-left quadrant; LR: lower-right quadrant.
Fig. 3. Apoptosis and cell cycle arrest are stimulated by TXA9 in A549 cells, n = 3. Means ± S.D. ⁄P < 0.05 and ⁄⁄P < 0.01 compared with the SubG0/G1 phase percentage in the groups not treated with TXA9; ##P < 0.01 compared with the G2/M phase percentage in the groups not treated with TXA9. 382 383 384 385 386 387 388 389 390 391 392 393
The mean values of the final tumor weights are shown in Fig. 5B. Clearly, the taxol and HTXA9 groups exhibited significantly inhibited tumor growth compared with the control group, while inhibitory rates of 62.5% (P = 0.045) and 64.2% (P = 0.037), respectively. However, the P values for the tumor weights of the LTXA9 and MTXA9 groups were approximately close to 0.05 (0.054 and 0.057, respectively). The inhibition rates of these two groups were calculated to be 58.4% for LTXA9 and 57.5% for MTXA9. Thus, TXA9 can induce tumor inhibition rates similar to those induced by taxol not only in the tumor volumes but also in the tumor weight. Photographs of representative tumors excised on Day 28 are presented in Fig. 6A.
In addition, according to the histopathological examination of the tumors under a microscope (Fig. 6B), closely spaced tumor cells with hyperchromatic nuclei could be clearly observed in the control group, which demonstrates the rapid growth of the tumor cells. However, the tumor cells in the taxol and HTXA9 groups exhibited marked degeneration, disruption, and death, especially in the HTXA9 group. Hence, the i.v. administration of HTXA9 resulted in a significant inhibition of tumor growth.
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3.6. Toxicity evaluation of TXA9 treatment
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Q3 3.6.1. Animal body weight and organ/body weight index The purpose of this study was not only to evaluate the in vivo anti-proliferation activity of TXA9 but also to investigate the potential toxic side effects of this compound. The mice body weights were monitored throughout the treatment period and were measured thrice a week. At the end of the research period, the organs were collected and weighed. The data obtained are expressed as the organ index, which is the ratio between the organ weight (mg) and the body weight (g). During the treatment, an obvious reduction in body weight could be observed in the taxol group (Fig. 7A) compared with the control group (Day 16, P = 0.046; Day 28, P = 0.051). In addition, the body weights of the mice in all of the TXA9-treated group slowly increased during the experimental period, which was similar to the trend observed for the control group. In particular, under the same dosage, the average body weight showed different variation trends between TXA9 and taxol, indicating that TXA9 does not exert any toxic effects. In contrast, small changes in the organ indices (including heart, liver, spleen, lung, and kidney) were observed in the mice in the control, taxol, and TXA9-treated groups (Fig. 7B), which demonstrates that the functions of these organs were not markedly
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Fig. 4. Death receptor apoptotic pathway is activated by TXA9. A549 cells were lysed after treatment with 600 nM TXA9 for different durations, and the whole-cell lysates were evaluated by Western blotting. The protein levels of Fas, FADD, and caspases-8 were examined after treatment with TXA9 (A). TXA9 treatment cleaved procaspase-3 and its death substrates (ICAD) in A549 cells (B). A549 cells were treated with 600 nM TXA9 for different durations, and the expression levels of caspase-3 and ICAD were examined by Western blotting analyses.
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affected by treatment with taxol or TXA9. Hence, the TXA9-treated mice did not exhibit a significant (P > 0.05) body weight loss or reduction in organ indices compared with the control mice. Additionally, these results illustrate that TXA9 is well tolerated at a dose that exhibits significant in vivo anti-tumor activity. 3.6.2. Histology of main organs The major mice organs were collected from mice in the HTXA9, taxol, and control groups for histopathological examination. The hepatic cells in these three groups all exhibited minor cloudy swellings and degenerations (Fig. 7C), which indicates that minor hepatic injury was caused by the vehicle (5% propylene glycol and 5% ethanol/saline). The analysis of the sections of the kidney revealed that the renal tubules of the mice in the taxol and control groups exhibited partial degeneration, whereas this effect was not observed in the HTXA9-treated mice (Fig. 7D), which suggested that TXA9 is safe to the bodies. The other organs, such as the heart, lung, and spleen, all appeared normal, and there were no obvious pathological changes between any of the groups (Fig. 7E–G). 3.6.3. Hematology and serum biochemistry Routine blood examinations were performed at the end of the experiment, and these examinations included counts of the red blood cells (RBCs), white blood cells (WBCs), and platelets (PLT). Both taxol and TXA9 had no effect on the RBC count compared with
Fig. 5. In vivo inhibitory activity of TXA9 on A549 tumor growth. TXA9 and taxol were dissolved in the vehicle (5% propylene glycol and 5% ethanol/saline), and mice were treated intravenously three times a week for four consecutive weeks with vehicle, taxol (5 mg kg 1), LTXA9 (5 mg kg 1), MTXA9 (10 mg kg 1), or HTXA9 (15 mg kg 1). Taxol was used as a positive control. Both HTXA9 and taxol significantly inhibited the tumor growth of A549 lung carcinoma xenografts (A). ⁄ P < 0.05, indicating that the HTXA9 group was significantly different from the control group; #P < 0.05, indicating that the taxol group is significantly different from the control group. End-point (28 days) tumor weight of the mice treated with taxol and different doses of TXA9 (B). The data are reported as the means ± SEM (A) or means ± SD (B). ⁄P < 0.05 compared with the control group. The P values for each group are the following: taxol, 0.045; LTXA9, 0.054; MTXA9, 0.057; and HTXA9, 0.037.
the control (Table 2), but these treatments caused small reductions in the WBC and PLT counts. However, only taxol induced a significant decrease in the PLT count, which decreased by 40.5%. This marked reduction in platelets may be due to marrow suppression corresponding to the well-known clinical side effect of taxol. The serum biochemical parameters of the mice were also evaluated. The levels of AST, ALT, BUN, Crea, TP, AKP, LDH, and TBIL clearly reflected the hepatic, renal, and cardiac functions, which is why these parameters are widely used in clinical diagnosis. No significant differences in the levels of these indices were observed between all of the groups (data not shown), showing that the treatments did not produce any significant changes in the main organ functions. This finding was also in accordance with the assessment of the organ/body weights described above.
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NSCLC, the main form of lung cancer, is the leading cause of death from cancer in the world [20,21]. Thus, the development of new drugs against NSCLC is of utmost importance in the field of drug discovery. Plant-based drug discovery has resulted mainly in the development of anticancer agents and continues to contribute to new leads in clinical trials. Taxol is a typical and excellent example of a widely used first-line broad-spectrum anticancer drug in clinical situations, including NSCLC. However, its wellknown side-effects, such as marrow suppression, anaphylaxis, and neurotoxicity, as well as the development of resistance to taxol, had limited its application.
