Downregulation Of Survivin Research Articles

Dihydromyricetin Induces Apoptosis and Reverses Drug Resistance in Ovarian Cancer Cells by p53-mediated Downregulation of Survivin

Ovarian cancer is one of the leading causes of death in gynecological malignancies, and the resistance to chemotherapeutic agents remains a major challenge to successful ovarian cancer chemotherapy. Dihydromyricetin (DHM), a natural flavonoid derived from Ampeopsis Grossdentata, has been widely applied in food industry and medicine for a long time. However, little is known about the effects of DHM on ovarian cancer and the underlying mechanisms. In this study, we demonstrated that DHM could effectively inhibit the proliferation of ovarian cancer cells and induce cell apoptosis. Survivin, an inhibitor of apoptosis (IAPs) family member, exhibited a decreased expression level after DHM treatment, which may be attributed to the activation of p53. Moreover, DHM markedly sensitized paclitaxel (PTX) and doxorubicin (DOX) resistant ovarian cancer cells to PTX and DOX by inhibiting survivin expression. Collectively, our findings highlight a previously undiscovered effect of DHM, which induces apoptosis and reverses multi-drug resistance against ovarian cancer cells through downregulation of survivin.

Oxymatrine inhibits proliferation of human bladder cancer T24 cells by inducing apoptosis and cell cycle arrest

Oxymatrine has been shown to exert an antitumor effect on several types of cancer cells. However, the role of oxymatrine in bladder cancer has not yet been evaluated. The present study was designed to investigate the potential anti-proliferative effect of oxymatrine on bladder cancer T24 cells and the possible mechanisms involved. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine cell growth, and the cell morphology was examined using hematoxylin and eosin staining, wrights' staining and electron microscopy. The caspase-3 and survivin mRNA and protein levels were assessed using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The expression of tumor protein p53 (p53), Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2) were analyzed using immunohistochemistry. Oxymatrine inhibited the proliferation of the T24 cells in a dose- and time-dependent manner. Oxymatrine also induced apoptosis and cell cycle arrest in the cells, in association with the upregulation of caspase-3 and Bax, and the downregulation of survivin, Bcl-2 and p53 expression. Overall, oxymatrine inhibits the proliferation of human bladder cancer cells by inducing apoptosis and cell cycle arrest via mechanisms that involve p53-Bax signaling and the downregulation of survivin expression.

Disulfiram suppresses cancer stem-like properties and STAT3 signaling in triple-negative breast cancer cells

In the presence of copper (Cu), disulfiram (DSF) suppresses properties associated with cancer stem cells (CSCs) in breast cancer, but the mechanism of action is poorly understood. In the present study, we observed that DSF/Cu treatment induced apoptosis, mediated by caspase-3 activation in triple-negative breast cancer (TNBC) cells. DSF/Cu treatment also specifically targeted CSC-like cell populations, marked by the inhibition of ALDH1 activity, the suppression of CD44+/CD24-and CD49f+/CD24 + subpopulations, and the subsequent impairment of mammosphere formation. These effects were functionally associated with a significant impact on the STAT3 signaling pathway, characterized by the downregulation of phospho-STAT3, cyclin D1 and survivin. In an MDA-MB-231-derived xenograft model, DSF administration significantly downregulated ALDH1A1, CD44 and phospho-STAT3 levels. These findings show for the first time that DSF suppresses stem-like properties in TNBC by targeting the STAT3 signaling pathway.

Targeting NF-kappa B Signaling by Artesunate Restores Sensitivity of Castrate-Resistant Prostate Cancer Cells to Antiandrogens

Androgen deprivation therapy (ADT) is the most preferred treatment for men with metastatic prostate cancer (PCa). However, the disease eventually progresses and develops resistance to ADT in majority of the patients, leading to the emergence of metastatic castration-resistant prostate cancer (mCRPC). Here, we assessed artesunate (AS), an artemisinin derivative, for its anticancer properties and ability to alleviate resistance to androgen receptor (AR) antagonists. We have shown AS in combination with bicalutamide (Bic) attenuates the oncogenic properties of the castrate-resistant (PC3, 22RV1) and androgen-responsive (LNCaP) PCa cells. Mechanistically, AS and Bic combination inhibits nuclear factor (NF)-κB signaling and decreases AR and/or AR-variant 7 expression via ubiquitin-mediated proteasomal degradation. The combination induces oxidative stress and apoptosis via survivin downregulation and caspase-3 activation, resulting in poly-ADP-ribose polymerase (PARP) cleavage. Moreover, preclinical castrate-resistant PC3 xenograft studies in NOD/SCID mice (n =28, seven per group) show remarkable tumor regression and significant reduction in lungs and bone metastases upon administering AS (50 mg/kg per day in two divided doses) and Bic (50 mg/kg per day) via oral gavage. Taken together, we for the first time provide a compelling preclinical rationale that AS could disrupt AR antagonist–mediated resistance observed in mCRPC. The current study also indicates that the therapeutic combination of Food and Drug Administration–approved AS or NF-κB inhibitors and AR antagonists may enhance the clinical efficacy in the treatment of mCRPC patients.

Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin.

HER2/neu positive breast tumors predict a high mortality and comprise 25%–30% of breast cancer. We have shown that Flavokawain A (FKA) preferentially reduces the viabilities of HER2-overexpressing breast cancer cell lines (i.e., SKBR3 and MCF7/HER2) versus those with less HER2 expression (i.e., MCF7 and MDA-MB-468). FKA at cytotoxic concentrations to breast cancer cell lines also has a minimal effect on the growth of non-malignant breast epithelial MCF10A cells. FKA induces G2M arrest in cell cycle progression of HER2-overexpressing breast cancer cell lines through inhibition of Cdc2 and Cdc25C phosphorylation and downregulation of expression of Myt1 and Wee1 leading to increased Cdc2 kinase activities. In addition, FKA induces apoptosis in SKBR3 cells by increasing the protein expression of Bim and BAX and decreasing expression of Bcl2, BclX/L, XIAP, and survivin. FKA also downregulates the protein expression of HER-2 and inhibits AKT phosphorylation. Herceptin plus FKA treatment leads to an enhanced growth inhibitory effect on HER-2 overexpressing breast cancer cell lines through downregulation of Myt1, Wee1, Skp2, survivin, and XIAP. Our results suggest FKA as a promising and novel apoptosis inducer and G2 blocking agent that, in combination with Herceptin, enhances for the treatment of HER2-overexpressing breast cancer.

PCSK9 regulates apoptosis in human lung adenocarcinoma A549 cells via endoplasmic reticulum stress and mitochondrial signaling pathways.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the subtilisin family of PCs that encodes a neural apoptosis-regulated convertase 1. However, the precise role of PCSK9 in lung cancer cell apoptosis has remained elusive. In the present study, A549 human lung adenocarcinoma cells were transfected with PCSK9 small interfering (si)RNA to investigate the underlying mechanisms of apoptosis. The results indicated that PCSK9 siRNA exhibited anti-tumor activity by inducing apoptosis as determined by a Cell Counting Kit-8 and Hoechst staining analysis. In addition, PCSK9 siRNA significantly increased apoptosis of A549 cells in part via activation of caspase-3 and downregulation of the anti-apoptotic proteins survivin and X-linked inhibitor of apoptosis protein. Moreover, the results demonstrated that perturbations in the mitochondrial membrane were associated with the deregulation of the Bax/Bcl-2 ratio, which led to the release of cytochrome c after PCSK9 siRNA transfection. In addition, PCSK9 siRNA also induced endoplasmic reticulum stress (ERS) by increasing the levels of 78 kDa glucose-regulated protein (GRP78), GRP94, phosphorylated protein kinase R-like ER kinase and phosphorylated eukaryotic initiation factor 2α. Therefore, these results demonstrated that PCSK9 siRNA may exert its anti-tumor activity through inducing mitochondrial dysfunction and ERS-associated cell death in A549 cells.

TrichostatinA induces bladder cancer cell death via intrinsic apoptosis at the earlyphase and Sp1‑survivin downregulation at the latephase of treatment.

Histone deacetylase (HDAC) inhibitors have been widely shown to result in cancer cell death. The present study investigated the mechanisms underlying the antitumor effects of the phytochemical trichostatinA (TSA), a classic pan-HDAC inhibitor, in 5,637 urinary bladder cancer cells. It was found that TSA caused cell cycle arrest at the G2/M and G1phase accompanied by reduced expression of cyclinD1 and upregulated induction of p21. In addition, TSA induced morphological changes, reduced cell viability and apoptotic cell death in 5,637cells through caspase-3 activation followed by PARP cleavage. The loss of mitochondrial membrane potential (MMP) indicated that TSA induced apoptosis in 5,637cells through the intrinsic mitochondrial pathway. TSA significantly suppressed Akt activity at 12h after treatment, suggesting that the apoptosis in the early phase was mediated by Akt inhibition. In addition, the protein level of transcription factor Sp1 was decreased at 24h after TSA treatment, which likely led to the downregulation of survivin gene expression, and then contributed to the antitumor activity of TSA. Taken together, the present study delineated that TSA-induced growth inhibition and apoptosis in 5,637cells was associated with pAKT inhibition and MMP loss at the early phase, followed by downregulation of Sp1 and survivin at the late phase of treatment.

