Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.

BACKGROUND: Breast cancer (BC) is the most common cancer among women and it is responsible for more than 40,000 deaths in the United States and more than 500,000 deaths worldwide each year. In previous decades, the development of improved screening, diagnosis and treatment methods has led to decreases in BC mortality rates. More recently, novel targeted therapeutic options, such as the use of monoclonal antibodies and small molecule inhibitors that target specific cancer cell-related components, have been developed. These components include ErbB family members (HER1, HER2, HER3 and HER4), Ras/MAPK pathway components (Ras, Raf, MEK and ERK), VEGF family members (VEGFA, VEGFB, VEGFC, VEGF and PGF), apoptosis and cell cycle regulators (BAK, BAX, BCL-2, BCL-X, MCL-1 and BCL-W, p53 and PI3K/Akt/mTOR pathway components) and DNA repair pathway components such as BRCA1. In addition, long noncoding RNA inhibitor-, microRNA inhibitor/mimic- and immunotherapy-based approaches are being developed for the treatment of BC. Finally, a novel powerful technique called CRISPR-Cas9-based gene editing is emerging as a precise tool for the targeted treatment of cancer, including BC.
CONCLUSIONS: Potential new strategies that are designed to specifically target BC are presented. Several clinical trials using these strategies are already in progress and have shown promising results, but inherent limitations such as off-target effects and low delivery efficiencies still have to be resolved. By improving the clinical efficacy of current therapies and exploring new ones, it is anticipated that novel ways to overcome BC may become attainable.

Over 95% of human genes are alternatively spliced, expressing splice isoforms that often exhibit antagonistic functions. We describe genes whose alternative splicing has been linked to prostate cancer; namely

The plasticity of solid tumors between the epithelial and mesenchymal states critically influences their malignant progression and metastasis. The epithelial-mesenchymal transition (EMT), which supports cancer cell invasion and metastasis, is promoted by pro-survival members (e.g., Bcl-2 and Bcl-X

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. However, the underlying mechanism of osteosarcoma carcinogenesis and progression remains unknown. In the present study, we evaluated the expression profile of miRNAs in osteosarcoma tissues and the adjacent normal tissues. We found that the expression of miR-422a was down-regulated in osteosarcoma tissues and cell lines. In addition, we observed significantly elevated levels of repressive H3K9me3 and H3K27me3 and decreased active H3K4me3 on the promote region of miR-422a in osteosarcoma cells and clinical samples. Furthermore, up-regulation of miR-422a exhibited both

LncRNA HOX transcript antisense RNA (HOTAIR) is involved in lots of cancers. The pro-survival protein Bcl-w is frequently found in cancer development. However, the effect of HOTAIR on Bcl-w in breast cancer is not well documented. In this study, we first evaluated the correlation between HOTAIR level and Bcl-w expression in clinical breast cancer tissues. We observed that the expression levels of Bcl-w were much higher in the breast cancer samples than that in their paired noncancerous tissues. Moreover, the levels of HOTAIR were positively associated with those of Bcl-w in clinical breast cancer samples. As expected, we observed that HOTAIR was able to up-regulate the expression of Bcl-w in breast cancer cells. Mechanistically, we found that miR-206 was capable of inhibiting the expression of Bcl-w by directly binding to the 3'UTR of Bcl-w mRNA. Interestingly, HOTAIR could increase the expression of Bcl-w through sequestering miR-206 at post-transcriptional level. Functionally, our data showed that HOTAIR-induced Bcl-w by miR-206 facilitated the proliferation of breast cancer cells. Thus, we conclude that HOTAIR up-regulates Bcl-w to enhance cell proliferation through sequestering miR-206 in breast cancer. Our finding provides new insights into the mechanism of breast cancer mediated by HOTAIR.

AIM: Previous observations have implicated miR-509-3p's ability in regulating cisplatin-triggered apoptosis in ovarian cancer. However, the underlying mechanisms were not fully understood.
MATERIALS & METHODS: The roles of miR-509-3p in cellular apoptosis were assessed through MTT and DAPI assays. The confirmation of the regulation of BCL2 family members by miR-509-3p was investigated by luciferase reporter assay, western blot, quantitative real-time PCR and rescue experiments.
RESULTS: MiR-509-3p can decrease the IC
CONCLUSION: Our study demonstrates that miR-509-3p could sensitize ovarian cancer cells to cisplatin treatment by targeting multiple anti-apoptosis genes including BCL2.