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Fig. 6. Photographs of typical tumors xenografts from the mice in the control, taxol, and HTXA9 groups (A). Microstructures of A549 cells in the control and treatment groups (B).
Fig. 7. Treatment with TXA9 does not cause any significant toxicity in nude mice bearing A549 lung carcinoma xenografts. Body weights of the mice during treatment from Days 0 to 28 (A). ⁄P < 0.05 compared with the control group. Organ indices of mice at the end of the treatment period (B). The data are reported as the means ± SD. Histopathological examination of visceral organs from mice at the end of the treatment period after staining with H&E. Photographs of liver (C), kidney (D), heart (E), lung (F), and spleen (G) sections are shown (magnification = 400). The arrow indicates partial degeneration in the cells.
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The current study was conducted to examine the anti-tumor activity and toxicological effects of TXA9, a cardiac glycoside, which was obtained from SJ through bioactivity-guided fractionation in a preliminary study. Prior to the present study, there had been no detailed investigation of its anti-tumor potential. The analysis of the in vitro anti-proliferative effect of TXA9 on many NSCLC cell lines was compared with that of taxol. The results revealed that TXA9 exhibits inhibitory effects against the A549, Ltep-a2,
and PC-9 cell lines similar to those of taxol, more potent effects on NCI-H1299 and Lu99 cell lines compared with taxol, and no toxic effects against normal cells. To identify whether it remains active in vivo, this compound was administered to nude mice inoculated with human lung A549 adenocarcinoma cells. Because there are no preliminary in vivo studies of TXA9, an acute toxicity test was first performed, to determine a safe dose. Due to its poor solubility, the highest dose possible in the vehicle (5% propylene
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Table 2 Hematological indices of the mice in the control, taxol, and TXA9 groups. Groups
WBC (109/L)
RBC (1012/L)
PLT (109/L)
Control Taxol LTXA9 MTXA9 HTXA9
8.34 ± 3.99 6.13 ± 2.54 6.78 ± 2.92 6.55 ± 1.40 7.37 ± 2.61
9.96 ± 0.34 9.24 ± 1.12 9.97 ± 0.33 9.54 ± 0.62 9.90 ± 0.43
637.57 ± 183.96 379.50 ± 75.31⁄ 499.25 ± 151.99 481.60 ± 195.99 560.25 ± 174.31
The values are expressed as means ± SD. ⁄P < 0.05 compared with the control group. Taxol was used as a positive control.
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glycol and 5% ethanol/saline) was 25 mg kg 1, which did not cause any deaths after 7 days. The maximum dosage of TXA9 administered i.v. was confirmed to be 25 mg kg 1. To investigate the in vivo anti-tumor effect of TXA9, three doses ranging from 1/5 to 3/5 of the maximum administration dosage were used, and the dose of taxol (5 mg kg 1) used is known to be safe in mice [22]. All of the treatments produced tumor growth inhibition with the following rates: 62.5% (taxol, P = 0.045), 58.4% (LTXA9, P = 0.054), 57.5% (MTXA9, P = 0.057), and 64.2% (HTXA9, P = 0.037). However, taxol had a significant reducing effect on the body weight growth and PLT count, which were effects that were not observed in the TXA9-treated mice. The results indicate that TXA9 can induce tumor inhibition rate similar to that induced by taxol not only in the tumor volumes but in the tumor weight with slight side effects. Although TXA9 exhibited anti-proliferative activity similar to that of taxol in vitro, the in vivo evaluation showed weaker effects with the same dose. This phenomenon is likely due to the pharmacokinetic characteristics of TXA9, and our preliminary study showed that TXA9 may be rapidly distributed and eliminated after i.v. injection (data not shown). Consequently, the administration of this treatment thrice a week was unable to maintain a sufficiently high plasma concentration of TXA9 to provide a significant effect in vivo. This finding agreed with the results of a previous report on the pharmacokinetics of periplocin (another cardiac glycoside) in rats, which showed its rapid distribution and elimination in rats after i.v. injection [23]. However, it is well known that CGs always have a significantly long biological half-life and accumulation in clinical use; for example, digitoxin has an average half-life of four to 6 days [24]. This finding shows that compounds with similar chemical structures may possess markedly different in vivo pharmacokinetic properties even if they exhibit similar in vitro activity. This interesting result will be confirmed through further research, and its better promising in vivo anti-tumor effect will be explored in detail. Regarding the anti-tumor mechanism, numerous studies have shown that CGs can induce either apoptosis or cell death in cancer cells through multiple pathways. Apoptosis is a cell-autonomous death process that requires the active participation of endogenous cellular enzymes for cell disassembly through two major pathways: the death-receptor-induced extrinsic pathway and the mitochondrial-apoptosome-mediated intrinsic pathway [25]. In the present study, it was shown that TXA9 can inhibit the proliferation of A549 cells in a dose- and time-dependent manner (Fig. 1B), and this inhibitory effect was related with apoptosis, through the extrinsic pathway, as was demonstrated by the following analyses. Apoptotic cells possess unique morphological and biochemical characteristics, including membrane blebbing, cytoplasmic shrinkage, chromatin condensation, and the formation of apoptotic bodies [26]. In our study, the typical apoptotic morphological changes in A549 cells could be observed clearly under the microscope (Fig. 2B and D). The flow cytometry analysis demonstrated that TXA9 can induce A549 cell apoptosis after treatment for 24 h (Fig. 2E) and also induce cell cycle arrest in SubG0/1 and G2/M periods (Fig. 3).
In contrast, Fas is a member of the TNF receptor family that activates apoptosis upon binding of the Fas ligand. The interaction between the Fas receptor and its ligands plays an important role in the regulation of apoptosis. In the extrinsic apoptotic pathway, Fas binds to the Fas ligand to activate caspase-8 and caspase-3 in order to execute cell apoptosis. This process is mediated by a death-inducing signaling complex that consists of FADD and procaspase-8, which produces caspase-8 and leads to the activation of downstream caspases [27]. Of these caspases, caspase-3 is one of the critical effector caspases in the downstream execution of apoptosis [28]. In our study, we found that A549 cells treated with TXA9 underwent apoptosis by activating a caspase cascade that involves the increased expression of Fas and FADD and the activation of the cleavage of procaspase-8 into caspase-8 (Fig. 4A). The procaspase-3 level then decreases, the caspase-3 levels increase and degrade ICAD (Fig. 4B). These findings indicate that TXA9 causes the activation of caspase-3, which cleaves the death substrate ICAD and ultimately leads to apoptosis. All of the abovedescribed results indicate that TXA9 induces apoptosis by activating the Fas death receptor pathway.
