Mol Biol Rep (2014) 41:2101–2108 DOI 10.1007/s11033-014-3059-z
Matrine inhibits proliferation and induces apoptosis of human colon cancer LoVo cells by inactivating Akt pathway Shujun Zhang • Binglin Cheng • Hali Li • Wei Xu • Bo Zhai • Shangha Pan • Lei Wang Ming Liu • Xueying Sun
•
Received: 19 January 2013 / Accepted: 4 January 2014 / Published online: 23 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The present study has investigated the antitumor activity and the underlying mechanisms of matrine on human colon cancer LoVo cells. Matrine inhibited the proliferation of the cells in dose- and time-dependent manners. The concentration required for 50 % inhibition (IC50) was 1.15, 0.738, and 0.414 mg/ml, when cell were incubated with matrine for 24, 48, and 72 h, respectively. Matrine induced cell cycle arrest at G1 phase by downregulating cyclin D1 and upregulating p27 and p21. Matrine induced cell apoptosis by reducing the ratio of Bcl-2/ Bax and increasing the activation of caspase-9 in a dosedependent manner. Matrine displayed its anti-tumor activity by inactivating Akt, the upstream factor of the above proteins. Matrine significantly reduced the protein levels of pAkt, and increased the protein levels of other downstream factors, pBad and pGSK-3b. Specific inhibition of pAkt induced cell apoptosis, and synergized with matrine to inhibit the proliferation of LoVo cells; whereas activation of Akt neutralized the inhibitory effect of S. Zhang Department of Pathology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China B. Cheng W. Xu Department of Integrated Medicine, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China H. Li B. Zhai S. Pan X. Sun (&) The Hepatosplenic Surgery Center, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China e-mail: [email protected] L. Wang M. Liu (&) Department of Oncology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China e-mail: [email protected]
matrine on cell proliferation. The present study has demonstrated that matrine inhibits proliferation and induces apoptosis of human colon cancer LoVo cells by inactivating Akt pathway, indicating matrine may be a potential anti-cancer agent for colon cancer. Keywords Matrine LoVo cells Apoptosis Cell cycle Akt pathway
Introduction Matrine (with a molecular formula of C15H24N2O) is one of the main components extracted from a traditional Chinese herb, Sophora flavescens Ait [1]. Matrine has displayed many promising pharmaceutical effects for many diseases in either clinical trials or animal models without showing any obvious side-effects [2–5]. Matrine has also exhibited anti-cancer activities against many types of cancers including cervical cancer [6], leukemia [1, 7–9], gastric cancer [10–12], lung cancer [13], breast cancer [14–16], melanoma [17], gallbladder carcinoma [18], prostate cancer [19], osteosarcoma [20] and hepatocellular carcinoma [13, 21]. We have previously reported that matrine induced programmed cell death by regulating relevant genes based on a PCR array analysis in C6 glioma cells [22]. More recently we have demonstrated that matrine inhibited the growth of primary and metastatic breast cancer in animal models [23] and pancreatic cancer cells in vitro and in vivo [24] by antiapoptotic, anti-proliferative and anti-angiogenic activities. However, the effects of matrine on colon cancer have not yet been investigated, though colorectal cancer is one of the leading causes of cancer-related death worldwide [25]. Therefore, we designed the present study to investigate the
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anti-cancer activity of matrine on human colon cancer LoVo cells and the underlying mechanisms.
Materials and methods Cell culture, reagents and antibodies Human colon cancer cell line LoVo was obtained from the Type Culture Collection Cell Bank (Chinese Academy of Sciences Committee, Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Shanghai, China), supplemented with 10 % fetal calf serum, 100 U/ml penicillin and 100 lg/ml streptomycin, at 37 °C in a humidified atmosphere of 5 % CO2. Matrine was purchased from Shaanxi Huike Botanical Development Co., Ltd, Xi’an, China (Batch No. MA20070302, 98 % purity). The Akt inhibitor V, triciribine, was purchased from Merk KGaA (Merk, Darmstadt, Germany). Recombinant human insulin-like growth factor (IGF)-1 was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies (Abs) against Bax, Bcl-2, Akt, phosphorylated Akt (pAkt), phosphorylated Bad (pBad)-Ser136, cyclin D1, cyclin E, p27 and p21 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA. USA). Abs against phosphorylated GSK (pGSK)-3b and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology, Danvers, MA, USA. Cell proliferation/viability assays Cell proliferation was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Cells (5 9 104) were seeded in 200 ll of medium in 96-well plates, and cultured overnight. Then the medium was replaced with fresh media containing testing agents. Untreated cells served as controls. At different time points, 20 ll of MTT was added to each well followed by a 4-h incubation. The medium was discarded and 150 ll of dimethyl sulphoxide (DMSO) was added into each well, and incubated for 20 min. The OD (optical density) 490 nm was measured, and the proliferation inhibition rate (%) or relative cell viability (%) was calculated. In addition, the cell proliferation index was also measured with a Cell Counting Kit-8 (CCK-8) kit (Dojindo Molecular Technologies, Gaithersburg, MD, USA) as described previously [26]. Assessment of cell cycle and apoptosis Cells were seeded at 5.0 9 105 cells/well in 6-well plates, cultured in media containing testing agents, and harvested. The percentage of cells at G1 and S phases was determined with a cell cycle detection kit (BD Biosciences, Beijing,
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Fig. 1 Matrine inhibits cell proliferation in dose- and time-dependent matters. Human colon cancer LoVo cells were incubated with matrine at a serial of concentrations (0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mg/ml). Cells cultured in the absence of matrine (0 mg/ml) served as controls. a The cell viability was assessed by the MTT assay at indicated time points (24, 48, and 72 h) and the inhibition of proliferation rate (%) was calculated. b The cell viability was also assessed by the CCK-8 assay and the proliferation index was calculated
China) using a Beckman Coulter Epics Altra II cytometer (Beckman Coulter, California, USA). The cells were incubated with 5 ll of Annexin V and 5 ll of propidium iodide (PI) for 15 min at room temperature in dark, according to the manufacturer’s instruction (BD Biosciences, San Jose, CA), and then subjected to flow cytometry to measure the apoptosis rate (%). The cells were also examined under a laser scanning confocal microscope (LSM-510 Meta, Carl Zeiss Jena GmbH, Jena, Germany), and apoptotic cells were counted to calculate the apoptosis rate (%). Immunoblotting analysis Protein concentrations in cell lysates were determined (Bio-Rad, Richmond, CA, USA). Lysates were resolved on
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SDS-PAGE gels, transferred to PVDF membranes, and immunoblotted as previously described [23, 24]. The density of each band was measured using a densitometric analysis program (FR200, Shanghai, China). The relative level of each protein was normalized with respect to GAPDH band density. In preliminary experiments, serial dilutions of lysates (containing 2.5, 5, 10, 20, 40 or 80 lg protein) were immunoblotted. Band intensities were measured and plotted against protein amounts to generate a standard curve, and the amount of protein for each immunoblot was determined. Statistical analysis All data were expressed as the mean values ± standard deviation (SD). The half maximal inhibitory concentration (IC50) was calculated with a logarithmic regression analysis. Comparisons were made with a one-way analysis of variance (ANOVA) followed by a Dunnett’s test using SPSS software (version 17.0). P \ 0.05 was considered statistically significant.