BACKGROUND: Accumulating evidence indicates that long non-coding RNAs (lncRNAs) play a crucial role in tumorigenesis. Here, we report a novel lncRNA, RP11-436H11.5, that regulates renal cell carcinoma (RCC) cell proliferation and invasion by sponging miR-335-5p.
METHODS: Expression of lncRNA RP11-436H11.5 was determined by a qRT-PCR assay in RCC tissues. RCC cell proliferation and invasion were measured by a cell proliferation assay and a transwell invasion assay. Expression of BCL-W was detected by a western blot assay. Interactions between lncRNA RP11-436H11.5 and miR-335-5p were measured by a luciferase reporter assay and a RNA-pull down assay. In vivo experiments were used to detect tumor formation.
RESULTS: In this study, the qRT-PCR results illustrated that lncRNA RP11-436H11.5 was more highly expressed in RCC tissues than in adjacent normal renal tissues. The results of survival analysis indicated that patients in the high lncRNA RP11-436H11.5 group presented significantly worse outcomes compared with those in the low lncRNA RP11-436H11.5 group. Downregulation of lncRNA RP11-436H11.5 suppressed RCC cell proliferation and invasion in vitro and in vivo. Luciferase reporter assay results demonstrated that lncRNA RP11-436H11.5 enhanced BCL-W expression by regulating miR-335-5p expression. LncRNA RP11-436H11.5 could function as a miR-335-5p decoy to derepress expression of BCL-W.
CONCLUSIONS: LncRNA RP11-436H11.5 could function as a competing endogenous RNA to promote RCC cell proliferation and invasion, which might serve as a therapeutic application to suppress RCC progression.

Gastric cancer continues to be the second most frequent cause of cancer deaths worldwide. However, the exact molecular mechanisms are still unclear. Further research to find potential targets for therapy is critical and urgent. In this study, we found that ARPC2 promoted cell proliferation and invasion in the human cancer cell line MKN-28 using a cell total number assay, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, cell colony formation assay, migration assay, invasion assay, and wound healing assay. For downstream pathways, CTNND1, EZH2, BCL2L2, CDH2, VIM, and EGFR were upregulated by ARPC2, whereas PTEN, BAK, and CDH1 were downregulated by ARPC2. In a clinical study, we examined the expression of ARPC2 in 110 cases of normal human gastric tissues and 110 cases of human gastric cancer tissues. ARPC2 showed higher expression in gastric cancer tissues than in normal gastric tissues. In the association analysis of 110 gastric cancer tissues, ARPC2 showed significant associations with large tumor size, lymph node invasion, and high tumor stage. In addition, ARPC2-positive patients exhibited lower RFS and OS rates compared with ARPC2-negative patients. We thus identify that ARPC2 plays an aneretic role in human gastric cancer and provided a new target for gastric cancer therapy.

MicroRNAs play a crucial role in gene expression regulation in various types of cancers. Previous studies show the expression level of miR‑146a‑5p is downregulated in epithelial ovarian cancer. Further investigations suggest this downregulation is responsible for apoptosis resistance in ovarian cancer cells. However, the mechanism of how miR‑146a‑5p promotes apoptosis remains unclear. In this study, the role of miR‑146a‑5p in cisplatin‑induced apoptosis of ovarian cancer cells was assessed by DAPI staining, MTT assays, and monitoring expression of XIAP, BCL2L2, BIRC2 and BIRC5 through a dual‑luciferase assay. Our results show that miR‑146a‑5p can regulate three important anti‑apoptotic genes including XIAP, BCL2L2 and BIRC5 via their 3'UTRs. Not only can overexpression of miR‑146a‑5p downregulate the expression of XIAP in SKOV3 cells, but it also lowers the IC50 values of cisplatin in OVCAR3 and SKOV3 cells and enhances the susceptibility of OVCAR3, SKOV3 and primary ovarian cancer cells to cisplatin‑induced apoptosis. The effect of XIAP rescuing cisplatin‑induced apoptosis accelerated by miR‑146a‑5p further supports our conclusion. Our results suggest that the regulation of three anti‑apoptotic genes by miR‑146‑5p enhances the therapeutic effects of cisplatin.

