ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas

BACKGROUND: Inhibitors of apoptosis (IAPs) are a family of cell death inhibitors found in viruses and metazoans. All IAPs have at least one baculovirus IAP repeat (BIR) motif that is essential for their anti-apoptotic activity. IAPs physically interact with a variety of pro-apoptotic proteins and inhibit apoptosis induced by diverse stimuli. This allows them to function as sensors and inhibitors of death signals that emanate from a variety of pathways. RESULTS: Here we report the characterization of ML-IAP, a novel human IAP that contains a single BIR and RING finger motif. ML-IAP is a powerful inhibitor of apoptosis induced by death receptors and chemotherapeutic agents, probably functioning as a direct inhibitor of downstream effector caspases. Modeling studies of the structure of the BIR domain revealed it to closely resemble the fold determined for the BIR2 domain of X-IAP. Deletion and mutational analysis demonstrated that integrity of the BIR domain was required for anti-apoptotic function. Tissue survey analysis showed expression in a number of embryonic tissues and tumor cell lines. In particular, the majority of melanoma cell lines expressed high levels of ML-IAP in contrast to primary melanocytes, which expressed undetectable levels. These melanoma cells were significantly more resistant to drug-induced apoptosis. CONCLUSIONS: ML-IAP, a novel human IAP, inhibits apoptosis induced by death receptors and chemotherapeutic agents. The BIR of ML-IAP possesses an evolutionarily conserved fold that is necessary for anti-apoptotic activity. Elevated expression of ML-IAP renders melanoma cells resistant to apoptotic stimuli and thereby potentially contributes to the pathogenesis of this malignancy.     

2-Deoxyglucose sensitizes melanoma cells to TRAIL-induced apoptosis which is reduced by mannose

While melanoma cell lines use aerobic glycolysis, addition of a competitive inhibitor such as 2-deoxyglucose (2DG) by itself achieved only modest killing. To overcome high levels of pro-survival proteins in melanoma cells, 2DG or glucose deprivation (GD) was combined with tumor necrosis factor-related apoptosis inducing-ligand (TRAIL). TRAIL treatment by itself also only induced modest killing, but combining TRAIL with 2DG or GD triggered a synergistic pro-apoptotic response in melanoma lines but not melanocytes. In melanoma cells, there was cleavage of caspases 3, 8 and Bid. Killing by combination treatments was completely blocked by a pan-caspase inhibitor, z-VAD. Mechanistically, 2DG and GD enhanced surface levels for both death receptors (DR4 and DR5); which was accompanied by reductions in levels of Mcl-1, Bcl-2 and survivin. Mannose pre-treatment reduced enhanced killing by combination treatments, accompanied by reduced DR5 levels. These results indicate melanoma cells in which there is altered glucose-related metabolomics can be exploited by interfering with glucose metabolism in combination with TRAIL; thereby overcoming the notorious death resistance of melanoma. Thus, a new therapeutic window is open for future clinical trials using agents targeting the glucose-related metabolome, in combination with agents triggering death receptors in patients with melanoma.     

Selenocystine potentiates cancer cell apoptosis induced by 5-fluorouracil by triggering reactive oxygen species-mediated DNA damage and inactivation of the ERK pathway

5-Fluorouracil (5-FU)-based chemotherapy as a first-line treatment is quite limited, because of its inefficiency and clinical resistance to it. The search for chemosensitizers that could augment its efficiency and overcome the drug resistance to 5-FU has kindled great interest among scientists. Selenocystine (SeC), a naturally occurring selenoamino acid, displayed broad-spectrum anticancer activity in our previous studies. This study demonstrates that SeC acts as an effective enhancer of 5-FU-induced apoptosis in A375 human melanoma cells through induction of mitochondria-mediated apoptosis with the involvement of DNA damage-mediated p53 phosphorylation and ERK inactivation. Pretreatment of the cells with SeC significantly enhanced 5-FU-induced loss of mitochondrial membrane potential (∆ψ_m) by regulating the expression levels of Bcl-2 family proteins. SeC and 5-FU in combination also triggered cell oxidative stress through regulation of the intracellular redox system and led to DNA damage and inactivation of ERK and AKT. Moreover, inhibitors of ERK and AKT effectively enhanced the apoptotic cell death induced by the combined treatment. However, pretreatment of the cells with glutathione reversed the apoptosis induced by SeC and 5-FU and recovered the expression of ERK and AKT inactivation, which revealed the important role of reactive oxygen species in cell apoptosis and regulation of ERK and AKT pathways. Taken together, our results suggest that a strategy of using SeC and 5-FU in combination could be a highly efficient way to achieve anticancer synergism.     

