凋亡素及其诱导肿瘤细胞凋亡的作用

凋亡素是一种来源于鸡贫血病毒的小分子蛋白,能够选择性地诱导肿瘤细胞和转化细胞的凋亡,而对正常细胞无作用.凋亡素的肿瘤细胞特异性与其在细胞中的核定位密切相关.它诱导的细胞凋亡既不依赖于p53,也不会被Bcl-2的过表达所抑制,但涉及Caspase-3的激活.因此,凋亡素可作为一种新型的肿瘤诊断工具和候选抗肿瘤治疗制剂.     

Apoptin induces apoptosis in an oral cancer mouse model

Apoptin, a chicken anemia virus-derived protein, induces apoptosis in various tumor cell lines and xenografted tumors. Its apoptotic activity is not hampered by tumor-suppressor p53 mutations or overexpression of anti-apoptosis proteins Bcl-2 or Bcl-x(L). We report for the first time the effects of apoptin expression in primary oral tumors, induced by the carcinogen 4-Nitroquinoline- 1-oxide in immunocompetent mice. In vivo a significant amount of primary oral tumor cells expressing apoptin cells underwent apoptosis, whereas synthesis of the LacZ control product did not. Ectopical expression of apoptin in passage 1 cell cultures derived from these oral tumors also resulted in apoptin-induced. Both in-vivo and in-vitro treated cells underwent apoptosis via the activation of caspase-3. The fact that apoptin induces apoptosis in primary squamous cell carcinoma cells indicates that apoptin is a potential therapeutic agent for treatment of head and neck squamous cell carcinoma.     

Differential sensitivity to Ad5 E1B-21kD and Bcl-2 proteins of apoptin-induced versus p53-induced apoptosis

Apoptin, a small protein derived from chicken anemia virus (CAV),induces apoptosis in human tumor cell lines regardless of whetherthese express p53 or not. We examined whether the small adenovirus5 E1B protein of 21 kDa (E1B-21KD, also called E1B-19kD) andBcl-2 could inhibit apoptin-induced apoptosis in human tumorcell lines and compared this with p53-induced apoptosis. E1B-21kD,but not Bcl-2, was found to inhibit apoptin-induced apoptosisin the osteosarcoma cell lines U2OS and Saos-2. However, neitherexpression of E1B-21kD nor of Bcl-2 resulted in inhibition ofapoptin-induced apoptosis in Hep3B hepatoma cells and kidneyrhabdoid tumor G401 cells. Both Bcl-2 and Ad5 E1B-21kD wereable to inhibit p53-induced apoptosis in the human tumor celllines Saos-2 and Hep3B. In Saos-2 and U2OS, but not in Hep3Band G401, expression of E1B-21kD leads to retention of apoptinin the cytoplasm, in that way preventing its nuclear function.These results indicate that proteins inhibiting the p53-inducedapoptotic pathway do not block apoptin-induced apoptosis ordo so only in a cell type-specific manner. The apoptin-inducedapoptotic pathway is distinct from that induced by p53 and,therefore, apoptin is a potential antitumor agent.     

P53 mediates ceramide-induced apoptosis in SKN-SH cells

Ceramide induces apoptotic cell death in a dose- and time-dependent manner in neuroblastoma SKN-SH cells. Pretreatment with caspase inhibitors blocks cell death, suggesting that a set of caspase activities including caspase 1, as well as caspase 3, are involved in ceramide-induced apoptosis in SKN-SH cells. Treatment with a caspase inhibitor 3 h after ceramide addition did not inhibit cell death, although caspase activity was substantially reduced. Ceramide-induced apoptosis is accompanied by accumulation of p53 followed by an increase of Bax and decrease of Bcl-2 levels. Inhibition of p53 expression with p53 antisense oligonucleotides inhibits apoptosis and prevents the increase in Bax and decrease in Bcl-2. Furthermore, pretreatment with p53 antisense oligonucleotides markedly inhibits the induction of caspase activity. These results suggest that p53 regulates the ratio Bcl-2/Bax and the expression/activation of caspases during ceramide-induced apoptosis in SKN-SH cells. Caspase inhibition did not alter the expression of p53, Bcl-2 and Bax. Thus ceramide-induced reduction in the Bcl-2/Bax ratio, increase in caspase activity, and apoptosis is dependent upon increases in cellular p53 levels which play a critical role in the regulation of apoptotic cell death.     

