Original Papers

79

Authors

Danqing Xu 1, 2*, Yuanzhi Lao 1, 2*, Naihan Xu 3, Hui Hu 1, 2, Wenwei Fu 1, 2, Hongsheng Tan 1, 2, Yunzhi Gu 1, 2, Zhijun Song 4, Peng Cao 5, Hongxi Xu 1, 2

Affiliations

The affiliations are listed at the end of the article

Key words " Garcinia l " Clusiaceae l " autophagy l " apoptosis l " natural compound l " caged prenylxanthones l

Abstract

Abbreviations

!

!

Natural compounds from medicinal plants are important resources for drug development. Active compounds targeting apoptosis and autophagy are candidates for anti‑cancer drugs. In this study, we collected Garcinia species from China and extracted them into water or ethanol fractions. Then, we performed a functional screen in search of novel apoptosis and autophagy regulators. We first characterized the anti‑proliferation activity of the crude extracts on multiple cell lines. HeLa cells expressing GFP‑LC3 were used to examine the effects of the crude extracts on autophagy. Their activities were confirmed by Western blots of A549 and HeLa cells. By using bioassay guided fractionation, we found that two caged prenylxanthones from Garcinia bracteata, neobractatin and isobractatin, can significantly induce apoptosis and inhibit autophagy. Our results suggest that different Garcinia species displayed various degrees of toxicity on different cancer cell lines. Furthermore, the use of a high content screening assay to screen natural products was an essential method to identify novel autophagy regulators.

ATGs: autophagy‑related proteins BCA: bicinchoninic acid BSA: bovine serum albumin ECL: enhanced chemiluminescence FRET: fluorescent resonance energy transfer GFP‑LC3: green fluorescent protein‑fused LC3 HCQ: hydroxychloroquine HCS: high content screening HRP: horseradish peroxidase HTS: high‑throughput screening LC3/MAP1LC3B: microtubule‑associated protein 1 light chain 3 PARP: poly(ADP‑ribose)‑polymerase PCD: programmed cell death PI: propidium iodide PPAPs: polycyclic polyprenylated acylphloroglucinols ppm: parts per million SQSTM1: poly‑ubiquitin binding protein p62 or sequestome 1 TBS/T: Tris‑buffered saline/Tween20 TCM: traditional Chinese medicine TMS: tetramethylsilane

received revised accepted

July 10, 2014 August 25, 2014 October 21, 2014

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383356 Published online December 5, 2014 Planta Med 2015; 81: 79–89 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Hongxi Xu School of Pharmacy Shanghai University of Traditional Chinese Medicine Cai Lun Road 1200 Shanghai 201203 China Phone: + 86 21 51 32 30 89 [email protected]

Supporting information available online at http://www.thieme‑connect.de/products

Introduction !

Dysregulated cell death is a common feature of cancer, and the modulation of this cellular response plays an important role in cancer therapy. Apoptosis, autophagy, and necroptosis are important cell death forms, and resistance to cell death is considered to be one of the hallmarks of cancer

* Authors contributed equally to this work.

[1]. Apoptosis, also called type I PCD, is mainly controlled by the integrity of the outer membranes of mitochondria in the cell [2]. The pro‑ and anti‑apoptosis Bcl‑2 family proteins are the key regulators for activating apoptotic pathways. When the anti‑apoptotic proteins are inhibited, the pro‑apoptotic Bax and Bak disrupt the integrity of the outer mitochondrial membrane, which causes the release of pro‑apoptotic signaling proteins (e.g., cytochrome c, Smac/DIABLO). Then, they initiate a cascade of proteolysis involving

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Identification and Characterization of Anticancer Compounds Targeting Apoptosis and Autophagy from Chinese Native Garcinia Species

Original Papers

caspases responsible for the execution phase of apoptosis [3]. Autophagy (type II PCD) is an evolutionarily conserved membrane process that results in the transporting of cellular contents to lysosomes for degradation [4]. During tumor development and in cancer therapy, autophagy plays paradoxical roles in promoting cell survival and cell death [5]. Autophagy functions as a tumor suppression mechanism by removing damaged organelles and proteins and preventing genomic instability that drives tumorigenesis. On the contrary, autophagy has been demonstrated to promote the survival of tumor cells under nutrient or chemical stress [6, 7]. Therefore, therapeutic modulation of autophagy may serve as an important and challenging endeavor in cancer treatment [8, 9]. The autophagy process involves initiation, elongation, closure, maturation, and degradation, which are controlled by highly conserved ATGs [10]. LC3/MAP1LC3B, a homologue of yeast protein ATG8, serves as a marker protein for autophagosomes [11]. The complex signaling pathways that control cell death have been proven to be targets for anti‑cancer drug discovery [12]. Cell‑based tests that quantify cell death are broadly used for drug screens. However, it is necessary to apply further assays to identify the biochemical cascades and the molecular targets of the active compounds. During the past decade, many conventional HTS cell death detection methods targeting apoptosis and autophagy have been developed. Caspase activation is a universal marker of mitochondria‑dependent apoptosis and can be quantified by immunoblotting methods using antibodies against active forms of these enzymes. Moreover, the FRET biosensor containing a caspase cleavage site can be applied to HTS for apoptotic inducers [13, 14]. GFP‑LC3 protein can be applied to screen autophagy regulators in live cells. The number of GFP‑LC3 puncta is very low under normal conditions but rapidly increases when autophagy is activated by rapamycin or stress [15]. However, the increase of GFP‑LC3 level is not necessarily dependent on autophagy induction. It may be the result of lysosome defects and associated with the inhibition of autophagy. To confirm the function of chemicals as either inducers or inhibitors of autophagy, more assay criteria, such as the monitoring of autophagic flux is required [16, 17]. SQSTM1 is selectively incorporated into autophagosomes through direct binding to LC3 and is efficiently degraded by autophagy. Thus, the total cellular expression levels of p62 correlate with autophagic activity [18]. The novel autophagy regulators are not only lead compounds for drug development but also provide researchers a useful tool to investigate the complex autophagy signaling pathways [19]. Compounds from natural plants are important resources for drugs against a wide variety of diseases, including cancer. Many TCMs containing active compounds exhibit antitumor effects and have been used for various types of cancer treatment. Garcinia species (Clusiaceae) have been studied for more than 70 years, and many bioactive compounds, including xanthones, caged xanthones, PPAPs, and benzophenones, have been identified with anticancer potentials [20]. Gambogic acid, a caged xanthone from G. hanburyi Hook. f., has been tested in vitro and in vivo as a novel anticancer agent that inhibits cell proliferation, angiogenesis, and metastasis [21–23]. Interestingly, many caged Garcinia xanthones have been identified in the past several decades, and most of them display high cytotoxic efficacy against various cancer cells [24]. These studies suggest that the plant metabolites from Garcinia species have unique chemical structures and potent bioactivities and can be a promising pharmacological target for drug design and development. Previously, we applied

