Downregulation Of XIAP Expression Research Articles

Sterigmatocystin induces autophagic and apoptotic cell death of liver cancer cells via downregulation of XIAP

XIAP, or the X-linked Inhibitor of Apoptosis Protein, is the most extensively studied member within the IAP gene family. It possesses the capability to impede apoptosis through direct inhibition of caspase activity. Various kinds of cancers overexpress XIAP to enable cancer cells to avoid apoptosis. Consequently, the inhibition of XIAP holds significant clinical implications for the development of anti-tumor medications and the treatment of cancer. In this study, sterigmatocystin, a natural compound obtained from the genus asperigillus, was demonstrated to be able to induce apoptotic and autophagic cell death in liver cancer cells. Mechanistically, sterigmatocystin induces apoptosis by downregulation of XIAP expression. Additionally, sterigmatocystin treatment induces cell cycle arrest, blocks cell proliferation, and slows down colony formation in liver cancer cells. Importantly, sterigmatocystin exhibits a remarkable therapeutic effect in a nude mice model. Our findings revealed a novel mechanism through which sterigmatocystin induces apoptotic and autophagic cell death of liver cancer cells by suppressing XIAP expression, this offers a promising therapeutic approach for treating liver cancer patients.

Gallic acid induces apoptosis and enhances the anticancer effects of cisplatin in human small cell lung cancer H446 cell line via the ROS-dependent mitochondrial apoptotic pathway.

Small cell lung cancer (SCLC) is the most aggressive lung cancer subtype and accounts for more than 15% of all lung cancer cases. Cisplatin [cis-diamminedichloroplatinum (CDDP)]-based combination chemotherapy is the cornerstone for all stages of SCLC. However, acquired multidrug resistance (MDR) and intolerable toxicities lead to a high mortality rate in SCLC patients. Gallic acid [3,4,5-trihydroxybenzoic acid (GA)] is a natural botanic phenolic compound which can induce cell apoptosis in several types of cancers. In the present study, we aimed to explore the anticancer effects of GA on human SCLC H446 cells and its promotive effects on the anticancer activities of cisplatin. The viability of the H446 cells was analyzed by MTT assay. Morphological changes in the H446 cells were observed under an inverted microscope. Apoptosis induction was determined by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The level of reactive oxygen species (ROS) was assessed by 2'7'-dichlorofluorescein diacetate (DCFH‑DA), mitochondrial membrane potential (MMP) by JC-1, and western blotting was used to examine the expression of mitochondrial apoptosis-related proteins. The results showed that both GA and cisplatin changed the morphology, inhibited the growth and induced apoptosis in the H446 cells by inducing generation of ROS, disruption of MMP, downregulation of XIAP expression, and upregulation of Bax, Apaf-1, DIABLO and p53 expression. More importantly, GA combined with cisplatin exhibited synergistic effects on inducing of these pro-apoptotic mediators and modulating the activation of apoptosis-related molecules. However, inhibition of the generation of ROS by N-acetyl-l-cysteine (NAC), a specific ROS inhibitor, reversed the cell apoptosis induced by cisplatin combined with GA. In conclusion, the results from the present study revealed that GA exhibited an anticancer effect on human SCLC H446 cells and enhanced the antitumor activities of cisplatin via the ROS-dependent mitochondrial apoptotic pathway.

Abstract 4893: XIAP downregulation accompanies inhibition of melanoma xenograft growth by mebendazole

Abstract Background: Melanoma's intrinsic resistance to therapy has been attributed to modulation of cellular survival pathways including apoptosis. We originally reported that mebendazole (MBZ), which acts as a microtubule-disrupting agent, is a potent inducer of apoptosis in melanoma cells in vitro via a Bcl-2 dependent mechanism (Molecular Cancer Research, 2008). Here, we show that MBZ is also able to effectively modulate another key anti-apoptotic factor, XIAP, to promote melanoma apoptosis both in vitro and in vivo. Methodology: Safety and efficacy of mebendazole was assessed using a human melanoma xenograft model established from M-14 cells. The effect of MBZ on XIAP expression and induction of apoptotic pathways was evaluated by immunoblotting and co-immunoprecipitation techniques along with densitometric quantification. The growth inhibitory effects of MBZ were determined in a panel of melanoma cell lines with the sulphorhodamine (SRB) assay. siRNA techniques were used to reduce expression of XIAP. Results: Oral MBZ is as effective as the current standard of care temozolomide (TMZ) in reducing melanoma growth in vivo, with no notable toxicities observed. Inhibition of melanoma growth in vivo is accompanied by MBZ specific reduced expression of XIAP and cleavage of caspase 9. In response to MBZ treatment, sensitive cell lines display reduced levels of XIAP expression whereas resistant cell lines do not. Moreover, down-regulation of XIAP expression by siRNA in the MBZ-resistant melanoma cell line UACC1097 was accompanied by a reduction in the IC50 for MBZ from >10 μM to 3.7 μM. Exposure of melanoma cells to MBZ promotes the interaction of SMAC/DIABLO with XIAP, thereby alleviating inhibition of apoptosis by XIAP. Conclusions: In addition to demonstrating efficacy and safety in vivo, our preclinical studies of the promising anti-melanoma agent mebendazole provide insight into characterization of an apoptotic promoting mechanism of action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4893. doi:1538-7445.AM2012-4893

Deguelin, a Novel Hsp90 Inhibitor, Suppresses STAT3 and STAT5 Activity and Induces Apoptosis in the Human T-Cell Leukemia Virus Type 1-Transformed Cells.

