Role of p53 in transcriptional repression of SVCT2.

SVCT2, Sodium-dependent Vitamin C Transporter 2, uniquely transports ascorbic acid (also known as vitamin C and ascorbate) into all types of cells. Vitamin C is an essential nutrient that must be obtained through the diet and plasma levels are tightly regulated by transporter activity. Vitamin C plays an important role in antioxidant defenses and is a cofactor for many enzymes that enable hormone synthesis, oxygen sensing, collagen synthesis and epigenetic pathways. Although SVCT2 has various functions, regulation of its expression/activity remains poorly understood. We found a p53-binding site, within the SVCT2 promoter, using a transcription factor binding-site prediction tool. In this study, we show that p53 can directly repress SVCT2 transcription by binding a proximal- (~-185 to -171 bp) and a distal- (~-1800 to -1787 bp) p53-responsive element (PRE), Chromatin immunoprecipitation assays showed that PRE-bound p53 interacts with the corepressor-histone deacetylase 3 (HDAC3), resulting in deacetylation of histones Ac-H4, at the proximal promoter, resulting in transcriptional silencing of SVCT2. Overall, our data suggests that p53 is a potent transcriptional repressor of SVCT2, a critical transporter of diet-derived ascorbic acid, across the plasma membranes of numerous essential tissue cell types.

Inhibition of JAK1/2 can overcome EGFR-TKI resistance in human NSCLC.

Non-small lung cancer (NSCLC) is the most common cancer in the world. The epidermal growth factor receptor (EGFR) gene is mutated in approximately 10% of lung cancer cases in the US and 50% of lung cancer in Asia. The representative target therapeutic agent, erlotinib (EGFR tyrosine kinase inhibitor; EGFR TKI), is effective in inactivating EGFR in lung cancer patients. However, approximately 50-60% of patients are resistant to EGFR TKI. These populations are associated with the EGFR mutation. To overcome resistance to EGFR TKI, we discovered a JAK1 inhibitor, CJ14939. We investigated the efficacy of CJ14939 in human NSCLC cell lines in vitro and in vivo. Our results showed that CJ14939 induced the inhibition of cell growth. Moreover, we demonstrated that combination treatment with erlotinib and CJ14939 induced cell death in vitro and inhibited tumor growth in vivo. In addition, we confirmed the suppression of phosphorylated EGFR, JAK1, and Stat3 expression in erlotinib and CJ14939-treated human NSCLC cell lines. Our results provide evidence that JAK inhibition overcomes resistance to EGFR TKI in human NSCLCs.

Chemosensitivity to HM90822, a novel synthetic IAP antagonist, is determined by p-AKT-inducible XIAP phosphorylation in human pancreatic cancer cells.

Inhibitor of apoptosis proteins (IAPs) are overexpressed in the majority of cancers and prevent apoptosis by inhibiting caspases. IAPs have therefore attracted considerable attention as potential targets for anticancer therapy. Here, we demonstrated that HM90822 (abbreviated HM822; a new synthetic IAP antagonist) induced apoptotic cell death via proteasome-dependent degradation of BIR2/3 domain-containing IAPs in human pancreatic cancer cells. HM822 inhibited the expression of XIAP and cIAP1/2 proteins in Panc-1 and BxPC-3 cells, which are sensitive to HM822. HM822 also induced IAP ubiquitination and promoted proteasome-dependent IAP degradation. However, cells expressing phospho-XIAP (Ser87) and AKT exhibited resistance to HM822. In other words, the overexpression of AKT-CA (constitutive active form for AKT) or AKT-WT induced resistance to HM822. In addition, in Panc-1 xenograft and orthotopic mouse models, we revealed that tumor growth was suppressed by the administration of HM822. Taken together, these results suggest that HM822 induces apoptosis through ubiquitin/proteasome-dependent degradation of BIR3 domain-containing IAPs. These findings suggest that phospho-XIAP and phospho-AKT may be used as biomarkers for predicting the efficacy of HM822 in pancreatic cancer patients.