Targeting glutamine metabolism sensitizes melanoma cells to TRAIL-induced death

Targeting specific metabolic pathways has emerged for cancer therapeutics. For melanoma, metabolic studies have solely focused on high glucose uptake. By contrast, little is known regarding addiction to glutamine. Using five melanoma lines and two normal cell types, addition of aminooxyacetate (AOA), an inhibitor of glutamate-dependent transaminase regulating glutaminolytic pathway, two lines underwent low levels of apoptosis (>30%), while the other three lines were resistant, as were normal cells to AOA. However, three resistant lines (but not normal cells), became sensitized to undergoing apoptosis when TRAIL was combined with AOA. TRAIL by itself had minimal effects on all cell lines and normal cells, and did not augment AOA-induced killing in the two sensitive melanoma lines. AOA plus TRAIL induced a caspase-dependent apoptotic response. AOA did not influence TRAIL DR4 or DR5 cell surface death receptor levels, but AOA enhanced pro-apoptotic protein levels of Noxa, while reducing pro-survival protein Mcl-1. To verify AOA was targeting glutamine pathway, depletion of glutamine produced similar results, because absence of glutamine sensitized three melanoma lines, but not fibroblasts to killing by TRAIL. Glutamine depletion also led to Noxa induction. These results indicate some lines are addicted to glutamine, and treatment with AOA or glutamine depletion sensitizes melanoma to TRAIL-mediated killing, while sparing normal cells. Future studies are indicated to translate these discoveries to metastatic melanoma as there is currently no treatment available to prolong survival.     

Ectopically expressed perforin-1 is proapoptotic in tumor cell lines by increasing caspase-3 activity and the nuclear translocation of cytochrome C

Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell's plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.) into the cytoplasm provides the proteases with access to numerous protein substrates that promote apoptosis after cleavage. These proteases are believed to be the main executioners of target cell apoptosis. Although the PRF and granzyme components are both critical to this process and in some way involved in inducing cell death in target cells, the inhibition of tumor growth could still be efficient in granzyme-deficient mice. It is unclear whether PRF alone can suppress tumors. In this study, we discovered that forced ectopic expression of PRF alone, in the absence of granzymes, could mediate cell death in cancer cells. Notably, transient expression of both full-length and truncated active-form PRF in human Hep G2, SK-BR-3, and HeLa cells was found to induce apparent cell growth inhibition and cell death, as evidenced by chromosome condensation and DNA fragmentation, increased caspase-3 activity, and the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. This PRF-induced cell death could be abrogated by pan-caspase inhibitor (Z-VAD) and mitochondria protector (TAT-BH4). The implication of these results is that ectopically expressed PRF has apoptosis-inducing abilities, and PRF alone is sufficient to induce apoptotic cell death in cells with ectopic expression. Taking this into consideration, our results suggest the possibility of using PRF as a pro-apoptotic gene for tumor therapeutics.     

