Capsaicin induces apoptosis in human osteosarcoma cells through AMPK-dependent and AMPK-independent signaling pathways

Recent studies have focused on the anti-tumor activity of capsaicin. However, the potential effects of capsaicin in osteosarcoma cells and the underlying mechanisms are not fully understood. In the current study, we observed that capsaicin-induced growth inhibition and apoptosis in cultured osteosarcoma cells (U2OS and MG63), which were associated with a significant AMP-activated protein kinase (AMPK) activation. AMPK inhibition by compound C or RNA interference suppressed capsaicin-induced cytotoxicity, while AMPK activators (AICAR and A769662) promoted osteosarcoma cell death. For the mechanism study, we found that AMPK activation was required for capsaicin-induced mTORC1 (mTOR complex 1) inhibition, B cell lymphoma 2 (Bcl-2) downregulation and Bax upregulation in MG63 cells. Capsaicin administration induced p53 activation, mitochondrial translocation and Bcl-2 killer association, such effects were dependent on AMPK activation. Interestingly, we observed a significant pro-apoptotic c-Jun NH2-terminal kinases activation by capsaicin in MG63 cells, which appeared to be AMPK independent. In conclusion, capsaicin possessed strong efficacy against human osteosarcoma cells. Molecular studies revealed that capsaicin activated AMPK-dependent and AMPK-independent signalings to mediate cell apoptosis. The results of this study should have significant translational relevance in managing this deadly malignancy.     

Induction of apoptosis in prostate tumor PC-3 cells and inhibition of xenograft prostate tumor growth by the vanilloid capsaicin

Capsaicin, the pungent ingredient of hot chilli pepper, has been recently shown to induce apoptosis in several cell lines through a not well known mechanism. Here, we investigated the role of the vanilloid capsaicin in the death regulation of the human cancer androgen-resistant cell line PC-3. Capsaicin inhibited the growth of PC-3 with an IC₅₀ of 20 μM cells and induced cell apoptosis, as assessed by flow cytometry and nuclei staining with DAPI. Capsaicin induced apoptosis in prostate cells by a mechanism involving reactive oxygen species generation, dissipation of the mitochondrial inner transmembrane potential (ΔΨ_m) and activation of caspase 3. Capsaicin-induced apoptosis was not reduced by the antagonist capsazepine in a dose range from 0.1 μM to 20 μM, suggesting a receptor-independent mechanism. To study the in vivo effects of capsaicinoids, PC-3 cells were grown as xenografts in nude mice. Subcutaneous injection of either capsaicin or capsazepine (5 mg/kg body weight) in nude mice suppressed PC-3 tumor growth in all tumors investigated and induced apoptosis of tumor cells. Our data show a role for capsaicin against androgen-independent prostate cancer cells in vitro and in vivo and suggest that capsaicin is a promising anti-tumor agent in hormone-refractory prostate cancer, which shows resistance to many chemotherapeutic agents.     

TRPV6 mediates capsaicin-induced apoptosis in gastric cancer cells--Mechanisms behind a possible new "hot" cancer treatment

Capsaicin is an organic compound in chili peppers which are consumed by over one quarter of the world's population daily. Studies have shown that capsaicin can induce apoptosis in some cancer cells by unknown mechanisms. In this study, both gastric cancer and normal epithelial cells were treated with capsaicin and examined for apoptosis by Annexin V binding. Our results showed that capsaicin induces apoptosis in both cells, although cancer cells are more susceptible. This susceptibility is dependent on the availability of TRPV6, a calcium-selective channel protein, as overexpression of TRPV6 in normal cells increased capsaicin-induced apoptosis and knockdown of TRPV6 in cancer cells suppressed this action. Our results further demonstrated that capsaicin increases mitochondrial permeability through activation of Bax and p53 in a JNK-dependent manner. Conclusions: (1) TRPV6, rather than TRPV1 (the well-known capsaicin receptor), mediates capsaicin-induced apoptosis in gastric cells; (2) abundance of TRPV6 in gastric cells determines their live or death under capsaicin treatment; and (3) capsaicin induces apoptosis by stabilization of p53 through JNK activation. Together, our data suggest that capsaicin may be a promising dietary candidate for cancer chemoprevention.     