Targeting ß-catenin overcomes MEK inhibition resistance in colon cancer with KRAS and PIK3CA mutations.

BACKGROUND: Mitogen-activated protein kinases (MEK 1/2) are central components of the RAS signalling pathway and are attractive targets for cancer therapy. These agents continue to be investigated in KRAS mutant colon cancer but are met with significant resistance. Clinical investigations have demonstrated that these strategies are not well tolerated by patients. METHODS: We investigated a biomarker of response for MEK inhibition in KRAS mutant colon cancers by LC-MS/MS analysis. We tested the MEK inhibitor in PIK3CA wild(wt) and mutant(mt) colon cancer cells. In addition, we tested the combinational effects of MEK and TNKS inhibitor in vitro and in vivo. RESULTS: We identified ß-catenin, a key mediator of the WNT pathway, in response to MEK inhibitor. MEK inhibition led to a decrease in ß-catenin in PIK3CA wt colon cancer cells but not in mt. Tumour regression was promoted by combination of MEK inhibition and NVP-TNS656, which targets the WNT pathway. Furthermore, inhibition of MEK promoted tumour regression in colon cancer patient-derived xenograft models expressing PIK3CA wt. CONCLUSIONS: We propose that inhibition of the WNT pathway, particularly ß-catenin, may bypass resistance to MEK inhibition in human PIK3CA mt colon cancer. Therefore, we suggest that ß-catenin is a potential predictive marker of MEK inhibitor resistance.

Cellular inhibitor of apoptosis protein 1 (cIAP1) stability contributes to YM155 resistance in human gastric cancer cells.

YM155, which blocks the expression of survivin, a member of the inhibitor of apoptosis (IAP) family, induces cell death in a variety of cancer types, including prostate, bladder, breast, leukemia, and non-small lung cancer. However, the mechanism underlying gastric cancer susceptibility and resistance to YM155 is yet to be specified. Here, we demonstrate that cIAP1 stability dictates resistance to YM155 in human gastric cancer cells. Treatment of human gastric cancer cells with YM155 differentially induced cell death dependent on the stability of cIAP1 as well as survivin. Transfection with cIAP1 expression plasmids decreased cell sensitivity to YM155, whereas knockdown of endogenous cIAP1 using RNA interference enhanced sensitivity to YM155. In addition, double knockdown of survivin and cIAP1 significantly induced cell death in the YM155-resistant cell line, MKN45. We also showed that YM155 induced autoubiquitination and proteasome-dependent degradation of cIAP1. Surprisingly, survivin affected the stability of cIAP1 through binding, contributing to cell sensitivity to YM155. Thus, our findings reveal that YM155 sensitizes human gastric cancer cells to apoptotic cell death by degrading cIAP1, and furthermore, cIAP1 in gastric cancer cells may act as a PD marker for YM155 treatment.

SAHA, an HDAC inhibitor, overcomes erlotinib resistance in human pancreatic cancer cells by modulating E-cadherin.

Pancreatic cancer is one of the most lethal cancers and remains a major unsolved health problem. Less than 20 % of patients are surgical candidates, and the median survival for non-resected patients is approximately 3 to 4 months. Despite the existence of many conventional cancer therapies, few targeted therapies have been developed for pancreatic cancer. Combination therapy using erlotinib and gemcitabine is an approved standard chemotherapy for advanced pancreatic cancer, but it has marginal therapeutic benefit. To try to improve the therapeutic outlook, we studied the efficacy of another combination treatment and the relevance to E-cadherin in human pancreatic cancer cells. We treated two human pancreatic cancer cell lines with the histone deacetylase inhibitor (HDACi) SAHA. Interestingly, in these Panc-1 and Capan1 cells, we observed that the expression levels of E-cadherin and phosphorylated EGFR were gradually upregulated after treatment with SAHA. Furthermore, these cells underwent induced cell death after exposure to the combination treatment of SAHA and erlotinib. In Panc-1 cells, overexpression of E-cadherin activated the phosphorylation of EGFR and increased the cell sensitivity to erlotinib. In Capan1 cells, knocking down E-cadherin decreased the expression of phosphorylated EGFR, and these cells did not respond to erlotinib. Therefore, we demonstrated the efficacy of the combined treatment with SAHA and erlotinib in human pancreatic cancer cells, and we determined that the increased efficacy was due, at least in part, to the effects of SAHA on the expression of E-cadherin. Our studies suggest that E-cadherin may be a potent biomarker for pancreatic cancer.