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

bioassay‑guided fractionation by using a caspase‑3 FRET sensor to identify many novel compounds targeting apoptosis in China native Garcinia species [25–29]. Furthermore, the mechanisms of action of some compounds involve the induction of apoptosis, inhibition of autophagic flux, cell cycle arrest, and modulation of oncogenes, etc. [30–33]. Recently, we constructed HeLa cells stably expressing GFP‑LC3 combined with HCS to identify autophagy regulators from microRNAs and natural compounds from Garcinia species [34, 35]. Our studies demonstrated that Garcinia species contain many bioactive compounds affecting apoptosis and autophagy pathways. To obtain a comprehensive understanding of the efficacy of different bioactive compounds, it is necessary to perform autophagy HCS from crude extracts of plants to look for novel autophagic regulators. In this study, we collected different parts (leaves, fruits, bulks, and bulks of roots) from China native Garcinia species, used 95% EtOH and water to obtain crude extracts, respectively, applied a cell viability assay using multiple cancer cell lines and selected the most potent fractions to perform bioassay‑guided fractionation on GFP‑LC3 HCS. Our results suggested that two active compounds, neobractatin and isobractatin, from G. bracteata C. Y. Wu ex Y. H. Li have strong autophagy inhibition effects.

Results !

Fourteen herbs from Garcinia species were collected and their different parts, including leaves, twigs, seeds, pericarps, barks, root barks, or fruits, were extracted by water or ethanol to make different types of plant extracts. Three cancer cell lines, including the human prostatic cancer cell line PC‑3, human lung adenocarcinoma epithelial cell line A549, and human colon carcinoma cell line HT29, were used to determine the anti‑proliferation activ" Table 1, ethanol extracts ities of the crude extracts. As shown in l are more active than water extracts against these cancer cell " Table 1. lines. The most potent fractions were highlighted in l We found that the ethanol extracts from G. xanthochymus Hook. f. ex T. Anders., G. bracteata C. Y. Wu ex Y. H. Li, G. lancilimba C. Y. Wu ex Y. H. Li, G. oligantha Merr., G. esculenta Y. H. Li, G. pedunculata Roxb., and G. yunnanensis Hu exhibited high growth inhibitory activity against all three cancer cell lines. We further tested the anti‑proliferation activity of the 11 selected Garcinia species extracts on 9 other cancer cell lines, and those fractions with IC50 " Table values less than 10 µg/mL were highlighted. As shown in l 2, they displayed different potencies against various cancer cell lines. After the assays, four Garcinia species extracts were selected for further analysis, including No. 6 (ethanol extract from leaves of G. bracteata), No. 18 (ethanol extract from leaves of G. oligantha), No. 60 (ethanol extract from root barks of G. pedunculata), and No. 72 (ethanol extract from root barks of G. esculenta). The overall average IC50 values were approximately 20 µg/mL, 30 µg/mL, and 10 µg/mL corresponding to colon cancer, breast " Fig. 1). Our prolifcancer, and leukemia cell lines, respectively (l eration assay suggested that most components with anti‑cancer potential were extracted by the ethanol solvent. In addition, different plants or different parts from the same plants contained various components that displayed a distinct effect on multiple cancer cell lines. To further confirm the cytotoxicity of the four chosen crude extracts, A549 cells were used to analyze their effects on cell cycle distribution and cell death by flow cytometry analysis. As shown " Fig. 2 A, four crude extracts caused Sub‑G1 fraction accumuin l

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

80

Original Papers

81

Table 1 Cytotoxicity of seventy six extracts from Garcinia species on three tumor cell lines. Cells were cultured and seeded in 96‑well plates, and extracts were treated for 72 h. Etoposide was used as positive control. Cell proliferation was detected using a CCK‑8 kit. Name

Parts

Extract

1 2 3 4 67 68 69 70 5 6 7 8 9 10 11 12 13 14 15 16 55 56 17 18 19 20 21 22 23 24 57 58 25 26 27 28 29 30 31 32 33 34 35 36 71 72 73 74 75 76 37 38 39 40 63 64 65 66 41 42 43 44

