Project description:Smac mimetics (SM) have been recently reported to kill cancer cells through the extrinsic apoptosis pathway mediated by autocrine tumor necrosis factor (TNF). SM also activates nuclear factor-kappaB (NF-kappaB). However, how SM induces NF-kappaB and the role of NF-kappaB in SM-induced cancer cell death has not been well elucidated. We found that effective blockage of NF-kappaB had no detectable effect on SM compound 3 (SMC3)-induced TNF secretion, suggesting that the induction of TNF by SMC3 is independent of NF-kappaB. Conversely, SMC3-induced NF-kappaB activation was found to be mediated by autocrine TNF because this effect of SMC3 was effectively inhibited when TNF was blocked with either a TNF neutralizing antibody or TNF small interfering RNA. In addition, although SMC3 dramatically reduced c-IAP1 level, it had marginal effect on c-IAP2 expression, TNF-induced RIP modification, NF-kappaB activation, and downstream antiapoptosis NF-kappaB target expression. Furthermore, blocking NF-kappaB by targeting IKKbeta or RelA substantially potentiated SMC3-induced cytotoxicity, suggesting that the NF-kappaB pathway inhibits SMC3-induced apoptosis in cancer cells. Our results show that through TNF autocrine, SM induces an IKKbeta-mediated NF-kappaB activation pathway that protects cancer cells against SM-induced apoptosis, and thus, NF-kappaB blockage could be an effective approach for improving the anticancer value of SM.
Project description:Vanadium exerts a variety of biological effects, including antiproliferative responses through activation of the respective signaling pathways and the generation of reactive oxygen species. As epidermal cells are exposed to environmental insults, human keratinocytes (HaCaT) were used to investigate the mechanism of the antiproliferative effects of vanadyl(IV) sulfate (VOSO(4)). Treatment of HaCaT cells with VOSO(4) inhibited proliferation and induced apoptosis in a dose-dependent manner. Inhibition of proliferation was associated with downregulation of cyclins D1 and E, E2F1, and the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1). Induction of apoptosis correlated with upregulation of the c-fos oncoprotein, changes in the expression of clusterin (CLU), an altered ratio of antiapoptotic to proapoptotic Bcl-2 protein family members, and poly(ADP-ribose) polymerase-1 cleavage. Forced overexpression of c-fos induced apoptosis in HaCaT cells that correlated with secretory CLU downregulation and upregulation of nuclear CLU (nCLU), a pro-death protein. Overexpression of Bcl-2 protected HaCaT cells from vanadium-induced apoptosis, whereas secretory CLU overexpression offered no cytoprotection. In contrast, nCLU sensitized HaCaT cells to apoptosis. Our data suggest that vanadium-mediated apoptosis was promoted by c-fos, leading to alterations in CLU isoform processing and induction of the pro-death nCLU protein.
Project description:Neuroblastoma is the most common pediatric extracranial solid tumor with a broad spectrum of clinical behavior and poor prognosis. Despite intensive multimodal therapy, ongoing clinical trials, and basic science investigations, neuroblastoma remains a complex medical challenge with a long-term survival rate less than 40%. In our study, we found that resveratrol (3, 5, 4'-trihydroxystilbene, RSV), a naturally occurring phytoalexin, possesses an anticancer activity through blocking cell growth and inducing apoptosis in neuroblastoma cell line Neuro-2a (N-2a) cells. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative proteomic analysis, we found that 395 proteins were up-regulated and 302 proteins were down-regulated in the nucleus of N-2a cells treated with RSV. Among these, the polycomb protein histone methyltransferase EZH2 was reduced significantly, which is aberrantly overexpressed in neuroblastoma and crucial to maintain the malignant phenotype of neuroblastoma by epigenetic repression of multiple tumor suppressor genes. EZH2 reduction further led to decreased H3K27me3 level and reactivation of neuroblastoma tumor suppressor genes CLU and NGFR. Disruption EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibition with DZNep triggered cellular apoptosis in N-2a cells. We found that the up-regulation of miR-137 level was responsible for reduced EZH2 level in tumor suppression induced by RSV. Inhibition of miR-137 expression rescued the cellular apoptosis phenotypes, EZH2 reduction, and CLU and NGFR reactivation, associated with RSV treatment. Taken together, our findings present for the first time, an epigenetic mechanism involving miR-137-mediated EZH2 repression in RSV-induced apoptosis and tumor suppression of neuroblastoma, which would provide a key potential therapeutic target in neuroblastoma treatment.