Down-Regulation of Tumor-Associated NADH Oxidase,tNOX (ENOX2), Enhances Capsaicin-Induced Inhibition of Gastric Cancer Cell Growth

Gastric cancer is a common human malignancy and a major contributor to cancer-related deaths worldwide. Unfortunately, the prognosis of most gastric cancer patients is poor because they are generally diagnosed at a late stage after the cancer has already metastasized. Most current research, therefore, emphasizes selective targeting of cancer cells by apoptosis-inducing agents. One such therapeutic agent is capsaicin, a component of chili peppers that has been shown to possess anti-growth activity against various cancer cell lines. Here, we examined the effect of capsaicin on SNU-1 and TMC-1 gastric cancer cells and found differing outcomes between the two cell lines. Our results show that capsaicin induced significant cytotoxicity with increases in oxidative stress, PARP cleavage, and apoptosis in sensitive SNU-1 cells. In contrast, TMC-1 cells were much less sensitive to capsaicin, exhibiting low cytotoxicity and very little apoptosis in response to capsaicin treatment. Capsaicin-induced apoptosis in SNU-1 cells was associated with down-regulation of tumor-associated NADH oxidase (tNOX) mRNA and protein. On the contrary, tNOX expression was scarcely affected by capsaicin in TMC-1 cells. We further showed that tNOX-knockdown sensitized TMC-1 cells to capsaicin-induced apoptosis and G1 phase accumulation, and led to decreased cell growth, demonstrating that tNOX is essential for cancer cell growth. Collectively, these results indicate that capsaicin induces divergent effects of the growth of gastric cancer cells that parallel its effects on tNOX expression, and demonstrate that forced tNOX down-regulation restored capsaicin-induced growth inhibition in TMC-1 cells.     

Apoptosis induction by dohevanil, a DHA substitutive analog of capsaicin, in MCF-7 cells

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient in a variety of red peppers of the genus Capsicum, is a type of vanilloid. It has been shown to induce apoptosis in many cell types. The effects of vanilloids on apoptosis induction are thought to be correlated with the length and degree of the unsaturation of the fatty acyl chains. In this study, we compared the effect of capsaicin and its docosahexaenoic acid (DHA, C22:6) analog (we named as dohevanil) on human breast cancer MCF-7 cells, which do not express caspase-3. Dohevanil, which was synthesized from DHA and vanillylamine, has longer and highly unsaturated fatty acyl chain than capsaicin. We showed that both vanilloids exhibit effects of growth inhibition and DNA fragmentation induction in MCF-7 cells. These effects of dohevanil were more potent than capsaicin. Because these effects were inhibited by z-VAD-fmk, a broad-spectrum caspase inhibitor, the vanilloids induced the apoptosis via caspase-dependent pathway not involving caspase-3. In conclusion, dohevanil has a more potent effect on apoptosis induction in MCF-7 cells than capsaicin.     

Dihydrocapsaicin (DHC), a saturated structural analog of capsaicin, induces autophagy in human cancer cells in a catalase-regulated manner

Although capsaicin, a pungent component of red pepper, is known to induce apoptosis in several types of cancer cells, the mechanisms underlying capsaicin-induced cytotoxicity are unclear. Here, we showed that dihydrocapsaicin (DHC), an analog of capsaicin, is a potential inducer of autophagy. DHC was more cytotoxic than capsaicin in HCT116, MCF-7 and WI38 cell lines. Capsaicin and DHC did not affect the sub-G(1) apoptotic peak, but induced G(0)/G(1) arrest in HCT116 and MCF-7 cells. DHC caused the artificial autophagosome marker GFP-LC3 to redistribute and upregulated expression of autophagy-related proteins. Blocking of autophagy by 3-methyladenine (3MA) as well as siRNA Atg5 induced a high level of caspase-3 activation. Although pretreatment with zVAD completely inhibited caspase-3 activation by 3MA, it did not prevent cell death. DHC-induced autophagy was enhanced by zVAD pretreatment, as shown by increased accumulation of LC3-II protein. DHC attenuated basal ROS levels through catalase induction; this effect was enhanced by antioxidants, which increased both LC3-II expression and caspase-3 activation. The catalase inhibitor 3-amino-1,2,4-triazole (3AT) abrogated DHC-induced expression of LC3-II, overexpression of the catalase gene increased expression of LC3-II protein, and knockdown decreased it. Additionally, DHC-induced autophagy was independent of p53 status. Collectively, DHC activates autophagy in a p53-independent manner and that may contribute to cytotoxicity of DHC.     