G. xanthochymus

leaves leaves twigs twigs seeds seeds pericarps pericarps leaves leaves twigs twigs leaves leaves twigs twigs leaves leaves twigs twigs barks barks leaves leaves twigs twigs leaves leaves twigs twigs barks barks leaves leaves twigs twigs leaves leaves twigs twigs leaves leaves twigs twigs root barks root barks barks barks fruits fruits leaves leaves twigs twigs root barks root barks barks barks leaves leaves twigs twigs

water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH

G. bracteata

G. multiflora

G. lancilimba

G. oligantha

G. oblongifolia

G. cowa

G. xipshuanbannaensis

G. esculenta

G. nujiangensis

G. paucinervis

IC50 (µg/ml) PC‑3

HT‑29

A549

> 100 49.47 > 100 92.28 > 100 35.08 > 100 > 100 > 100 22.17 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 31.13 86.90  8.48 > 100 11.10 > 100 > 100 > 100 > 100 > 100 92.69 > 100 > 100 > 100 95.14 > 100 > 100 > 100 > 100 > 100 74.35 > 100 > 100 > 100 62.70 > 100 53.63 > 100 > 100 > 100 > 100 > 100 93.50 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100

> 100 61.26 > 100 75.20 > 100 73.08 > 100 > 100 > 100 29.84 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 31.08 82.58 12.68 > 100 15.35 > 100 > 100 > 100 > 100 > 100 93.25 > 100 > 100 > 100 98.39 > 100 > 100 > 100 > 100 > 100 82.98 > 100 > 100 > 100 41.34 > 100 47.79 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100

> 100 38.85 > 100 68.64 > 100 67.99 > 100 > 100 > 100 28.80 > 100 > 100 > 100 > 100 > 100 97.65 > 100 > 100 > 100 > 100 > 100 16.63 41.06  9.95 > 100  9.97 > 100 > 100 > 100 > 100 72.01 57.04 > 100 > 100 > 100 60.67 > 100 > 100 > 100 > 100 > 100 61.20 > 100 99.28 > 100 19.16 > 100 20.64 > 100 > 100 > 100 > 100 > 100 67.34 > 100 > 100 > 100 > 100 > 100 > 100 > 100 > 100

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

No.

continued

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

Original Papers

Table 1 Continued No.

45 46 47 48 59 60 61 62 49 50 51 52 53 54

Name

Parts

G. pedunculata

leaves leaves twigs twigs root barks root barks barks barks twigs twigs fruits fruits pericarps pericarps

G. yunnanensis

G. mangostana

IC50 (µg/ml)

Extract

water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH water EtOH

Etoposide

PC-3

HT-29

A549

> 100 > 100 > 100 > 100 > 100 28.37 > 100 >100 > 100 35.05 > 100 > 100 > 100 > 100 13.04

> 100 > 100 > 100 > 100 > 100 18.52 98.63 99.27 > 100 40.61 > 100 97.98 > 100 74.52 21.36

> 100 > 100 > 100 > 100 > 100 26.69 41.72 51.17 > 100 34.54 > 100 84.58 > 100 62.33 2.22

Table 2 Cytotoxicity of eleven extracts from Garcinia species on eleven tumor cell lines. Cells were cultured and seeded in 96‑well plates, and extracts were treated for 72 h. Etoposide was used as positive control. Cell proliferation was detected using a CCK‑8 kit. IC50 (µg/mL) No.

Prostate

Colon cancer

Lung

cancer PC‑3

Breast cancer

cancer HT‑29

Colo

HCT‑15

A549

Cervical

Leukemia

cancer MCF7

205

MDA-

HeLa

tfMB-

RPM-

K562

Molt‑4

25.62 12.84  6.34 11.17 25.92 19.0 18.7 58.8 25.09 19.27 23.13 2.18

11.13  4.76  3.36  4.19  8.57  5.17 15.75 25.16 34.36  9.90  9.20 2.05

I‑8226

231 2 6 18 20 50 56 60 62 68 72 74 ETO

49.47 22.17  8.48 11.10 35.05 31.13 28.37 > 100 35.08 62.70 53.63 13.04

61.26 29.84 12.68 15.35 40.61 31.08 18.52 99.27 73.08 41.34 47.79 21.36

42.46 23.07  9.12 13.95 43.0 16.9 33.71 38.2 37.27 18.74 22.32 25.01

31.89 26.87 12.05 18.24 37.7 30.12 31.44 33.52 37.76 23.93 30.28 26.67

38.85 28.80  9.95  9.97 34.54 16.63 26.69 51.17 67.99 19.16 20.64 2.22

78.84 34.73 18.99 28.46 81.94 32.96 39.91 > 100 > 100 87.50 90.14 3.07

66.21 19.36 14.71 16.37 80.09 36.42 42.49 > 100 65.7 56.42 63.89 6.11

28.35 25.13  9.29 13.93 31.13 18.11 19.24 19.46 35.49 10.14 11.10 1.02

21.57  6.20  4.25  4.34 18.3 14.0 13.82 17.25 32.91 16.44 11.74 3.39

Fig. 1 Cytotoxicity effects of EtOH extracts from four Garcinia species on eleven tumor cell lines. Cells were cultured and seeded in 96‑well plates, and extracts were treated for 72 h. Etoposide was used as positive control. Cell proliferation was detected using a CCK‑8 kit.

