Downregulation Of Survivin Research Articles

Effect of allicin on proliferation and apoptosis of KG-1 cells and its molecular mechanism

Allicin is one of the main bioactive substances in garlic, with antibacterial, hypolipidemic and other pharmacological effects. In this study, apoptosis-related indicators were detected to explore the molecular mechanism of allicin on KG-1 cell proliferation inhibition. The apoptosis rate of KG-1 cells induced by allicin was detected by flow cytometry. The effect of allicin on the expressions of Bax, Bcl-2, survivin and ERK mRNA in KG-1 cells was detected by RT-qPCR. Western blot was used to detect the expressions of caspase 3, cleaved caspase 3, ERK1/2, p-ERK1/2 and survivin protein in KG-1 cells. According to the findings, compared with the control group, allicin could significantly inhibit the proliferation activity of KG-1 cells in a concentration-dependent and time-dependent manner. Flow cytometry showed that allicin could induce the apoptosis of KG-1 cells, which was mainly late apoptosis. The results of RT-qPCR showed that the expressions of Bax mRNA, Bcl-2, survivin and ERK mRNA in KG-1 cells increased after treatment with allicin. The results of Western-blot showed that after KG-1 cells were treated with allicin, the expressions of caspase 3 and its active form cleaved caspase 3 increased, the expressions of survivin, ERK1/2 and its active form p-ERK1/2 were decreased, of which p-ERK1/2 was down-regulated in a dose-dependent manner. The above results suggest that allicin inhibited the proliferation of KG-1 cells primarily by inducing late apoptosis; the execution of apoptosis involved cleaved caspase 3; the induction of apoptosis involved the protein expression, the decrease of ERK1/2andexpression of survivin and the dose-dependent decrease of p-ERK1/2; the mRNA expression involved the increase of Bax, and the down-regulation of survivin, Bcl-2 and ERK1/2.

Trametinib downregulates survivin expression in RB1-positive KRAS-mutant lung adenocarcinoma cells

High expression levels of survivin in KRAS-mutant lung adenocarcinomas are linked with unfavorable patient outcomes, suggesting that survivin is a promising target for tumor treatment. We found that trametinib, a MEK inhibitor, downregulates survivin expression in the RB1-positive KRAS-mutant lung adenocarcinoma cell lines H358 and H441. In these cell lines, trametinib treatment induced p21 expression and dephosphorylated RB1, leading to sustained suppression of survivin. Knockdown of p21 or RB1 restored survivin expression in trametinib-treated cells, at least partially, which supports the contribution of these molecules to trametinib-mediated survivin suppression. In RB1-negative KRAS-mutant lung adenocarcinoma H2009 cells, survivin downregulation by trametinib was only slight and transient, and trametinib-resistant (TR) cells developed within 1 month of treatment. H2009 TR cells depended much more on survivin for survival than its parental cells, as evidenced by apoptosis induction when survivin was depleted. These findings collectively suggest that trametinib is effective for the treatment of RB1-positive KRAS-mutant lung adenocarcinomas through sustained survivin suppression, but not for RB1-negative lung adenocarcinomas. Thus, the RB1 status could be a biomarker for trametinib application in KRAS-mutant lung adenocarcinomas.

Corilagin induces the apoptosis of hepatocellular carcinoma cells through the mitochondrial apoptotic and death receptor pathways.

Corilagin, a gallotannin, is one of the major active components of many ethnopharmacological plants and exhibits antitumor, anti-inflammatory and antioxidative properties. In recent years, corilagin has provoked much attention due to its antitumor activity, yet the mechanisms attributed to its anticancer actions are largely unknown. In our previous research, our group reported that corilagin could inhibit the proliferation of hepatocellular carcinoma (HCC) cells by inducing G2/M phase arrest. In the present study, observation of the morphological changes showed that corilagin induced the apoptosis of HCC cells as determined by AO/EB and Hoechst 33258 staining assays. Furthermore, flow cytometric analysis was carried out to calculate the apoptotic rate which was 24.1% following treatment with corilagin (37.5 µM). At the molecular level, mitochondrial membrane potential assay and western blot analysis showed that the mitochondrial transmembrane potential was reduced and the rate of release of cytochrome c was increased, which led to the activation of caspase-9, caspase-3 and cleavage of PARP in the cytoplasm indicating activation of the mitochondrial apoptotic pathway. Moreover, following treatment with corilagin, we noted upregulation of Fas and FasL and activation of caspase-8 which represented activation of the death receptor pathway, and we also observed downregulation of Bcl-2 and survivin which was also attributed to the antitumor effect of corilagin. These results suggest that corilagin significantly induced the apoptosis of HCC cells through both the mitochondrial apoptotic pathway and the death receptor pathway, and corilagin is a potential complementary anticancer herbal drug for HCC therapy.