Bax and Bak are required for apoptosis induction by sulforaphane, a cruciferous vegetable-derived cancer chemopreventive agent

Sulforaphane, a constituent of many edible cruciferous vegetables, including broccoli, effectively suppresses proliferation of cancer cells in culture and in vivo by causing apoptosis induction, but the sequence of events leading to cell death is poorly defined. Here, we show that multidomain proapoptotic Bcl-2 family members Bax and Bak play a critical role in apoptosis induction by sulforaphane. This conclusion is based on the following observations: (a) sulforaphane treatment caused a dose- and time-dependent increase in the protein levels of both Bax and Bak and conformational change and mitochondrial translocation of Bax in SV40-transformed mouse embryonic fibroblasts (MEF) derived from wild-type mice to trigger cytosolic release of apoptogenic molecules (cytochrome c and Smac/DIABLO), activation of caspase-9 and caspase-3, and ultimately cell death; (b) MEFs derived from Bax or Bak knockout mice resisted cell death by sulforaphane, and (c) MEFs derived from Bax and Bak double knockout mice exhibited even greater protection against sulforaphane-induced cytochrome c release, caspase activation, and apoptosis compared with wild-type or single knockout cells. Interestingly, sulforaphane treatment also caused a dose- and time-dependent increase in the protein level of Apaf-1 in wild-type, Bax-/-, and Bak-/- MEFs but not in double knockout, suggesting that Bax and Bak might regulate sulforaphane-mediated induction of Apaf-1 protein. A marked decline in the protein level of X-linked inhibitor of apoptosis on treatment with sulforaphane was also observed. Thus, it is reasonable to postulate that sulforaphane-induced apoptosis is amplified by a decrease in X-linked inhibitor of apoptosis level, which functions to block cell death by inhibiting activities of caspases. In conclusion, the results of the present study indicate that Bax and Bak proteins play a critical role in initiation of cell death by sulforaphane.     

Grape seed proanthocyanidins induce apoptosis and inhibit metastasis of highly metastatic breast carcinoma cells

The strategies available for the treatment of metastatic breast cancer are limited. Dietary botanicals may have a better protective effect on this disease. We therefore investigated the effects of grape seed proanthocyanidins (GSPs) on a highly metastatic mouse mammary carcinoma cell line. In vitro treatment of breast cancer cells, 4T1, MCF-7 and MDA-MB-468, with GSPs resulted in significant inhibition of cellular proliferation and viability, and induction of apoptosis in 4T1 cells in a time- and dose-dependent manner. Further analysis indicated an alteration in the ratio of Bax/Bcl-2 proteins in favor of apoptosis, and the knockdown of Bax using Bax siRNA transfection of 4T1 cells resulted in blocking of GSPs-induced apoptosis. Induction of apoptosis was associated with the release of cytochrome c, increased expression of Apaf-1 and activation of caspase 3 and poly (ADP-ribose) polymerase. Treatment with the pan-caspase inhibitor (Z-VAD-FMK) resulted in partial but significant inhibition of apoptosis in 4T1 cells suggesting the involvement of both caspase activation-dependent and activation-independent pathways in the apoptosis of 4T1 cells induced by GSPs. The effects of dietary GSPs were then examined using an in vivo model in which 4T1 cells were implanted subcutaneously in Balb/c mice. Dietary GSPs (0.2 and 0.5%, w/w) significantly inhibited the growth of the implanted 4T1 tumor cells and increased the ratio of Bax:Bcl-2 proteins, cytochrome c release, induction of Apaf-1 and activation of caspase 3 in the tumor microenvironment. Notably, the metastasis of tumor cells to the lungs was inhibited significantly and the survival of the mice enhanced. These data suggest that GSPs possess chemotherapeutic efficacy against breast cancer including inhibition of metastasis.     

Grape seed proanthocyanidins induce apoptosis through p53, Bax, and caspase 3 pathways