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

82

Original Papers

83

lation in a dosage‑dependent manner which reflects the number of dying cells. The effects on G0/G1, S, and G2/M phases were also analyzed. Tests indicated that 50 µg/mL crude extract from the leaves of G. bracteata caused G2/M arrest. Notably, 16.7 µg/mL extract from G. oligantha induced G0/G1 arrest; however, a higher concentration (50 µg/mL) of extract activated S and G2/M arrest. The effects of these fractions on apoptosis were examined " Fig. 2 C, A549 cells were senby Western blotting. As shown in l sitive to extracts from G. bracteata, G. oligantha, and G. esculenta as the cleavage of PARP and caspase‑3 was detected in high concentration treatment. In addition, the crude extract from G. pedunculata was able to activate apoptosis in HeLa cells at a 50 µg/ mL concentration. Autophagy plays an important role in tumorigenesis and chemotherapy. We recently reported that compounds from Garcinia species might regulate autophagic flux and be beneficial for anti‑cancer efficacy [35]. Here, we used HeLa‑GFP‑LC3 cells and the HCS platform to investigate whether the crude extracts can " Fig. 3 A, the regulate autophagy in cancer cells. As shown in l GFP‑LC3 signal was distributed in the cytosol without significant puncta formation in the control and low concentration treatment groups. In high concentration extract‑treated cells, the GFP‑LC3 displayed puncta accumulation, which suggested that the autophagy pathway was influenced. During autophagy, the amount of LC3B‑II positively correlates with the number of autophagosomes. Therefore, the conversion from endogenous LC3B–I to LC3B‑II can be used to monitor the autophagic activity. We examined the effect of the four crude extracts on LC3B conversion in both A549 and HeLa cells. All tested crude extracts could increase " Fig. 3 B). the LC3B‑II conversion in these two cancer cell lines (l Both induction and suppression of autolysosomal maturation resulted in increased numbers of autophagosomes. To distinguish

whether autophagosome accumulation is due to autophagy induction or inhibition, we performed an autophagic flux assay. p62 serves as a link between LC3 and ubiquitinated substrates. Inhibition of autophagy correlates with increased levels of p62 in mammals and vice versa. We then examined the total cellular amount of p62 that was delivered to the lysosomes for degradation. Immunoblot analysis revealed remarkable changes of p62, which were detected at 48 h after 50 µg/mL drug treatments " Fig. 3 B). A significant increase of p62 was observed in G. brac(l teata‑ and G. oligantha‑treated cells, which suggests that the autophagy procedure was suppressed. On the contrary, extracts from G. esculenta could induce autophagy, which was reflected by a reduction of p62. Taken together, our results indicate that different extracts might contain distinct active compounds targeting different signaling pathways, such as the cell cycle, cell death, and autophagy. Therefore, the use of phytochemistry to identify single compounds from the crude extracts with bioassay‑guided fractionation is necessary, and it is helpful to elucidate the mechanism of action of active compounds. Using these bioactivity screen platforms, we isolated two caged prenylxanthones, neobractatin and isobractatin, from the etha" Fig. 4 A). It has been nol fraction of the leaves of G. bracteata (l reported that isobractatin demonstrated cytotoxicity against KB cells (nasopharyngeal carcinoma), A549 cells (lung adenocarcinoma), MCF7 cells (breast cancer), and PC3 cells (prostate cancer) [36, 37]. However, the bioactivity of neobractatin has not been previously studied. We investigated the effects of these two compounds on cell proliferation and cell death. In CCK8 cell proliferation assays, neobractatin and isobractatin displayed strong inhibition of both A549 and HeLa cells, with IC50 values of approximately 2 µM. To examine whether these two compounds could

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Fig. 2 Herbal extracts‑induced apoptosis and cell cycle arrest. A549 cells were treated with 4 Garcinia extracts with indicated concentrations. After 24 h of treatment, the cells were harvested, fixed in 70 % EtOH, and stained with PI. The cell cycle and cell death were detected by FACS. A Sub‑G1 fraction in different concentration treatment. B Cell cycle distribution in different concentration treatment. C Western blot showed the apoptosis‑related proteins upon the 4 Garcinia extracts treatment for 48 h.

Original Papers

Fig. 3 Herbal extracts‑regulated autophagy. HeLa cells stably expressing GFP‑LC3 were seeded in 96well plates and treated with extracts for 48 h. The nucleus was stained with Hoechst. The images were acquired with an Opera (GFP ex and em) using a 40 x‑H2O objective. A Upper panel: Image from DMSO treated cells as control. Lower panel: Cells were treated with indicated extracts, and images were acquired after 48 h treatment. B Western blot showed the autophagy‑related proteins upon the 4 Garcinia extracts treatment for 48 h. The quantities of p62 and LC3B‑II were quantified in the histograms. The intensity was measured by ImageJ software. (Color figure available online only.)

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

84

induce cell death, we performed flow cytometry analysis to " Fig. 5 A, 4 µM of quantify the sub‑G1 population. As shown in l neobractatin and isobractatin treatment significantly increased the sub‑G1 fraction in A549 cells. In addition, we used Western blotting to check the apoptotic‑related proteins, such as caspase‑3 and PARP, upon treatment with these compounds. In both A549 cells and HeLa cells, 48 h of treatment with neobractatin

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

and isobractatin caused a decrease of pro‑caspase 3 and cleavage of PARP, which suggests the activation of apoptosis. Therefore, our data indicate that these two caged prenylxanthones could suppress cancer cell growth by inducing apoptosis. " Fig. 3 B, we show that the crude extract of G. bracteata (6#) In l inhibited autophagic flux in cancer cells. It was of interest to explore whether neobractatin and isobractatin were the effective

Original Papers

85

Fig. 5 Neobractatin and isobractatin‑induced apoptosis in cancer cells. A549 cells were treated with neobractatin and isobractatin. After 24 h of treatment, the cells were harvested, fixed in 70% EtOH and stained with PI. The cell cycle and cell death were detected by FACS. A Cell cycle attribution of A549 cells under different concentration treatments. Lower and left panel show the Sub‑G1 faction data. B Western blot showed the apoptosis related proteins upon neobractatin and isobractatin treatment for 48 h. (Color figure available online only.)