Reversal of drug resistance by planetary ball milled (PBM) nanoparticle loaded with resveratrol and docetaxel in prostate cancer

The folate receptor (FR) is a valued target that is highly expressed in various cancers, which will expedite the development of ligand-receptor binding based cancer therapeutics. In the present investigation, through tissue microarray analysis, we report higher levels of folate receptor expression in prostate cancer (PCa) tissue derived from patients, which were minimal in normal tissue. For folate-receptor based targeted therapy of PCa, we generated novel planetary ball milled (PBM) nanoparticles (NPs) encapsulated with resveratrol (RES), and in combination with docetaxel (DTX) and conjugated with folic acid (FA) on the surface. The cytotoxic effect of FA-conjugated DTX-nanoparticles was found effectual that reduced the concentration of free drug (DTX) to 28 times. Flow cytometry analysis showed a significant increase in the number of apoptotic cells by 30.92% and 65.9% in the FA-conjugated RES and in combination with DTX nanoparticle formulation respectively. However, only 8.9% apoptotic cells were found with control (empty NP). The expressions of NF-kB p65, COX-2, pro (BAX, BAK) and anti-apoptotic (BCL-2, BCL-XL) genes were significantly reduced after treatment with FA-RES + DTX-NP. In addition, the FA-conjugated DTX formulation exhibited additional cytotoxic effects with the down-regulation of survivin and an increased expression of Cleaved Caspase-3 in PCa cells. Further, we observed that treating DTX resistant PCa cells with FA-RES + DTX-NP exhibited a reversal of the ABC-transporter markers thereby limiting the multidrug resistance phenotype of the cancer cells. Our results strongly suggested that FA conjugated nanoparticle drugs acted as effective inhibitors of drug efflux that effectually enhances the intracellular concentration of the drug to exhibit their cytotoxic effect.

In vitro and in vivo suppression of hepatocellular carcinoma by Amorphophallus konjac tuber through regulation of survivin and bax

Background: Amorphophallus konjac tuber (AKT) is used in traditional Chinese medicine (TCM) to treat various types of cancer. Our study aims to evaluate the antitumor activity of AKT against hepatocellular carcinoma both in vitro and in vivo , as well as the underlying mechanisms. Methods: AKT was extracted by aqua and organic solutions. The antitumor activity of the organic extract was tested in hepatocellular carcinoma cell lines Huh7 and H22, the latter was also used to establish mouse tumor model. Cell apoptotic rate was analyzed by flow cytometry. In vivo expression of apoptosis associated proteins survivin and bax were analyzed by immunohistochemistry (IHC). Results: AKT exhibited antitumor activity against hepatocellular carcinoma cell lines. In vivo suppression of H22 was comparable with 5-fluorouracil (5-FU). AKT could induce apoptosis through up-regulation of bax and down-regulation of survivin. Conclusions: Our data showed that AKT suppressed hepatocellular carcinoma, and the mechanism was associated with regulation of survivin and bax expression, providing support to the clinical use of AKT-based medication to achieve optimized outcome in hepatocellular carcinoma patients.

Survivin-Based Treatment Strategies for Squamous Cell Carcinoma.