Results Matrine inhibits cell proliferation Matrine inhibited the proliferation of LoVo cells in doseand time-dependent manners as measured by an MTT assay (Fig. 1a). With a logarithmic regression analysis, the values of IC50 were calculated to be 1.15 mg/ml (R2 = 0.905), 0.738 mg/ml (R2 = 0.983), and 0.414 mg/ml (R2 = 0.905), when the cells were incubated with matrine for 24, 48 and 72 h, respectively (Fig. 1a). The inhibitory effect of matrine on cell proliferation was further confirmed by CCK-8 assay (Fig. 1b).
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controls (Fig. 2e, f). Furthermore, the alterations of protein levels of cyclin D1, p27 and p21 upon matrine treatment showed a dose-dependent manner. Matrine induces cell apoptosis LoVo cells were incubated with matrine for 48 h, and flow cytometric analysis was used to measure the apoptosis rates. The representative dot histograms of cytometry show the rates of early-staged apoptotic cells were 1.2, 5.8, 14.9 or 17.6 %; and that of late-staged apoptotic cells were 0.1, 0.5, 2.7 or 3.6 %; when cells were treated with matrine at concentrations of 0, 0.4, 0.8 or 1.6 mg/ml, respectively (Fig. 3a–d). Matrine induced apoptosis of LoVo cells in a dose-dependent manner, and the rates of both early- and late-staged apoptotic cells in matrine-treated cells were significantly increased, compared with untreated controls (Fig. 3e). The cells were further examined under microscopy, and apoptotic cells were counted. Similarly, matrine induced cell apoptosis in a dose-dependent matter. Thus incubation of matrine at concentrations of 0.4, 0.8, and 1.6 mg/ml led to apoptosis rates of 8.6, 16.7, and 24.4 %, respectively (Fig. 3f). Only a small number of apoptotic cells were detected among untreated cells, and most of them were at the early phase of apoptosis (Fig. 3g). However, among the matrine-treated cells, we could detect more apoptotic cells including late-staged apoptotic cells, which had their nuclei stained red by PI as the plasma membrane becomes increasingly permeable during the later stage of apoptosis (Fig. 3h–j). We further detected the changes of apoptosis-related proteins in the above cells. Matrine treatment upregulated expression of Bax and downregulated expression of Bcl-2, thus reduced the ratio of Bcl-2/Bax, and also increased the activation of caspase-9, in a dose-dependent manner (Fig. 3k–m).
Matrine induces cell cycle arrest Matrine inactivates Akt pathway We next investigated the effects of matrine on the cell cycle as analyzed by flow cytometry. The fraction of cells at the G1 phase was highly significantly (P \ 0.001) increased in matrine-treated cells, compared with untreated controls, in a dose-dependent manner (Fig. 2a–d). To elucidate the mechanisms accounting for the cell cycle arrest by matrine, we examined the expression of regulators of the G1/S phase transition. As shown in Fig. 2e, f, the protein levels of cyclin D1 but not cyclin E was highly significantly (P \ 0.001) decreased in matrine-treated cells compared with those in untreated controls. In contrast, the levels of p27 and p21 were highly significantly (P \ 0.001) elevated in matrine-treated cells compared with untreated
Given that the above proteins examined are linked to Akt pathways, thus we hypothesized that matrine might display its activities on LoVo cells through effecting Akt activation. As shown in Fig. 4a, b, matrine had little effect on the expression of Akt, while highly significantly reduced the protein levels of pAkt in a dose-dependent matter. Matrine also significantly increased the protein levels of pBad and pGSK-3b (Fig. 4a, b), which are negatively regulated by Akt [27]. The data drove us to investigate whether inhibition of Akt activation could induce apoptosis of LoVo cells as Akt inhibitors have been shown to induce apoptosis of cancer
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Fig. 2 Matrine induces cell cycle arrest. LoVo cells were incubated with matrine for 48 h, and then subjected to flow cytometry to measure the stages of cell cycle. a–c Representative histograms were taken from cytometrically analyzed cells cultured in the medium in the absence (0 mg/ml, control) (a) or presence of matrine at concentrations of 0.4 (b) or 0.8 (c) mg/ml. d The percentages of cells in the G1 phase were plotted. e Expression of cyclin D1, cyclin E, p27 and p21 proteins was detected using immunoblotting. f The density of each protein band was measured and normalized to that of GAPDH. A significant (P \ 0.05) difference from respective control is denoted by asterisk, a highly significant (P \ 0.001), by double asterisk
cells [28]. Here, we could also confirm that triciribine highly significantly reduced the level of pAkt (Fig. 4c, d), and increased the apoptosis rates of LoVo cells (Fig. 4e). We further investigated that whether triciribine could synergize with matrine to inhibit the growth of LoVo cells. As shown in Fig. 4f, triciribine highly significant reduced the viability of LoVo cells, and the combination of triciribine and matrine resulted in even lower viability of the cells, compared with triciribine or matrine alone. We further investigated whether activation of Akt could neutralize the inhibitory effect of matrine by using IGF-1, an activator of Akt [29]. As shown in Fig. 4g, IGF-1 significant stimulated the growth of LoVo cells, and the combination of IGF-1 and matrine resulted in a significantly higher proliferation index than matrine alone.