NVP-BEZ235, a dual PI3K/mTOR inhibitor, induces cell death through alternate routes in prostate cancer cells depending on the PTEN genotype.

Deregulation of the PI3K-AKT/mTOR pathway due to mutation of the tumor suppressor gene PTEN frequently occurs in human prostate cancer and is therefore considered to be an attractive therapeutic target. Here, we investigated how the PTEN genotype affected the antitumor effect of NVP-BEZ235 in human prostate cancer cells. In this setting, NVP-BEZ235 induced cell death in a PTEN-independent manner. NVP-BEZ235 selectively induced apoptotic cell death in the prostate cancer cell line DU145, which harbors wild-type PTEN; however, in the PC3 cell line, which is PTEN-null, treatment with NVP-BEZ235 resulted in autophagic cell death. Consistently, NVP-BEZ235 treatment did not result in the cleavage of caspase-3; instead, it resulted in the conversion of LC3-I to LC3-II, indicating autophagic cell death; these results suggest that an alternate mechanism of cell death is induced by NVP-BEZ235 in PTEN-null prostate cancer cells. Based on our findings, we conclude that the PTEN/PI3K/Akt pathway is critical for prostate cancer survival, and targeting PI3K signaling by NVP-BEZ235 may be beneficial in the treatment of prostate cancer, independent of the PTEN genotype.

NPS-1034, a novel MET inhibitor, inhibits the activated MET receptor and its constitutively active mutants.

The MET proto-oncogene product, which is the receptor for hepatocyte growth factor (HGF), has been implicated in tumorigenesis and metastatic progression. Point mutations in MET lead to the aberrant activation of the receptor in many types of human malignancies, and the deregulated activity of MET has been correlated with tumor growth, invasion, and metastasis. MET has therefore attracted considerable attention as a potential target in anticancer therapy. Here, we report that a novel MET kinase inhibitor, NPS-1034, inhibits various constitutively active mutant forms of MET as well as HGF-activated wild-type MET. NPS-1034 inhibited the proliferation of cells expressing activated MET and promoted the regression of tumors formed from such cells in a mouse xenograft model through anti-angiogenic and pro-apoptotic actions. NPS-1034 also inhibited HGF-stimulated activation of MET signaling in the presence or absence of serum. Furthermore, when tested on 27 different MET variants, NPS-1034 inhibited 15 of the 17 MET variants that exhibited autophosphorylation with nanomolar potency; only the F1218I and M1149T variants were not inhibited by NPS-1034. Notably, NPS-1034 inhibited three MET variants that are resistant to the MET inhibitors SU11274, NVP-BVU972, and PHA665752. Together, these results suggest that NPS-1034 can be used as a potent therapeutic agent for human malignancies bearing MET point mutations or expressing activated MET.

The Immune Suppressor IGSF1 as a Potential Target for Cancer Immunotherapy.