Survivin, an anti-apoptotic molecule abundantly expressed in most human neoplasms, has been reported to contribute to cancer initiation and drug resistance in a wide variety of human tumors. Efficient downregulation of survivin can sensitize tumor cells to various therapeutic interventions, generating considerable efforts in its validation as a new target in cancer therapy. This review thoroughly analyzes up-to-date information on the potential of survivin as a therapeutic target for new anticancer treatments. The literature dealing with the therapeutic targeting of survivin will be reviewed, discussing specifically squamous cell carcinomas (SCCs), and with emphasis on the last clinical trials. This review gives insight into the recent developments undertaken in validating various treatment strategies that target survivin in SCCs and analyze the translational possibility, identifying those strategies that seem to be the closest to being incorporated into clinical practice. The most recent developments, such as dominant-negative survivin mutants, RNA interference, anti-sense oligonucleotides, small-molecule inhibitors, and peptide-based immunotherapy, seem to be helpful for effectively downregulating survivin expression and reducing tumor growth potential, increasing the apoptotic rate, and sensitizing tumor cells to chemo- and radiotherapy. However, selective and efficient targeting of survivin in clinical trials still poses a major challenge.

BIX-01294 sensitizes renal cancer Caki cells to TRAIL-induced apoptosis through downregulation of survivin expression and upregulation of DR5 expression

BIX-01294 (BIX), a G9a histone methyltransferase inhibitor, has been reported for its anti-proliferative and anticancer activities against various cancer cell lines. In this study, we investigated whether BIX could sensitize TRAIL-mediated apoptosis in various cancer cells. Combined treatment with BIX and TRAIL markedly induced apoptosis in human renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MCF-7), and lung carcinoma (A549) cells. In contrast, BIX and TRAIL co-treatment did not induce apoptosis in normal cells, specifically mouse kidney cell (TCMK-1) and human skin fibroblast (HSF). BIX downregulated protein expression levels of XIAP and survivin at the post-translational level. Overexpression of survivin markedly blocked combined BIX and TRAIL treatment-induced apoptosis, but XIAP had no effect. Furthermore, BIX induced upregulation of DR5 expression at the transcriptional levels, and knockdown of DR5 expression using small interfering RNAs (siRNAs) markedly attenuated BIX and TRAIL-induced apoptosis. Interestingly, siRNA-mediated G9a histone methyltransferase knockdown also enhanced TRAIL-induced apoptosis in Caki cells. However, knockdown of G9a did not change expression levels of XIAP, survivin, and DR5. Therefore, BIX-mediated TRAIL sensitization was independent of histone methyltransferase G9a activity. Taken together, these results suggest that BIX facilitates TRAIL-mediated apoptosis via downregulation of survivin and upregulation of DR5 expression in renal carcinoma Caki cells.▶ BIX facilitates TRAIL-mediated apoptosis in human renal carcinoma Caki cells.▶ Downregulation of survivin contributes to BIX plus TRAIL-induced apoptosis.▶ Upregulation of DR5 is involved in BIX plus TRAIL-mediated apoptosis.▶ BIX-mediated TRAIL sensitization is independent of ROS production.

Honokiol Radiosensitizes Squamous Cell Carcinoma of the Head and Neck by Downregulation of Survivin.

Purpose: Previous studies revealed diverging results regarding the role of survivin in squamous cell carcinoma of the head and neck (SCCHN). This study aimed to evaluate the clinical significance of survivin expression in SCCHN; the function of survivin in DNA-damage repair following ionizing radiation therapy (RT) in SCCHN cells; and the potential of honokiol to enhance RT through downregulation of survivin.Experimental Design: Expression of survivin in SCCHN patient primary tumor tissues (n = 100) was analyzed and correlated with clinical parameters. SCCHN cell lines were used to evaluate the function of survivin and the effects of honokiol on survivin expression in vitro and in vivoResults: Overexpression of survivin was significantly associated with lymph nodes' metastatic status (P = 0.025), worse overall survival (OS), and disease-free survival (DFS) in patients receiving RT (n = 65, OS: P = 0.024, DFS: P = 0.006) and in all patients with SCCHN (n = 100, OS: P = 0.002, DFS: P = 0.003). In SCCHN cells, depletion of survivin led to increased DNA damage and cell death following RT, whereas overexpression of survivin increased clonogenic survival. RT induced nuclear accumulation of survivin and its molecular interaction with γ-H2AX and DNA-PKCs. Survivin specifically bound to DNA DSB sites induced by I-SceI endonuclease. Honokiol (which downregulates survivin expression) in combination with RT significantly augmented cytotoxicity in SCCHN cells with acquired radioresistance and inhibited growth in SCCHN xenograft tumors.Conclusions: Survivin is a negative prognostic factor and is involved in DNA-damage repair induced by RT. Targeting survivin using honokiol in combination with RT may provide novel therapeutic opportunities. Clin Cancer Res; 24(4); 858-69. ©2017 AACR.