compounds producing the autophagic flux inhibition effect. We then performed Western blotting to examine the effects of the two compounds on the autophagic proteins LC3B and p62. " Fig. 6 A indicates that both neobractatin and isobractatin could l increase LC3B–I to LC3B‑II conversion in A549 and HeLa cells in a dosage‑dependent manner. In addition, the accumulation of p62 was observed in high‑concentration treatment samples, suggesting that both compounds were able to inhibit autophagic flux in

cancer cells. We also used the HCS platform to measure the influence of these compounds on GFP‑LC3 puncta formation. As " Fig. 6 B, 2 µM neobractatin and isobractatin treatshown in l ment for 48 h induced significant puncta accumulation. To quantify the puncta number, we used Hoechst to stain the nucleus and software to identify the cytosolic fraction in each cell (for details, see Materials and Methods). The GFP‑LC3 puncta in single cells could be automatically counted by the software. The statistical

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Fig. 4 Two compounds purified from Garcinia bracteata induced cell death in cancer cells. A Chemical structures of isobractatin and neobractain. B Cell proliferation curve under compounds treatment measured by CCK‑8 kit. IC50 was calculated by Prism software.

Original Papers

Fig. 6 Neobractatin and isobractatin-regulated autophagy in cancer cells. A A549 (left) and HeLa (right) cells were treated with compounds at different concentrations. After 48 h of treatment, the cells were harvested, and the indicated proteins were tested by Western blot. The quantities of p62 and LC3B-II were quantified in histograms. The intensity was measured by ImageJ software. B HeLa cells stably expressing GFP‑LC3 were seeded in a 96well dish and treated with compounds for 48 h. 20 µM HCQ was used as positive control. The images were acquired by an Opera (GFP ex and em) with a 40 x-H2O objective. C The number of GFP‑LC3 puncta in each cell was calculated by Columbus software. 20 µM HCQ was used as positive control. (Color figure available online only.)

analysis confirmed that neobractatin and isobractatin caused " Fig. 6 C). Our results suggest that both neopuncta formation (l bractatin and isobractatin were able to regulate autophagic signaling pathways, and they are most likely autophagic flux inhibitors. In summary, we collected Chinese native Garcinia species and obtained ethanol and water fractions for each sample. We first used PC‑3, HT‑29, and A549 cancer cell lines to test the cytotoxicity of these crude extracts. Second, we chose the fractions with high activity and confirmed their anti‑proliferation ability using more cancer cell lines, including those for colon, lung, breast, and cervical cancer, as well as leukemia. Our results suggest that most active compounds remained in the ethanol fractions. Later, we selected four ethanol fractions from different plants to investigate their effects on cell death and autophagy. Finally, we isolated two caged prenylxanthones, neobractatin and isobractatin, and provided evidence that they could activate apoptosis and suppress autophagic flux.

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

Discussion !

Natural compounds have served as a major source of drugs, and more than 50 % of pharmaceuticals are derived from natural products. Bioassay‑guided fractionation proved to be an efficient approach to identify potent components from effective decoctions or plants. Our results indicate that these fractions contained active components inhibiting the proliferation of multiple cancer cell lines. Interestingly, these fractions displayed differential activities for different cell lines. For instance, the extract from G. esculenta exhibited strong inhibition of lung cancer (A549) and cervical cancer (HeLa) cells but only minor toxicity to breast cancer cells (MCF and MDA‑MB‑231). These findings suggest that the components in this fraction could activate different signaling pathways. To further investigate the function of these fractions, we chose ethanol extracts from four Garcinia species to analyze their effects on cell cycle distribution, apoptosis‑related proteins, and autophagy pathways in the following study. As we expected, all of the samples had the potential to affect the cell cycle, apoptosis, and autophagy. By analyzing the protein level of p62, an autophagic flux marker, we found that some fractions contained autophagic flux inhibitors (fraction 6# and 18#), whereas others

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

86

Original Papers

Materials and Methods !

Drugs and general procedure The positive control compound etoposide (E1383, purity ≥98 %) and HCQ (H0195, purity ≥98 %) were bought from Sigma‑Aldrich. The powder was dissolved in DMSO to make a 10 mM solution stored at − 20 °C before usage. All NMR spectra were recorded on a Bruker AV‑400 spectrometer at 400 MHz for 1H NMR, HSQC, and HMBC, and at 100 MHz for 13C NMR in DMSO‑d6. Chemical shifts are reported in ppm relative to TMS.

Plant material The 38 samples of 14 Garcinia species were collected from Yunnan, Guangxi, Hainan, and Guangdong provinces in China " Table 1). The plant materials were authenticated by Professor (l Zhao Yiming, Guangxi Medicinal Garden, and Prof. Wang Hong, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. All voucher specimens (Herbarium No. GAR-01 to GAR038) were deposited in the Innovative Research Laboratory of TCM, Shanghai University of TCM.

Preparation of crude extracts Each dried plant sample was ground into a fine powder using a pulverizer. A sample of 10.0 g of fine powder was placed in a 250‑mL round‑bottom flask in a water bath and extracted for 30 min twice under reflux with 10 volumes of 95% ethanol or distilled water and then filtered. The filtrate was evaporated to dryness under vacuum. These extracts were dissolved in DMSO and further diluted with cell culture medium. The final DMSO concentration was below 1 % of total volume of the medium in all treatments and controls.