Backgroud:Glioblastoma is a kind of highly malignant and aggressive tumours in the central nervous system. Previously, we found that neurotensin (NTS) and its high-affinity receptor 1 (NTSR1) had essential roles in cell proliferation and invasiveness of glioblastoma. Unexpectedly, cell death also appeared by inhibition of NTSR1 except for cell cycle arrest. However, the mechanisms were remained to be further explored.
METHODS: Cells treated with SR48692, a selective antagonist of NTSR1, or NTSR1 shRNA were stained with Annexin V-FITC/PI and the apoptosis was assessed by flow cytometry. Cytochrome c release was detected by using immunofluorescence. Mitochondrial membrane potential (MMP, ΔΨm) loss was stained by JC-1 and detected by immunofluorescence or flow cytometry. Apoptosis antibody array and microRNA microarray were performed to seek the potential regulators of NTSR1 inhibition-induced apoptosis. Interaction between let-7a-3p and Bcl-w 3'UTR was evaluated by using luciferase assay.
RESULTS: SR48692 induced massive apoptosis, which was related to mitochondrial cytochrome c release and MMP loss. Knockdown of NTSR1 induced slight apoptosis and significant MMP loss. In addition, NTSR1 inhibition sensitised glioblastoma cells to actinomycin D or doxorubicin-induced apoptosis. Consistently, NTSR1 inhibition-induced mitochondrial apoptosis was accompanied by downregulation of Bcl-w and Bcl-2. Restoration of Bcl-w partly rescued NTSR1 deficiency-induced apoptosis. In addition, NTSR1 deficiency promoted higher let-7a-3p expression and inhibition let-7a-3p partly rescued NTSR1 inhibition-induced apoptosis. In addition, let-7a-3p inhibition promoted 3'UTR activities of Bcl-w and the expression of c-Myc and LIN28, which were the upstream of let-7a-3p, decreased after NTSR1 inhibition.
CONCLUSIONS: NTSR1 had an important role in protecting glioblastoma from intrinsic apoptosis via c-Myc/LIN28/let-7a-3p/Bcl-w axis.

Cervical cancer is one of the most common cancers in females, with a high incidence and mortality around the world. However, the pathogenesis in cervical cancer is not completely known. In the present study, we investigated the role of miR-126-5p and Bcl2l2 in cervical cancer cells. First, miR-126-5p expression was aberrantly downregulated in human cervical cancer tumor tissues in comparison with normal tissues, as evaluated by RT-PCR. Consistently, the levels of miR-126-5p were also significantly reduced in cervical cancer cell lines when compared to normal cervical epithelial cells. Flow cytometric analysis showed that the rate of apoptosis of cervical cancer cells was significantly increased by miR-126-5p overexpression but inhibited by miR-126-5p inhibitor. A similar change pattern was observed in the expression of apoptosis-regulated protein caspase 3 in cervical cancer cells transfected with miR-126-5p mimic or inhibitor. By bioinformatic prediction with online databases and verification using luciferase reporter assay, we then identified that Bcl2l2 is a direct target of miR-126-5p in cervical cancer cells. The expression of Bcl2l2 was strongly downregulated by the miR-126-5p mimic but upregulated by the miR-126-5p inhibitor in cervical cancer cells, and Bcl2l2 expression was significantly increased in human cervical cancer tumor tissues, which was negatively correlated with miR-126-5p levels. Furthermore, we confirmed that the rate of apoptosis was significantly increased by Bcl2l2 silencing in cervical cancer cells, which was not affected by the miR-126-5p inhibitor. In addition, the increased apoptosis of cells by the miR-126-5p mimic was inhibited by Bcl2l2 overexpression. In summary, miR-126-5p plays an inhibitory role in human cervical cancer progression, regulating the apoptosis of cancer cells via directly targeting Bcl2l2. This might provide a potential therapeutic target for cervical cancer.