The development of first-generation immune-checkpoint inhibitors targeting PD-1/PD-L1 and CTLA-4 ushered in a new era in anticancer therapy. Although immune-checkpoint blockade therapies have shown clinical success, a substantial number of patients yet fail to benefit. Many studies are under way to discover next-generation immunotherapeutic targets. Immunoglobulin superfamily member 1 (IGSF1) is a membrane glycoprotein proposed to regulate thyroid function. Despite containing 12 immunoglobin domains, a possible role for IGSF1, in immune response, remains unknown. Here, our studies revealed that IGSF1 is predominantly expressed in tumors but not normal tissues, and increased expression is observed in PD-L1low non-small cell lung cancer (NSCLC) cells as compared with PD-L1high cells. Subsequently, we developed and characterized an IGSF1-specific human monoclonal antibody, WM-A1, that effectively promoted antitumor immunity and overcame the limitations of first-generation immune-checkpoint inhibitors, likely via a distinct mechanism of action. We further demonstrated high WM-A1 efficacy in humanized peripheral blood mononuclear cells (PBMC), and syngeneic mouse models, finding additive efficacy in combination with an anti-PD-1 (a well-characterized checkpoint inhibitor). These findings support IGSF1 as an immune target that might complement existing cancer immunotherapeutics.

Ring finger protein 149 is an E3 ubiquitin ligase active on wild-type v-Raf murine sarcoma viral oncogene homolog B1 (BRAF).

Members of the RAF family (ARAF, BRAF, and CRAF/RAF-1) are involved in a variety of cellular activities, including growth, survival, differentiation, and transformation. An oncogene encodes BRAF, the function of which is linked to MEK activation. BRAF is the most effective RAF kinase in terms of induction of MEK/ERK activity. However, the mechanisms involved in BRAF regulation remain unclear. In the present work, we used a tandem affinity purification approach to show that RNF149 (RING finger protein 149) interacts with wild-type BRAF. The latter protein is a RING domain-containing E3 ubiquitin ligase involved in control of gene transcription, translation, cytoskeletal organization, cell adhesion, and epithelial development. We showed that RNF149 bound directly to the C-terminal kinase-containing domain of wild-type BRAF and induced ubiquitination, followed by proteasome-dependent degradation, of the latter protein. Functionally, RNF149 attenuated the increase in cell growth induced by wild-type BRAF. However, RNF149 did not bind to mutant BRAF or induce ubiquitination thereof. Thus, we show that RNF149 is an E3 ubiquitin ligase active on wild-type BRAF.

Targeting isoforms of RON kinase (MST1R) drives antitumor efficacy.

Recepteur d'origine nantais (RON, MST1R) is a single-span transmembrane receptor tyrosine kinase (RTK) aberrantly expressed in numerous cancers, including various solid tumors. How naturally occurring splicing isoforms of RON, especially those which are constitutively activated, affect tumorigenesis and therapeutic response, is largely unknown. Here, we identified that presence of activated RON could be a possible factor for the development of resistance against anti-EGFR (cetuximab) therapy in colorectal cancer patient tissues. Also, we elucidated the roles of three splicing variants of RON, RON Δ155, Δ160, and Δ165 as tumor drivers in cancer cell lines. Subsequently, we designed an inhibitor of RON, WM-S1-030, to suppress phosphorylation thereby inhibiting the activation of the three RON variants as well as the wild type. Specifically, WM-S1-030 treatment led to potent regression of tumor growth in solid tumors expressing the RON variants Δ155, Δ160, and Δ165. Two mechanisms for the RON oncogenic activity depending on KRAS genotype was evaluated in our study which include activation of EGFR and Src, in a trimeric complex, and stabilization of the beta-catenin. In terms of the immunotherapy, WM-S1-030 elicited notable antitumor immunity in anti-PD-1 resistant cell derived mouse model, likely via repression of M1/M2 polarization of macrophages. These findings suggest that WM-S1-030 could be developed as a new treatment option for cancer patients expressing these three RON variants.