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Discussion Since the discovery of matrine as a potential anti-cancer drug, many lines of studies have tested its anti-cancer activities in various types of cancer cells [1, 6–24], but little is known about its effects on colon cancer. The present study has demonstrated that matrine inhibited the growth of human colon cancer LoVo cells by inactivating Akt pathways. Matrine inhibited the proliferation of LoVo cells in dose- and time-dependent manners, induced cell cycle arrest at G1 phase by downregulating cyclin D1, and upregulating p27 and p21. Matrine increased cell apoptosis by reducing the ratio of Bcl-2/Bax and increasing activation of caspase-9. Matrine inhibited activation of Akt, evidenced by reduced protein levels of pAkt, and increased
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Fig. 3 Matrine induces cell apoptosis. LoVo cells were incubated with matrine for 48 h, and then cell apoptosis was examined by cytometry and microscopy. a–d Representative dot plots were from cytometrically analyzed cells cultured in the medium in the absence (0 mg/ml, control) (a) or presence of matrine at concentrations of 0.4 (b), 0.8 (c) or 1.6 (d) mg/ml. EA, early-staged apoptosis; LA, latestaged apoptosis. The apoptosis rates measured by cytometry (e) or microscopy (f) were plotted. g–j Representative photographs were
taken from Annexin V/PI-stained cells incubated in the absence (0 mg/ml, control) (g) or presence of matrine at concentrations of 0.4 (h), 0.8 (i) or 1.6 mg/ml (j). k The above cells were lysed and immunoblotted. l, m The density of each band in k was measured and normalized to that of GAPDH, respectively, and the ratio of Bcl-2/ Bax was calculated (l). A significant (P \ 0.05) difference from respective control is denoted by asterisk, a highly significant (P \ 0.001), by double asterisk
expression of pBad and pGSK-3b. Furthermore, specific inhibition of Akt induced apoptosis and synergized with matrine to inhibit the viability of LoVo cells; while activation of Akt neutralized the inhibitory effect of matrine. Akt is the central mediator of the Akt pathway with numerous downstream molecules, such as GSK-3, Bad,
p21, p27 and cyclin D1, which regulate cell proliferation and apoptosis [27, 30]. The activation of Akt was positively correlated with cancer stages, indicating that pAkt is an independent prognostic maker for colon cancer patients [31]. Blocking Akt activation has been shown to inhibit proliferation and induce apoptosis in cells of many types of
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Fig. 4 Matrine inhibits activation of Akt. a LoVo cells were cultured in the medium in the absence (0 mg/ml, control) or presence of matrine at concentrations of 0.4 or 0.8 mg/ml, and then subjected to immunoblotting for detecting expression of Akt, pAkt, pBad and pGSK-3b. b The density of each protein band in a was measured and normalized to that of GAPDH. c–e Cells were cultured in the medium in the absence or presence of triciribine (1 or 3 lM) for 4 h, and then subjected to immunoblotting (c) or cytometry to measure apoptosis rates (e). d The density of each protein band in c was measured and normalized to that of GAPDH. f Cells were incubated with matrine (0.4 mg/ml), triciribine (1 lM), or the combination for 8 h, and then cell viability was measured by MTT assay. g Cells were incubated with matrine (0.8 mg/ml), IGF-1 (50 ng/ml), or the combination for 24 h and then cell proliferation index was measured by CCK-8 assay. Untreated cells served as controls. A significant (P \ 0.05) difference from respective control is denoted by asterisk, a highly significant (P \ 0.001), by double asterisk. Dagger indicates a significant (P \ 0.05) reduction from the cells treated with matrine or triciribine alone. Double dagger indicates a significant (P \ 0.05) increase from the cells treated with matrine alone
cancers including colorectal cancer [32]. Those reports together with the present results indicate that Akt plays an important role in regulating proliferation and apoptosis of colon cancer cells. Further, we have demonstrated that matrine inhibited the activation of Akt, and thereby regulated the expression of proteins controlling cell proliferation and apoptosis in LoVo cells. In accord, it has been reported that matrine inactivates Akt in human acute myeloid leukemia [1] or breast cancer [15] cells. P21 and p27 are implicated in the negative regulation of cell cycle progression from G1 to S phase by binding to and modulating the activity of CDKs [33]. Akt
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negatively regulates p21 and p27 and opposes p27mediated G1 arrest [34]. It has reported that matrine upregulated p21 and p27 by dephosphorylating Akt in human breast cancer MCF-7 cells [15]. Akt positively regulates cyclin D1 expression through inactivation of GSK3b [27]. Conversely, cyclin D1 promotes progression from G1 to S phase by triggering the initiation of DNA replication, regulating genes that orchestrate cell proliferation and G1 to S phase transition [35]. Therefore, matrine induced cell cycle arrest at G1 phase by downregulating cyclin D1 and upregulating p27 and p21 via inactivation of Akt.
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Matrine has been shown to induce apoptosis of cells of gastric cancer [10], leukemia [8, 9], lung cancer and hepatocellular carcinoma [13], and C6 glioma [22], by regulating molecules of Bcl-2 family and activating caspases. In accord, the present study has demonstrated that matrine-induced apoptosis was accompanied by a decrease of the ratio of Bcl-2/Bax, and increased activation of caspase-9. Although not investigated herein, matrine may also induce cell apoptosis via death receptor-mediated apoptosis pathways, as it has also been reported that matrine induced apoptosis of human gastric cancer [11] and osteosarcoma [20] cells through upregulating Fas/FasL. We have previously reported that matrine upregulated expression of 14 members of TNF gene family in a PCR array in glioma cells, indicating that the death receptor pathway might be involved in matrine-induced cell apoptosis [22]. In summary, the present data have shown that matrine displayed anti-cancer activity against human colon cancer LoVo cells by inactivating Akt pathway. Although not investigated in the present study, matrine may also inhibit cell proliferation via other cellular signal pathways. For instance, it has been reported that matrine modulated mitogen-activated protein kinase (MAPK) pathway in human Jurkat T cells [36]. However, in another study, matrine induced cell apoptosis of leukemia U937 cells in an MAPK-independent pathway [7]. The controversy could be explained by that matrine regulates MAPK pathway in a cell specific manner. But the effect of matrine on MAPK pathway in colon cancer cells needs further investigation. Compared to conventional chemotherapeutic agents, matrine does not have serious adverse cytotoxicity [2–5], and has displayed synergistic effects with cytotoxic agents, in inhibiting proliferation of human breast cancer cells [16]. Importantly, clinical trials in China have demonstrated that administration of matrine improved immune function and life quality of cancer patients who received conventional therapies [37, 38]. These studies suggest that matrine may be a potential anti-cancer agent and warrant future investigation for colon cancer. Acknowledgments This work was supported by grants from the National Natural Scientific Foundation of China (30973474 and 81272467), and Postdoctoral Foundation of Heilongjiang Provincial Government (LRB09-404). Zhang S and Cheng B contributed equally to this work.