Plagioneurin B, a potent isolated compound induces apoptotic signalling pathways and cell cycle arrest in ovarian cancer cells.

Plagioneurin B belongs to acetogenin group has well-established class of compounds. Acetogenin group has attracted worldwide attention in the past few years due their biological abilities as inhibitors for several types of tumour cells. Plagioneurin B was isolated via conventional chromatography and tested for thorough mechanistic apoptosis activity on human ovarian cancer cells (CAOV-3). Its structure was also docked at several possible targets using Autodock tools software. Our findings showed that plagioneurin B successfully inhibits the growth of CAOV-3 cells at IC50 of 0.62µM. The existence of apoptotic bodies, cell membrane blebbing and chromatin condensation indicated the hallmark of apoptosis. Increase of Annexin V-FITC bound to phosphatidylserine confirmed the apoptosis induction in the cells. The apoptosis event was triggered through the extrinsic and intrinsic pathways via activation of caspases 8 and 9, respectively. Stimulation of caspase 3 and the presence of DNA ladder suggested downstream apoptotic signalling were initiated. Further confirmation of apoptosis was conducted at the molecular levels where up-regulation in Bax, as well as down-regulation of Bcl-2, Hsp-70 and survivin were observed. Plagioneurin B was also seen to arrest CAOV-3 cells cycle at the G2/M phase. Docking simulation of plagioneurin B with CD95 demonstrated that the high binding affinity and hydrogen bonds formation may explain the capability of plagioneurin B to trigger apoptosis. This study is therefore importance in finding the effective compound that may offer an alternative drug for ovarian cancer treatment.

Dihydroartemisinin suppresses STAT3 signaling and Mcl-1 and Survivin expression to potentiate ABT-263-induced apoptosis in Non-small Cell Lung Cancer cells harboring EGFR or RAS mutation

Non-small cell lung cancer (NSCLC) is the most common malignancy worldwide. A significant fraction of NSCLC carries activating mutations in epidermal growth factor receptor (EGFR) or RAS oncogene. Dihydroartemisinin (DHA) is a semisynthetic derivative of the herbal antimalarial drug artemisinin that has been recently reported to exhibit anti-cancer activity. To develop new therapeutic strategies for NSCLC, we investigated the interactions between DHA and ABT-263 in NSCLC cells harboring EGFR or RAS mutation. Our data indicated that DHA synergized with ABT-263 to trigger Bax-dependent apoptosis in NSCLC cells in culture. DHA treatment antagonized ABT-263-induced Mcl-1 upregulation and sensitized NSCLC cells to ABT-263-triggered apoptosis. Additionally, DHA treatment caused downregulation of Survivin and upregulation of Bim, which also contribute to cotreatment-induced cytotoxicity. Moreover, DHA effectively suppressed STAT3 phosphorylation, and STAT3 inactivation resulted in the downregulation of Mcl-1 and Survivin, functioning to enhance ABT-263-induced cytotoxicity. Finally, cotreatment of DHA and ABT-263 significantly inhibited xenograft growth in nude mice. Together, DHA effectively inhibits STAT3 activity and modulates expression of Mcl-1, Survivin and Bim, thereby synergizing with ABT-263 to trigger apoptosis in NSCLC cells harboring EGFR or RAS mutation. Our data provide a novel therapeutic strategy for EGFR or RAS mutant NSCLC treatment.

Survivin-targeting miR-542-3p overcomes HER3 signaling-induced chemoresistance and enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer