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5. Conclusions
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In summary, the present study provides the first demonstration of the remarkable in vitro and in vivo anti-tumor effects of TXA9 against NSCLC. The findings show that TXA9 exhibits more selective anti-proliferative activity against NSCLC cell lines than taxol in vitro. The in vivo studies demonstrate that a threefold higher dose of TXA9 shows a tumor inhibition rate similar to that obtained with taxol and is safer. The mechanistic studies show that TXA9 can stimulate apoptosis through the extrinsic pathway and cell cycle arrest in the SubG0/1 and G2/M periods. Therefore, therapy with strong anti-tumor activity and without any significant toxicity can be obtained using TXA9, and this is a promising development that will be followed up in clinical trials against NSCLC.
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Acknowledgments
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This work was financially supported by Grants from the National Natural Science and Technology Major Project ‘‘Key New Drug Creation and Manufacturing Program’’ (No. 2010ZX09401304) and the Natural Science Foundation of China (No. 30973958).
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Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids 6 7
TXA9, a cardiac glycoside from Streptocaulon juventas, exerts a potent anti-tumor activity against human non-small cell lung cancer cells in vitro and in vivo
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a Development and Utilization Key Laboratory of Northeast Plant Materials, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China b School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China c The Animal Experimental Center, Shenyang Pharmaceutical University, Shenyang 110016, China d The People’s Liberation Army 463 Hospital, Shenyang 110042, China e Department of Bioscience, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama, Japan
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Rui Xue a, Na Han a, Mingyu Xia b, Chun Ye a, Zhihui Hao c, Lihui Wang b, Yu Wang d, Jingyu Yang b, Ikuo Saiki e, Jun Yin a,⇑
a r t i c l e Q2
i n f o
Article history: Received 6 May 2014 Received in revised form 2 October 2014 Accepted 18 December 2014 Available online xxxx Keywords: Acovenosigenin A 3-O-b-D-glucoside Anti-tumor NSCLC Streptocaulon juventas Cardiac glycosides Apoptosis
a b s t r a c t Non-small cell lung cancer is the most common type of lung cancer and the most common cause of cancer-related death in humans. TXA9, which is a natural product separated from an anti-tumor-active fraction of the roots of Streptocaulon juventas, may possess potent anti-proliferative activity according to the present study. In this study, the anti-tumor effects and toxicity of TXA9 were tested against human nonsmall cell lung cancer cell lines (A549, NCI-H1299, Ltep-a2, PC-9, and Lu99) and a normal human lung embryonic fibroblast cell (HE-lung) in vitro, and then toward A549 cells in vivo in a murine xenograft model. The results show that TXA9 exhibits potent cytotoxic activities against non-small lung cancer cells and has no toxic effect on the normal human lung embryonic fibroblast cells. The mechanistic studies demonstrate that TXA9 can induce the apoptosis of A549 cells through the extrinsic pathway. The in vivo study results reveal that the intravenous administration of TXA9 at high-dose (15 mg kg 1) induces significant tumor growth inhibition of non-small cell lung cancer xenografts with tumor inhibition rate up to 64.2%, compared with mice in the control group. The inhibitory effect was similar to that of taxol (62.5%). In particular, no significantly adverse effects were exerted by TXA9, which suggests that it is well tolerated. This promising natural product may be useful as a potential novel anti-tumor candidate. Ó 2014 Published by Elsevier Inc.
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1. Introduction Non-small cell lung cancer (NSCLC), accounts for approximately 85% of all lung cancers, and almost 65–70% of NSCLC patients preAbbreviations: NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; CGs, cardiac glycosides; SJ, Streptocaulon juventas; MTT, 3-(4,5-dimethylibiazol-2yl)-2,5-diphenyl-tetrazolium bromide; AO, acridine orange; PI, propidium iodide; i.v., intravenous; LTXA9, TXA9 at a dose of 5 mg kg 1; MTXA9, TXA9 at a dose of 10 mg kg 1; HTXA9, TXA9 at a dose of 15 mg kg 1; RBC, red blood cell; WBC, white blood cell; PLT, platelets; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; LDH, lactic dehydrogenase; BUN, blood urea nitrogen; TBIL, total bilirubin; Crea, creatinine; TP, total protein; FADD, Fasassociated death domain. ⇑ Corresponding author at: School of Traditional Chinese Materia Medica 48#, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, China. Tel./fax: +86 24 23986491. E-mail address: [email protected] (J. Yin).
sent with advanced disease at the time of diagnosis [1]. The prognosis for NSCLC patients is very poor with a five-year survival rate of only 15%, and this malignancy remains difficult to treat [2]. Due to the limited therapeutic effect of standard chemotherapy, radiotherapy and surgery, no effective treatment has been developed to date. For many years, natural products have played a very important role in anticancer drug discovery and development because they offer a large structural diversity [3]. As a result, both terrestrial and marine organisms are presently considered important sources of novel lead compounds. Well known for their positive inotropic effect, cardiac glycosides (CGs) have been used clinically for many years for the treatment of heart failure and atrial arrhythmias. Since the middle of the 1960s, initial observations from epidemiological studies have shown that cancer patients receiving cardiac glycosides exhibit significantly lower mortality rates than patients without treatment
http://dx.doi.org/10.1016/j.steroids.2014.12.015 0039-128X/Ó 2014 Published by Elsevier Inc.
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with such drugs. Consequently, it is generally accepted that CGs have a putative anti-cancer effect, and the anti-proliferative activity of CGs, such as digitoxin on human cancer cells has been widely reported [4–8]. However, it is well known that the most significant adverse effect of digitoxin, which is frequently encountered in clinical treatment, is cardiotoxicity [9–10]. The narrow therapeutic window dramatically limits the application of digitoxin, because its toxicity is a major concern whenever it is considered for therapy [11]. Therefore, it is necessary to discover new candidate drugs that exhibit a significant anti-tumor effect but low toxicity. Streptocaulon juventas (SJ) (Asclepiadaceae), which is mainly distributed in Southeast Asia, is used in folk medicine to stimulate the spleen, tonify the body, and strengthen the kidney [12]. We previously reported that the 75% ethanol extract of SJ exhibits potent inhibitory activity in vivo on the growth of the human lung A549 adenocarcinoma cell line with only mild adverse effects [13]. A subsequent phytochemical study of the active extract confirmed that cardiac glycosides contribute to the observed anti-tumor activity [14–15]. Among the numerous cardiac glycosides obtained, TXA9 was chosen as the lead compound for further research due to its potent in vitro anti-proliferative activity and relatively simple structure. In the present study, the potential anti-proliferative activities of TXA9 against NSCLC in vitro and in vivo were evaluated, and any possible treatment-related toxic effects were assessed through a series of hematology and serum biochemistry tests, investigations of the visceral organs, and monitoring of body weight. Additionally, the apoptosis mechanism of TXA9 on A549 cells was investigated.