References 1. Zhang S, Zhang Y, Zhuang Y, Wang J, Ye J, Zhang S, Wu J, Yu K, Han Y (2012) Matrine induces apoptosis in human acute myeloid leukemia cells via the mitochondrial pathway and Akt inactivation. PLoS One 7:e46853 2. Long Y, Lin XT, Zeng KL, Zhang L (2004) Efficacy of intramuscular matrine in the treatment of chronic hepatitis B. Hepatobiliary Pancreat Dis Int 3:69–72
2107 3. Zhang JP, Zhang M, Zhou JP, Liu FT, Zhou B, Xie WF, Guo C (2001) Antifibrotic effects of matrine on in vitro and in vivo models of liver fibrosis in rats. Acta Pharmacol Sin 22:183–186 4. Liu JY, Hu JH, Zhu QG, Li FQ, Wang J, Sun HJ (2007) Effect of matrine on the expression of substance P receptor and inflammatory cytokines production in human skin keratinocytes and fibroblasts. Int Immunopharmacol 7:816–823 5. Li X, Chu W, Liu J, Xue X, Lu Y, Shan H, Yang B (2009) Antiarrhythmic properties of long-term treatment with matrine in arrhythmic rat induced by coronary ligation. Biol Pharm Bull 32:1521–1526 6. Zhang L, Wang T, Wen X, Wei Y, Peng X, Li H, Wei L (2007) Effect of matrine on HeLa cell adhesion and migration. Eur J Pharmacol 563:69–76 7. Liu XS, Jiang J, Jiao XY, Wu YE, Lin JH (2006) Matrineinduced apoptosis in leukemia U937 cells: involvement of caspases activation and MAPK-independent pathways. Planta Med 72:501–506 8. Liu XS, Jiang J (2006) Molecular mechanism of matrine- induced apoptosis in leukemia K562 Cells. Am J Chin Med 34:1095–1103 9. Jiang H, Hou C, Zhang S, Xie H, Zhou W, Jin Q, Cheng X, Qian R, Zhang X (2007) Matrine upregulates the cell cycle protein E2F-1 and triggers apoptosis via the mitochondrial pathway in K562 cells. Eur J Pharmacol 559:98–108 10. Luo C, Zhu Y, Jiang T, Lu X, Zhang W, Jing Q, Li J, Pang L, Chen K, Qiu F, Yu X, Yang J, Huang J (2007) Matrine induced gastric cancer MKN45 cells apoptosis via increasing pro-apoptotic molecules of Bcl-2 family. Toxicology 229:245–252 11. Dai ZJ, Gao J, Ji ZZ, Wang XJ, Ren HT, Liu XX, Wu WY, Kang HF, Guang H (2009) Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3. J Ethnopharmacol 123:91–96 12. Luo C, Zhong HJ, Zhu LM, Wu XG, Ying JE, Wang XH, Lu¨ WX, Xu Q, Zhu YL, Huang J (2012) Inhibition of matrine against gastric cancer cell line MNK45 growth and its anti-tumor mechanism. Mol Biol Rep 39:5459–5464 13. Zhang Y, Zhang H, Yu P, Liu Q, Liu K, Duan H, Luan G, Yagasaki K, Zhang G (2009) Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration. Cytotechnology 59:191–200 14. Yu P, Liu Q, Liu K, Yagasaki K, Wu E, Zhang G (2009) Matrine suppresses breast cancer cell proliferation and invasion via VEGF-Akt-NF-kappaB signaling. Cytotechnology 59:219–229 15. Li LQ, Li XL, Wang L, Du WJ, Guo R, Liang HH, Liu X, Liang DS, Lu YJ, Shan HL, Jiang HC (2012) Matrine Inhibits Breast Cancer Growth Via miR-21/PTEN/Akt Pathway in MCF-7 Cells. Cell Physiol Biochem 30:631–641 16. Yu P, Liu Q, Liu K, Yagasaki K, Wu E (2009) Matrine suppresses breast cancer cell proliferation and invasion via VEGFAkt-NF-kappaB signaling. Cytotechnology 59:219–229 17. Liu XY, Fang H, Yang ZG, Wang XY, Ruan LM, Fang DR, Ding YG, Wang YN, Zhang Y, Jiang XL, Chen HC (2008) Matrine inhibits invasiveness and metastasis of human malignant melanoma cell line A375 in vitro. Int J Dermatol 47:448–456 18. Zhang Z, Wang X, Wu W, Wang J, Wang Y, Wu X, Fei X, Li S, Zhang J, Dong P, Gu J, Liu Y (2012) Effects of matrine on proliferation and apoptosis in gallbladder carcinoma cells (GBCSD). Phytother Res 26:932–937 19. Zhang P, Wang Z, Chong T, Ji Z (2012) Matrine inhibits proliferation and induces apoptosis of the androgen-independent prostate cancer cell line PC-3. Mol Med Rep 5:783–787 20. Liang CZ, Zhang JK, Shi Z, Liu B, Shen CQ, Tao HM (2012) Matrine induces caspase-dependent apoptosis in human osteosarcoma cells in vitro and in vivo through the upregulation of Bax and Fas/FasL and downregulation of Bcl-2. Cancer Chemother Pharmacol 69:317–331
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2108 21. Ma L, Wen S, Zhan Y, He Y, Liu X, Jiang J (2008) Anticancer effects of the Chinese medicine matrine on murine hepatocellular carcinoma cells. Planta Med 74:245–251 22. Zhang S, Qi J, Sun L, Cheng B, Pan S, Zhou M, Sun X (2009) Matrine induces programmed cell death and regulates expression of relevant genes based on PCR array analysis in C6 glioma cells. Mol Biol Rep 35:421–429 23. Li H, Tan G, Jiang X, Qiao H, Pan S, Jiang H, Kanwar JR, Sun X (2010) Matrine inhibits growth of primary and metastatic breast cancer in mice. Am J Chin Med 38:1115–1130 24. Liu T, Song Y, Chen H, Pan S, Sun X (2010) Matrine inhibits proliferation and induces apoptosis of pancreatic cancer cells in vitro and in vivo. Biol Pharm Bull 33:1740–1745 25. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. CA Cancer J Clin 61:69–90 26. He C, Sun XP, Qiao H, Jiang X, Wang D, Jin X, Dong X, Wang J, Jiang H, Sun X (2012) Downregulating Hypoxia-inducible factor2a improves the efficacy of doxorubicin to treat hepatocellular carcinoma. Cancer Sci 103:528–534 27. Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream. Cell 129:1261–1274 28. Dieterle A, Orth R, Daubrawa M, Grotemeier A, Alers S, Ullrich S, Lammers R, Wesselborg S, Stork B (2009) The Akt inhibitor triciribine sensitizes prostate carcinoma cells to TRAIL-induced apoptosis. Int J Cancer 125:932–941 29. Zheng WH, Quirion R (2006) Insulin-like growth factor-1 (IGF-1) induces the activation/phosphorylation of Akt kinase and cAMP responseelement-binding protein (CREB) by activating different signaling pathways in PC12 cells. BMC Neurosci 7:51 30. Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-kinaseAKT pathway in human cancer. Nat Rev Cancer 2:489–501
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Mol Biol Rep (2014) 41:2101–2108 31. Zhang Y, Liu X, Zhang J, Li L, Liu C (2012) The expression and clinical significance of PI3 K, pAkt and VEGF in colon cancer. Oncol Lett 4:763–766 32. Ericson K, Gan C, Cheong I, Rago C, Samuels Y, Velculescu VE, Kinzler KW, Huso DL, Vogelstein B, Papadopoulos N (2010) Genetic inactivation of AKT1, AKT2, and PDPK1 in human colorectal cancer cells clarifies their roles in tumor growth regulation. Proc Natl Acad Sci USA 107:2598–2603 33. Lu Z, Hunter T (2010) Ubiquitylation and proteasomal degradation of the p21 Cip1, p27 Kip1 and p57 Kip2 CDK inhibitors. Cell Cycle 9:2342–2352 34. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM (2002) PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 8:1153–1160 35. Alao JP (2007) The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention. Mol Cancer 6:24 36. Li T, Wong VK, Yi XQ, Wong YF, Zhou H, Liu L (2010) Matrine induces cell anergy in human Jurkat T cells through modulation of mitogen-activated protein kinases and nuclear factor of activated T-cells signaling with concomitant up-regulation of anergy-associated genes expression. Biol Pharm Bull 33:40–46 37. Chen J, Mei Q, Xu YC, Du J, Wei Y, Xu ZM (2006) Effects of matrine injection on T-lymphocyte subsets of patients with malignant tumor after gamma knife radiosurgery]. [Article in Chinese]. Zhong Xi Yi Jie He Xue Bao 4:78–79 38. Huang S, Fan W, Liu P, Tian J (2011) Meta analysis of compound matrine injection combined with cisplatin chemotherapy for advanced gastric cancer]. [Article in Chinese]. Zhongguo Zhong Yao Za Zhi 36:3198–3202
Matrine inhibits proliferation and induces apoptosis of human colon cancer LoVo cells by inactivating Akt pathway Shujun Zhang • Binglin Cheng • Hali Li • Wei Xu • Bo Zhai • Shangha Pan • Lei Wang Ming Liu • Xueying Sun
•