Capsaicin binds to prohibitin 2 and displaces it from the mitochondria to the nucleus

Capsaicin is widely used as a food additive and as an analgesic agent. Besides its well-known role in nociception, which is mediated by vanilloid receptor 1 specifically expressed in dorsal root ganglion neurons, capsaicin has also been considered as a potential anticancer agent, as it inhibits cell proliferation and induces apoptosis in various types of cancer cells. Here we identified a new molecular target of capsaicin from human myeloid leukemia cells. We show that capsaicin binds to prohibitin (PHB) 2, which is normally localized to the inner mitochondrial membrane, and induces its translocation to the nucleus. PHB2 is implicated in the maintenance of mitochondrial morphology and the control of apoptosis. We also provide evidence suggesting that capsaicin causes apoptosis directly through the mitochondria and that PHB2 contributes to capsaicin-induced apoptosis at multiple levels. This work will serve as an important foundation for further understanding of anticancer activity of capsaicin.     

辣椒素对舌鳞癌细胞增殖和凋亡影响的初步研究

目的:研究辣椒素对人舌鳞癌细胞增殖和存活的影响。方法:不同浓度辣椒素处理TSCCA和Tca8113细胞24,48和72h,MTS法检测细胞增殖,Western Blot法检测细胞周期调控和细胞凋亡相关分子的表达。结果:随着辣椒素浓度的增加,p53,p21和p27表达上调,Bcl-2表达未受到明显影响,Bcl-XL和Mcl-1表达抑制,caspase7和PARP剪切体表达上调。结论:辣椒素呈剂量依赖和时间依赖性抑制TSCCA和Tca8113细胞增殖,其机制可能与细胞周期和细胞凋亡相关分子表达的改变相关。     

Matrine inhibits proliferation and induces apoptosis of human colon cancer LoVo cells by inactivating Akt pathway

The present study has investigated the anti-tumor 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 dose-dependent 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-3β. 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 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.     

Combination of Gefitinib and DNA Methylation Inhibitor Decitabine Exerts Synergistic Anti-Cancer Activity in Colon Cancer Cells

Despite recent advances in the treatment of human colon cancer, the chemotherapy efficacy against colon cancer is still unsatisfactory. In the present study, effects of concomitant inhibition of the epidermal growth factor receptor (EGFR) and DNA methyltransferase were examined in human colon cancer cells. We demonstrated that decitabine (a DNA methyltransferase inhibitor) synergized with gefitinib (an EGFR inhibitor) to reduce cell viability and colony formation in SW1116 and LOVO cells. However, the combination of the two compounds displayed minimal toxicity to NCM460 cells, a normal human colon mucosal epithelial cell line. The combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. In addition, the combination of decitabine with gefitinib markedly inhibited colon cancer cell migration. Furthermore, gefitinib synergistically enhanced decitabine-induced cytotoxicity was primarily due to apoptosis as shown by Annexin V labeling that was attenuated by z-VAD-fmk, a pan caspase inhibitor. Concomitantly, cell apoptosis resulting from the co-treatment of gefitinib and decitabine was accompanied by induction of BAX, cleaved caspase 3 and cleaved PARP, along with reduction of Bcl-2 compared to treatment with either drug alone. Interestingly, combined treatment with these two drugs increased the expression of XIAP-associated factor 1 (XAF1) which play an important role in cell apoptosis. Moreover, small interfering RNA (siRNA) depletion of XAF1 significantly attenuated colon cancer cells apoptosis induced by the combination of the two drugs. Our findings suggested that gefitinib in combination with decitabine exerted enhanced cell apoptosis in colon cancer cells were involved in mitochondrial-mediated pathway and induction of XAF1 expression. In conclusion, based on the observations from our study, we suggested that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating colon cancer.     