Elevated expression of HER3, which interacts with HER2 in breast cancer cells, confers chemoresistance via phosphoinositide 3-kinase (PI-3K)/Akt-dependent upregulation of Survivin. However, the underlying mechanism is not clear. Ectopic expression or specific knockdown of HER3 in HER2-overexpressing breast cancer cells did not alter Survivin mRNA levels and Survivin protein stability, supporting the notion that HER3 signaling may regulate specific miRNAs that target Survivin to alter its protein translation. Here we showed that overexpression and specific knockdown of HER3 reduced and enhanced expression of two Survivin-targeting miRNAs, miR-203 and miR-542-3p, in breast cancer cells, respectively. While the specific inhibitor of either miR-203 or miR-542-3p attenuated an anti-HER3 antibody-induced downregulation of Survivin, inhibition of miR-542-3p exhibited a better efficacy than miR-203 inhibition did. Consistently, miR-542-3p mimic was much more effective than miR-203 mimic not only in inhibition of Survivin, but also in enhancement of paclitaxel-induced apoptosis in HER2-overexpressing breast cancer cells. Moreover, the combination of miR-542-3p mimic and paclitaxel, as compared with either agent alone, significantly inhibited in vivo tumor growth of HER2-overexpressing breast cancer cells. Collectively, our data indicated that the HER3/PI-3K/Akt signaling upregulates Survivin via suppression of miR-203 and miR-542-3p. Because miR-542-3p has three binding sites on the 3′-UTR of Survivin mRNA, its mimic was able to effectively downregulate Survivin in vitro and in vivo. Thus, miR-542-3p-replacement therapy is an excellent approach to overcome HER3-mediated paclitaxel resistance and significantly enhances the antitumor activity of paclitaxel against HER2-overexpressing breast cancer.

Survivin is a novel transcription regulator of KIT and is downregulated by miRNA-494 in gastrointestinal stromal tumors.

Gain‐of‐function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT‐independent targets of KIT‐regulating mRNAs. We sought to investigate how miR‐494 inhibits GIST proliferation and to identify novel target gene. We used microarray‐based gene expression analyses to identify pathways and target genes affected by miR‐494. The expressional relationship between survivin and miR‐494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR‐494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR‐494, and its expression showed an inverse correlation with miR‐494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = −0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR‐494‐mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR‐494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT.

MiR-133b reverses cisplatin resistance by targeting GSTP1 in cisplatin-resistant lung cancer cells.

MicroRNAs play a critical role in chemoresistance and are implicated in various biological and pathological processes of cells. The objective of the present study was to explore the role of miR-133b and its mechanism in the regulation of cisplatin resistance and tumor progression in cisplatin-resistant non-small cell lung cancer (NSCLC) cells. Reverse transcription-quantitative polymerase chain reaction and western blot assays of the cisplatin-resistant cell lines A549/DPP and H1299/DDP displayed the reduced expression of miR-133b and increased expression of glutathione-S-transferase P1 (GSTP1) in the resistant cells compared with the respective parental cell lines A549 and H1299. Cell Counting kit-8, flow cytometry, colony formation and Transwell migration assays indicated that the overexpression of miR-133b increased the chemosensitivity to cisplatin and attenuated the proliferation and migration capacities of the cisplatin-resistant NSCLC cell lines in vitro. A dual-luciferase reporter assay demonstrated that miR-133b negatively regulated the expression of GSTP1 by targeting its 3′-untranslated region. In addition, the knockdown of GSTP1 by transfection with small interfering RNA exerted similar effects on cell chemosensitivity, proliferation and migration as did ectopic miR-133b expression, in addition to the upregulation of Bax and down-regulation of Bcl-2, survivin and matrix metalloproteinase expression. In conclusion, the present study findings provide the insights that miR-133b reduces cisplatin resistance and its overexpression contributes to the suppression of the malignant growth and aggressiveness of cisplatin-resistant NSCLC cells by targeting GSTP1. This could potentially be exploited as a novel therapeutic strategy for the reversal of cisplatin resistance.

MiR-494 Sensitizes Gastric Cancer Cells to TRAIL Treatment Through Downregulation of Survivin