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2.1. Ethics statement
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All of the animal care and experimental procedures were approved by the Animal Ethical Committee of Shenyang Pharmaceutical University. All tumor implantations, blood collections, and sacrifices were performed under anesthesia and all efforts were made to minimize suffering.
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2.2. Plant material
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2.4. Cell culture
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The human lung A549 adenocarcinoma cell line, human nonsmall cell lung carcinoma NCI-H1299 cell line, human lung PC-9 adenocarcinoma cell line, and lung giant cell Lu99 carcinoma cell line were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). The human lung Ltep-a2 adenocarcinoma cell line was ordered from Typical Culture Preservation Commission Cell Bank (Chinese Academy of Sciences, Shanghai, China). The HE-lung embryonic fibroblast cell line was provided by Prof. Ikuo Saiki (Department of Bioscience, Institute of Natural Medicine, University of Toyama, Sugitani, Toyama, Japan) as a gift. All of the cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in an atmosphere of 5% CO2 and 95% air.
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2.5. Cell proliferation
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The effect of TXA9 on cell proliferation was assessed using the 3-(4,5-dimethylibiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, 96-well microtitre plates were seeded with 5 103 cells per well and incubated for 24 h. Culture media containing different concentrations of the samples were then added. After incubation for 12 h, 24 h, and 48 h, 100 lL of MTT from a stock solution (0.5 mg/mL) was added to each well, and the plate was incubated for 4 h. The absorbance of the resulting formazan product was measured at 492 nm using a microplate reader (Tecan, Austria).
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2.6. Observation of cell morphological changes and acridine orange staining analysis
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The morphological changes were observed as described previously [16]. Briefly, A549 cells were cultured at a density of 8 105 cells/flask in 25-mm2 cell culture flasks. After 24 h of incubation, the cells were treated with 600 nM TXA9 and incubated for an additional 24 h. At the end of the incubation period, the morphological changes in the cells were observed through photomicrography using a phase-contrast microscope. Acridine orange (AO) staining was used as an indicator of cell apoptosis. A549 cells were plated at 3 105 cells/well in six-well plates. After 24 h of incubation, the cells were treated with TXA9 at a concentration of 600 nM and incubated for 24 h. The prepared cells were washed once with PBS. After 10 mg/L AO was added to the cells, the cells were incubated for 30 min at 37 °C and then observed under a fluorescence microscope at an excitation wavelength of 488 nm and an emission wavelength of 515 nm.
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Roots of SJ were collected in Simao, Yunnan Province in 2009. No specific permissions were required for the collections because the crude drug is wild and is not an endangered or protected species. The roots were identified by Prof. Jun Yin (Division of Pharmacognosy, Shenyang Pharmaceutical University), and voucher samples (SPU 2162-2165) were deposited at the herbarium of Shenyang Pharmaceutical University, Shenyang, China. 2.3. Preparation of TXA9
2.7. Flow cytometry analysis by annexin V and propidium iodide staining
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TXA9 was isolated from the roots of SJ collected in Yunnan Province of China in 2009. The air-dried roots were extracted with EtOH–H2O (75:25, v/v) for 1.5 h, and this process was repeated three times. The combined solution was concentrated under reduced pressure until there was no trace of alcohol and then extracted with n-butanol four times. The concentrated extract was then subjected to silica-gel column chromatography with CH2Cl2–MeOH (25:1, 12:1, 1:1, v/v) to yield three fractions (A, B and C). Fraction B was applied to a silica-gel column, eluted with EtOAc–Me2CO–H2O (10:1:0.1, v/v/v), subjected to ODS silica-gel column chromatography with MeOH–H2O (50:50, v/v), and recrystallized using MeOH to obtain TXA9 (purity: 99%).
Annexin V-FITC/PI double staining was employed for the quantitative determination of the percentage of apoptotic cells. A549 cell at a density of 3 106 cells in each dish (ID: 6 cm) were incubated overnight, and then treated with sample or vehicle. After incubation for 6, 12, or 24 h with TXA9, the cells were digested with trypsin and suspended with PBS. After centrifugation, the PBS was removed, and the cells were stained with FITC-conjugated annexin V and propidium iodide (PI) for 15 min at 20 °C in a Ca2+enriched binding buffer (apoptosis detection kit, R&D Systems, Abingdon, UK; 20). The cells were immediately analyzed with a flow cytometer in their staining solution (Becton Dickinson FACScan, Franklin Lakes, NJ, USA).
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2.8. Cell cycle analysis by flow cytometry
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The degree of cell apoptosis was determined through forward/ side scatter analysis and PI staining using a FACScan flow cytometer (Becton Dickinson) equipped with an argon-ion laser as described previously [17]. Briefly, A549 cells were cultured in cell culture flasks and treated with 600 nM TXA9 for 12, 24, 36, and 48 h. The prepared cells were washed once with PBS, fixed in 70% ethanol overnight, washed once with PBS, and stained with PI solution (50 lg/mL PI and 50 lg/mL RNase A in PBS) for 30 min in the dark. The samples were then analyzed by flow cytometry.
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2.9. Western blotting analysis
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A549 cells were treated with 600 nM TXA9 for different time periods. Both adherent and floating cells were collected, and a Western blotting analysis was then performed as previously described [17]. Equal amounts of total proteins were separated by 12% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk in NaCl/Tris-T (10 mM Tris/HCl, pH 8.0, 150 mM NaCl and 0.05% Tween 20) buffer for 1 h at room temperature and then incubated at 4 °C overnight with primary antibodies against caspase 3, caspase 8, Fas, and FADD (Santa Cruz, CA, USA). After several washes, the membranes were then incubated with the corresponding HRP-conjugated secondary antibodies at room temperature for 1 h and visualized with the enhanced chemiluminescence detection system (GE Healthcare, USA).
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of treatment, all of the mice were killed, and their tumors and organs were removed, weighed, and then fixed in 10% formalin, embedded in paraplast, cut into 4-lm-thick sections, and stained with hematoxylin and eosin (H&E) for histopathological examination. The slices were investigated under the microscope.