Capsaicin induces apoptosis through ubiquitin–proteasome system dysfunction

Capsaicin is an active component of red pepper having an antiproliferative effect in a variety of cancer cells, which recent evidence suggests due to its ability to induce apoptosis. However, the molecular mechanisms through which capsaicin induces apoptosis are not well understood. Here we demonstrate that capsaicin‐induced apoptosis is mediated via the inhibition cellular proteasome function. Treatment of capsaicin to mouse neuro 2a cells results in the inhibition of proteasome activity in a dose‐ and time‐dependent manner that seems to correlate with its effect on cell death. The effect of capsaicin on cellular proteasome function is indirect and probably mediated via the generation of oxidative stress. Exposure of capsaicin also causes increased accumulation of ubiquitinated proteins as wells as various target substrates of proteasome like p53 and Bax and p27. Like many other classical proteasome inhibitors, capsaicin also triggers the intrinsic pathway of apoptosis involving mitochondria and induces neurite outgrowth. Our results strongly support for the use of capsaicin as an anticancer drug. J. Cell. Biochem. 109: 933–942, 2010. © 2010 Wiley‐Liss, Inc.     

Selective induction of apoptosis by capsaicin in transformed cells: the role of reactive oxygen species and calcium

Capsaicin is a vanilloid quinone analog that inhibits the plasma membrane electron transport (PMOR) system and induces apoptosis in transformed cells. Using a cytofluorimetric approach we have determined that capsaicin induces a rapid increase of reactive oxygen species (ROS) followed by a subsequent disruption of the transmembrane mitochondrial potential (DeltaPsim) and DNA nuclear loss in transformed cell lines and in mitogen activated human T cells. This apoptotic pathway is biochemically different from the typical one induced by either ceramide or edelfosine where, in our system, the DeltaPsim dissipation precedes the generation of reactive oxygen species. Neither production of ROS nor apoptosis was found in capsaicin-treated resting T cells where the activity of the PMOR system is minimal when compared with mitogen activated or transformed T cells. Capsaicin also induces Ca2+ mobilization in activated but not in resting T cells. However, preincubation of cells with BAPTA-AM, which chelate cytosolic free calcium, did not prevent ROS generation or apoptosis induced by capsaicin, suggesting that ROS generation in capsaicin treated cells is not a consequence of calcium signaling and that the apoptotic pathway may be separated from the one that mobilizes calcium. Moreover, we present data for the implication of a possible vanilloid receptor in calcium mobilization, but not in ROS generation. These results provide evidence that the PMOR system may be an interesting target to design antitumoral and anti-inflammatory drugs.     

The role of Hsp70 chaperone in the reaction of human leukemic cells to anticancer drugs

Most of anti-tumor factors are designed to kill selectively cancer cells; in most cases this action is related to the ability of the above substances to induce apoptosis. One of potent anti-apoptotic mechanisms is based on Hsp70 protein. Since the level of this protein is often higher in malignant tumors than in normal tissues, the aim of this study was to establish whether the elevated Hsp70 content may influence the process of apoptosis induced by anti-tumor drugs in cancer cells population. The increase of intracellular content of Hsp70 in human leukemia U-937 cells was attained by a mild heat stress or by transfection of cells with the human hsp70 gene. The elevation of Hsp70 quantity, irrespective of the way it was performed, leads to the inhibition of apoptosis in cells treated with two substances, etoposide or adriamycin. The inhibition of apoptosis was accompanied with the reducing of the share of cells with fragmented nuclei and with the delay in caspase activation. It is suggested that in addition to the previously discovered targets, whose activity is suppressed by the Hsp70 chaperone, this protein can inhibit the activity of caspase-3 and -7; this delays the onset of apoptosis in part of a cancer cell population.     