Background/Aims: TNF-related apoptosis-inducing ligand (TRAIL) is a novel and low-toxic anti-tumor drug used for various cancers. However, cancer cells usually develop mechanisms to acquire the resistance against TRAIL. Among these changes, dysregulation of microRNAs (miRNAs) usually occurs in cancer cells and is responsible for induction of drug resistance. Methods: Expression of miR-494 in gastric cancer tissues and cell lines was detected by quantitative reverse transcriptase real time PCR (qRT-PCR) analysis. Effect of miR-494 on regulating the TRAIL sensitivity to gastric cancer cell lines was evaluated by MTT assays. Bioinformatics and luciferase reporter assays were used to confirm the regulation of miR-494 on survivin. Mitochondrial apoptosis pathway in gastric cancer cells was tested by western blot and flow cytometry analysis. Results: Obvious downregulation of miR-494 was observed in gastric cancer cells. Furthermore, we found that expression profile of miR-494 was associated with TRAIL-sensitivity in gastric cancer. Enforced expression of miR-494 was found to sensitize the gastric cancer cells to TRAIL-induced cytotoxicity. Mechanically, Luciferase reporter assays proved that survivin was the target of miR-494 in gastric cancer cells. Enforced expression of miR-494 decreased the expression of survivin, and thus promoted the TRAIL-induced mitochondria collapse and apoptosis pathway. Conclusion: MiR-494/survivin axis represents a potential mechanism which is responsible for TRAIL resistance in gastric cancer cells. Increasing the miR-494 expression may serve as a novel therapeutic strategy to sensitize gastric cancer cells to TRAIL treatment.

Bone Marrow Cell Therapy on 1,2-Dimethylhydrazine (DMH)-Induced Colon Cancer in Rats

Background/Aims: Stem cell based therapies are being under focus due to their possible role in treatment of various tumors. Bone marrow stem cells believed to have anticancer potential and are preferred for their activities by stimulating the immune system, migration to the site of tumor and ability for inducting apoptosis in cancer cells. The current study was aimed to investigate the tumor suppressive effects of bone marrow cells (BMCs) in 1,2-dimethylhydrazine (DMH)-induced colon cancer in rats. Methods: The rats were randomly allocated into four groups: control, BMCs alone, DMH alone and BMCs with DMH. BMCs were injected intrarectally while DMH was injected subcutaneously at 20 mg/kg body weight once a week for 15 weeks. Histopathological examination and gene expression of survivin, β-catenin and multidrug resistance-1 (MDR-1) by real-time reverse transcription-polymerase chain reaction (RT-PCR) in rat colon tissues. This is in addition to oxidative stress markers in colon were performed across all groups. Results: The presence of aberrant crypt foci was reordered once histopathological examination of colon tissue from rats which received DMH alone. Administration of BMCs into rats starting from zero-day of DMH injection improved the histopathological picture which showed a clear improvement in mucosal layer, few inflammatory cells infiltration periglandular and in the lamina propria. Gene expression in rat colon tissue demonstrated that BMCs down-regulated survivin, β-catenin, MDR-1 and cytokeratin 20 genes expression in colon tissues after colon cancer induction. Amelioration of the colon status after administration of MSCs has been evidenced by a major reduction of lipid peroxidation, nitric oxide, and increasing of glutathione content and superoxide dismutase along with catalase activities. Conclusion: Our findings demonstrated that BMCs have tumor suppressive effects in DMH-induced colon cancer as evidenced by down-regulation of survivin, β-catenin, and MDR-1 genes and enhancing the antioxidant activity.

Abstract B088: Identification of a novel regulator in GIST tumorigenesis

Abstract Purpose: Gain-of-function mutations of KIT are pathognomonic in the majority of sporadic gastrointestinal stromal tumors (GISTs). Several micro-RNAs have been shown to be dysregulated in GISTs and impact KIT expression levels. Little is known, though, about KIT-independent targets of KIT-regulating mRNAs. We sought to investigate how miR-494 inhibits GIST proliferation and to identify novel target gene. Experimental Design: We used gene expression analyses to identify pathways and target genes affected by miR-494. The expressional relationship between Survivin and miR-494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, and promoter assays were performed to clarify the roles of Survivin in GIST progression. Results: Gene expression microarray analysis revealed that miR-494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin was a key target gene of miR-494. Analysis of the 35 GISTs revealed that there was an inverse correlation between Survivin and miR-494 expression (Pearson’s correlation coefficient, r = -0.418, P = 0.012). Downregulation of Survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced Survivin overexpression relieved miR-494 mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of Survivin. Survivin regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GIST tissues revealed that Survivin expression was significantly correlated with overall survival of GIST patients (P = 0.004). Conclusion: Our findings indicated that miR-494 synergistically suppressed GISTs by concomitantly targeting Survivin and KIT, with accompanying vertical blockade of the PI3K pathways. Citation Format: Seongju Yun, Won Kyu Kim, Hoguen Kim. Identification of a novel regulator in GIST tumorigenesis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B088.