Extraction and isolation The air‑dried trunks of G. bracteata (4.0 kg) was pulverized and extracted with 95 % (v/v) ethanol (3 × 8 L) at room temperature, filtered and concentrated to give a crude extract (754.8 g). The EtOAc‑soluble fraction (262.2 g) was subjected to Si gel column chromatography (Φ10 × 75 cm, 3.0 kg) with a gradient of petroleum ether‑acetone as the eluent, and ten fractions (A–J) were collected. Fraction B (24.8 g) and fraction D (14.5 g) were further separated on Si gel, Sephadex LH‑20, and RP‑C18 Si gel columns to give pure compounds 1–2 (Supporting Information). Compound 1 (isobractatin) and compound 2 (neobractatin) were identified based on MS and NMR spectroscopy analysis and by comparison of their spectroscopic data with published values [36, 40]. The purity of these two compounds was greater than 98 %.

Cell lines and cell culture All cell lines from ATCC and the cell bank of the Shanghai Institutes of Biochemistry and Cell Biology, Chinese Academy of Sciences, were maintained at 37 °C with a 5% CO2 humidified atmosphere in growth medium as recommended by the providers and subjected to in vitro assays between passages 8 ~ 15.

Cell proliferation assay Cell proliferation assays were performed as previously described [41]. Briefly, each cell line was seeded in a 96‑well tissue culture plate (Corning) at a predetermined density in 180 µL of complete medium, attached overnight and treated using natural products or compounds for another 72 h. Then, the medium was discarded and replaced with 10 % CCK‑8 (Dojindo) in complete medium, and the plates were incubated for another 2 h. The OD450 was measured with SpectraMAX 190 spectrophotometer (MDS). A background absorbance of the ODblank was subtracted from all wells. The inhibition rate (IR) was determined with following formula: IR (%) = (ODDMSO − ODcompound)/ODDMSO × 100 %. The CCK‑8 assay as above was used for determination of cell viability.

Propidium iodide staining for flow cytometry A549 cells were collected and washed with PBS twice and then fixed with 75% alcohol overnight. After being washed with PBS, RNase (10 µg/mL) was added and incubated for 15 min at 37 °C to eliminate the interference of RNA. Cells were then treated with PI (Sigma) for another 30 min. Cells were washed, and the DNA contents were detected by FACSCalibur [42].

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

contained an autophagy inducer (fraction 72#). As autophagy contributes to tumorigenesis and cancer cell sensitivity under stress, the discovery of novel autophagy regulators might be beneficial to anti‑cancer drug discovery. Through further isolation and chemical structure identification, we obtained two caged prenylxanthones, neobractatin and isobractatin, and characterized their bioactivities in regards to apoptosis and autophagy. Our data clearly indicate that these two compounds could induce apoptosis and inhibit autophagic flux at a low dosage compared with the crude extracts. Taken together, our study findings suggest that using cancer cells stably expressing GFP‑LC3 to screen autophagy modulators was an effective way to search for novel compounds targeting autophagy. Therefore, it would be interesting to continue to study other fractions, such as extracts from G. oligantha (18#) and G. esculenta (72#), to identify components that affect autophagy signaling pathways. Cell‑based HCS, which analyzes biological events at subcellular resolution, is widely used in pharmacological research [38]. Previously, we used GFP‑LC3‑expressing HeLa cells to screen our own pure compounds library from Garcinia species and identified that oblongifolin C was an effective autophagic flux inhibitor [35]. In this study, we used the same cell line to screen our crude extracts library. The screening setup was optimized in a single compound treatment, and the increase of GFP‑LC3 puncta was quantified automatically. However, the HCS system had difficulties in processing the images under the crude extracts treatment. " Fig. 3 B, the cellular morphology and size changed As shown in l dramatically upon treatment. Therefore, it was difficult to differentiate the nuclear and cytoplasmic regions and to calculate the cell size. This might be due to the complexity of the crude extracts since their active components could have activated multiple cellular signaling pathways. To develop a suitable screen platform for crude extracts, researchers may consider combining imaging‑based screenings with Western blots of autophagy-related proteins, such as LC3 and p62 [16]. Alternatively, a novel HTS method based on ATG protein (e.g., ATG4B) activity can be considered for application [39].

87

Original Papers

Western blotting

Affiliations

The cell lysate was prepared in RIPA buffer and quantified by the BCA method (Pierce). Thirty micrograms of protein per sample was loaded onto a 4 ~ 12 % NuPAGE® Novex SDS gel (Invitrogen). The protein was transferred by an iBlot® dry blotting device (Invitrogen) onto nitrocellulose membranes. After blocking nonspecific binding with TBS/T (0.1 %)containing 5 % non‑fat milk for 1 h at room temperature, the membrane was incubated in LC3B (sigma, L7543), SQSTM1/p62 (MBL, PM045), PARP (Cell Signaling Technology, CST #9542P), or caspase‑3 (Cell Signaling Technology, CST #9662P) (1 : 1000 in TBS/T containing 3% BSA and gently shaken at 4 °C overnight. The membrane was washed with TBS/T three times to remove the unbound antibody and then incubated with the secondary antibody (HRP‑conjugated goat anti‑mouse IgG or goat anti‑rabbit IgG, 1 : 5000; KangChen Biotech) for 1 h at room temperature. Protein bands were visualized with an ECL kit (Pierce).