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2.12. Efficacy and toxicity of TXA9
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The evaluation of efficacy and toxicity of TXA9 in nude mice was based on the analyses of tumor growth curves, tumor weight, organ indices as described previously [13]. Blood of each mouse was collected, then the hematological parameters were determined using a Y-159 blood analyzer (Nihon Kehden, Japan), and the serum was used to measure various biochemical indexes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactic dehydrogenase (LDH), blood urea nitrogen (BUN), total bilirubin (TBIL), creatinine (Crea), and total protein (TP), using the appropriate reagent kits and an ELISA reader (Tecan, Australia).
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2.13. Statistical analyses
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The in vitro experiments were repeated at least three times. The results are expressed as the means ± SD or means ± SEM. The statistical analyses were performed using one-way analysis of variance followed by the LSD test under the condition of variance homogeneity and Dunnett’s T3 test under the condition of variance heterogeneity. Statistical significance was considered to be obtained at P < 0.05.
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2.10. Animal and tumor xenograft model
3. Results
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3.1. TXA9 shows potent anti-proliferative activity against human nonsmall cell lung cancer cell lines
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2.11. Treatment
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When the average tumor size reached 100 mm3, the animals were divided into five groups (ten animals per group) and administered the following treatments: (A) control [vehicle (5% propylene glycol and 5% ethanol/saline), i.v.]; (B) taxol (5 mg kg 1, i.v.); (C) low dose of TXA9 (LTXA9, 5 mg kg 1, i.v.); (D) medial dose of TXA9 (MTXA9, 10 mg kg 1, i.v.) and (E) high dose of TXA9 (HTXA9, 15 mg kg 1, i.v.). Taxol and TXA9 were dissolved in 5% propylene glycol and 5% ethanol/saline. All of the compounds were injected three times a week for four consecutive weeks, starting on the 14th day after the tumor graft. Three times a week, each mouse was weighed, and the tumor volume was measured. After 28 days
On the basis of spectroscopic studies and comparison with a reference [18], the structure of TXA9 was elucidated as acovenosigenin A 3-O-b-D-glucoside (Fig. 1A). In the initial study, the antiproliferative activity of TXA9 against five types of human nonsmall cell lung cancer cell lines and one normal human lung cell line was first evaluated through the MTT assay, using taxol as a positive control. TXA9 exhibited inhibitory effects against A549 (IC50: 0.11 lM), Ltep-a2 (IC50: 0.03 lM), and PC-9 (IC50: 0.44 lM) cell lines similar to those of taxol (IC50: A549, 0.16 lM; Ltep-a2, 0.01 lM; and PC-9, 1.25 lM). However, the test compound showed more potent growth inhibition activities than taxol on the NCIH1299 (TXA9: 0.006 lM and taxol: 0.48 lM) and Lu99 (TXA9: 0.32 lM and taxol: 25.6 lM) cell lines. This promising result demonstrates that TXA9 can selectively inhibit the growth of tumor cell lines but has no toxic effect against normal cells (IC50 > 100 lM) (Table 1). Moreover, this finding also illustrates that TXA9 exhibits potent, dose- and time-dependent anti-proliferative activity at 12 h, 24 h, and 48 h (Fig. 1B). Based on these results, 0.53 lM was calculated as the IC50 value for TXA9 at 24 h and 600 nM was chosen for the subsequent mechanism studies. The analysis of the morphological changes in A549 cells [Fig. 2(I)] showed that shrinking or multi-blebbing cell shapes and the presence of apoptotic bodies are clearly observed in the cells treated with TXA9 at a concentration of 600 nM (Fig. 2B), which were quite different to those of the control (Fig. 2A). These morphological changes are typical of apoptosis [19]. Furthermore, the morphological changes in the cell nuclei were monitored by AO staining. The cell nuclei exposed to TXA9 at a concentration of 600 nM displayed distinct karyopyknosis and dense fluorescence, and the crescent bodies could be easily observed at the edge of
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Six-week-old female BALB/c nu/nu mice (weight: 18–20 g) purchased from the Animal Experiment Center of Shenyang Pharmaceutical University were maintained under pathogen-free conditions and fed irradiated chow. After an acclimation period of approximately 7 days, tumor inoculation was performed. A549 cells were harvested and resuspended at a final concentration of 3 107 cells/mL in PBS and Matrigel™ Matrix (BD Biosciences, Two Oak Park, Bedford, MA, USA) at a 1:1 ratio. The mice were inoculated s.c. into the right flank with 3 106 cells (0.1 mL). The tumors were measured with a vernier caliper every 3 days, and their volumes were calculated as described previously [13]. Before administration, TXA9 was subjected to an acute toxicity study to determine the optimal dose. This dose was determined through the intravenous (i.v.) administration of a single dose to healthy mice that had not received a tumor graft. The survival of the animals was monitored up to 7 days. Four different doses of TXA9 (i.e., 4.2, 10, 15 and 25 mg kg 1) were used to determine the index. The maximum administration dosage of TXA9 via the i.v. route was found to be 25 mg kg 1.
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Fig. 1. Chemical structure of TXA9 (A) and its in vitro growth-inhibitory activities against A549 cells (B). TXA9 was administered at six concentrations ranging from 3 to 480 nM, and each concentration was analyzed three times. The cells were cultured for 12 h, 24 h, and 48 h. The results are presented as the growth inhibitory rate of the cells. All of the experiments were performed three times to validate the results.
Table 1 The cytotoxic activities of TXA9 and taxol in vitro. Compounds
TXA9 Taxol
IC50 (lM) A549
NCI-H1299
Ltep-a2
PC-9
Lu99
HE-lung
0.11 0.16
0.006 0.48
0.03 0.01
0.44 1.25
0.32 25.6
>100 80
All of the IC50 values were acquired from a 48-h MTT assay with at least three independent experiments. A549: human lung adenocarcinoma cell; NCI-H1299: human non-small cell lung carcinoma cell; Ltep-a2: human lung adenocarcinoma cell; PC-9: human lung adenocarcinoma cell; Lu99: lung giant cell carcinoma cell; HE-lung: HE-lung embryonic fibroblast cell. Taxol was used as a positive control.
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the nuclear membrane (Fig. 2D), whereas the cell nuclei without TXA9 treatment exhibited a smooth surface and homogeneous fluorescence (Fig. 2C). All of these changes indicate that TXA9 may induce apoptosis in A549 cells. 3.2. TXA9 can induce apoptosis as determined by flow cytometry analysis
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To confirm the cell death type, the cells were stained with Annexin V and PI [Fig. 2(II)], and the cell death at 6 h, 12 h, and 24 h was analyzed by flow cytometry (Fig. 2E). As a result, the percentages of cells in the lower-right quadrant were 3.60%, 4.59%, and 25.99% after 6 h, 12 h, and 24 h respectively of treatment, respectively, showing that TXA9 can induce a significant increase in the percentage of apoptotic cells.