CJ14939, a Novel JAK Inhibitor, Increases Oxaliplatin-induced Cell Death Through JAK/STAT Pathway in Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer is reported to have the highest mortality rate among human malignancies. Although many research results for the treatment of colorectal cancer have been reported, there is no suitable treatment when resistance has developed. Therefore, it is necessary to develop new therapeutic agents. Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling plays an essential role in cell differentiation, proliferation, and survival. Abnormal activation of the JAK/STAT signaling pathway, by gene mutation or amplification, may induce cancer development, and sustained JAK/STAT activation is involved in chemoresistance. While many therapeutic agents have been developed to treat colon cancer, there remains no drug to overcome resistance to chemotherapies. The purpose of this study was to determine the potential of CJ14939 as a novel JAK inhibitor for the treatment of colorectal cancer. MATERIALS AND METHODS: In this study, cell culture, cell death assay, 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, colony formation assay, immunoblot analysis and tumor xenograft were applied. RESULTS: CJ14939 induced cell death, and inhibited phosphorylation of JAK1 and STAT3 in colorectal cancer cells. Furthermore, CJ14939 also promoted oxaliplatin-induced cell death, up-regulated expression of cleaved caspase-3, and down-regulated expression of phospho-JAK1 and phospho-STAT3. In vivo, co-treatment with CJ14939 and oxaliplatin notably reduced tumor growth when compared with CJ14939 or oxaliplatin treatment alone. CONCLUSION: This study identifies the important potential of CJ14939 in colorectal cancer treatment and suggests that combining CJ14939 with oxaliplatin might be a novel therapeutic strategy for patients with colorectal cancer.

Foxp3 expression in p53-dependent DNA damage responses.

The forkhead transcription factor, Foxp3, is thought to act as a master regulator that controls (suppresses) expression of the breast cancer oncogenes, SKP2 and HER-2/ErbB2. However, the mechanisms that regulate Foxp3 expression and thereby modulate tumor development remain largely unexplored. Here, we demonstrate that Foxp3 up-regulation requires p53 function, showing that Foxp3 expression is directly regulated by p53 upon DNA damage responses in human breast and colon carcinoma cells. Treatment with the genotoxic agents, doxorubicin or etoposide, induced Foxp3 expression in p53-positive carcinoma cells, but not in cells lacking p53 function. Furthermore, knock down of endogenous wild-type p53 using RNA interference abrogated Foxp3 induction by genotoxic agents, and exogenous expression of p53 in cells lacking p53 restored the responsiveness of Foxp3 to DNA-damaging stresses. In addition, Foxp3 knock down blunted the p53-mediated growth inhibitory response to DNA-damaging agents. These results suggest that induction of Foxp3 in the context of tumor suppression is regulated in a p53-dependent manner and implicate Foxp3 as a key determinant of cell fate in p53-dependent DNA damage responses.

Mutation SVCT2 promotes cell proliferation, invasion and migration in colorectal cancer.

The sodium-dependent vitamin C transporter 2 (SVCT2) surface glycoprotein regulates ascorbate accumulation in the plasma, often resulting in the induction of cancer cell death. Therefore, high expression of this gene associates with increased overall survival in several cancers. However, in colorectal cancer (CRC), high (likely mutated) SVCT2 expression relates to poor overall survival, and its functional significance has not been studied. Thus, we hypothesize that mutant SVCT2 expression could affect CRC patient survival. According to biological databases, SVCT2 has been found to be mutated frequently, and SVCT2 E264K has a particularly high pathogenic score (0.98), compared to other SVCT2 mutant sites, in CRC patients. Interestingly, our results reveal expression of SVCT2 E264K in many CRC tissues and cells. Also, we found wild-type SVCT2 expression to be largely localized to the cytoplasm and membrane, while SVCT2 E264K was restricted to the cytoplasm. We further found that SVCT2 E264K overexpression increases cell growth. By contrast, SVCT2 E264K knockdown significantly reduced cell proliferation and promoted cell apoptosis, resulting in inhibition of cell invasion and migration. Taken together, SVCT2 E264K plays a critical role in proliferation in CRC. Our results suggest that SVCT2 E264K could be a promising novel therapeutic target in CRC.

Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells.