Received: 19 January 2013 / Accepted: 4 January 2014 / Published online: 23 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The present study has investigated the antitumor activity and the underlying mechanisms of matrine on human colon cancer LoVo cells. Matrine inhibited the proliferation of the cells in dose- and time-dependent manners. The concentration required for 50 % inhibition (IC50) was 1.15, 0.738, and 0.414 mg/ml, when cell were incubated with matrine for 24, 48, and 72 h, respectively. Matrine induced cell cycle arrest at G1 phase by downregulating cyclin D1 and upregulating p27 and p21. Matrine induced cell apoptosis by reducing the ratio of Bcl-2/ Bax and increasing the activation of caspase-9 in a dosedependent manner. Matrine displayed its anti-tumor activity by inactivating Akt, the upstream factor of the above proteins. Matrine significantly reduced the protein levels of pAkt, and increased the protein levels of other downstream factors, pBad and pGSK-3b. Specific inhibition of pAkt induced cell apoptosis, and synergized with matrine to inhibit the proliferation of LoVo cells; whereas activation of Akt neutralized the inhibitory effect of S. Zhang Department of Pathology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China B. Cheng W. Xu Department of Integrated Medicine, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China H. Li B. Zhai S. Pan X. Sun (&) The Hepatosplenic Surgery Center, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China e-mail: [email protected] L. Wang M. Liu (&) Department of Oncology, The Fourth Affiliated Hospital of Harbin Medical University, Harbin 150001, China e-mail: [email protected]
matrine on cell proliferation. The present study has demonstrated that matrine inhibits proliferation and induces apoptosis of human colon cancer LoVo cells by inactivating Akt pathway, indicating matrine may be a potential anti-cancer agent for colon cancer. Keywords Matrine LoVo cells Apoptosis Cell cycle Akt pathway
Introduction Matrine (with a molecular formula of C15H24N2O) is one of the main components extracted from a traditional Chinese herb, Sophora flavescens Ait [1]. Matrine has displayed many promising pharmaceutical effects for many diseases in either clinical trials or animal models without showing any obvious side-effects [2–5]. Matrine has also exhibited anti-cancer activities against many types of cancers including cervical cancer [6], leukemia [1, 7–9], gastric cancer [10–12], lung cancer [13], breast cancer [14–16], melanoma [17], gallbladder carcinoma [18], prostate cancer [19], osteosarcoma [20] and hepatocellular carcinoma [13, 21]. We have previously reported that matrine induced programmed cell death by regulating relevant genes based on a PCR array analysis in C6 glioma cells [22]. More recently we have demonstrated that matrine inhibited the growth of primary and metastatic breast cancer in animal models [23] and pancreatic cancer cells in vitro and in vivo [24] by antiapoptotic, anti-proliferative and anti-angiogenic activities. However, the effects of matrine on colon cancer have not yet been investigated, though colorectal cancer is one of the leading causes of cancer-related death worldwide [25]. Therefore, we designed the present study to investigate the
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anti-cancer activity of matrine on human colon cancer LoVo cells and the underlying mechanisms.
Materials and methods Cell culture, reagents and antibodies Human colon cancer cell line LoVo was obtained from the Type Culture Collection Cell Bank (Chinese Academy of Sciences Committee, Shanghai, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Shanghai, China), supplemented with 10 % fetal calf serum, 100 U/ml penicillin and 100 lg/ml streptomycin, at 37 °C in a humidified atmosphere of 5 % CO2. Matrine was purchased from Shaanxi Huike Botanical Development Co., Ltd, Xi’an, China (Batch No. MA20070302, 98 % purity). The Akt inhibitor V, triciribine, was purchased from Merk KGaA (Merk, Darmstadt, Germany). Recombinant human insulin-like growth factor (IGF)-1 was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies (Abs) against Bax, Bcl-2, Akt, phosphorylated Akt (pAkt), phosphorylated Bad (pBad)-Ser136, cyclin D1, cyclin E, p27 and p21 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA. USA). Abs against phosphorylated GSK (pGSK)-3b and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology, Danvers, MA, USA. Cell proliferation/viability assays Cell proliferation was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Cells (5 9 104) were seeded in 200 ll of medium in 96-well plates, and cultured overnight. Then the medium was replaced with fresh media containing testing agents. Untreated cells served as controls. At different time points, 20 ll of MTT was added to each well followed by a 4-h incubation. The medium was discarded and 150 ll of dimethyl sulphoxide (DMSO) was added into each well, and incubated for 20 min. The OD (optical density) 490 nm was measured, and the proliferation inhibition rate (%) or relative cell viability (%) was calculated. In addition, the cell proliferation index was also measured with a Cell Counting Kit-8 (CCK-8) kit (Dojindo Molecular Technologies, Gaithersburg, MD, USA) as described previously [26]. Assessment of cell cycle and apoptosis Cells were seeded at 5.0 9 105 cells/well in 6-well plates, cultured in media containing testing agents, and harvested. The percentage of cells at G1 and S phases was determined with a cell cycle detection kit (BD Biosciences, Beijing,
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Fig. 1 Matrine inhibits cell proliferation in dose- and time-dependent matters. Human colon cancer LoVo cells were incubated with matrine at a serial of concentrations (0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mg/ml). Cells cultured in the absence of matrine (0 mg/ml) served as controls. a The cell viability was assessed by the MTT assay at indicated time points (24, 48, and 72 h) and the inhibition of proliferation rate (%) was calculated. b The cell viability was also assessed by the CCK-8 assay and the proliferation index was calculated
China) using a Beckman Coulter Epics Altra II cytometer (Beckman Coulter, California, USA). The cells were incubated with 5 ll of Annexin V and 5 ll of propidium iodide (PI) for 15 min at room temperature in dark, according to the manufacturer’s instruction (BD Biosciences, San Jose, CA), and then subjected to flow cytometry to measure the apoptosis rate (%). The cells were also examined under a laser scanning confocal microscope (LSM-510 Meta, Carl Zeiss Jena GmbH, Jena, Germany), and apoptotic cells were counted to calculate the apoptosis rate (%). Immunoblotting analysis Protein concentrations in cell lysates were determined (Bio-Rad, Richmond, CA, USA). Lysates were resolved on
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SDS-PAGE gels, transferred to PVDF membranes, and immunoblotted as previously described [23, 24]. The density of each band was measured using a densitometric analysis program (FR200, Shanghai, China). The relative level of each protein was normalized with respect to GAPDH band density. In preliminary experiments, serial dilutions of lysates (containing 2.5, 5, 10, 20, 40 or 80 lg protein) were immunoblotted. Band intensities were measured and plotted against protein amounts to generate a standard curve, and the amount of protein for each immunoblot was determined. Statistical analysis All data were expressed as the mean values ± standard deviation (SD). The half maximal inhibitory concentration (IC50) was calculated with a logarithmic regression analysis. Comparisons were made with a one-way analysis of variance (ANOVA) followed by a Dunnett’s test using SPSS software (version 17.0). P \ 0.05 was considered statistically significant.