Ceramide-induced apoptosis of D283 medulloblastoma cells requires mitochondrial respiratory chain activity but occurs independently of caspases and is not sensitive to Bcl-xL overexpression

Ceramides are potent lipid second messengers that are involved in apoptotic and hypoxic/ischaemic neurone death. We investigated the role of mitochondria and the mitochondrial apoptosis pathway in ceramide-induced cell death using human D283 medulloblastoma cells with a reduced mitochondrial DNA copy number (rho- cells) and a corresponding defect in mitochondrial respiration. Treatment with the complex I inhibitor rotenone, C2- or C8-ceramide induced cell death in D283 control cells, while rho- cells were significantly protected. In contrast, activation of the mitochondrial apoptosis pathway by transient overexpression of the pro-apoptotic Bax protein or exposure to the kinase inhibitor staurosporine induced apoptosis to a similar extent in control and rho- cells. Overexpression of the antiapoptotic protein Bcl-xL failed to inhibit the toxic effect of C2-ceramide in D283 control cells, and no significant increase in caspase-3-like protease activity could be detected during the death process. Despite this, C2-ceramide induced significant chromatin condensation and cell shrinkage in D283 control cells, reminiscent of apoptosis. These morphological alterations were associated with the activation of calpains. Both apoptotic morphology and calpain activation were attenuated in rho- cells. Our data indicate that the apoptosis-inducing effect of C2-ceramide may require mitochondrial respiratory chain activity and can occur independently of the mitochondrial apoptosis pathway, but involves the activation of calpains.     

Interaction between caspase-3 and caspase-5 in the stretch-induced programmed cell death in the human periodontal ligament cells

In our previous studies, programmed cell death (PCD) was induced in human periodontal ligament (PDL) cells, through activation of caspase-3 and upregulation of CASP5 gene (encoding caspase-5 protein), in response to mechanical stretch loading. The aim of this study is to explore the relationship between the inflammatory caspase, caspase-5, and the apoptotic executioner protein, caspase-3, in human PDL cells. Here, we found that cyclic stretching upregulated the activity and the protein expression level of caspase-3 and -5 and the addition of the caspase-3 inhibitor or caspase-5 inhibitor significantly inhibited the stretch-induced PCD. Meanwhile, the inhibition of caspase-5 inhibited the activation of caspase-3 and vice versa. The result of coimmunoprecipitation also demonstrated that the expression of caspase-3 was immunoprecipitated with caspase-5. Thus, our study revealed that the in vitro application of cyclic stretching induced PCD by activation of caspase-3 and -5 in human PDL cells, and these two caspases could interact with each other after mechanical stretch loading. The study may facilitate further studies on the mechanism of stretch-induced PCD and help us understand the force-related periodontal homeostasis and remodeling better.     

BMRP is a Bcl-2 binding protein that induces apoptosis

Members of the Bcl-2 family of proteins play important roles in the regulation of cell death by apoptosis. The yeast Two-Hybrid system was utilized to identify a protein that interacts with the anti-apoptotic protein Bcl-2, designated BMRP. This protein corresponds to a previously known mitochondrial ribosomal protein (MRPL41). Binding experiments confirmed the interaction of BMRP to Bcl-2 in mammalian cells. Subcellular fractionation by differential centrifugation studies showed that both Bcl-2 and BMRP are localized to the same fractions (fractions that are rich in mitochondria). Northern blot analysis revealed a major bmrp mRNA band of approximately 0.8 kb in several human tissues. Additionally, a larger 2.2 kb mRNA species was also observed in some tissues. Western blot analysis showed that endogenous BMRP runs as a band of 16-17 kDa in SDS-PAGE. Overexpression of BMRP induced cell death in primary embryonic fibroblasts and NIH/3T3 cells. Transfection of BMRP showed similar effects to those observed by overexpression of the pro-apoptotic proteins Bax or Bad. BMRP-stimulated cell death was counteracted by co-expression of Bcl-2. The baculoviral caspase inhibitor p35 also protected cells from BMRP-induced cell death. These findings suggest that BMRP is a mitochondrial ribosomal protein involved in the regulation of cell death by apoptosis, probably affecting pathways mediated by Bcl-2 and caspases.     