ABT-737 is a BH3 mimetic inhibitor of Bcl-xL, Bcl-2, and Bcl-w, and it has been reported for anti-cancer effects in various types of cancer cells. However, ABT-737 fails to induce apoptosis in cancer cell with high levels of Mcl-1 expression. The pharmacological survivin inhibitor YM155 has been reported to induce downregulation of Mcl-1 expression. Therefore, we investigated the effect of YM155 to sensitize resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. We found that ABT-737 alone and YM155 alone did not induce apoptosis, but YM155 markedly sensitized ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma cells (U251MG), and human lung carcinoma cells (A549). In contrast, combined treatment with ABT-737 and YM155 did not increase apoptosis in normal mouse kidney cells (TCMK-1) and human mesangial cells (MC). YM155 induced lysosome-dependent downregulation of Mcl-1 expression in Mcl-1-overexpressed Caki cells. In addition, combined treatment with ABT-737 and YM155 induced loss of mitochondrial membrane potential and inhibited interaction of Bcl-xL and Bax. Taken together, our results suggested that YM155 effectively improves sensitivity to ABT-737 through downregulation of Mcl-1 expression.

Compromised apoptotic signaling is a prerequisite for tumorigenesis. The design of effective therapies for cancer treatment depends on a comprehensive understanding of the mechanisms that govern cell survival. The antiapoptotic proteins of the BCL-2 family are key regulators of cell survival and are frequently overexpressed in malignancies, leading to increased cancer cell survival. Unlike BCL-2 and BCL-XL, the closest antiapoptotic relative BCL-W is required for spermatogenesis, but was considered dispensable for all other cell types. Here, however, we have exposed a critical role for BCL-W in B cell survival and lymphomagenesis. Loss of Bcl-w conferred sensitivity to growth factor deprivation-induced B cell apoptosis. Moreover, Bcl-w loss profoundly delayed MYC-mediated B cell lymphoma development due to increased MYC-induced B cell apoptosis. We also determined that MYC regulates BCL-W expression through its transcriptional regulation of specific miR. BCL-W expression was highly selected for in patient samples of Burkitt lymphoma (BL), with 88.5% expressing BCL-W. BCL-W knockdown in BL cell lines induced apoptosis, and its overexpression conferred resistance to BCL-2 family-targeting BH3 mimetics. Additionally, BCL-W was overexpressed in diffuse large B cell lymphoma and correlated with decreased patient survival. Collectively, our results reveal that BCL-W profoundly contributes to B cell lymphoma, and its expression could serve as a biomarker for diagnosis and aid in the development of better targeted therapies.

Glioblastoma (GB) is the most frequent and biologically the most aggressive primary brain tumor in adults. Standard treatment for newly diagnosed GB consists of surgical resection, radiotherapy and chemotherapy. Resistance to therapy is a major obstacle, even with optimal treatment with a survival median of only 12-15 months. The heterogeneity and treatment response of GB makes this tumor type a challenging area of research. The aim of our study was to study the response of normal human astrocyte (HA) and human GB (T98G) cell lines to apoptosis inhibitors in vitro. ABT-737 is an inhibitor of anti-apoptotic proteins Bcl-2, Bcl-xL, Bcl-w, while MIM-1 is an Mcl-1 protein inhibitor. The viability of the cells was assayed biochemically using the cytotoxic methyl thiazolyl tetrazolium (MTT) assay. Changes in the expression of apoptosis-associated genes (n=93) in two human brain cell lines after treatment with the apoptosis inhibitors ABT-737 and MIM-1 (individually), between the apoptosis inhibitor treated group and the control group, were determined using a commercially pre-designed microfluidic array. Significant changes in apoptotic gene expression with more than a 2.0-fold difference in their expression levels were obtained in both cell lines; the most altered genes were in the HA cell line after MIM-1 treatment (n=42). These results contribute to the importance of apoptosis in normal and cancerous brain tissues and provide information on the effect of apoptosis inhibitors on cell viability and gene expression. Despite extensive investigations, a cure for GB is currently not available. The identification of an apoptotic gene panel and determining the sensitivity of normal and GB brain cells to individual apoptosis inhibitors could help to improve clinical practice and increase our understanding of brain tumor cell metabolism and apoptosis inhibitors in GB cells and astrocytes. Recognizing expression changes in pro-apoptotic and anti-apoptotic genes could contribute to the development of new treatments.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is an aggressive neoplasm with a poor survival and novel therapies are urgently needed. The study of deregulated micro- RNAs (dereg-miRs) could constitute a strategy helping to detect specific genes playing a relevant role in the disease. Thus, the oncoproteins encoded by these genes could be exploited as novel therapeutic targets to be inhibited by small molecules, aptamers, or monoclonal antibodies.
METHODS: The present review is focused on candidate genes having convincing biological evidences to be both bona fide targets for dereg-miRs and playing a role in NSCLC progression. These genes were evaluated according to the molecular pathway they belong. Moreover, in the attempt to provide an even broader list of candidate therapeutic targets for NSCLC, the full list of genes was analyzed using the online tool Interactome DB.
RESULTS: Among the identified targets, some of them belong to p53 or MAP kinase signaling pathways, and others include caspases, MCL1, and BCL2L2 (playing a role in apoptosis), ZEB1, ZEB2, and USP25 (epithelial-to-mesenchymal transition), EZH2, SOX9, and HOXA5 (differentiation), Paxillin, LIMK1 and MTDH (cytoskeleton remodeling), and HDGF (angiogenesis). In addition, other targets, such as TIMP-2, PIM-1, and components of the IGF-signaling pathways were suggested following the interactome analysis.
CONCLUSION: Studies on dereg-miRs helped to identify a set of genes whose encoded proteins could constitute candidates for future therapeutic approaches.