Results Matrine inhibits cell proliferation Matrine inhibited the proliferation of LoVo cells in doseand time-dependent manners as measured by an MTT assay (Fig. 1a). With a logarithmic regression analysis, the values of IC50 were calculated to be 1.15 mg/ml (R2 = 0.905), 0.738 mg/ml (R2 = 0.983), and 0.414 mg/ml (R2 = 0.905), when the cells were incubated with matrine for 24, 48 and 72 h, respectively (Fig. 1a). The inhibitory effect of matrine on cell proliferation was further confirmed by CCK-8 assay (Fig. 1b).
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controls (Fig. 2e, f). Furthermore, the alterations of protein levels of cyclin D1, p27 and p21 upon matrine treatment showed a dose-dependent manner. Matrine induces cell apoptosis LoVo cells were incubated with matrine for 48 h, and flow cytometric analysis was used to measure the apoptosis rates. The representative dot histograms of cytometry show the rates of early-staged apoptotic cells were 1.2, 5.8, 14.9 or 17.6 %; and that of late-staged apoptotic cells were 0.1, 0.5, 2.7 or 3.6 %; when cells were treated with matrine at concentrations of 0, 0.4, 0.8 or 1.6 mg/ml, respectively (Fig. 3a–d). Matrine induced apoptosis of LoVo cells in a dose-dependent manner, and the rates of both early- and late-staged apoptotic cells in matrine-treated cells were significantly increased, compared with untreated controls (Fig. 3e). The cells were further examined under microscopy, and apoptotic cells were counted. Similarly, matrine induced cell apoptosis in a dose-dependent matter. Thus incubation of matrine at concentrations of 0.4, 0.8, and 1.6 mg/ml led to apoptosis rates of 8.6, 16.7, and 24.4 %, respectively (Fig. 3f). Only a small number of apoptotic cells were detected among untreated cells, and most of them were at the early phase of apoptosis (Fig. 3g). However, among the matrine-treated cells, we could detect more apoptotic cells including late-staged apoptotic cells, which had their nuclei stained red by PI as the plasma membrane becomes increasingly permeable during the later stage of apoptosis (Fig. 3h–j). We further detected the changes of apoptosis-related proteins in the above cells. Matrine treatment upregulated expression of Bax and downregulated expression of Bcl-2, thus reduced the ratio of Bcl-2/Bax, and also increased the activation of caspase-9, in a dose-dependent manner (Fig. 3k–m).
Matrine induces cell cycle arrest Matrine inactivates Akt pathway We next investigated the effects of matrine on the cell cycle as analyzed by flow cytometry. The fraction of cells at the G1 phase was highly significantly (P \ 0.001) increased in matrine-treated cells, compared with untreated controls, in a dose-dependent manner (Fig. 2a–d). To elucidate the mechanisms accounting for the cell cycle arrest by matrine, we examined the expression of regulators of the G1/S phase transition. As shown in Fig. 2e, f, the protein levels of cyclin D1 but not cyclin E was highly significantly (P \ 0.001) decreased in matrine-treated cells compared with those in untreated controls. In contrast, the levels of p27 and p21 were highly significantly (P \ 0.001) elevated in matrine-treated cells compared with untreated
Given that the above proteins examined are linked to Akt pathways, thus we hypothesized that matrine might display its activities on LoVo cells through effecting Akt activation. As shown in Fig. 4a, b, matrine had little effect on the expression of Akt, while highly significantly reduced the protein levels of pAkt in a dose-dependent matter. Matrine also significantly increased the protein levels of pBad and pGSK-3b (Fig. 4a, b), which are negatively regulated by Akt [27]. The data drove us to investigate whether inhibition of Akt activation could induce apoptosis of LoVo cells as Akt inhibitors have been shown to induce apoptosis of cancer
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Fig. 2 Matrine induces cell cycle arrest. LoVo cells were incubated with matrine for 48 h, and then subjected to flow cytometry to measure the stages of cell cycle. a–c Representative histograms were taken from cytometrically analyzed cells cultured in the medium in the absence (0 mg/ml, control) (a) or presence of matrine at concentrations of 0.4 (b) or 0.8 (c) mg/ml. d The percentages of cells in the G1 phase were plotted. e Expression of cyclin D1, cyclin E, p27 and p21 proteins was detected using immunoblotting. f The density of each protein band was measured and normalized to that of GAPDH. A significant (P \ 0.05) difference from respective control is denoted by asterisk, a highly significant (P \ 0.001), by double asterisk
cells [28]. Here, we could also confirm that triciribine highly significantly reduced the level of pAkt (Fig. 4c, d), and increased the apoptosis rates of LoVo cells (Fig. 4e). We further investigated that whether triciribine could synergize with matrine to inhibit the growth of LoVo cells. As shown in Fig. 4f, triciribine highly significant reduced the viability of LoVo cells, and the combination of triciribine and matrine resulted in even lower viability of the cells, compared with triciribine or matrine alone. We further investigated whether activation of Akt could neutralize the inhibitory effect of matrine by using IGF-1, an activator of Akt [29]. As shown in Fig. 4g, IGF-1 significant stimulated the growth of LoVo cells, and the combination of IGF-1 and matrine resulted in a significantly higher proliferation index than matrine alone.