1

Green fluorescent protein‑fused LC3 translocation and quantitative analysis HeLa cells stably expressing GFP‑LC3 were generated as previously described [34]. Briefly, HeLa cells were transfected with pEGFP‑LC3 plasmid using lipofectamine 2000 (Invitrogen, 11 668–019). One day after transfection, the cells were treated with 800 µg/mL G418 for 7 days. The surviving cells were continually cultured with 800 µg/mL G418 and named HeLa‑GFP‑LC3 cells. The HeLa‑GFP‑LC3 cells were seeded in a 96‑well plate (clear bottom, black; PerkinElmer) overnight. Cells were then treated with different concentrations of natural products or compounds in triplicate. After 48 h, the cells were fixed with 4% paraformaldehyde and washed 3 times with PBS. Image acquisition was performed using an Opera High Content Screening System (PerkinElmer) using a 40 x‑H2O objective. Data were analyzed by Columbus 2.3, which is software created by Perkin‑Elmer. To quantify the GFP‑LC3 spots, the following procedures were performed: 1. using the Hoechst channel to define the nuclei region (method A; common threshold 0.45 with area>100 µM2); 2. using the GFP channel to define the cellular cytoplasm region (method A; individual threshold 0.15); and 3. spot calculation in each cellʼs exclusive nuclear regions (method C; radius ≤ 5.0 µM; contrast > 0.13; spot to region intensity > 1.0; distance ≥ 2.3 px; peak radius 1.0 px).

Supporting information Details about the isolation and identification of isobractatin and neobractatin, 13C NMR and 1H NMR spectra of isobractatin as well as 13C NMR, DEPT135, 1H NMR, HSQC, and HSBC spectra of neobractatin are available as Supporting Information.

Acknowledgements !

This work was supported by the National Natural Science Foundation of China (No. 81 303 188, 81 273 403, and 81 173 485).

Conflict of Interest !

The authors declare no conflict of interest.

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

2

3

4 5

School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, P. R. China Engineering Research Center of Shanghai Colleges for TCM New Drug Discovery, Shanghai, P. R. China Key Lab in Healthy Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, P. R. China Guangxi Botanic Garden of Medicinal Plants, Nanning, Guangxi, P. R. China Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, Nanjing, Jiangsu, P. R. China

References 1 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144: 646–674 2 Llambi F, Green DR. Apoptosis and oncogenesis: give and take in the BCL‑2 family. Curr Opin Genet Dev 2011; 21: 12–20 3 Tait SW, Green DR. Mitochondria and cell signalling. J Cell Sci 2012; 125: 807–815 4 Xie Z, Klionsky DJ. Autophagosome formation: core machinery and adaptations. Nat Cell Biol 2007; 9: 1102–1109 5 Rosenfeldt MT, Ryan KM. The multiple roles of autophagy in cancer. Carcinogenesis 2011; 32: 955–963 6 Levine B. Cell biology: autophagy and cancer. Nature 2007; 446: 745– 747 7 Mathew R, Karantza‑Wadsworth V, White E. Role of autophagy in cancer. Nat Rev Cancer 2007; 7: 961–967 8 Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther 2011; 10: 1533–1541 9 Carew JS, Kelly KR, Nawrocki ST. Autophagy as a target for cancer therapy: new developments. Cancer Manag Res 2012; 4: 357–365 10 Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev 2011; 25: 1999–2010 11 Mizushima N, Klionsky DJ. Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr 2007; 27: 19–40 12 Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G. Cell death assays for drug discovery. Nat Rev Drug Discov 2011; 10: 221–237 13 Tyas L, Brophy VA, Pope A, Rivett AJ, Tavare JM. Rapid caspase‑3 activation during apoptosis revealed using fluorescence‑resonance energy transfer. EMBO Rep 2000; 1: 266–270 14 Luo KQ, Yu VC, Pu Y, Chang DC. Application of the fluorescence resonance energy transfer method for studying the dynamics of caspase‑3 activation during UV‑induced apoptosis in living HeLa cells. Biochem Biophys Res Commun 2001; 283: 1054–1060 15 Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 2004; 15: 1101–1111 16 Zhang L, Yu J, Pan H, Hu P, Hao Y, Cai W, Zhu H, Yu AD, Xie X, Ma D, Yuan J. Small molecule regulators of autophagy identified by an image‑based high‑throughput screen. Proc Natl Acad Sci U S A 2007; 104: 19023– 19028 17 Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell 2010; 140: 313–326 18 Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, Stenmark H, Johansen T. p 62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin‑induced cell death. J Cell Biol 2005; 171: 603–614 19 Shen S, Niso‑Santano M, Adjemian S, Takehara T, Malik SA, Minoux H, Souquere S, Marino G, Lachkar S, Senovilla L, Galluzzi L, Kepp O, Pierron G, Maiuri MC, Hikita H, Kroemer R, Kroemer G. Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity. Mol Cell 2012; 48: 667– 680 20 Han QB, Xu HX. Caged Garcinia xanthones: development since 1937. Curr Med Chem 2009; 16: 3775–3796 21 Anantachoke N, Tuchinda P, Kuhakarn C, Pohmakotr M, Reutrakul V. Prenylated caged xanthones: chemistry and biology. Pharm Biol 2012; 50: 78–91 22 Yi T, Yi Z, Cho SG, Luo J, Pandey MK, Aggarwal BB, Liu M. Gambogic acid inhibits angiogenesis and prostate tumor growth by suppressing vascular endothelial growth factor receptor 2 signaling. Cancer Res 2008; 68: 1843–1850 23 Wang X, Chen W. Gambogic acid is a novel anti‑cancer agent that inhibits cell proliferation, angiogenesis and metastasis. Anticancer Agents Med Chem 2012; 12: 994–1000