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment.

Downregulation of Survivin contributes to cell-cycle arrest during postnatal cardiac development in a severe spinal muscular atrophy mouse model.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, characterized by progressive degeneration of spinal-cord motor neurons, leading to atrophy of skeletal muscles. However, accumulating evidence indicates that it is a multi-system disorder, particularly in its severe forms. Several studies delineated structural and functional cardiac abnormalities in SMA patients and mouse models, yet the abnormalities have been primarily attributed to autonomic dysfunction. Here, we show in a severe mouse model that its cardiomyocytes undergo G0/G1 cell-cycle arrest and enhanced apoptosis during postnatal development. Microarray and real-time RT-PCR analyses revealed that a set of genes associated with cell cycle and apoptosis were dysregulated in newborn pups. Of particular interest, the Birc5 gene, which encodes Survivin, an essential protein for heart development, was down-regulated even on pre-symptomatic postnatal day 0. Interestingly, cultured cardiomyocytes depleted of SMN recapitulated the gene expression changes including downregulation of Survivin and abnormal cell-cycle progression; and overexpression of Survivin rescued the cell-cycle defect. Finally, increasing SMN in SMA mice with a therapeutic antisense oligonucleotide improved heart pathology and recovered expression of deregulated genes. Collectively, our data demonstrate that the cardiac malfunction of the severe SMA mouse model is mainly a cell-autonomous defect, caused by widespread gene deregulation in heart tissue, particularly of Birc5, resulting in developmental abnormalities through cell-cycle arrest and apoptosis.

Sensitization of rhTRAIL-resistant Triple-negative Breast Carcinoma Through Silibinin Co-Treatment.

Triple-negative breast cancer (TNBC) is the most fatal form of breast cancer due to the shortcomings of therapies. However, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is a promising anticancer therapeutic that possesses the capability to promote the induction of apoptosis in cancer cells, but some TNBCs are resistant to rhTRAIL's pro-apoptotic effects. Therefore, a combinatorial treatment approach with silibinin and rhTRAIL was considered in order to sensitize rhTRAIL-resistant TNBCs. The co-treatment of rhTRAIL and silibinin's impact on apoptosis induction in rhTRAIL-resistant TNBC BT-20 and HCC1937 cells was inspected via application of Annexin V/PI assays and western blot analysis. Silibinin possessed the ability to sensitize the examined rhTRAIL-resistant TNBC cells to rhTRAIL-induced apoptosis through the up-regulation of death receptors 4 and 5 and the down-regulation of survivin transcriptionally. Silibinin is a good sensitizing agent for rhTRAIL-resistant TNBCs.

Correlation between E-cadherin interactions, survivin expression, and apoptosis in MDCK and ts-Src MDCK cell culture models

Survivin, a member of inhibitor of apoptosis (IAP) protein family, is a multifunctional protein expressed in most cancers. In addition to inhibition of apoptosis, it regulates proliferation and promotes migration. Its presence and function in cells is strongly regulated via transcription factors, intracellular localization, and degradation. We analyzed the presence of survivin at protein level in various culture environments and under activation of Src tyrosine kinase in epithelial canine kidney MDCK cells in order to elucidate factors controlling survivin ‘lifespan’. We used untransformed and temperature sensitive ts-Src MDCK cells as a model and forced them to grow in suspension (1D), in 2D on hard and soft surfaces and in soft 3D Matrigel environment with or without EGTA. In addition, we tested the effect of stressful conditions by cultivating the cells in the presence of an anti-cancer drug and a generator of reactive oxygen species (ROS), piperlongumine (PL) with or without an antioxidant, N-acetylcysteine (NAC). We could confirm that inhibition of apoptosis and simultaneous downregulation of survivin in MDCK cells required both intact cell–cell junctions, trans-interactions of E-cadherin and soft 3D matrix environment. In ts-Src-transformed MDCK cells, survivin was upregulated as soon as the cell–cell junctions were disintegrated. ROS generation with PL-induced cell death of ts-Src MDCK cells concomitantly with survivin downregulation. NAC rescued the ts-Src MDCK cells from ROS-induced apoptosis without upregulation of survivin resulting in a situation resembling untransformed MDCK cells in 3D environment and E-cadherin delineating the lateral cell walls.