ATP-柠檬酸裂解酶在黑素瘤维罗非尼治疗抵抗中的作用机制研究

目的:探讨ATP-柠檬酸裂解酶(ATP-citrate lyase,ACLY)在黑素瘤维罗非尼治疗抵抗中的作用及其可能机制。方法:通过Western blot实验检测5μM维罗非尼处理前后黑素瘤细胞中ACLY的表达水平,采用shRNA干涉黑素瘤细胞中ACLY的表达后再给予5μM维罗非尼处理,通过流式细胞术检测细胞凋亡水平;通过Western blot实验检测黑素瘤特异性转录因子MITF的表达水平,以及抗凋亡蛋白BCL-2和促凋亡蛋白BAX的表达水平。结果:1)维罗非尼处理后黑素瘤细胞ACLY的表达水平较处理前明显升高;2)沉默ACLY可以显著增加维罗非尼处理后黑素瘤细胞的凋亡水平;3)在维罗非尼处理下,沉默ACLY可以导致MITF和BCL-2表达水平较对照组显著降低,BAX表达水平升高。结论:ACLY表达水平升高参与了黑素瘤维罗非尼治疗抵抗,可能与其调控MITF表达的作用有关。     

Transient expression of wild-type or mitochondrially targeted Bcl-2 induces apoptosis, whereas transient expression of endoplasmic reticulum-targeted Bcl-2 is protective against Bax-induced cell death

Bcl-2 protein family members function either to promote or inhibit programmed cell death. Bcl-2, typically an inhibitor of apoptosis, has also been demonstrated to have pro-apoptotic activity (Cheng, E. H., Kirsch, D. G., Clem, R. J., et al. (1997) Science 278, 1966-1968). The pro-apoptotic activity has been attributed to the cleavage of Bcl-2 by caspase-3, which converts Bcl-2 to a pro-apoptotic molecule. Bcl-2 is a membrane protein that is localized in the endoplasmic reticulum (ER) membrane, the outer mitochondrial membrane, and the nuclear envelope. Here, we demonstrate that transient expression of Bcl-2 at levels comparable to those found in stably transfected cells induces apoptosis in human embryonic kidney 293 cells and in the human breast cell line MDA-MB-468 cells. Furthermore, we have targeted Bcl-2 specifically to either the ER or the outer mitochondrial membrane to test whether induction of apoptosis by Bcl-2 is dependent upon its localization within either of these membranes. Our findings indicate that Bcl-2 specifically targeted to the mitochondria induces cell death, whereas Bcl-2 that is targeted to the ER does not. The expression of Bcl-2 does result in its cleavage to a 20-kDa protein; however, mutation of the caspase-3 cleavage site (D34A) does not inhibit its ability to induce cell death. Additionally, we find that transiently expressed ER-targeted Bcl-2 inhibits cell death induced by Bax overexpression. In conclusion, the ability of Bcl-2 to promote apoptosis is associated with its localization at the mitochondria. Furthermore, the ability of ER-targeted Bcl-2 to protect against Bax-induced apoptosis suggests that the ER localization of Bcl-2 may play an important role in its protective function.     

The hepatitis C virus NS2 protein is an inhibitor of CIDE-B-induced apoptosis

Chronic hepatitis C virus (HCV) infection frequently leads to liver cancer. To determine the viral factor(s) potentially involved in viral persistence, we focused our work on NS2, a viral protein of unknown function. To assign a role for NS2, we searched for cellular proteins that interact with NS2. Performing a two-hybrid screen on a human liver cDNA library, we found that NS2 interacted with the liver-specific pro-apoptotic CIDE-B protein. Binding specificity of NS2 for CIDE-B was confirmed by cell-free assays associated with colocalization studies and coprecipitation experiments on human endogenous CIDE-B. CIDE-B, a member of the novel CIDE family of apoptosis-inducing factors, has been reported to show strong cell death-inducing activity in its C-terminal domain. We show that this CIDE-B killing domain is involved in the NS2 interaction. NS2 binding was sufficient to inhibit CIDE-B-induced apoptosis because an NS2 deletion mutant unable to interact with CIDE-B in vitro lost its capacity to interfere with CIDE-B cell death activity. Although it has been reported that CIDE-B-induced apoptosis is characterized by mitochondrial localization, the precise apoptotic mechanism remained unknown. Here, we show that CIDE-B induced cell death in a caspase-dependent manner through cytochrome c release from mitochondria. Furthermore, we found that NS2 counteracted the cytochrome c release induced by CIDE-B. In vivo, the CIDE-B protein level was extremely low in adenovirus-infected transgenic mice expressing the HCV polyprotein compared with that in wild-type mice. We suggest that NS2 interferes with the CIDE-B-induced death pathway and participates in HCV strategies to subvert host cell defense.     