Aberrant expression of miR-15a was recently reported in several types of cancers; however, its role in HPV-positive hypopharyngeal squamous cell carcinoma (HSCC) remains obscure. In the present study, we investigated the mechanism by which miR-15a induces HPV-positive HSCC apoptosis. Synthetic miR-15a mimics were transfected into FaDu cells (HPV-negative), and the miR-15a inhibitor was transfected into HPV-positive HSCC cells. miR-15a expression was analyzed by RT-PCR, and BCL2L2 and BCL2 were analyzed by western blotting. The Hochest 33342/propidium iodide (PI) and caspase-3/-9 assays, and Annexin V staining were used to assess the effect of miR-15a on apoptosis. After transfection, overexpression of miR-15a in the FaDu cells was associated with significantly decreased BCL2L2 and BCL2 expression and a significant increase in the apoptosis rate. The opposite results were observed in HPV-positive HSCC, where downregulation of miR-15a suppressed apoptosis. These findings indicate that miR-15a acts as a tumor suppressor in HPV-positive HSCC.

Colorectal cancer (CRC) is one of the most common human malignant tumors worldwide. Arising from the transformation of epithelial cells in the colon and/or rectum into malignant cells, the foundation of CRC pathogenesis lies in the progressive accumulation of mutations in oncogenes and tumor-suppressor genes, such as KRAS and APC. Resistance to apoptosis is one of the key mechanisms in the development of CRC as it is for any other kind of cancer. Natural products have been shown to induce the expression of apoptosis regulators that are blocked in cancer cells. In the present study, a series of in vitro assays were employed to study the apoptosis-inducing attributes of Isoledene rich sub-fraction (IR-SF) collected from the oleo-gum resin of M. ferrea. Data obtained, showed that IR-SF inhibited cell proliferation and induced typical apoptotic changes in the overall morphology of all the CRC cell lines tested. Fluorescent staining assays revealed characteristic nuclear condensation, and marked decrease in mitochondrial outer membrane potential in the treated cells. In addition, an increment in the levels of ROS, caspase-8, -9 and -3 was observed. Proteomic analysis revealed that IR-SF up-regulated the expression of pro-apoptotic proteins, i.e., Bid, Bim and cytochrome c. Cytochrome c in turn activated caspases cascade resulting in the induction of apoptosis. Moreover, IR-SF significantly down-regulated Bcl-2, Bcl-w, survivin, xIAP and HSPs pro-survival proteins and induced DNA fragmentation and G0/G1-phase arrest in HCT 116 cells. Chemical characterization of IR-SF by GC-MS and HPLC methods identified Isoledene as one of the major compounds. Altogether, results of the present study demonstrate that IR-SF may induce apoptosis in human colorectal carcinoma cells through activation of ROS-mediated apoptotic pathways.