Project description:Metastasis is responsible for the majority of prostate cancer-related deaths; however, little is known about the molecular mechanisms that underlie this process. Here we identify an oncogene-tumor suppressor cascade that promotes prostate cancer growth and metastasis by coordinately activating the small GTPase Ras and nuclear factor-kappaB (NF-kappaB). Specifically, we show that loss of the Ras GTPase-activating protein (RasGAP) gene DAB2IP induces metastatic prostate cancer in an orthotopic mouse tumor model. Notably, DAB2IP functions as a signaling scaffold that coordinately regulates Ras and NF-kappaB through distinct domains to promote tumor growth and metastasis, respectively. DAB2IP is suppressed in human prostate cancer, where its expression inversely correlates with tumor grade and predicts prognosis. Moreover, we report that epigenetic silencing of DAB2IP is a key mechanism by which the polycomb-group protein histone-lysine N-methyltransferase EZH2 activates Ras and NF-kappaB and triggers metastasis. These studies define the mechanism by which two major pathways can be simultaneously activated in metastatic prostate cancer and establish EZH2 as a driver of metastasis.
Project description:Melanoma is a lethal skin cancer prone to progression and metastasis, and resistant to therapy. Metastasis and therapy resistance of melanoma and other cancers are driven by tumor cell plasticity, largely via acquisition/loss of stem-like characteristics and transitions between epithelial and mesenchymal phenotypes (EMT/MET). NME1 is a metastasis suppressor gene that inhibits metastatic potential when its expression is enforced in melanoma and other cancers. Herein, we have unmasked a novel role for NME1 as a driver of melanoma growth distinct from its canonical function as a metastasis suppressor. NME1 promotes expansion of stem-like melanoma cells that exhibit elevated expression of stem cell markers (e.g., Sox2, Sox10, Oct-4, KLF4, and Ccnb-1), enhanced growth as melanoma spheres in culture, and enhanced tumor growth and lung colonizing activities in vivo. In contrast, NME1 expression did not affect the proliferation of melanoma cell lines in monolayer culture conditions. Silencing of NME1 expression resulted in a dramatic reduction in melanoma sphere size, and impaired tumor growth and metastatic activities of melanoma sphere cells when xenografted in immunocompromised mice. Individual cells within melanoma sphere cultures displayed a wide range of NME1 expression across multiple melanoma cell lines. Cell subpopulations with elevated NME1 expression were fast cycling and displayed enhanced expression of stem cell markers. IMPLICATIONS: Our findings suggest the current model of NME1 as a metastasis-suppressing factor requires refinement, bringing into consideration its heterogeneous expression within melanoma sphere cultures and its novel role in promoting the expansion and tumorigenicity of stem-like cells.
Project description:Defects in apoptosis contribute to treatment resistance and poor outcome of pancreatic cancer, calling for novel therapeutic strategies. Here, we provide the first evidence that nuclear factor (NF) ?B is required for Smac mimetic-mediated sensitization of pancreatic carcinoma cells for gemcitabine-induced apoptosis. The Smac mimetic BV6 cooperates with gemcitabine to reduce cell viability and to induce apoptosis. In addition, BV6 significantly enhances the cytotoxicity of several anticancer drugs against pancreatic carcinoma cells, including doxorubicin, cisplatin, and 5-fluorouracil. Molecular studies reveal that BV6 stimulates NF-?B activation, which is further increased in the presence of gemcitabine. Importantly, inhibition of NF-?B by overexpression of the dominant-negative I?B? superrepressor significantly decreases BV6- and gemcitabine-induced apoptosis, demonstrating that NF-?B exerts a proapoptotic function in this model of apoptosis. In support of this notion, inhibition of tumor necrosis factor ? (TNF?) by the TNF? blocking antibody Enbrel reduces BV6- and gemcitabine-induced activation of caspase 8 and 3, loss of mitochondrial membrane potential, and apoptosis. By demonstrating that BV6 and gemcitabine trigger a NF-?B-dependent, TNF?-mediated loop to activate apoptosis signaling pathways and caspase-dependent apoptotic cell death, our findings have important implications for the development of Smac mimetic-based combination protocols in the treatment of pancreatic cancer.