Hic‐5 affects proliferation,migration and invasion of B16 murine melanoma cells

Hic‐5 is a shuttling protein between the cell membrane and the nucleus which functions as a focal adhesion adaptor protein and a nuclear receptor coactivator. Although several studies have shown its involvement in other types of cancer, the role of Hic‐5 in melanoma is unknown. Herein, we show for the first time that Hic‐5 is expressed in B16‐F1 murine melanoma cells. To determine its function in melanoma cells, we used shRNA‐mediated RNA interference and established stable clones with down‐regulated Hic‐5 expression. These clones had impaired growth and metastatic potential compared with controls in vivo, which correlated with decreased proliferation, migration and invasion in vitro. Moreover, silencing of Hic‐5 expression in B16‐F1 activated RhoA with an amoeboid phenotypic change, indicating that Hic‐5 is a key regulator of B16‐F1 metastasis in the context of Rho‐dependent motility. These results provide new evidence that Hic‐5 is a possible molecular target for treatment of melanoma.     

Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells

Silibinin, a flavonolignan isolated from the milk thistle plant (Silybum marianum), possesses anti-neoplastic properties. In vitro and in vivo studies have recently shown that silibinin inhibits the growth of colorectal cancer (CRC). The present study investigates the mechanisms of silibinin-induced cell death using an in vitro model of human colon cancer progression, consisting of primary tumor cells (SW480) and their derived metastatic cells (SW620) isolated from a metastasis of the same patient. Silibinin induced apoptotic cell death evidenced by DNA fragmentation and activation of caspase-3 in both cell lines. Silibinin enhanced the expression (protein and mRNA) of TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR4/DR5) at the cell surface in SW480 cells, and induced their expression in TRAIL-resistant SW620 cells normally not expressing DR4/DR5. Caspase-8 and -10 were activated demonstrating the involvement of the extrinsic apoptotic pathway in silibinin-treated SW480 and SW620 cells. The protein Bid was cleaved in SW480 cells indicating a cross-talk between extrinsic and intrinsic apoptotic pathway. We demonstrated that silibinin activated also the intrinsic apoptotic pathway in both cell lines, including the perturbation of the mitochondrial membrane potential, the release of cytochrome c into the cytosol and the activation of caspase-9. Simultaneously to apoptosis, silibinin triggered an autophagic response. The inhibition of autophagy with a specific inhibitor enhanced cell death, suggesting a cytoprotective function for autophagy in silibinin-treated cells. Taken together, our data show that silibinin initiated in SW480 and SW620 cells an autophagic-mediated survival response overwhelmed by the activation of both the extrinsic and intrinsic apoptotic pathways.     

Suppression of FOXM1 sensitizes human cancer cells to cell death induced by DNA-damage

Irradiation and DNA-damaging chemotherapeutic agents are commonly used in anticancer treatments. Following DNA damage FOXM1 protein levels are often elevated. In this study, we sought to investigate the potential role of FOXM1 in programmed cell death induced by DNA-damage. Human cancer cells after FOXM1 suppression were subjected to doxorubicin or γ-irradiation treatment. Our findings indicate that FOXM1 downregulation by stable or transient knockdown using RNAi or by treatment with proteasome inhibitors that target FOXM1 strongly sensitized human cancer cells of different origin to DNA-damage-induced apoptosis. We showed that FOXM1 suppresses the activation of pro-apoptotic JNK and positively regulates anti-apoptotic Bcl-2, suggesting that JNK activation and Bcl-2 down-regulation could mediate sensitivity to DNA-damaging agent-induced apoptosis after targeting FOXM1. Since FOXM1 is widely expressed in human cancers, our data further support the fact that it is a valid target for combinatorial anticancer therapy.