Adult T-cell leukemia (ATL) cells are remarkably resistant to conventional chemotherapy. Therefore, new agents that effectively kill these cells are needed. Deguelin is a rotenoid from African plant Mundulea sericea which has been recently shown to suppress the lung tumorigenesis via inhibiting the activation of PI3kinase-Akt signaling. More recently, deguelin has been shown to bind to ATP-binding pocket of heat shock protein (Hsp) 90 and inhibit functions of Hsp90 client proteins. However, the effect of deguelin on ATL cells is not known. We investigated the effects of deguelin on the cell growth and cell death of human T-cell leukemia virus type 1 (HTLV-1)- transformed cells, KUT-1 and MT-2 (kindly provided by Dr. Hanada, Kagoshima Medical Center, Kagoshima and Dr. Kibata, Hayashibara Biochemical Laboratory Inc., Okayama, Japan, respectively) by trypan-blue exclusion and MTT assays, and annexin V/PI staining. Deguelin inhibited the proliferation of KUT-1 and MT-2 cells in a time-and a dose-dependent manner and had much less cytotoxic effects on normal human peripheral blood mononuclear cells. In addition, flow cytometric analysis revealed a higher frequency of annexin V-positive cells in deguelin treated cells compared to that in untreated cells. These results indicate that deguelin induces the cell-growth arrest and cell death in ATL cells. To better understand the mechanism of these effects, we examined the intracellular signaling and the expression of apoptosis associated proteins by immunoblot analysis. We found that deguelin prevented the tyrosine phosphorylation of the transcription factor signal transducer and activator of transcription (STAT) 3 and STAT5, both of which are constitutively activated in these cells. In addition, the inhibitor of apoptosis protein, XIAP expression decreased at 24 hours after deguelin treatment, indicating that deguelin induces the cell growth arrest and apoptosis via at least in part the inhibition of STAT3 and STAT5 phosphorylation and the downregulation of XIAP expression. We next examined whether deguelin suppresses janus kinase (JAK) 3 phosphorylation in KUT-1 cells using immunoprecipitation assay. However, deguelin treatment did not inhibit JAK3 phosphorylation, suggesting that the inhibition of STAT5 phosphorylation by deguelin is independent of JAK3 in KUT-1 cells. Since deguelin has been shown to inhibit the function of Hsp90 client proteins, we examined the protein expression of cyclin dependent kinase (Cdk) 4, which is known as one of the Hsp90 client proteins. Deguelin treatment reduced Cdk4 expression in both cells, suggesting that deguelin may destabilize Cdk4 via inhibiting the Hsp90 function. Taken together, deguelin appears to inhibit the proliferation and induce apoptosis of ATL cells via preventing the tyrosine phosphorylation of STAT proteins and destabilizing Cdk4. Thus, deguelin merits further investigation as a potential therapeutic agent for this incurable disease.

Evaluation of XIAP as a Molecular Target To Increase Sensitivity to Imatinib.