Grape seed proanthocyanidins (GSP) have been shown to inhibit skin chemical carcinogenesis and photocarcinogenesis in mice. The mechanisms responsible for the anticarcinogenic effects of GSP are not clearly understood. Here, we report that treatment of JB6 C141 cells (a well-developed cell culture model for studying tumor promotion in keratinocytes) and p53+/+ fibroblasts with GSP resulted in a dose-dependent induction of apoptosis. GSP-induced (20-80 g/ml) apoptosis was observed by using immunofluorescence (27-90% apoptosis) and flow cytometry (18-87% apoptosis). The induction of apoptosis by GSP was p53-dependent because it occurred mainly in cells expressing wild-type p53 (p53+/+; 15-80%) to a much greater extent than in p53-deficient cells (p53-/-; 6-20%). GSP-induced apoptosis in JB6 C141 cells was associated with increased expression of the tumor-suppressor protein, p53, and its phosphorylation at Ser15. The antiapoptotic proteins, Bcl-2 and Bcl-xl, were downregulated by GSP, whereas the expression of the pro-apoptotic protein, Bax, and the levels of cytochrome c release, Apaf-1, caspase-9, and cleaved caspase 3 (p19 and p17) were markedly increased in JB6 C141 cells. The downregulation of Bcl-2 and upregulation of Bax were also observed in wild-type p53 (p53+/+) fibroblasts but was not observed in their p53-deficient counterparts. These data clearly demonstrate that GSP-induced apoptosis is p53-dependent and mediated through the Bcl-2, Bax, and caspase 3 pathways.     

Growth inhibitory and antimetastatic effect of green tea polyphenols on metastasis-specific mouse mammary carcinoma 4T1 cells in vitro and in vivo systems.

PURPOSE: Breast cancer is the second leading cause of cancer-related deaths among females. Dietary habits may have a role in breast cancer risk and prevention as well. Here, we examined the effect of green tea polyphenols (GTP) on growth and metastasis of highly metastatic mouse mammary carcinoma 4T1 cells in vitro and in vivo systems. EXPERIMENTAL DESIGN: 4T1 cells were treated with (-)-epigallocatechin-3-gallate (EGCG), and the effect was determined on cellular proliferation, induction of apoptosis, proapoptosis, and antiapoptotic proteins of Bcl-2 family, and caspase 3 and poly(ADP-ribose) polymerase activation following 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and Western blot analysis. Anticarcinogenic and antimetastatic effect of GTP in 4T1 cells was assessed in immunocompetent BALB/c mice. RESULTS: Treatment of 4T1 cells with EGCG resulted in inhibition of cell proliferation, induction of apoptosis in dose- and time-dependent manner. The increase in apoptosis was accompanied with decrease in the protein expression of Bcl-2 concomitantly increase in Bax, cytochrome c release, Apaf-1, and cleavage of caspase 3 and PARP proteins. Treatment of EGCG-rich GTP in drinking water to 4T1 cells bearing BALB/c mice resulted in reduction of tumor growth accompanied with increase in Bax/Bcl-2 ratio, reduction in proliferating cell nuclear antigen and activation of caspase 3 in tumors. Metastasis of tumor cells to lungs was inhibited and survival period of animals was increased after green tea treatment. CONCLUSION: This study suggests that GTP have the ability to prevent the development of breast cancer and its metastasis; however, further in vivo studies are required to identify the molecular targets.     

Acacetin induces apoptosis in human T cell leukemia Jurkat cells via activation of a caspase cascade

Flavonoids are naturally occurring antioxidants, with several flavonoids shown to have chemopreventive effects on cancer. We investigated the effects of the flavonoid acacetin on human T cell leukemia Jurkat cells. Acacetin inhibited the proliferation of Jurkat cells by inducing apoptosis in a concentration- and time-dependent manner. Acacetin-induced cell death was characterized by changes in nuclear and cell morphology. Treatment of Jurkat cells with acacetin also induced caspase-3, -8 and -9 activities in a time-dependent manner. Acacetin-induced apoptosis was blocked by a broad-spectrum caspase inhibitor, a caspase-3 inhibitor and a caspase-8 inhibitor, but not by a caspase-9 inhibitor. In addition, acacetin promoted the expression of FAF1, phosphor-FADD, Apaf-1 and cytochrome c. Acacetin-induced apoptosis was also accompanied by upregulation of Bax, and downregulation of Bcl-2. Taken together, these results suggest that acacetin may induce apoptosis in T cell leukemia cells, possibly by activating the Fas-mediated pathway. These findings may help in designing cancer therapeutic and chemopreventive agents.     

Smac-mediated sensitization of human B-lymphoma cells to staurosporine- and lactacystin-triggered apoptosis is apoptosome-dependent.

