IFN-gamma induces apoptosis in ovarian cancer cells in vivo and in vitro.

PURPOSE: The purpose of this study was to compare in vitro and in vivo responses of primary human tumor cells to IFN-gamma. EXPERIMENTAL DESIGN: IFN-gamma may have therapeutic activity in patients with ovarian cancer. We showed previously that this cytokine had direct antiproliferative activity against human ovarian cancer cell lines and xenografts in nude mice. To further understand the role of IFN-gamma in ovarian cancer, we compared its action on 8 ovarian cancer cell lines with the response of 14 primary cultures of ovarian tumor cells isolated from patients with ascitic disease. A pilot clinical study was then conducted to see whether IFN-gamma would also induce apoptosis in human tumor cells in vivo. Six patients with ascites and advanced disease were given IFN-gamma by i.p. injection, and sequential samples of ascites were analyzed. RESULTS: IFN-gamma had antiproliferative activity in 8 of 8 ovarian cancer cell lines and 11 of 14 primary cultures. This activity was dose related, and cleaved poly(ADP-ribose) polymerase in protein isolates provided evidence of apoptosis. In the clinical study, there was a 3 log(10) pharmacokinetic advantage in peritoneal compared with plasma levels of IFN-gamma. In two of six patients, there was a 90% reduction in tumor cells in ascites after IFN-gamma treatment, and this was related to clinical benefit as assessed by intervals between paracentesis. In all six patients, there were increased amounts of cleaved poly(ADP-ribose) polymerase in protein extracts of ascitic cells sampled during IFN-gamma treatment. CONCLUSIONS: IFN-gamma induces apoptosis in vitro and in vivo in human epithelial ovarian cancer.     

ZNF23 induces apoptosis in human ovarian cancer cells

We recently reported that the level of ZNF23, a KRAB-containing zinc finger protein, is reduced in human cancers and it inhibits cell growth by inducing cell cycle arrest. Here we showed that ZNF23 also induces apoptosis in ovarian cancer cells. The protein level of ZNF23 in ovarian cancers was greatly down-regulated compared with that in the normal ovaries. Introduction of ZNF23 into ovarian cancer cells led to apoptosis as demonstrated by activation of caspase-3, nuclear condensation and formation of a sub-G1 peak. This apoptotic process was correlated with loss in mitochondrial membrane potential, cytochrome c release and caspase-9 activation. Furthermore, ZNF23 induced apoptosis partially via down-regulation of Bcl-XL. Thus, our results suggest that ZNF23 may also induce apoptosis to suppress tumor cell growth and points to the possibility that its down-regulation might facilitate ovarian cancer cell survival.     

沉默半乳糖凝集素-3体外诱导卵巢癌细胞凋亡和内质网应激的实验研究

目的:研究沉默半乳糖凝集素-3(Galectin-3)对卵巢癌细胞凋亡和内质网应激的影响。方法:卵巢癌SKOV3细胞感染Galectin-3 siRNA 重组慢病毒,Real time PCR和Western blot检测沉默效果。MTT方法测定细胞增殖变化,平板克隆实验检测细胞克隆形成能力变化,流式细胞术检测细胞凋亡变化,Western blot检测细胞中c-caspase-3、CHOP、ATF4蛋白水平变化。结果:与感染阴性对照慢病毒和未感染的细胞比较,Galectin-3 siRNA 重组慢病毒感染后的SKOV3细胞中Galectin-3 mRNA和蛋白表达水平均降低。与感染阴性对照慢病毒和未感染的细胞比较,Galectin-3 siRNA 重组慢病毒可以明显下调SKOV3细胞的增殖、克隆形成能力,提高细胞凋亡率,促进细胞中c-caspase-3、CHOP、ATF4蛋白水平。结论:沉默Galectin-3抑制卵巢癌细胞生长,诱导卵巢癌细胞凋亡,促进细胞内质网应激。     

Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells

Cisplatin-centered chemotherapy is a key treatment for ovarian cancer, but resistance to chemotherapeutic agents remains a major cause of treatment failure. Multiple factors are known to contribute to the development of this chemoresistance. Although it has been demonstrated that X-linked inhibitor of apoptosis protein (Xiap) prevents apoptosis by inhibiting effector caspases, if and how it is important in chemoresistance in ovarian cancer has not been studied. The effects of Xiap down-regulation and/or restoration of wild type p53 by recombinant adenovirus infection were examined on four ovarian epithelial cancer cell lines [C13*, A2780-s (wild type p53), A2780-cp (mutant p53), and SKOV3 (null p53)]. Apoptosis and protein expression (e.g., Xiap, caspase-3, p53, MDM2, and p21waf1) were assessed by Hoechst 33258 stain and Western blot, respectively. We demonstrated that Xiap down-regulation following adenoviral antisense expression induces apoptosis in the wild-type p53 cells, but not in the mutated or null cells. Xiap down-regulation resulted in caspase-3 activation, caspase-mediated MDM2 processing, and p53 accumulation. Restoration of wild type p53 in the p53-mutated or -null cells significantly enhanced the proapoptotic effect of Xiap antisense expression. Down-regulation of Xiap induced apoptosis in chemoresistant ovarian cancer cells, a process dependent on p53 status.     

雄黄抑制卵巢癌细胞株COC1增殖和诱导凋亡的体外研究

目的:研究雄黄对卵巢癌细胞株COC1的抑制增殖及诱导凋亡作用。方法:用不同浓度的雄黄溶液作用于人卵巢癌细胞株COC1,于不同时间点收集细胞,MTT法检测肿瘤细胞生长抑制率;流式细胞仪检测细胞凋亡率及分析细胞周期的变化;原位末端标记法(TUNEL)观察细胞凋亡形态学特征。结果:不同浓度雄黄溶液对COC1细胞有不同程度的生长抑制作用。其生长抑制率具有浓度和时间依赖性。并有明显周期特异性生长抑制作用。结论:雄黄溶液对于COC1细胞具有诱导凋亡、抑制增殖的作用,诱导肿瘤细胞发生G1期阻滞是雄黄抑制卵巢癌细胞生长作用的可能机制之一。     

Parthenolide induces proliferation inhibition and apoptosis of pancreatic cancer cells in vitro

Background

To explore the anti-tumor effects of parthenolide in human pancreatic cancer.

Methods

BxPC-3 cell, a human pancreatic cancer, was treated with parthenolide at different concentrations. The MTT assay was used to analyze cell viability. Flow cytometry and DNA fragmentation analysis were applied to evaluate apoptosis after parthenolide treatment. The wound closure and cell invasion assay were also employed in the study. Western blotting was used to demonstrate Bad, Bcl-2, Bax, caspase-9 and pro-caspase-3 expression.

Results

The MTT assay indicated that the pancreatic cancer growth could be dose-dependently inhibited by parthenoolide. This phenomenon was confirmed by flow cytometry and DNA fragmentation analysis. The wound closure assay and cell invasion assay showed that BxPC-3 cell was significantly suppressed by parthenolide at 7.5 μM and 15 μM. Western Blotting demonstrated the Bcl-2 and pro-caspase-3 were down-regulated while the Bax and caspase-9 were up-regulated. No alteration in Bad expression was found after treatment.

Conclusions

The parthenolide can inhibit the cell growth, migration, and induce the apoptosis in human pancreatic cancer. These findings may provide a novel approach for pancreatic cancer treatment.     

Noscapine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo

Purpose

Noscapine plays an important role in the regulation of cell growth and death. It has been reported to potentiate the anti-tumor effect by inducing apoptosis in various malignant cells. However, the mechanism of inducing apoptosis in gastric cancer cells by this agent remains to be clarified.

Methods

In the study, we investigated the signaling pathways by which noscapine induces apoptosis in gastric cancer cell lines. Apoptosis of four human gastric cancer cell lines was induced by treatment with noscapine.

Results

Our results indicate that noscapine induced a dose-dependent apoptosis of these cells. The treatment with noscapine upregulated Bax and Cytochrome c (Cyt-c) protein, downregulated Bcl-2 protein. Caspase-3 and caspase-9 were activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, in xenograft tumor mouse model, noscapine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay.

Conclusions

These data of the study suggest that noscapine induces apoptosis in gastric cancer cells via mitochondrial pathways.     