PURPOSE: Endometrial cancer is the most common gynecological malignancy in developed countries. The role of tumor suppressor genes (TSG) in endometrioid endometrial adenocarcinoma (EEC) has an important impact on patient survival prognosis. Thus, it is important to identify TSG transcripts that differentiate endometrial adenocarcinoma into various pathomorphological grades. The aim of this study was to analyze the expression profile of tumor suppressor genes related to the cell cycle in patients with endometrial adenocarcinoma across histological differentiation and to identify transcripts which differentiate endometrium into various pathomorphological grades.
MATERIAL AND METHODS: Gene expression analysis was completed for 19 endometrial endometrioid adenocarcinomas and 5 normal specimens (obtained from women with diagnosed uterine fibroids, benign ovarian tumors and a prolapsed uterus with histopathologically confirmed endometrium in the proliferative phase) using Affymetrix HG-U133A oligonucleotide microarrays. The statistical analysis was performed using the GeneSpring13.0 software and PANTHER classification system.
RESULTS: Significant changes in gene expression were observed across histological differentiation. The WT-1, CYR 61, TSPYL5 genes were statistically and biologically significant in all cancer grades, and were considered to be primary for the G1 grade in endometrial cancer. The G2 cancer specific genes were BCL2L2 and HNRNPA0, whereas in G3 there was only BAK.
CONCLUSION: In conclusion, the WT-1, CYR61 and TSPYL5 gene expressions are potentially correlated with patient survival in all endometrial cancer grades. The TSGs identified are considered to be important in EEC pathogenesis and further research is needed to confirm this.

Chemotherapeutic resistance is the main reason of the failure in clinical treatment of gastric cancer. Berberine (BER) is the active compound of traditional Chinese medicine Huang Lian. The aim of this present study is to evaluate the effect of BER on cisplatin resistance in gastric cancer cells and to investigate its possible mechanism. Gastric cancer cell lines SGC-7901 and BGC-823 and their respective cisplatin-resistant variants SGC-7901/DDP and BGC-823/DDP were used in this study. We found that BER treatment significantly reversed cisplatin sensitivity and induced caspase-dependent apoptosis in SGC-7901/DDP and BGC-823/DDP cells; BER treatment induced miR-203 expression, and overexpression of miR-203 mimicked the cisplatin-sensitizing effect of BER. Importantly, we showed that miR-203 was able to target the 3'UTR of Bcl-w. Therefore, we conclude that BER treatment reduces cisplatin resistance of gastric cancer cells by modulating the miR-203/Bcl-w apoptotic axis. BER may be a novel agent to enhance chemotherapeutic responses in cisplatin-resistant gastric cancer patients.

BACKGROUND: Leiomyosarcoma is an aggressive soft tissue sarcoma with a 5-year survival rate of 15 to 60%. Treatment options for inoperable or metastatic patients are limited owing to frequent resistance of tumours to chemotherapy and radiation. In this study, we hypothesised that antiapoptotic Bcl-2 family proteins might contribute to leiomyosarcoma chemoresistance and therefore inhibition of Bcl-2 family proteins might sensitise leiomyosarcomas to conventional chemotherapy.
METHODS: Expression of the Bcl-2 family proteins Bcl-xL, Bcl-w and Bcl-2 was investigated using immunohistochemistry on a tissue microarray containing 43 leiomyosarcomas. Furthermore, we investigated whether ABT-737, a potent BH3 mimetic, sensitises leiomyosarcoma cells to doxorubicin treatment in vitro.
RESULTS: Seventy-seven per cent, 84% and 42% of leiomyosarcomas demonstrated high expression of Bcl-2, Bcl-xL and Bcl-w, respectively. Single-agent treatment with ABT-737 resulted in a minor reduction of cell viability. However, combination treatment of ABT-737 and doxorubicin revealed synergism in all four cell lines, by inducing apoptosis.
CONCLUSIONS: In conclusion, Bcl-2 family proteins contribute to soft tissue leiomyosarcoma chemoresistance. Antiapoptotic proteins are highly expressed in leiomyosarcoma of soft tissue, and inhibition of these proteins using a BH3 mimetic increases leiomyosarcoma sensitivity to doxorubicin.