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3.3. TXA9 can induce cell cycle arrest in the SubG0/1 and G2/M periods
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The flow cytometric analysis of the cell cycle distribution displayed that the percentages of control cells in the SubG0/G1 phase was only 1.68%, and this percentage was obviously increased to
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3.51%, 13.29%, 25.33%, and 36.33% after treatment with 600 nM TXA9 for 12, 24, 36, and 48 h, respectively (Fig. 3). This finding further confirms that TXA9 can induce time-dependent apoptosis in A549 cells. In addition, compared with the percentage of control cells in the G2/M phase (12.78%), treatment with TXA9 for 12 and 24 h significantly increased the percentages of cells in this phase to 28.08% and 28.50%, respectively. This phenomenon indicates that TXA9 can cause a marked lever of G2/M cycle arrest in A549 cells. In association with the increased apoptosis rates, the G2/M phase percentage was decreased by treatment with TXA9 for 36 and 48 h. These results showed that TXA9 can obviously induce A549 cell cycle arrest at the G2/M periods within 24 h.
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3.4. TXA9 can induce apoptosis in A549 cells through the death receptor pathway
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Analyses of the cell lysates by Western blotting were performed to evaluate the protein expression of A549 cells after treatment with TXA9. After treatment with 600 nM TXA9 for 12, 24, 36, and 48 h, the protein levels of Fas and Fas-associated death domain (FADD), which can activated the cascade reaction of apoptosis via the extrinsic pathway, were up-regulated in a time-dependent manner. Additionally, TXA9 was found to time-dependently cleave procaspase-8 and procaspase-3 to the active forms, namely caspase-8 and caspase-3, respectively, as well as its death substrates (ICAD) (Fig. 4A and B). All of these lines of evidence prove that TXA9 activates the extrinsic apoptotic pathway. Such an obvious in vitro anti-proliferative effect encouraged us to conduct an in vivo study on nude mice inoculated with A549 cells.
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3.5. TXA9 inhibits the growth of lung carcinoma xenografts
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The potential in vivo anti-tumor efficacy of TXA9 was investigated in s.c. xenografts of human lung A549 adenocarcinoma cells in nude mice. When the average tumors reached 100 mm3, the mice were treated with different doses of TXA9 thrice a week for four consecutive weeks, and taxol was used as a positive control. The tumor volume and body weight of each mouse were measured every 3 days, and a tumor growth curve was constructed for each group. The tumor volumes of the mice in the control group exhibited a rapid growth, with the mean value increasing from 142.2 ± 30.4 mm3 on Day 1 to 1011.1 ± 108.9 mm3 on Day 28. Compared with the control group, taxol (the positive control) produced a markedly slow growth, with the mean value increasing from 170.7 ± 25.0 mm3 on Day 1 to 424.2 ± 43.4 mm3 on Day 28. Similar results were obtained for the mice treated with HTXA9 (15 mg kg 1 of TXA9): the mean tumor volume increased from 152.3 ± 18.7 mm3 to 343.9 ± 98.0 mm3. In addition, the tumor volume of the mice in the HTXA9 group was significantly different compared with that of the control on Day 13 and from Days 19 to 28, whereas the tumor volume in the positive control group displayed a significant difference from Days 19 to 28. These results confirm the time- and dose-dependent inhibitory effect of TXA9 on tumor volume over the intervention period of 28 days. According to the curve (Fig. 5A), HTXA9 exhibited slightly better anti-proliferation activity compared with taxol, although the difference was not significant. Although the volumes in the LTXA9 (5 mg kg 1 of TXA9) and MTXA9 (10 mg kg 1 of TXA9) groups exhibited slow proliferation, with the mean volumes increasing from 146.7 ± 26.4 mm3 to 503.0 ± 124.1 mm3 and from 3 156.7 ± 21.5 mm to 467.7 ± 67.5 mm3, respectively, there was no significant difference compared with the control. This finding indicates that the i.v. administration of HTXA9 was effective.
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Fig. 2. The morphological changes (I) and flow cytometry analysis by annexin V and propidium iodide staining of A549 cells treated with TXA9 (II). The cells were incubated in the medium alone for 24 h (A), and in medium containing 600 nM TXA9 for 24 h (B) (magnification = 200). An arrow indicates the multi-blebbing cells and apoptotic bodies. The scale bar represents 100 lm. A549 cells incubated in the medium alone for 24 h (C) (magnification = 200) and in medium containing 600 nM TXA9 for 24 h (D) (magnification = 200) were subjected to AO staining. The scale bar represents 200 lm. Two white arrows note the distinct karyopyknosis and dense fluorescence of the cell nuclei, and crescent bodies are observed at the edge of the nuclear membrane. Annexin V and propidium iodide double assay for A549 cells treated with TXA9 for 0 h, 6 h, 12 h, and 24 h (E). The X-axis indicates the fluorescence intensity of the tumor cells bound with FITC-conjugated Annexin V, and the Y-axis indicates the fluorescence intensity of tumor cells bound with PI. UL: upper-left quadrant; UR: upper-right quadrant; LL: lower-left quadrant; LR: lower-right quadrant.
Fig. 3. Apoptosis and cell cycle arrest are stimulated by TXA9 in A549 cells, n = 3. Means ± S.D. ⁄P < 0.05 and ⁄⁄P < 0.01 compared with the SubG0/G1 phase percentage in the groups not treated with TXA9; ##P < 0.01 compared with the G2/M phase percentage in the groups not treated with TXA9. 382 383 384 385 386 387 388 389 390 391 392 393
The mean values of the final tumor weights are shown in Fig. 5B. Clearly, the taxol and HTXA9 groups exhibited significantly inhibited tumor growth compared with the control group, while inhibitory rates of 62.5% (P = 0.045) and 64.2% (P = 0.037), respectively. However, the P values for the tumor weights of the LTXA9 and MTXA9 groups were approximately close to 0.05 (0.054 and 0.057, respectively). The inhibition rates of these two groups were calculated to be 58.4% for LTXA9 and 57.5% for MTXA9. Thus, TXA9 can induce tumor inhibition rates similar to those induced by taxol not only in the tumor volumes but also in the tumor weight. Photographs of representative tumors excised on Day 28 are presented in Fig. 6A.