Artemisinin induces apoptosis in human cancer cells

BACKGROUND: Artemisinin is a chemical compound extracted from the wormwood plant, Artemisia annua L. It has been shown to selectively kill cancer cells in vitro and retard the growth of implanted fibrosarcoma tumors in rats. In the present research, we investigated its mechanism of cytotoxicity to cancer cells. MATERIALS AND METHODS: Molt-4 cells, in complete RPMI-1640 medium, were first incubated with 12 microM of human holotransferrin at 37 degrees C in a humid atmosphere of 5% CO2 for one hour. This enhanced the iron supply to the cells. The cells were then pelleted and transferred to a complete RPMI-1640 containing 200 microM of an analog dihydroartemisinin (DHA) and incubation was started (0 h). In addition, some culture samples were treated with holotransferrin alone and some (controls) were assayed without neither holotransferrin nor DHA treatment. Cells were counted and DNA diffusion assay was used to evaluate apoptosis and necrosis in each sample at 0 h and at 1, 2, 4 and 8 h of incubation. RESULTS: DHA treatment significantly decreased cell counts and increased the proportion of apoptosis in cancer cells compared to controls (chi2=4.5, df=1, p<0.035). Addition of holotransferrin significantly further decreased cell counts (chi2=4.5, df=1, p<0.035) and increased apoptosis (chi2=4.5, df=1, p<0.035). No necrotic cells were observed. CONCLUSION: This rapid induction of apoptosis in cancer cells after treatment with DHA indicates that artemisinin and its analogs may be inexpensive and effective cancer agents.     

Adherence of ovarian cancer cells induces pleural mesothelial cell (PMC) permeability

Malignant pleural effusion (MPE) carries a grave prognosis with median survival after diagnosis being 5 months.The major causes of MPE are lung, breast, ovary,and gastric cancer. It is still unclear how cancer cells penetrate the pleural mesothelial monolayer and reach the pleural space. In this study we examined the effect of ovarian epithelial cancer cells on a confluent pleural mesothelial cell (PMC) monolayer. We demonstrate that ovarian cancer cells adhere to the mesothelial monolayer in a time-dependent manner and induce PMC barrier dysfunction as evidenced by a drop in electrical resistance on electrical cell substrate impedance-sensing system (ECIS) and increased protein permeability. Barrier dysfunction is attenuated by addition of vascular endothelial growth factor (VEGF) antibody. Significant release of VEOF was noted when ovarian cancer cells were cocultured with PMC. Electron microscopy demonstrated gap formation in PMC monolayer only at the site of cancer cell attachment with surrounding areas remaining confluent.     

博安霉素诱导人鼻咽癌细胞凋亡作用

目的：探讨博安霉素（博苯霉素Ａ６）抗人鼻咽癌作用的机理。方法：应用荧光显微镜、流式细胞仪及琼脂糖凝胶电泳等方法检测博安霉素诱导人鼻咽癌ＳＵＮＥ－１细胞凋亡的作用。结果：当博安霉素浓度为５０μｇ／ｍｌ，作用于ＳＵＮＥ－１细胞１０小时，荧光显微镜下ＳＮＵＥＬ０１细胞出现典型的ＤＮＡ梯状条带。结论：博安霉素具有体外诱导鼻咽癌细胞凋亡的作用，而诱导凋亡为其抗肿瘤作用的机制之一。     

Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells

Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.     

右旋柠烯诱导人胃癌细胞凋亡

目的　探讨右旋柠烯 (D limonene)诱导人胃癌细胞株凋亡作用的机制。方法　采用噻唑蓝 (MTT)比色法、电镜、流式细胞术以及免疫细胞化学法 ,对p5 3和bcl 2在肿瘤细胞内的表达以及细胞凋亡的定性与定量指标进行检测。结果　经D limonene处理的BGC 82 3细胞出现核固缩 ,染色质边集 ,凋亡小体形成。凋亡细胞发生率与药物浓度呈正相关 ,即药物浓度为 0 .2 5 μg/ml(4 8h) ,凋亡细胞的发生率从 (2 .71± 0 .78) %上升至 (31.6 2± 7.81) %。经D limonene处理的BGC 82 3细胞内p5 3蛋白表达较对照组明显增加 ,bcl 2蛋白表达较对照组降低。结论　D limonene对人胃癌细胞的杀伤主要是通过诱导细胞凋亡 ,升提p5 3及降低bcl 2的蛋白表达为其诱发肿瘤细胞凋亡的机制之一。     