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

88

24 Chantarasriwong O, Batova A, Chavasiri W, Theodorakis EA. Chemistry and biology of the caged Garcinia xanthones. Chemistry 2010; 16: 9944–9962 25 Han QB, Tian HL, Yang NY, Qiao CF, Song JZ, Chang DC, Luo KQ, Xu HX. Polyprenylated xanthones from Garcinia lancilimba showing apoptotic effects against HeLa‑C3 cells. Chem Biodiversity 2008; 5: 2710–2717 26 Huang SX, Feng C, Zhou Y, Xu G, Han QB, Qiao CF, Chang DC, Luo KQ, Xu HX. Bioassay‑guided isolation of xanthones and polycyclic prenylated acylphloroglucinols from Garcinia oblongifolia. J Nat Prod 2009; 72: 130–135 27 Liu X, Yu T, Gao XM, Zhou Y, Qiao CF, Peng Y, Chen SL, Luo KQ, Xu HX. Apoptotic effects of polyprenylated benzoylphloroglucinol derivatives from the twigs of Garcinia multiflora. J Nat Prod 2010; 73: 1355–1359 28 Xu G, Kan WL, Zhou Y, Song JZ, Han QB, Qiao CF, Cho CH, Rudd JA, Lin G, Xu HX. Cytotoxic acylphloroglucinol derivatives from the twigs of Garcinia cowa. J Nat Prod 2010; 73: 104–108 29 Gao XM, Yu T, Cui MZ, Pu JX, Du X, Han QB, Hu QF, Liu TC, Luo KQ, Xu HX. Identification and evaluation of apoptotic compounds from Garcinia oligantha. Bioorg Med Chem Lett 2012; 22: 2350–2353 30 Feng C, Zhou LY, Yu T, Xu G, Tian HL, Xu JJ, Xu HX, Luo KQ. A new anticancer compound, oblongifolin C, inhibits tumor growth and promotes apoptosis in HeLa cells through Bax activation. Int J Cancer 2012; 131: 1445–1454 31 Fu WM, Zhang JF, Wang H, Tan HS, Wang WM, Chen SC, Zhu X, Chan TM, Tse CM, Leung KS, Lu G, Xu HX, Kung HF. Apoptosis induced by 1, 3, 6, 7tetrahydroxyxanthone in hepatocellular carcinoma and proteomic analysis. Apoptosis 2012; 17: 842–851 32 Fu WM, Zhang JF, Wang H, Xi ZC, Wang WM, Zhuang P, Zhu X, Chen SC, Chan TM, Leung KS, Lu G, Xu HX, Kung HF. Heat shock protein 27 mediates the effect of 1, 3, 5‑trihydroxy‑13, 13‑dimethyl‑2H‑pyran [7, 6‑b] xanthone on mitochondrial apoptosis in hepatocellular carcinoma. J Proteomics 2012; 75: 4833–4843 33 Kan WL, Yin C, Xu HX, Xu G, To KK, Cho CH, Rudd JA, Lin G. Antitumor effects of novel compound, guttiferone K, on colon cancer by

34

35

36

37

38

39

40 41

42

p 21Waf1/Cip1‑mediated G(0)/G(1) cell cycle arrest and apoptosis. Int J Cancer 2013; 132: 707–716 Wan G, Xie W, Liu Z, Xu W, Lao Y, Huang N, Cui K, Liao M, He J, Jiang Y, Yang BB, Xu H, Xu N, Zhang Y. Hypoxia‑induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway. Autophagy 2014; 10: 70–79 Lao Y, Wan G, Liu Z, Wang X, Ruan P, Xu W, Xu D, Xie W, Zhang Y, Xu H, Xu N. The natural compound oblongifolin C inhibits autophagic flux and enhances antitumor efficacy of nutrient deprivation. Autophagy 2014; 10: 736–749 Thoison O, Fahy J, Dumontet V, Chiaroni A, Riche C, Tri MV, Sevenet T. Cytotoxic prenylxanthones from Garcinia bracteata. J Nat Prod 2000; 63: 441–446 Shen T, Li W, Wang YY, Zhong QQ, Wang SQ, Wang XN, Ren DM, Lou HX. Antiproliferative activities of Garcinia bracteata extract and its active ingredient, isobractatin, against human tumor cell lines. Arch Pharmacal Res 2014; 37: 412–420 Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS. High‑throughput screening assays for the identification of chemical probes. Nat Chem Biol 2007; 3: 466–479 Shu CW, Madiraju C, Zhai D, Welsh K, Diaz P, Sergienko E, Sano R, Reed JC. High‑throughput fluorescence assay for small‑molecule inhibitors of autophagins/Atg4. J Biomol Screen 2011; 16: 174–182 Na Z, Hu HB, Fan QF. A novel caged‑prenylxanthone from Garcinia bracteata. Chin Chem Lett 2010; 21: 443–445 Zhang C, Wu X, Zhang M, Zhu L, Zhao R, Xu D, Lin Z, Liang C, Chen T, Chen L, Ren Y, Zhang J, Qin N, Zhang X. Small molecule R1498 as a well‑tolerated and orally active kinase inhibitor for hepatocellular carcinoma and gastric cancer treatment via targeting angiogenesis and mitosis pathways. PloS One 2013; 8: e65264 Xu D, Cao J, Qian S, Li L, Hu C, Weng Q, Lou J, Zhu D, Zhu H, Hu Y, He Q, Yang B. 5 k, a novel beta‑O‑demethyl‑epipodophyllotoxin analogue, inhibits the proliferation of cancer cells in vitro and in vivo via the induction of G2 arrest and apoptosis. Invest New Drugs 2011; 29: 786–799

Xu D et al. Identification and Characterization …

Planta Med 2015; 81: 79–89

89

This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Original Papers

Copyright of Planta Medica is the property of Georg Thieme Verlag Stuttgart and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.

Identification and characterization of anticancer compounds targeting apoptosis and autophagy from Chinese native Garcinia species.

Natural compounds from medicinal plants are important resources for drug development. Active compounds targeting apoptosis and autophagy are candidate...
493KB Sizes 1 Downloads 4 Views