Breast cancer is the leading cause of cancer-associated deaths in women worldwide. Clinical biomarkers give information on disease progression and identify relevant biological pathways. A confounding factor that uncouples markers from disease outcome is the ability of tumor cells to mutate and evade clinical intervention. Therefore, we focussed on apoptotic genes that modulate tumor regression. Using gene and tissue microarray analyses, we identified an association of Bcl-2 interacting killer (Bik) with poor breast cancer prognosis. Bik prognostic ability was independent of Estrogen Receptor/Progesterone Receptor and Her2 status. Additionally, Bik was independent of anti-apoptotic Bcl-2, Bcl-xL, Mcl-1 and Bcl-w suggesting a complex mechanism of tumor promotion identified by Bik high tumors. Bik also stimulates autophagy, which can contribute to enhanced tumor fitness. We found a significant association between the autophagy marker ATG5 and Bik. Combined high expression level of ATG5 and Bik was a stronger predictor of outcome than either alone. Thus, our study identifies Bik as a novel, independent prognostic biomarker for poor outcomes in breast cancer and suggests that Bik-mediated autophagy contributes to disease recurrence.

MCL-1 (BCL-2 family anti-apoptotic protein) is responsible for melanoma's resistance to therapy. Cancer initiating cells also contribute to resistance and relapse from treatments. Here we examined the effects of the MCL-1 inhibitor SC-2001 in killing non melanoma-initiating-cells (bulk of melanoma), and melanoma-initiating-cells (MICs). By itself, SC-2001 significantly kills melanoma cells under monolayer conditions in vitro and in a conventional mouse xenograft model. However, even at high doses (10μM), SC-2001 does not effectively eliminate MICs. In contrast, the combination of SC-2001 with ABT-737 (a BCL-2/BCL-XL/BCL-W inhibitor) significantly decreases ALDH+ cells, disrupts primary spheres, and inhibits the self-renewability of MICs. These results were observed in multiple melanomas, including short term cultures of relapsed tumors from current treatments, independent of the mutation status of BRAF or NRAS. Using a low-cell-number mouse xenograft model, we examined the effects of these treatments on the tumor initiating ability of MIC-enriched cultures. The combination therapy reduces tumor formation significantly compared to either drug alone. Mechanistic studies using shRNA and the CRISPR-Cas9 technology demonstrated that the upregulation of pro-apoptotic proteins NOXA and BIM contribute to the combination-induced cell death. These results indicate that the MCL-1 inhibitor SC-2001 combined with ABT-737 is a promising treatment strategy for targeting melanoma.

Aberrant expression of the miR-129 family has been found in several types of cancer, yet its expression and potential biologic role in breast cancer remain largely unknown. In the present study, we found that miR-129-2 was consistently downregulated in the breast cancer specimens and cell lines. Overexpression of miR-129-2-3p markedly suppressed breast cancer cell proliferation and induced its apoptosis. In addition, a luciferase reporter assay revealed that miR-129-2-3p suppressed BCL2L2 expression. Furthermore, BCL2L2 was able to reverse miR-129-2-3p-mediated cell apoptosis, indicating that BCL2L2 plays a crucial role in mediating the tumor-suppressive role of miR-129-2-3p. Moreover, bisulfite DNA sequencing PCR (BSP) analysis identified that promoter hypermethylation was responsible for the downregulation of miR-129-2 in breast cancer. Collectively, our findings indicate that miR-129-2 is downregulated in breast cancer cells by promoter hypermethylation. Moreover, downregulation of miR-129-2 results in BCL2L2 overexpression and disease progression in breast cancer patients.