Second mitochondrial activator of caspase (Smac)-derived peptides have previously been shown to facilitate apoptosis of various types of cancer cells. However, it remains unclear whether the effects of such Smac agonists are dependent on apoptotic protease-activating factor-1 (Apaf-1), a key component of the apoptosome. Here, we explored the role of Apaf-1 through overexpression of this protein in the B-lymphoma cell line Raji that is defective for cytosolic Apaf-1 expression. Enforced expression of Apaf-1 rendered Raji cells sensitive to staurosporine as well as to the proteasome inhibitor, lactacystin. Importantly, co-treatment with Smac peptides resulted in a threefold higher degree of apoptosis in Apaf-1-expressing Raji cells, but not in mock-transfected cells. Smac peptides also potentiated apoptosis of the DG-75 cell line following liberation of endogenous Apaf-1 from the plasma membrane, but were ineffective when added alone. Furthermore, we observed high levels of expression in several B-lymphoma cell lines of cellular inhibitor of apoptosis protein-2 (cIAP2), and immunodepletion of cIAP2 (a target of Smac) was found to sensitize Apaf-1-overexpressing Raji cells to cytochrome c-dependent caspase activation. Collectively, these results demonstrate the importance of Apaf-1 in Smac-mediated potentiation of apoptosis of B-lymphoma-derived cells.     

Interferon-alpha-induced apoptosis in U266 cells is associated with activation of the proapoptotic Bcl-2 family members Bak and Bax

We have recently reported that the cytokine interferon-alpha (IFNalpha), commonly used in the treatment of cancer, induced a caspase-dependent apoptosis in tumor cell lines. The signaling mechanisms involved have not been defined. Here, we show that both proapoptotic Bcl-2 family members Bak and Bax were activated by IFNalpha, strictly in correlation with the induction of apoptosis. Using double stainings, we demonstrated that Bak was activated prior to cytochrome c (cyt c) release and caspase-3 activation, whereas activated Bax was only found in cells with released cyt c, mitochondrial depolarization, as well as activated caspase-3. Furthermore, IFNalpha-induced activation of Bak, and to a large extent also of Bax, was dependent on caspase activity. With the use of a panel of specific caspase inhibitors we found, however, that none of caspases-1 to -10 were responsible for this activation. Neither was the Ca(2+)-dependent protease calpain nor the stress-activated p38 SAPK pathway significantly involved. Overexpression of Bcl-2 blocked apoptosis induced by IFNalpha totally abolished Bak activation, as well as decreased the amount of activated Bax. We conclude that IFNalpha induces Bak and Bax activation via distinct mechanisms involving an unknown protease, and that their activation is regulated by Bcl-2.     

凋亡素特异性诱导肿瘤细胞凋亡分子机制

来源于鸡贫血病毒(CAV)的凋亡素(apoptin)能选择性地诱导肿瘤细胞和转化细胞的凋亡,对正常人二倍体细胞无影响,而且它诱导的凋亡不依赖p53介导,不被bcl-2抑制,使其成为一种有前景的抗肿瘤制剂.该文就凋亡素诱导细胞凋亡的作用及其特异性诱导肿瘤细胞凋亡的分子机制进行综述.     

Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine

Hydroxychloroquine (HCQ) is a lysosomotropic amine with cytotoxic properties. Here, we show that HCQ induces signs of lysosomal membrane permeabilization (LMP), such as the decrease in the lysosomal pH gradient and the release of cathepsin B from the lysosomal lumen, followed by signs of apoptosis including caspase activation, phosphatidylserine exposure, and chromatin condensation with DNA loss. HCQ also induces mitochondrial membrane permeabilization (MMP), as indicated by the insertion of Bax into mitochondrial membranes, the conformational activation of Bax within mitochondria, the release of cytochrome c from mitochondria, and the loss of the mitochondrial transmembrane potential. To determine the molecular order among these events, we introduced inhibitors of LMP (bafilomycin A(1)), MMP (Bcl-X(L), wild-type Bcl-2, mitochondrion-targeted Bcl-2, or viral mitochondrial inhibitor of apoptosis from cytomegalovirus), and caspases (Z-VAD.fmk) into the system. Our data indicate that caspase-independent MMP is rate-limiting for LMP-mediated caspase activation. Mouse embryonic fibroblasts lacking the expression of both Bax and Bak are resistant against hydroxychloroquine-induced apoptosis. Such Bax(-/-) Bak(-/-) cells manifest normal LMP, yet fail to undergo MMP and subsequent cell death. The data reported herein indicate that LMP does not suffice to trigger caspase activation and that Bax/Bak-dependent MMP is a critical step of LMP-induced cell death.     