In addition, according to the histopathological examination of the tumors under a microscope (Fig. 6B), closely spaced tumor cells with hyperchromatic nuclei could be clearly observed in the control group, which demonstrates the rapid growth of the tumor cells. However, the tumor cells in the taxol and HTXA9 groups exhibited marked degeneration, disruption, and death, especially in the HTXA9 group. Hence, the i.v. administration of HTXA9 resulted in a significant inhibition of tumor growth.
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3.6. Toxicity evaluation of TXA9 treatment
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Q3 3.6.1. Animal body weight and organ/body weight index The purpose of this study was not only to evaluate the in vivo anti-proliferation activity of TXA9 but also to investigate the potential toxic side effects of this compound. The mice body weights were monitored throughout the treatment period and were measured thrice a week. At the end of the research period, the organs were collected and weighed. The data obtained are expressed as the organ index, which is the ratio between the organ weight (mg) and the body weight (g). During the treatment, an obvious reduction in body weight could be observed in the taxol group (Fig. 7A) compared with the control group (Day 16, P = 0.046; Day 28, P = 0.051). In addition, the body weights of the mice in all of the TXA9-treated group slowly increased during the experimental period, which was similar to the trend observed for the control group. In particular, under the same dosage, the average body weight showed different variation trends between TXA9 and taxol, indicating that TXA9 does not exert any toxic effects. In contrast, small changes in the organ indices (including heart, liver, spleen, lung, and kidney) were observed in the mice in the control, taxol, and TXA9-treated groups (Fig. 7B), which demonstrates that the functions of these organs were not markedly
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Fig. 4. Death receptor apoptotic pathway is activated by TXA9. A549 cells were lysed after treatment with 600 nM TXA9 for different durations, and the whole-cell lysates were evaluated by Western blotting. The protein levels of Fas, FADD, and caspases-8 were examined after treatment with TXA9 (A). TXA9 treatment cleaved procaspase-3 and its death substrates (ICAD) in A549 cells (B). A549 cells were treated with 600 nM TXA9 for different durations, and the expression levels of caspase-3 and ICAD were examined by Western blotting analyses.
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affected by treatment with taxol or TXA9. Hence, the TXA9-treated mice did not exhibit a significant (P > 0.05) body weight loss or reduction in organ indices compared with the control mice. Additionally, these results illustrate that TXA9 is well tolerated at a dose that exhibits significant in vivo anti-tumor activity. 3.6.2. Histology of main organs The major mice organs were collected from mice in the HTXA9, taxol, and control groups for histopathological examination. The hepatic cells in these three groups all exhibited minor cloudy swellings and degenerations (Fig. 7C), which indicates that minor hepatic injury was caused by the vehicle (5% propylene glycol and 5% ethanol/saline). The analysis of the sections of the kidney revealed that the renal tubules of the mice in the taxol and control groups exhibited partial degeneration, whereas this effect was not observed in the HTXA9-treated mice (Fig. 7D), which suggested that TXA9 is safe to the bodies. The other organs, such as the heart, lung, and spleen, all appeared normal, and there were no obvious pathological changes between any of the groups (Fig. 7E–G). 3.6.3. Hematology and serum biochemistry Routine blood examinations were performed at the end of the experiment, and these examinations included counts of the red blood cells (RBCs), white blood cells (WBCs), and platelets (PLT). Both taxol and TXA9 had no effect on the RBC count compared with
Fig. 5. In vivo inhibitory activity of TXA9 on A549 tumor growth. TXA9 and taxol were dissolved in the vehicle (5% propylene glycol and 5% ethanol/saline), and mice were treated intravenously three times a week for four consecutive weeks with vehicle, taxol (5 mg kg 1), LTXA9 (5 mg kg 1), MTXA9 (10 mg kg 1), or HTXA9 (15 mg kg 1). Taxol was used as a positive control. Both HTXA9 and taxol significantly inhibited the tumor growth of A549 lung carcinoma xenografts (A). ⁄ P < 0.05, indicating that the HTXA9 group was significantly different from the control group; #P < 0.05, indicating that the taxol group is significantly different from the control group. End-point (28 days) tumor weight of the mice treated with taxol and different doses of TXA9 (B). The data are reported as the means ± SEM (A) or means ± SD (B). ⁄P < 0.05 compared with the control group. The P values for each group are the following: taxol, 0.045; LTXA9, 0.054; MTXA9, 0.057; and HTXA9, 0.037.
the control (Table 2), but these treatments caused small reductions in the WBC and PLT counts. However, only taxol induced a significant decrease in the PLT count, which decreased by 40.5%. This marked reduction in platelets may be due to marrow suppression corresponding to the well-known clinical side effect of taxol. The serum biochemical parameters of the mice were also evaluated. The levels of AST, ALT, BUN, Crea, TP, AKP, LDH, and TBIL clearly reflected the hepatic, renal, and cardiac functions, which is why these parameters are widely used in clinical diagnosis. No significant differences in the levels of these indices were observed between all of the groups (data not shown), showing that the treatments did not produce any significant changes in the main organ functions. This finding was also in accordance with the assessment of the organ/body weights described above.
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4. Discussion
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NSCLC, the main form of lung cancer, is the leading cause of death from cancer in the world [20,21]. Thus, the development of new drugs against NSCLC is of utmost importance in the field of drug discovery. Plant-based drug discovery has resulted mainly in the development of anticancer agents and continues to contribute to new leads in clinical trials. Taxol is a typical and excellent example of a widely used first-line broad-spectrum anticancer drug in clinical situations, including NSCLC. However, its wellknown side-effects, such as marrow suppression, anaphylaxis, and neurotoxicity, as well as the development of resistance to taxol, had limited its application.
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Fig. 6. Photographs of typical tumors xenografts from the mice in the control, taxol, and HTXA9 groups (A). Microstructures of A549 cells in the control and treatment groups (B).
Fig. 7. Treatment with TXA9 does not cause any significant toxicity in nude mice bearing A549 lung carcinoma xenografts. Body weights of the mice during treatment from Days 0 to 28 (A). ⁄P < 0.05 compared with the control group. Organ indices of mice at the end of the treatment period (B). The data are reported as the means ± SD. Histopathological examination of visceral organs from mice at the end of the treatment period after staining with H&E. Photographs of liver (C), kidney (D), heart (E), lung (F), and spleen (G) sections are shown (magnification = 400). The arrow indicates partial degeneration in the cells.