Project description:Interleukin-24 (IL-24), a member of the IL-10 cytokine family whose physiological function remains largely unknown, has been shown to induce apoptosis when expressed in an adenoviral background. It is yet little understood, why IL-24 alone induced apoptosis only in a limited number of tumor cell lines. Analyzing an influenza A virus vector expressing IL-24 for its oncolytic potential revealed enhanced pro-apoptotic activity of the chimeric virus compared with virus or IL-24 alone. Interestingly, IL-24-mediated enhancement of influenza-A-induced apoptosis did not require viral replication but critically depended on toll-like receptor 3 (TLR3) and caspase-8. Immunoprecipitation of TLR3 showed that infection by influenza A virus induced formation of a TLR3-associated signaling complex containing TRIF, RIP1, FADD, cFLIP and pro-caspase-8. Co-administration of IL-24 decreased the presence of cFLIP in the TLR3-associated complex, converting it into an atypical, TLR3-associated death-inducing signaling complex (TLR3 DISC) that induced apoptosis by enabling caspase-8 activation at this complex. The sensitizing effect of IL-24 on TLR3-induced apoptosis, mediated by influenza A virus or the TLR3-specific agonist poly(I:C), was also evident on tumor spheroids. In conclusion, rather than acting as an apoptosis inducer itself, IL-24 sensitizes cancer cells to TLR-mediated apoptosis by enabling the formation of an atypical DISC which, in the case of influenza A virus or poly(I:C), is associated with TLR3.
Project description:EBV latent antigen EBNA3C is indispensible for in vitro B-cell immortalization resulting in continuously proliferating lymphoblastoid cell lines (LCLs). EBNA3C was previously shown to target pRb for ubiquitin-proteasome mediated degradation, which facilitates G1 to S transition controlled by the major transcriptional activator E2F1. E2F1 also plays a pivotal role in regulating DNA damage induced apoptosis through both p53-dependent and -independent pathways. In this study, we demonstrate that in response to DNA damage LCLs knocked down for EBNA3C undergo a drastic induction of apoptosis, as a possible consequence of both p53- and E2F1-mediated activities. Importantly, EBNA3C was previously shown to suppress p53-induced apoptosis. Now, we also show that EBNA3C efficiently blocks E2F1-mediated apoptosis, as well as its anti-proliferative effects in a p53-independent manner, in response to DNA damage. The N- and C-terminal domains of EBNA3C form a stable pRb independent complex with the N-terminal DNA-binding region of E2F1 responsible for inducing apoptosis. Mechanistically, we show that EBNA3C represses E2F1 transcriptional activity via blocking its DNA-binding activity at the responsive promoters of p73 and Apaf-1 apoptosis induced genes, and also facilitates E2F1 degradation in an ubiquitin-proteasome dependent fashion. Moreover, in response to DNA damage, E2F1 knockdown LCLs exhibited a significant reduction in apoptosis with higher cell-viability. In the presence of normal mitogenic stimuli the growth rate of LCLs knockdown for E2F1 was markedly impaired; indicating that E2F1 plays a dual role in EBV positive cells and that active engagement of the EBNA3C-E2F1 complex is crucial for inhibition of DNA damage induced E2F1-mediated apoptosis. This study offers novel insights into our current understanding of EBV biology and enhances the potential for development of effective therapies against EBV associated B-cell lymphomas.
Project description:At present, chemotherapy is still the main treatment for cervical cancer. However, the drug resistance of chemotherapy drugs seriously restricts its use, so it is urgent to develop new drugs for cervical cancer. Some studies have shown that gambogic acid has a strong anti-tumor effect, while the anti-tumor effect and molecular mechanism of gambogic acid on cervical cancer need to be studied. Our study confirms that the cytotoxic effect of gambogic acid on cervical cancer cells depends on the expression of TR3 protein. Moreover, gambogic acid-induced apoptosis requires TR3 expression. In the mechanism, gambogic acid promoted nuclear export of TR3, resulting in up-regulation of p53, which leads to the decrease of mitochondrial membrane potential, eventually inducing apoptosis. These results suggest that the nuclear export of TR3 mediated gambogic acid-induced apoptosis through a p53-dependent apoptosis pathway.
Project description:Radiation-induced optic neuropathy (RION) is a devastating complication following external beam radiation therapy (EBRT) that leads to acute vision loss. To date, no efficient, available treatment for this complication, due partly to the lack of understanding regarding the developmental processes behind RION. Here, we report radiation caused changes in mitochondrial dynamics by regulating the mitochondrial fission proteins dynamin-related protein 1 (Drp1) and fission-1 (Fis1). Concurrent with an excessive production of reactive oxygen species (ROS), both neuronal injury and visual dysfunction resulted. Further, our findings delineate an important mechanism by which cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of Drp1 (Ser616) regulates defects in mitochondrial dynamics associated with neuronal injury in the development of RION. Both the pharmacological inhibition of Cdk5 by roscovitine and the inhibition of Drp1 by mdivi-1 inhibited mitochondrial fission and the production of ROS associated with radiation-induced neuronal loss. Taken together, these findings may have clinical significance in preventing the development of RION.