The use of human mesenchymal stem cells (hMSCs) in clinical applications requires large-scale cell expansion prior to administration. However, the prolonged culture of hMSCs results in cellular senescence, impairing their proliferation and therapeutic potentials. To understand the role of microRNAs (miRNAs) in regulating cellular senescence in hMSCs, we globally depleted miRNAs by silencing the DiGeorge syndrome critical region 8 (DGCR8) gene, an essential component of miRNA biogenesis. DGCR8 knockdown hMSCs exhibited severe proliferation defects and senescence-associated alterations, including increased levels of reactive oxygen species (ROS). Transcriptomic analysis revealed that the antioxidant gene superoxide dismutase 2 (SOD2) was significantly downregulated in DGCR8 knockdown hMSCs. Moreover, we found that DGCR8 silencing in hMSCs resulted in hypermethylation in CpG islands upstream of SOD2. 5-aza-2'-deoxycytidine treatment restored SOD2 expression and ROS levels. We also found that these effects were dependent on the epigenetic regulator DNA methyltransferase 3 alpha (DNMT3A). Using computational and experimental approaches, we demonstrated that DNMT3A expression was regulated by miR-29a-3p and miR-30c-5p. Overexpression of miR-29a-3p and/or miR-30c-5p reduced ROS levels in DGCR8 knockdown hMSCs and rescued proliferation defects, mitochondrial dysfunction, and premature senescence. Our findings provide novel insights into hMSCs senescence regulation by the miR-29a-3p/miR-30c-5p/DNMT3A/SOD2 axis.

Sulindac induces apoptotic cell death in susceptible human breast cancer cells through, at least in part, inhibition of IKKbeta.

Sulindac is a non-steroidal anti-inflammatory agent with anti-tumor activities that include the induction of apoptosis in various cancer cells and the inhibition malignant transformation. However, the molecular mechanisms underlying these effects are unclear. Recently, it has been shown that sulindac can inhibit NF-kappaB activation. Here, we demonstrate that sulindac induces apoptotic cell death in susceptible human breast cancer cells through, at least in part, inhibition of IKKbeta activity. More specifically, when we compared two different human breast cancer cell lines, Hs578T, which has relatively low basal IKKbeta activity, and MDA-MB231, which has relatively high basal IKKbeta activity, we found that MDA-MB231 was markedly more sensitive to sulindac-induced apoptosis than Hs578T. This was associated with greater caspase-3 and -9 activity in sulindac-treated MDA-MB231 cells. Using a combination of chemical kinase inhibitors and siRNA-mediated knockdown of specific kinases, we found that sulindac inhibits IKKbeta, which, in turn, leads to the p38 MAPK-dependent activation of JNK1. Together, these findings suggest that sulindac induces apoptosis in susceptible human breast cancer cells through, at least in part, the inhibition of IKKbeta and the subsequent p38 MAPK-dependent activation of JNK1.

Serum starvation induces G1 arrest through suppression of Skp2-CDK2 and CDK4 in SK-OV-3 cells.

Recent studies have suggested that Skp2, an SCF-type ubiquitin ligase, positively regulates cell cycle through degradation of p27, which is an inhibitor of cyclin-dependent kinase 2 (CDK2), which drives cells from the G1 to S phase of cell cycles. In the present study, we examined key regulatory proteins involved in serum starvation-induced cell cycle arrest in human ovarian cancer cells, SK-OV-3. Cell cycle analysis showed that cells were arrested at the G1 phase after serum starvation. Western blot analysis showed that the protein levels of CDK4 and CDK2 were significantly decreased in SK-OV-3 cells. Consistently, Roscovitine, an inhibitor of CDK2, induced cell cycle arrest in normally proliferating cells and a chemical inhibitor of CDK4, 3-ATA [3-Amino-9-thio(10H)-acridone], was found to induce growth arrest. We also found that the protein level of Skp2 was dramatically decreased in response to serum starvation. Moreover, CDK2 protein, which allows cell cycle transit from the G1 to the S phase, was decreased when the Skp2 expression was inhibited by specific siRNA of Skp2, but CDK4 was not decreased. Therefore, these results suggest that serum starvation induces G1 arrest through suppression of Skp2-dependent CDK2 activity and Skp2-independent CDK4 activity in human SK-OV-3 ovarian cancer cells.