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Discussion Since the discovery of matrine as a potential anti-cancer drug, many lines of studies have tested its anti-cancer activities in various types of cancer cells [1, 6–24], but little is known about its effects on colon cancer. The present study has demonstrated that matrine inhibited the growth of human colon cancer LoVo cells by inactivating Akt pathways. Matrine inhibited the proliferation of LoVo cells in dose- and time-dependent manners, induced cell cycle arrest at G1 phase by downregulating cyclin D1, and upregulating p27 and p21. Matrine increased cell apoptosis by reducing the ratio of Bcl-2/Bax and increasing activation of caspase-9. Matrine inhibited activation of Akt, evidenced by reduced protein levels of pAkt, and increased
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Fig. 3 Matrine induces cell apoptosis. LoVo cells were incubated with matrine for 48 h, and then cell apoptosis was examined by cytometry and microscopy. a–d Representative dot plots were from cytometrically analyzed cells cultured in the medium in the absence (0 mg/ml, control) (a) or presence of matrine at concentrations of 0.4 (b), 0.8 (c) or 1.6 (d) mg/ml. EA, early-staged apoptosis; LA, latestaged apoptosis. The apoptosis rates measured by cytometry (e) or microscopy (f) were plotted. g–j Representative photographs were
taken from Annexin V/PI-stained cells incubated in the absence (0 mg/ml, control) (g) or presence of matrine at concentrations of 0.4 (h), 0.8 (i) or 1.6 mg/ml (j). k The above cells were lysed and immunoblotted. l, m The density of each band in k was measured and normalized to that of GAPDH, respectively, and the ratio of Bcl-2/ Bax was calculated (l). A significant (P \ 0.05) difference from respective control is denoted by asterisk, a highly significant (P \ 0.001), by double asterisk
expression of pBad and pGSK-3b. Furthermore, specific inhibition of Akt induced apoptosis and synergized with matrine to inhibit the viability of LoVo cells; while activation of Akt neutralized the inhibitory effect of matrine. Akt is the central mediator of the Akt pathway with numerous downstream molecules, such as GSK-3, Bad,
p21, p27 and cyclin D1, which regulate cell proliferation and apoptosis [27, 30]. The activation of Akt was positively correlated with cancer stages, indicating that pAkt is an independent prognostic maker for colon cancer patients [31]. Blocking Akt activation has been shown to inhibit proliferation and induce apoptosis in cells of many types of
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Fig. 4 Matrine inhibits activation of Akt. a LoVo cells were cultured in the medium in the absence (0 mg/ml, control) or presence of matrine at concentrations of 0.4 or 0.8 mg/ml, and then subjected to immunoblotting for detecting expression of Akt, pAkt, pBad and pGSK-3b. b The density of each protein band in a was measured and normalized to that of GAPDH. c–e Cells were cultured in the medium in the absence or presence of triciribine (1 or 3 lM) for 4 h, and then subjected to immunoblotting (c) or cytometry to measure apoptosis rates (e). d The density of each protein band in c was measured and normalized to that of GAPDH. f Cells were incubated with matrine (0.4 mg/ml), triciribine (1 lM), or the combination for 8 h, and then cell viability was measured by MTT assay. g Cells were incubated with matrine (0.8 mg/ml), IGF-1 (50 ng/ml), or the combination for 24 h and then cell proliferation index was measured by CCK-8 assay. Untreated cells served as controls. A significant (P \ 0.05) difference from respective control is denoted by asterisk, a highly significant (P \ 0.001), by double asterisk. Dagger indicates a significant (P \ 0.05) reduction from the cells treated with matrine or triciribine alone. Double dagger indicates a significant (P \ 0.05) increase from the cells treated with matrine alone
cancers including colorectal cancer [32]. Those reports together with the present results indicate that Akt plays an important role in regulating proliferation and apoptosis of colon cancer cells. Further, we have demonstrated that matrine inhibited the activation of Akt, and thereby regulated the expression of proteins controlling cell proliferation and apoptosis in LoVo cells. In accord, it has been reported that matrine inactivates Akt in human acute myeloid leukemia [1] or breast cancer [15] cells. P21 and p27 are implicated in the negative regulation of cell cycle progression from G1 to S phase by binding to and modulating the activity of CDKs [33]. Akt
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negatively regulates p21 and p27 and opposes p27mediated G1 arrest [34]. It has reported that matrine upregulated p21 and p27 by dephosphorylating Akt in human breast cancer MCF-7 cells [15]. Akt positively regulates cyclin D1 expression through inactivation of GSK3b [27]. Conversely, cyclin D1 promotes progression from G1 to S phase by triggering the initiation of DNA replication, regulating genes that orchestrate cell proliferation and G1 to S phase transition [35]. Therefore, matrine induced cell cycle arrest at G1 phase by downregulating cyclin D1 and upregulating p27 and p21 via inactivation of Akt.
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Matrine has been shown to induce apoptosis of cells of gastric cancer [10], leukemia [8, 9], lung cancer and hepatocellular carcinoma [13], and C6 glioma [22], by regulating molecules of Bcl-2 family and activating caspases. In accord, the present study has demonstrated that matrine-induced apoptosis was accompanied by a decrease of the ratio of Bcl-2/Bax, and increased activation of caspase-9. Although not investigated herein, matrine may also induce cell apoptosis via death receptor-mediated apoptosis pathways, as it has also been reported that matrine induced apoptosis of human gastric cancer [11] and osteosarcoma [20] cells through upregulating Fas/FasL. We have previously reported that matrine upregulated expression of 14 members of TNF gene family in a PCR array in glioma cells, indicating that the death receptor pathway might be involved in matrine-induced cell apoptosis [22]. In summary, the present data have shown that matrine displayed anti-cancer activity against human colon cancer LoVo cells by inactivating Akt pathway. Although not investigated in the present study, matrine may also inhibit cell proliferation via other cellular signal pathways. For instance, it has been reported that matrine modulated mitogen-activated protein kinase (MAPK) pathway in human Jurkat T cells [36]. However, in another study, matrine induced cell apoptosis of leukemia U937 cells in an MAPK-independent pathway [7]. The controversy could be explained by that matrine regulates MAPK pathway in a cell specific manner. But the effect of matrine on MAPK pathway in colon cancer cells needs further investigation. Compared to conventional chemotherapeutic agents, matrine does not have serious adverse cytotoxicity [2–5], and has displayed synergistic effects with cytotoxic agents, in inhibiting proliferation of human breast cancer cells [16]. Importantly, clinical trials in China have demonstrated that administration of matrine improved immune function and life quality of cancer patients who received conventional therapies [37, 38]. These studies suggest that matrine may be a potential anti-cancer agent and warrant future investigation for colon cancer. Acknowledgments This work was supported by grants from the National Natural Scientific Foundation of China (30973474 and 81272467), and Postdoctoral Foundation of Heilongjiang Provincial Government (LRB09-404). Zhang S and Cheng B contributed equally to this work.