Capsaicin stimulates glucose uptake in C2C12 muscle cells via the reactive oxygen species (ROS)/AMPK/p38 MAPK pathway

Capsaicin has been reported to regulate blood glucose levels and to ameliorate insulin resistance in obese mice. This study demonstrates that capsaicin increases glucose uptake directly by activating AMP-activated protein kinase (AMPK) in C2C12 muscle cells, which manifested as an attenuation of glucose uptake when compound C, an AMPK inhibitor, was co-administered with capsaicin. However, the insulin signaling molecules insulin receptor substrate-1 (IRS-1) and Akt were not affected by capsaicin. Additional results showed that p38 mitogen-activated protein kinase (MAPK) is also involved in capsaicin-induced glucose transport downstream of AMPK because capsaicin increased p38 MAPK phosphorylation significantly and its specific inhibitor SB203580 inhibited capsaicin-mediated glucose uptake. Treatment with an AMPK inhibitor reduced p38 MAPK phosphorylation, but the p38 MAPK inhibitor had no effect on AMPK. Capsaicin stimulated ROS generation in C2C12 muscle cells, and when ROS were captured using the nonspecific antioxidant NAC, the increase in both capsaicin-induced AMPK phosphorylation and capsaicin-induced glucose uptake was attenuated, suggesting that ROS function as an upstream activator of AMPK. Taken together, these results suggest that capsaicin, independent of insulin, increases glucose uptake via ROS generation and consequent AMPK and p38 MAPK activations.     

羊栖菜多糖诱导lovo细胞凋亡及其机理探讨

本课题研究羊栖菜多糖(Sargassum Fusiforme Polysaccharides,SFPS)诱导人大肠癌lovo细胞凋亡及凋亡过程中Caspase-3的变化及其意义。MTT法检测SFPS对大肠癌细胞增殖的抑制率;通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡;应用Western blot法测定Caspase-3酶原的变化; RT-PCR检测Caspase-3 mRNA表达。结果显示:SFPS作用lovo细胞24,36,48和72h的IC_(50)分别为375,355,178和60mg/L,表明对lovo细胞具有显著生长抑制作用。在电镜下,可见明显的细胞凋亡特征:细胞膜表面微绒毛减少、染色质固缩、边集,凋亡小体形成。在琼脂糖凝胶电泳中,药物浓度为5-300mg/L作用24h后,显示有凋亡细胞特有的DNA梯状条带;而500mg/L处理后梯状条带模糊,开始出现“涂片状”,表明在高药物浓度的作用下,细胞有坏死。流式细胞仪测得细胞凋亡率有剂量的依赖性;DNA直方图出现亚G1峰,但细胞周期时相的分布无明显改变。SFPS处理lovo细胞后,发现Caspase-3酶原蛋白表达降低,Caspase-3的mRNA高表达,并具有剂量和时间的依赖性。实验结果提示,SFPS在体外能够诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一,而Caspase-3的活化参与了SFPS诱导lovo细胞凋亡的调控。     

羊栖菜多糖诱导lovo细胞凋亡及其机理探讨

本课题研究羊栖菜多糖（Sargassum Fusforme Polysaccharides．SFPS）诱导人大肠癌lovo细胞凋亡及凋亡过程中Caspase-3的变化及其意义。MTT法检测SFPS对大肠癌细胞增殖的抑制率：通过电镜、琼脂糖凝胶电泳、流式细胞术鉴定细胞凋亡：应用Westernblot法测定Caspase-3酶原的变化：RT—PCR检测Caspase-3mRNA表达。结果显示：SFPS作用lovo细胞24,36,48和72h的IC50分别为375,355,178和60mg／L,表明对lovo细胞具有显著生长抑制作用。在电镜下,可见明显的细胞凋亡特征：细胞膜表面微绒毛减少、染色质固缩、边集,凋亡小体形成。在琼脂糖凝胶电泳中,药物浓度为5—300mg／L作用24h后,显示有凋亡细胞特有的DNA梯状条带：而500mg／L处理后梯状条带模糊,开始出现“涂片状”,表明在高药物浓度的作用下,细胞有坏死。流式细胞仪测得细胞凋亡率有剂量的依赖性;DNA直方图出现亚G1峰,但细胞周期时相的分布无明显改变。SFPS处理lovo细胞后,发现Caspase-3酶原蛋白表达降低,Caspase-3的mRNA高表达,并具有剂量和时间的依赖性。实验结果提示,SFPS在体外能够诱导lovo细胞凋亡,这可能是SFPS抑制肿瘤增殖的机制之一．而Caspase-3的活化参与了SFPS诱导lovo细胞凋亡的调控。