Bax translocation is crucial for the sensitivity of leukaemic cells to etoposide-induced apoptosis

Bax translocation from cytosol to mitochondria is believed to be a crucial step for triggering cytochrome c release from mitochondria. However, it is unclear whether Bax translocation is associated with Bax induction by DNA damaging agents. The induction of Bax in response to DNA damaging agents has been considered to be linked with p53. In this study, we used the p53 negative human chronic myeloid leukaemia K562 cell line. Bax up-regulation occurred at the whole cell level after DNA damage induced by etoposide. However, after incubation with etoposide, Bax failed to translocate to mitochondria and as a result, the apoptotic process was blocked. A Bax stable transfectant, the K/Bax cell line, expressed more Bax protein in the cytosol, mitochondria and nuclei. This Bax overexpression induced cytochrome c release, a reduction of cytochrome c oxidase activity and mitochondrial membrane potential (Delta(Psi)m). However, Bax-induced apoptosis was blocked downstream of mitochondria in K562 cells. The increased levels of mitochondrial Bax sensitized cells to etoposide-induced activation of caspases-2, -3 and -9 and apoptosis. However, after transient transfection with the Apaf-1 gene, K/Bax cells were sensitized to etoposide-induced caspase activation and apoptosis to a larger extent compared with Bax or Apaf-1 transfection alone. We therefore conclude that two mechanisms contribute to the resistance of K562 cells to etoposide-induced apoptosis; firstly failure of Bax targeting to mitochondria and, secondly, deficiency of Apaf-1. Uncoupling of Bax translocation from Bax induction can occur in response to etoposide-induced DNA damage.     

Evodiamine,a constituent of Evodiae Fructus,induces anti-proliferating effects in tumor cells

We found that evodiamine, a major alkaloidal component of Evodiae Fructus (Goshuyu in Japan), inhibited proliferation of several tumor cell lines, but had less effect on human peripheral blood mononuclear cells (PBMC). We used human cervical cancer cells, HeLa, as a model to elucidate the molecular mechanisms of evodiamine-induced tumor cell death. The results showed that evodiamine induced oligonucleosomal fragmentation of DNA in HeLa cells and increased the activity of caspase-3, but not that of caspase-1, in vitro . Both evodiamine-induced DNA fragmentation and caspase-3 activity were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-Asp-Glu-Val-Asp-fmk). In addition, evodiamine increased the expression of the apoptosis inducer Bax, but decreased the expression of the apoptosis suppressor Bcl-2 in mitochondria. Taken together, our data indicated that evodiamine alters the balance of Bcl-2 and Bax gene expression and induces apoptosis through the caspase pathway in HeLa cells. (Cancer Sci 2003; 94: 92–98)     

Aspirin induces apoptosis through release of cytochrome c from mitochondria

Nonsteroidal anti-inflammatory drugs (NSAID) reduce the risk for cancer, due to their antiproliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and triggers release of cytochrome c into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.     

Rituximab modifies the cisplatin-mitochondrial signaling pathway, resulting in apoptosis in cisplatin-resistant non-Hodgkin's lymphoma.

PURPOSE: Rituximab (chimeric anti-CD20) can reverse the cisplatin-resistant phenotype of AIDS-related non-Hodgkin's lymphoma cell lines and results in cisplatin-mediated apoptosis. The mechanism by which apoptosis is achieved by the combination treatment was examined. EXPERIMENTAL DESIGN: The AIDS-related lymphoma (ARL) cell line 2F7 was treated with rituximab, cisplatin, and a combination of the two and analyzed by Western blot analyses for signaling proteins involved in the death receptor-mediated and mitochondrial pathways. RESULTS: Rituximab selectively inhibited the expression of Bcl-2 in the ARL cells. However, other proteins analyzed [namely, Apaf-1, Bax, Bid, caspase-3, caspase-8, caspase-9, X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2, cytochrome c, Fas, Fas ligand, FLIP, p53, and poly(ADP-ribose) polymerase] were not affected by either rituximab or cisplatin. Treatment with cisplatin induced the generation of mitochondrial reactive oxygen species, specifically intracellular peroxides. Furthermore, cisplatin alone was unable to induce the mitochondrial apoptotic events; however, the rituximab-cisplatin combination was able to synergistically induce significant apoptosis and mitochondria-mediated apoptotic events [mitochondrial membrane depolarization (DeltaPsi(m)), cytochrome c release from mitochondria, and caspase-3 and -9 activation]. The combination treatment facilitated the down-regulation of Bcl-2 by rituximab at an early time point. Decreased expression of additional proteins (Apaf-1, cIAP-1, cIAP-2, and XIAP) paralleled apoptosis detected at 24 h. CONCLUSIONS: These findings show that the selective down-regulation of Bcl-2 by rituximab leading to apoptosis in ARL cells by cisplatin is through the mitochondria-dependent caspase pathway.