Imatinib, a potent and selective inhibitor of BCR-ABL, has emerged as an effective drug for targeted therapy of CML. However, resistance to this drug has been arising, mainly due to an overexpression of BCR-ABL, to mutations in the tyrosine-kinase domain of BCR-ABL or to an overexpression of the multidrug resistance protein (Pgp). Cytotoxic drugs induce cellular apoptosis and resistance to these drugs is often due to resistance to apoptosis. Cancer cells may escape apoptosis due to an overexpression of anti-apoptotic proteins such as XIAP, which binds caspases 3, 7 and 9 and inhibits their action.Downregulation of XIAP expression has been documented to increase sensitivity of several cell lines to some conventional drugs. In the current study we investigated if downregulation of XIAP expression, following treatment with XIAP-siRNAs, would increase sensitivity to Imatinib in: i) a sensitive CML cell line (K562), when compared to treatment with control siRNAs and ii) a resistant cell line which overexpresses Pgp (K562Dox), when compared to treatment with Pgp-siRNAs or with control siRNAs. To confirm if the siRNAs were capable of downregulating the expression of their targets, Western Blots were carried out at 24, 48 and 72h after transfection. The cells had been: i) treated with medium only (Blank), ii) transfected with a control-siRNAs, iii) transfected with siRNAs for XIAP and iv) tranfected with siRNAs for Pgp (in the K562Dox cell line only). It was verified that the XIAP-siRNAs decreased XIAP protein levels in both cell lines 48h following transfection and Pgp-siRNAs decreased Pgp protein levels in the resistant cell line (K562Dox) 24h following transfection, when compared to the control-siRNA treatments. To further investigate if the downregulation of XIAP expression (and Pgp in the resistant cell line) caused sensitization of cells to1μM Imatinib, Imatinib was added to the cells previously transfected with siRNAs (for P-gp or XIAP or control siRNAs) and to cells in exponential growth (Blank cells). Viable cell number was counted by the Trypan Blue exclusion assay and apoptosis was verified with the TUNEL assay. Results of viable cell numbers from several experiments indicate that Imatinib treatment on its own, as expected, decreased more the viable cell number of the sensitive cell line than of the resistant cell line (from 100% in the Blank cells to 63 % in the treated K562 cells and to 86 % in the treated K562Dox cells). Furthermore, downregulation of XIAP expression on its own decreased the viable cell number of both cell lines (from 100% in the Blank cells to 62% in the K562 XIAP-siRNA treated cells and to 80% in the K562Dox XIAP-siRNA treated cells). Concomitant treatment with both XIAP-siRNAs and Imatinib further reduced the number of viable cells to 36% in the K562 cells and to 57% in the K562Dox cells. In the resistant cell line, downregulation of Pgp expression on its own reduced the viable cell number from 100% in Blank cells to 84% in the Pgp-siRNA treated cells. Concomitant treatment with Pgp-siRNAs and Imatinib further reduced the viable cell number to 60%. The reduction of viable cell number coincided with an increase in apoptosis. It was concluded that downregulation of XIAP expression may be a good approach to enhance sensitivity to Imatinib, even in the case of existing Pgp overexpression.

Downregulation of xIAP Expression by Small Interfering RNA Inhibits Cellular Viability and Increases Chemosensitivity to Methotrexate in Human Hepatoma Cell Line HepG2

The aim of this study was to investigate whether downregulating the expression of xIAP by RNAi (RNA interference) technology can induce the apoptosis of HepG2 cells, inhibit cellular viability and increase chemosensitivity of cancer cells. HepG2 cells were transfected with U6 promoter plasmids coding for short interfering RNAs (siRNAs) targeting xIAP. RT-PCR and western blot analysis were used to assess the mRNA and protein levels of xIAP expression. The suppression efficiency of xIAP by RNAi was evaluated using the MTT assay for cellular viability and Annexin V-PI binding assay for apoptosis. These results showed that siRNAs reduced cellular viability and increased cellular apoptosis. Moreover, downregulation of xIAP expression enhanced the chemosensitivity of HepG2 cells to methotrexate. These results suggest that the downregulation of xIAP by RNAi could potentially be used as a therapeutic strategy for human hepatocellular carcinoma.

Chemosensitization Effects of XIAP Downregulation in K562 Leukemia Cells

The effect of downregulation of the expression of the antiapoptotic protein XIAP with antisense oligonucleotides was evaluated in the K562 chronic myeloid leukemia (CML) cell line. This was carried out by studying the effects of downregulation of XIAP expression on cellular viability, cellular apoptosis and on the response to two chemotherapeutical drugs, etoposide and doxorubicin. We document that downregulation of XIAP expression decreased cellular viability, increased cellular apoptosis and enhanced the effects of doxorubicin.

Nuclear factor-kappaB-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulosa cells from tumor necrosis factor alpha-induced apoptosis.

Although X-linked inhibitor of apoptosis protein (Xiap) is an important intracellular suppressor of apoptosis in a variety of cell types and is present in ovary, its physiological role in follicular development remains unclear. The purpose of the present studies was to examine the modulatory role of Xiap in the proapoptotic action of tumor necrosis factor-alpha (TNFalpha) in rat granulosa cells. Granulosa cells from equine CG-primed immature rats were plated in RPMI 1640 medium containing 10% FCS and subsequently cultured in serum-free RPMI in the absence or presence of TNFalpha (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 microM), and/or adenoviral Xiap sense or antisense complementary DNA. TNFalpha alone failed to induce granulosa cell death, but in the presence of cycloheximide, it markedly increased the number of apoptotic granulosa cells (as assessed by in situ terminal deoxynucleotidyl transferase-mediated deox-UTPbiotin end labeling and DNA fragmentation analysis). Western analysis indicated that TNFalpha alone increased the Xiap protein level, a response significantly reduced by adenoviral Xiap antisense expression. Down-regulation of Xiap expression by antisense complementary DNA induced granulosa cell apoptosis, which was potentiated by the cytokine. Inhibition of nuclear factor-kappaB activation by N-acetyl-cysteine and SN50 suppressed Xiap protein expression and enhanced apoptosis induced by TNFalpha. The latter phenomenon was readily attenuated by adenoviral Xiap sense expression. In conclusion, these findings suggest that Xiap is an important intracellular modulator of the TNFalpha death signaling pathway in granulosa cells. Its expression is regulated by the TNFalpha via a nuclear factor-kappaB-mediated mechanism.