Vitamin C inhibits p53-induced replicative senescence through suppression of ROS production and p38 MAPK activity.

We previously reported that tumor cells expressing p53 increase intracellular levels of reactive oxygen species (ROS). In this study, we described an inhibitory effect of vitamin C on replicative senescence. Vitamin C was found to inhibit p53-induced senescence in human bladder cancer EJ cells. The senescence-like phenotype (SLP) induced by p53, which showed a morphological change and an irreversible cell cycle arrest, was not observed in vitamin C-treated EJ cells. In addition, vitamin C did not significantly affect normal cell proliferation. We investigated the molecular mechanisms of the inhibitory effect of vitamin C on the development of replicative senescence in EJ cells. We found that vitamin C inhibited this p53-induced ROS generation. Moreover, p38 kinase which was activated during p53-induced senescence was not observed in vitamin C-treated EJ cells. SB203580, a chemical inhibitor of p38 kinase, was found to consistently inhibit p53-induced senescence. Therefore, it is suggested that vitamin C inhibits p53-induced senescence by preventing ROS generation, which in turn leads to the activation of p38 MAPKinase. These results reveal the inhibitory mechanism of vitamin C on cellular senescence.

Human breast cancer cells display different sensitivities to ABT-263 based on the level of survivin.

ABT-263 (navitoclax), a Bcl-2 family protein inhibitor, was clinically tested as an anti-cancer agent. However, the clinical trials were limited given the occurrence of resistance to monotherapy in breast cancer cells. Our study investigates the mechanisms for overcoming navitoclax resistance by combining it with an mTOR inhibitor to indirectly target survivin. The apoptotic effects of navitoclax occurred in MDA-MB-231 breast cancer cells in a time- and dose-dependent fashion, but MCF-7 cells were resistant to navitoclax treatment. The expression of Bcl-2 family genes was not altered by navitoclax, but the expression of survivin, a member of the inhibitors of apoptosis proteins (IAP) family, was downregulated, which increased death signaling in MDA-MB-231 cells. In MCF-7 cells, a navitoclax-resistant cell line, combined treatment with navitoclax and everolimus synergistically reduced survivin expression and induced cell death. These data indicate that navitoclax induces cell death in MDA-MB-231 cells but not in MCF-7 cells. Decreased survivin expression in MDA-MB-231 cells may be a primary pathway for death signaling. Combined navitoclax and everolimus treatment induces cell death by reducing the stability of survivin in MCF-7 cells. Given that survivin-targeted therapy overcomes resistance to navitoclax, this strategy could be used to treat breast cancer patients.

Combined treatment with vitamin C and sulindac synergistically induces p53- and ROS-dependent apoptosis in human colon cancer cells.

Sulindac has anti-neoplastic properties against colorectal cancers; however, its use as a chemopreventive agent has been limited due to toxicity and efficacy concerns. Combinatorial treatment of colorectal cancers has been attempted to maximize anti-cancer efficacy with minimal side effects by administrating NSAIDs in combination with other inhibitory compounds or drugs such as l-ascorbic acid (vitamin C), which is known to exhibit cytotoxicity towards various cancer cells at high concentrations. In this study, we evaluated a combinatorial strategy utilizing sulindac and vitamin C. The death of HCT116 cells upon combination therapy occurred via a p53-mediated mechanism. The combination therapeutic resistance developed in isogenic p53 null HCT116 cells and siRNA-mediated p53 knockdown HCT116 cells, but the exogenous expression of p53 in p53 null isogenic cells resulted in the induction of cell death. In addition, we investigated an increased level of intracellular ROS (reactive oxygen species), which was preceded by p53 activation. The expression level of PUMA (p53-upregulated modulator of apoptosis), but not Bim, was significantly increased in HCT116 cells in response to the combination treatment. Taken together, our results demonstrate that combination therapy with sulindac and vitamin C could be a novel anti-cancer therapeutic strategy for p53 wild type colon cancers.