Lovastatin induces apoptosis of ovarian cancer cells and synergizes with doxorubicin: potential therapeutic relevance

Background  

Ovarian carcinoma is a rarely curable disease, for which new treatment options are required. As agents that block HMG-CoA reductase and the mevalonate pathway, the statin family of drugs are used in the treatment of hypercholesterolemia and have been shown to trigger apoptosis in a tumor-specific manner. Recent clinical trials show that the addition of statins to traditional chemotherapeutic strategies can increase efficacy of targeting statin-sensitive tumors. Our goal was to assess statin-induced apoptosis of ovarian cancer cells, either alone or in combination with chemotherapeutics, and then determine these mechanisms of action.     

The synthetic retinoid adapalene inhibits proliferation and induces apoptosis in colorectal cancer cells in vitro

Chemotherapy of advanced stages of colorectal carcinoma is unsatisfactory. Retinoids inhibit cell growth and induce apoptosis in a variety of human malignancies. We compared the effect of the synthetic retinoid adapalene (ADA) and 9-cis-retinoic acid (CRA) on carcinoma cell lines in vitro. Colon carcinoma cell lines CC-531, HT-29 and LOVO as well as human foreskin fibroblasts were exposed to different concentrations of ADA and CRA for 3-72 hr. Proliferation was assessed by BrdU incorporation and apoptosis by FACS analysis. Breakdown of DeltaPsi(m) was determined by JC-1 staining and activity of caspases 3 and 8, by a colorimetric assay. Quantitative Western blots were performed to detect changes in bax, bcl-2 and caspase-3. Both retinoic derivatives suppressed DNA synthesis and induced apoptosis in all tested cell lines time- and dose-dependently. While the natural retinoid CRA showed moderate antiproliferative and proapoptotic effects only at the highest concentration (10(-4) M), the synthetic retinoic ADA was significantly more effective, showing remarkable effects even at 10(-5) M. ADA and CRA disrupt DeltaPsi(m) and induce caspase-3 activity in responsive tumor cells. Quantitative Western blots showed a shift of the bax:bcl-2 ratio toward proapoptotic bax in ADA-treated cells. Our results clearly indicate the superiority of ADA compared to CRA. Therefore, we suggest that ADA may be far more suitable as an adjunctive therapeutic agent for treatment of colon cancer in vivo.     

Hyperthermia induces apoptosis in malignant fibrous histiocytoma cells in vitro

The effect of mild hyperthermia on a cultured rat malignant fibrous histiocytoma (MFH) cell line, MFH-2NR, was investigated. MFH cells in log-phase (growing phase) were heated at 41°–44°C for 1 hr. Hyperthermic treatment at 41°C did not substantially affect cell proliferation and treatment at 44°C caused necrosis. After hyperthermic treatment at 42° or 43°C, proliferation of MFH cells was arrested and morphological changes characteristic of apoptosis, cell shrinkage accompanying apoptotic bodies and chromatin condensation, became apparent. Hyperthermia-induced apoptosis was further confirmed by terminal deoxynucleotidyl transferase staining and a ladder pattern on agarose gel electrophoresis. Flow cytometric analysis indicated that the population in the G₁ phase of the cell cycle significantly decreased with a concomitant increase in apoptotic cells, indicating that apoptosis might occur mainly in the G₁ phase population. © 1996 Wiley-Liss, Inc.     