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The current study was conducted to examine the anti-tumor activity and toxicological effects of TXA9, a cardiac glycoside, which was obtained from SJ through bioactivity-guided fractionation in a preliminary study. Prior to the present study, there had been no detailed investigation of its anti-tumor potential. The analysis of the in vitro anti-proliferative effect of TXA9 on many NSCLC cell lines was compared with that of taxol. The results revealed that TXA9 exhibits inhibitory effects against the A549, Ltep-a2,
and PC-9 cell lines similar to those of taxol, more potent effects on NCI-H1299 and Lu99 cell lines compared with taxol, and no toxic effects against normal cells. To identify whether it remains active in vivo, this compound was administered to nude mice inoculated with human lung A549 adenocarcinoma cells. Because there are no preliminary in vivo studies of TXA9, an acute toxicity test was first performed, to determine a safe dose. Due to its poor solubility, the highest dose possible in the vehicle (5% propylene
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Table 2 Hematological indices of the mice in the control, taxol, and TXA9 groups. Groups
WBC (109/L)
RBC (1012/L)
PLT (109/L)
Control Taxol LTXA9 MTXA9 HTXA9
8.34 ± 3.99 6.13 ± 2.54 6.78 ± 2.92 6.55 ± 1.40 7.37 ± 2.61
9.96 ± 0.34 9.24 ± 1.12 9.97 ± 0.33 9.54 ± 0.62 9.90 ± 0.43
637.57 ± 183.96 379.50 ± 75.31⁄ 499.25 ± 151.99 481.60 ± 195.99 560.25 ± 174.31
The values are expressed as means ± SD. ⁄P < 0.05 compared with the control group. Taxol was used as a positive control.
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glycol and 5% ethanol/saline) was 25 mg kg 1, which did not cause any deaths after 7 days. The maximum dosage of TXA9 administered i.v. was confirmed to be 25 mg kg 1. To investigate the in vivo anti-tumor effect of TXA9, three doses ranging from 1/5 to 3/5 of the maximum administration dosage were used, and the dose of taxol (5 mg kg 1) used is known to be safe in mice [22]. All of the treatments produced tumor growth inhibition with the following rates: 62.5% (taxol, P = 0.045), 58.4% (LTXA9, P = 0.054), 57.5% (MTXA9, P = 0.057), and 64.2% (HTXA9, P = 0.037). However, taxol had a significant reducing effect on the body weight growth and PLT count, which were effects that were not observed in the TXA9-treated mice. The results indicate that TXA9 can induce tumor inhibition rate similar to that induced by taxol not only in the tumor volumes but in the tumor weight with slight side effects. Although TXA9 exhibited anti-proliferative activity similar to that of taxol in vitro, the in vivo evaluation showed weaker effects with the same dose. This phenomenon is likely due to the pharmacokinetic characteristics of TXA9, and our preliminary study showed that TXA9 may be rapidly distributed and eliminated after i.v. injection (data not shown). Consequently, the administration of this treatment thrice a week was unable to maintain a sufficiently high plasma concentration of TXA9 to provide a significant effect in vivo. This finding agreed with the results of a previous report on the pharmacokinetics of periplocin (another cardiac glycoside) in rats, which showed its rapid distribution and elimination in rats after i.v. injection [23]. However, it is well known that CGs always have a significantly long biological half-life and accumulation in clinical use; for example, digitoxin has an average half-life of four to 6 days [24]. This finding shows that compounds with similar chemical structures may possess markedly different in vivo pharmacokinetic properties even if they exhibit similar in vitro activity. This interesting result will be confirmed through further research, and its better promising in vivo anti-tumor effect will be explored in detail. Regarding the anti-tumor mechanism, numerous studies have shown that CGs can induce either apoptosis or cell death in cancer cells through multiple pathways. Apoptosis is a cell-autonomous death process that requires the active participation of endogenous cellular enzymes for cell disassembly through two major pathways: the death-receptor-induced extrinsic pathway and the mitochondrial-apoptosome-mediated intrinsic pathway [25]. In the present study, it was shown that TXA9 can inhibit the proliferation of A549 cells in a dose- and time-dependent manner (Fig. 1B), and this inhibitory effect was related with apoptosis, through the extrinsic pathway, as was demonstrated by the following analyses. Apoptotic cells possess unique morphological and biochemical characteristics, including membrane blebbing, cytoplasmic shrinkage, chromatin condensation, and the formation of apoptotic bodies [26]. In our study, the typical apoptotic morphological changes in A549 cells could be observed clearly under the microscope (Fig. 2B and D). The flow cytometry analysis demonstrated that TXA9 can induce A549 cell apoptosis after treatment for 24 h (Fig. 2E) and also induce cell cycle arrest in SubG0/1 and G2/M periods (Fig. 3).
In contrast, Fas is a member of the TNF receptor family that activates apoptosis upon binding of the Fas ligand. The interaction between the Fas receptor and its ligands plays an important role in the regulation of apoptosis. In the extrinsic apoptotic pathway, Fas binds to the Fas ligand to activate caspase-8 and caspase-3 in order to execute cell apoptosis. This process is mediated by a death-inducing signaling complex that consists of FADD and procaspase-8, which produces caspase-8 and leads to the activation of downstream caspases [27]. Of these caspases, caspase-3 is one of the critical effector caspases in the downstream execution of apoptosis [28]. In our study, we found that A549 cells treated with TXA9 underwent apoptosis by activating a caspase cascade that involves the increased expression of Fas and FADD and the activation of the cleavage of procaspase-8 into caspase-8 (Fig. 4A). The procaspase-3 level then decreases, the caspase-3 levels increase and degrade ICAD (Fig. 4B). These findings indicate that TXA9 causes the activation of caspase-3, which cleaves the death substrate ICAD and ultimately leads to apoptosis. All of the abovedescribed results indicate that TXA9 induces apoptosis by activating the Fas death receptor pathway.
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In summary, the present study provides the first demonstration of the remarkable in vitro and in vivo anti-tumor effects of TXA9 against NSCLC. The findings show that TXA9 exhibits more selective anti-proliferative activity against NSCLC cell lines than taxol in vitro. The in vivo studies demonstrate that a threefold higher dose of TXA9 shows a tumor inhibition rate similar to that obtained with taxol and is safer. The mechanistic studies show that TXA9 can stimulate apoptosis through the extrinsic pathway and cell cycle arrest in the SubG0/1 and G2/M periods. Therefore, therapy with strong anti-tumor activity and without any significant toxicity can be obtained using TXA9, and this is a promising development that will be followed up in clinical trials against NSCLC.
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Acknowledgments
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This work was financially supported by Grants from the National Natural Science and Technology Major Project ‘‘Key New Drug Creation and Manufacturing Program’’ (No. 2010ZX09401304) and the Natural Science Foundation of China (No. 30973958).
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Please cite this article in press as: Xue R et al. TXA9, a cardiac glycoside from Streptocaulon juventas, exerts a potent anti-tumor activity against human nonsmall cell lung cancer cells in vitro and in vivo. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.12.015
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