Oral cancer is usually preceded by pre-cancerous lesion and related to tobacco abuse. Tobacco carcinogens damage DNA and cells harboring such damaged DNA normally undergo apoptotic death, but cancer cells are exceptionally resistant to apoptosis. Here we studied association between sequence and expression variations in apoptotic pathway genes and risk of oral cancer and precancer. Ninety nine tag SNPs in 23 genes, involved in mitochondrial and non-mitochondrial apoptotic pathways, were genotyped in 525 cancer and 253 leukoplakia patients and 538 healthy controls using Illumina Golden Gate assay. Six SNPs (rs1473418 at BCL2; rs1950252 at BCL2L2; rs8190315 at BID; rs511044 at CASP1; rs2227310 at CASP7 and rs13010627 at CASP10) significantly modified risk of oral cancer but SNPs only at BCL2, CASP1and CASP10 modulated risk of leukoplakia. Combination of SNPs showed a steep increase in risk of cancer with increase in "effective" number of risk alleles. In silico analysis of published data set and our unpublished RNAseq data suggest that change in expression of BID and CASP7 may have affected risk of cancer. In conclusion, three SNPs, rs1473418 in BCL2, rs1950252 in BCL2L2 and rs511044 in CASP1, are being implicated for the first time in oral cancer. Since SNPs at BCL2, CASP1 and CASP10 modulated risk of both leukoplakia and cancer, so, they should be studied in more details for possible biomarkers in transition of leukoplakia to cancer. This study also implies importance of mitochondrial apoptotic pathway gene (such as BCL2) in progression of leukoplakia to oral cancer.

The current study investigated the mechanisms by which licochalcone C induces apoptosis of T24 human malignant bladder cancer cells. Cell viability was evaluated using an MTT assay. Apoptosis was investigated using a morphological assay, flow cytometry and a caspase‑3 activity assay. Alterations in the gene expression levels of Bcl‑2 family members were measured by semi‑quantitative reverse transcription‑polymerase chain reaction assays. The protein levels of pro‑caspase‑3 and cleaved poly(ADP ribose) polymerase were measured using western blotting. The results indicated that licochalcone C induced T24 cell apoptosis in a concentration‑dependent manner. Licochalcone C treatment reduced the levels of the anti‑apoptotic mRNAs (Bcl‑2, Bcl‑w and Bcl‑XL) and increased expression of the pro‑apoptotic mRNAs (Bax and Bim). The Bcl‑2 family inhibitor (ABT‑737) reduced apoptosis induced by licochalcone C in T24 cells. The current study demonstrated that licochalcone C may be a potential adjuvant therapeutic agent for bladder cancer.

There is currently a requirement for effective treatment strategies for human hepatocellular carcinoma (HCC), a leading cause of cancer‑associated mortality. MicroRNA-122 (miR-122), a repressor of the endogenous apoptosis regulator Bcl‑w, is frequently downregulated in HCC. Thus, it is hypothesized that the activation of miR‑122 may induce selective hepatocellular apoptosis via caspase activation in a model of HCC. In the present study, an miR‑122 mimic transfection was performed in HepG2 cells, and used to investigate the role and therapeutic potential of miR‑122 in the regulation of HCC‑derived cell lines. The apoptotic rates of HepG2 cells were significantly increased following miR‑122 mimic transfection. Reverse transcription‑polymerase chain reaction analysis revealed that Bcl‑w mRNA was significantly reduced, while the mRNA levels of caspase‑9 and caspase‑3 were markedly increased. The immunocytochemistry results supported the mRNA trends. Collectively, the present results suggest that endogenous miR‑122 contributes to HepG2 apoptosis and that transfection of mimic miR‑122 normalizes apoptotic levels in a model of HCC.

BACKGROUND: miR-630 has been reported to be a modulator of several cancers, but the mechanism by which is it influences radioresistance remains unknown. We aimed to identify the molecular function of miR-630 and its regulatory mechanism in colorectal cancer (CRC) cell lines.
METHODOLOGY: Overexpression and loss-of-function analyses of miR-630 were performed in CRC cell lines by measuring their levels of growth and apoptosis after ionic radiation (IR). Target genes were detected via a dual-luciferase assay and Western blot. Chromatin immunoprecipitation assay was carried out to identify the transcription factor regulating miR-630, and a demethylation experiment was also conducted.
RESULTS: miR-630 expression was found to be positively correlated with radiosensitivity in CRC cell lines (p<0.05). After IR treatment, miR-630 induced apoptosis in cells; however, the opposite was observed when miR-630 was downregulated (p<0.05). BCL2L2 and TP53RK were identified as the target genes of miR-630, and the function of miR-630 was found to depend on these two genes (p<0.05). In addition, evidence showed that CREB regulates the level of miR-630, and demethylation can elevate miR-630 levels (p<0.05).
CONCLUSION: CREB-miR-630-BCL2L2 and TP53RK comprise a novel signaling cascade regulating radiosensitivity in CRC cell lines by inducing cell apoptosis and death.