Downregulation of XIAP expression in ovarian cancer cells induces cell death in vitro and in vivo

A hallmark of cancer cells is an ability to evade apoptosis. Overexpression and/or activating mutations of prosurvival molecules such as the X-linked inhibitor of apoptosis (XIAP) contribute to this inappropriate cell survival. Our objectives were to investigate the effects of downregulation of XIAP in ovarian cancer cells in vitro and in vivo using the clinical candidate antisense oligonucleotide against XIAP, AEG35156 (AS XIAP). Three ovarian cancer cell lines were transfected with AS XIAP in vitro, and the effects on cell survival were assessed. Downregulation of XIAP resulted in significant apoptosis. To investigate the in vivo efficacy of AS XIAP, CD-1 nude mice were xenografted intraperitoneally with A2780-cp cells, treated with intraperitoneal AS XIAP and evaluated for survival time and tumor histology. Mice treated with 10 mg/kg/day AS XIAP showed a significant improvement in survival time compared to animals treated with control oligonucleotides. Histological analysis of the tumors showed significantly fewer viable cells in the AS XIAP-treated tumors. Downregulation of XIAP expression in ovarian cancer cells resulted in apoptosis in vitro and a prolonged survival time of ovarian cancer-bearing mice, which indicate that XIAP may be a valuable therapeutic target in ovarian cancers, and supports the ongoing clinical investigation of AEG35156.     

Apogossypolone induces autophagy and apoptosis in breast cancer MCF-7 cells in vitro and in vivo

Objective

Apogossypolone (ApoG2), a new derivative of gossypol, is a potent cell-growth inhibitor. ApoG2 has been demonstrated to have superior anti-tumor activity than gossypol in Bcl-2 transgenic mice. The purpose of this study was to investigate the inhibitory effect of ApoG2 on breast cancer cell line MCF-7 in vitro and in vivo, and to investigate its anti-tumor mechanism.

Methods

MCF-7 cell line in culture was treated with ApoG2. The inhibitory effects of ApoG2 on cell growth were measured by MTT and colony-formation assay. The cell apoptotic rate and cell cycle were analyzed by use of flow cytometry (FCM). The ultrastructural changes were observed by transmission electron microscopy. Autophagy was detected by acridine orange staining. Expression of Bcl-2, Bax, and Beclin 1 proteins was measured by western blot analysis.

Results

The inhibitory effect of ApoG2 on MCF-7 cell proliferation was dose and time-dependent. The maximum effect was observed when cells were incubated for 72 h with 40 μM ApoG2. ApoG2 at 5 μM also inhibited colony formation. FCM assay indicated that ApoG2 induced cell apoptosis and caused cell arrest in the S phase and G2/M phase. Transmission electron microscopic examination and acridine orange staining showed that ApoG2 induced intracellular autolysosome formation. Furthermore, ApoG2 reduced Bcl-2 expression, and enhanced expression of Bax and Beclin 1. Xenografting of MCF-7 cells in mice can also be inhibited by ApoG2.

Conclusion

ApoG2, a novel anti-apoptotic Bcl-2 agent, inhibits proliferation of breast cancer cell line MCF-7 by inducing cell apoptosis and autophagy.     

Glyfoline induces mitotic catastrophe and apoptosis in cancer cells

Glyfoline exhibits cytotoxic activity in vitro and antitumor activity in mice bearing murine or human solid tumors, but the underlying mechanisms are unknown. In our study, we found that glyfoline inhibited cell growth and induced accumulation of mitotic cells in human cancer cell lines. Glyfoline induced the appearance of spindle abnormalities, chromosome mis‐segregation, multipolar cell division and multiple nuclei, all of which are indicative of mitotic catastrophe. However, glyfoline did not bind to DNA and did not inhibit or stabilize tubulin polymerization, but slightly increased the resistance of mitotic spindles to nocodazole‐induced disassembly. In addition, microtubule aster formation was significantly enhanced in the extract prepared from glyfoline‐arrested mitotic cells compared to that from synchronized mitotic cells. When Eg5, a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, was inhibited using S‐trityl‐cysteine in glyfoline‐treated cells, formation of spindle multipolarity, multipolar cell division, and multinuclei was significantly reduced. After glyfoline‐mediated arrest of cells at mitosis, considerable poly(ADP‐ribose) polymerase degradation was induced and the number of annexin V‐positive cells significantly increased, indicating that glyfoline ultimately induces apoptosis. Small interfering RNA‐mediated silencing of the spindle checkpoint proteins BUBR1 and MAD2 markedly reduced induction of mitotic cell accumulation, but did not affect glyfoline‐induced mitotic catastrophe and apoptosis. Thus, glyfoline induces mitotic catastrophe probably by enhancing microtubule aster formation and subsequent apoptosis in cancer cells independently of spindle checkpoint function.