References 1. Zhang S, Zhang Y, Zhuang Y, Wang J, Ye J, Zhang S, Wu J, Yu K, Han Y (2012) Matrine induces apoptosis in human acute myeloid leukemia cells via the mitochondrial pathway and Akt inactivation. PLoS One 7:e46853 2. Long Y, Lin XT, Zeng KL, Zhang L (2004) Efficacy of intramuscular matrine in the treatment of chronic hepatitis B. Hepatobiliary Pancreat Dis Int 3:69–72
2107 3. Zhang JP, Zhang M, Zhou JP, Liu FT, Zhou B, Xie WF, Guo C (2001) Antifibrotic effects of matrine on in vitro and in vivo models of liver fibrosis in rats. Acta Pharmacol Sin 22:183–186 4. Liu JY, Hu JH, Zhu QG, Li FQ, Wang J, Sun HJ (2007) Effect of matrine on the expression of substance P receptor and inflammatory cytokines production in human skin keratinocytes and fibroblasts. Int Immunopharmacol 7:816–823 5. Li X, Chu W, Liu J, Xue X, Lu Y, Shan H, Yang B (2009) Antiarrhythmic properties of long-term treatment with matrine in arrhythmic rat induced by coronary ligation. Biol Pharm Bull 32:1521–1526 6. Zhang L, Wang T, Wen X, Wei Y, Peng X, Li H, Wei L (2007) Effect of matrine on HeLa cell adhesion and migration. Eur J Pharmacol 563:69–76 7. Liu XS, Jiang J, Jiao XY, Wu YE, Lin JH (2006) Matrineinduced apoptosis in leukemia U937 cells: involvement of caspases activation and MAPK-independent pathways. Planta Med 72:501–506 8. Liu XS, Jiang J (2006) Molecular mechanism of matrine- induced apoptosis in leukemia K562 Cells. Am J Chin Med 34:1095–1103 9. Jiang H, Hou C, Zhang S, Xie H, Zhou W, Jin Q, Cheng X, Qian R, Zhang X (2007) Matrine upregulates the cell cycle protein E2F-1 and triggers apoptosis via the mitochondrial pathway in K562 cells. Eur J Pharmacol 559:98–108 10. Luo C, Zhu Y, Jiang T, Lu X, Zhang W, Jing Q, Li J, Pang L, Chen K, Qiu F, Yu X, Yang J, Huang J (2007) Matrine induced gastric cancer MKN45 cells apoptosis via increasing pro-apoptotic molecules of Bcl-2 family. Toxicology 229:245–252 11. Dai ZJ, Gao J, Ji ZZ, Wang XJ, Ren HT, Liu XX, Wu WY, Kang HF, Guang H (2009) Matrine induces apoptosis in gastric carcinoma cells via alteration of Fas/FasL and activation of caspase-3. J Ethnopharmacol 123:91–96 12. Luo C, Zhong HJ, Zhu LM, Wu XG, Ying JE, Wang XH, Lu¨ WX, Xu Q, Zhu YL, Huang J (2012) Inhibition of matrine against gastric cancer cell line MNK45 growth and its anti-tumor mechanism. Mol Biol Rep 39:5459–5464 13. Zhang Y, Zhang H, Yu P, Liu Q, Liu K, Duan H, Luan G, Yagasaki K, Zhang G (2009) Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration. Cytotechnology 59:191–200 14. Yu P, Liu Q, Liu K, Yagasaki K, Wu E, Zhang G (2009) Matrine suppresses breast cancer cell proliferation and invasion via VEGF-Akt-NF-kappaB signaling. Cytotechnology 59:219–229 15. Li LQ, Li XL, Wang L, Du WJ, Guo R, Liang HH, Liu X, Liang DS, Lu YJ, Shan HL, Jiang HC (2012) Matrine Inhibits Breast Cancer Growth Via miR-21/PTEN/Akt Pathway in MCF-7 Cells. Cell Physiol Biochem 30:631–641 16. Yu P, Liu Q, Liu K, Yagasaki K, Wu E (2009) Matrine suppresses breast cancer cell proliferation and invasion via VEGFAkt-NF-kappaB signaling. Cytotechnology 59:219–229 17. Liu XY, Fang H, Yang ZG, Wang XY, Ruan LM, Fang DR, Ding YG, Wang YN, Zhang Y, Jiang XL, Chen HC (2008) Matrine inhibits invasiveness and metastasis of human malignant melanoma cell line A375 in vitro. Int J Dermatol 47:448–456 18. Zhang Z, Wang X, Wu W, Wang J, Wang Y, Wu X, Fei X, Li S, Zhang J, Dong P, Gu J, Liu Y (2012) Effects of matrine on proliferation and apoptosis in gallbladder carcinoma cells (GBCSD). Phytother Res 26:932–937 19. Zhang P, Wang Z, Chong T, Ji Z (2012) Matrine inhibits proliferation and induces apoptosis of the androgen-independent prostate cancer cell line PC-3. Mol Med Rep 5:783–787 20. Liang CZ, Zhang JK, Shi Z, Liu B, Shen CQ, Tao HM (2012) Matrine induces caspase-dependent apoptosis in human osteosarcoma cells in vitro and in vivo through the upregulation of Bax and Fas/FasL and downregulation of Bcl-2. Cancer Chemother Pharmacol 69:317–331
123
2108 21. Ma L, Wen S, Zhan Y, He Y, Liu X, Jiang J (2008) Anticancer effects of the Chinese medicine matrine on murine hepatocellular carcinoma cells. Planta Med 74:245–251 22. Zhang S, Qi J, Sun L, Cheng B, Pan S, Zhou M, Sun X (2009) Matrine induces programmed cell death and regulates expression of relevant genes based on PCR array analysis in C6 glioma cells. Mol Biol Rep 35:421–429 23. Li H, Tan G, Jiang X, Qiao H, Pan S, Jiang H, Kanwar JR, Sun X (2010) Matrine inhibits growth of primary and metastatic breast cancer in mice. Am J Chin Med 38:1115–1130 24. Liu T, Song Y, Chen H, Pan S, Sun X (2010) Matrine inhibits proliferation and induces apoptosis of pancreatic cancer cells in vitro and in vivo. Biol Pharm Bull 33:1740–1745 25. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. CA Cancer J Clin 61:69–90 26. He C, Sun XP, Qiao H, Jiang X, Wang D, Jin X, Dong X, Wang J, Jiang H, Sun X (2012) Downregulating Hypoxia-inducible factor2a improves the efficacy of doxorubicin to treat hepatocellular carcinoma. Cancer Sci 103:528–534 27. Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream. Cell 129:1261–1274 28. Dieterle A, Orth R, Daubrawa M, Grotemeier A, Alers S, Ullrich S, Lammers R, Wesselborg S, Stork B (2009) The Akt inhibitor triciribine sensitizes prostate carcinoma cells to TRAIL-induced apoptosis. Int J Cancer 125:932–941 29. Zheng WH, Quirion R (2006) Insulin-like growth factor-1 (IGF-1) induces the activation/phosphorylation of Akt kinase and cAMP responseelement-binding protein (CREB) by activating different signaling pathways in PC12 cells. BMC Neurosci 7:51 30. Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-kinaseAKT pathway in human cancer. Nat Rev Cancer 2:489–501
123
Mol Biol Rep (2014) 41:2101–2108 31. Zhang Y, Liu X, Zhang J, Li L, Liu C (2012) The expression and clinical significance of PI3 K, pAkt and VEGF in colon cancer. Oncol Lett 4:763–766 32. Ericson K, Gan C, Cheong I, Rago C, Samuels Y, Velculescu VE, Kinzler KW, Huso DL, Vogelstein B, Papadopoulos N (2010) Genetic inactivation of AKT1, AKT2, and PDPK1 in human colorectal cancer cells clarifies their roles in tumor growth regulation. Proc Natl Acad Sci USA 107:2598–2603 33. Lu Z, Hunter T (2010) Ubiquitylation and proteasomal degradation of the p21 Cip1, p27 Kip1 and p57 Kip2 CDK inhibitors. Cell Cycle 9:2342–2352 34. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, Slingerland JM (2002) PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 8:1153–1160 35. Alao JP (2007) The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention. Mol Cancer 6:24 36. Li T, Wong VK, Yi XQ, Wong YF, Zhou H, Liu L (2010) Matrine induces cell anergy in human Jurkat T cells through modulation of mitogen-activated protein kinases and nuclear factor of activated T-cells signaling with concomitant up-regulation of anergy-associated genes expression. Biol Pharm Bull 33:40–46 37. Chen J, Mei Q, Xu YC, Du J, Wei Y, Xu ZM (2006) Effects of matrine injection on T-lymphocyte subsets of patients with malignant tumor after gamma knife radiosurgery]. [Article in Chinese]. Zhong Xi Yi Jie He Xue Bao 4:78–79 38. Huang S, Fan W, Liu P, Tian J (2011) Meta analysis of compound matrine injection combined with cisplatin chemotherapy for advanced gastric cancer]. [Article in Chinese]. Zhongguo Zhong Yao Za Zhi 36:3198–3202