Survivin downregulation using siRNA nanoliposomes inhibits cell proliferation and promotes the apoptosis of MHCC-97H hepatic cancer cells: An in vitro and in vivo study.

At present, survivin is one of the most cancer-specific proteins that has been identified. The present study aimed to investigate the antitumor effects of novel survivin small interfering RNA (siRNA) nanoliposomes targeting survivin in human hepatocellular carcinoma MHCC-97H cells and xenograft mouse models. Survivin-targeted siRNA nanoliposomes were prepared and transfected into MHCC-97H cells and MHCC-97H-bearing nude mice. Survivin expression was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Cell viability was analyzed using an MTT assay and apoptosis was evaluated using Hoechst and Annexin V-fluorescein isothiocyanate/propidium iodide staining. Tumor growth in MHCC-97H-bearing mice was monitored following treatment and tumor samples were obtained for survivin expression analysis using RT-qPCR, western blotting and immunohistochemistry staining. Survivin expression levels were significantly downregulated by nanoliposome-mediated survivin siRNA delivery and this was associated with a significant inhibition of cell growth and an increase in the apoptosis of MHCC-97H cells. Downregulation of survivin expression using survivin siRNA nanoliposomes inhibited tumor growth in the MHCC-97H xenograft models without significant treatment-associated toxicity. Therefore, a cationic nanoliposome-based survivin siRNA delivery system was constructed and demonstrated to be efficient for survivin siRNA delivery in in vitro and in vivo studies. These results demonstrate that survivin downregulation was able to significantly attenuate cell proliferation and induce the apoptosis of MHCC-97H cells, as well as inhibit tumor cell growth in MHCC-97H xenograft models, indicating that survivin suppression using siRNA may contribute to the inhibition of tumor development by suppressing cell proliferation and promoting apoptosis.

Combined MEK and Pi3'-kinase inhibition reveals synergy in targeting thyroid cancer in vitro and in vivo.

Anaplastic thyroid cancers and radioiodine resistant thyroid cancer are posing a major treat since surgery combined with Iodine131 therapy is ineffective on them. Small-molecule inhibitors are presenting a new hope for patients, but often lead to drug resistance in many cancers. Based on the major mutations found in thyroid cancer, we propose the combination of a MEK inhibitor and a Pi3′-kinase inhibitor in pre-clinical models. We used human thyroid cancer cell lines and genetically engineered double mutant BRAFV600E PIK3CAH1047R mice to evaluate the effect of both inhibitors separately or in combination in terms of proliferation and signaling in vitro; tumor burden, histology, cell death induction and tumor markers expression in vivo. The combination of MEK and Pi’3-kinase inhibition shows a synergistic effect in term of proliferation and apoptosis induction through Survivin down-regulation in vitro. We show for the first time the effects of the combination of a MEK inhibitor and Pi3′-kinase inhibitor in a genetically engineered mouse model of aggressively lethal thyroid cancer. In fine, the two drugs cooperate to promote tumor shrinkage by inducing a proliferation arrest and an elevation of apoptosis in vivo. Moreover, a phenotypic reversion is also observed with a partial restoration of normal thyroid marker transcription, and thyroid cancer marker expression reduction.In conclusion, combination therapy of MEK and Pi3′-kinase inhibition synergizes to target double mutant thyroid cancer in vitro and in vivo. This multidrug approach could readily be translated into clinical practice and bring new perspectives for the treatment of incurable thyroid carcinoma.

Effect of the histone deacetylase inhibitor resminostat on head and neck squamous cell carcinoma cell lines.

Carcinogenesis is determined by various epigenetic events, such as histone deacetylation. The purpose of this study was to investigate the effect of the new histone deacetylase inhibitor resminostat on head and neck squamous cell carcinoma (HNSCC) cell lines. The cytotoxicity of resminostat and cisplatin on HNSCC cell lines SCC25, CAL27, and FaDu was determined using CCK-8 cell proliferation assay and combination index analysis. Cells were irradiated with 2 to 8 Gray. Apoptosis was measured using flow cytometry and expression of Mcl-1, p-AKT, and survivin was investigated. Treatment with resminostat showed a decrease of cell proliferation of HNSCC cell lines. In addition, a synergistic effect with cisplatin as well as with radiation treatment could be observed. Induction of cell death and dose-dependent downregulation of survivin was evident in all cell lines. Resminostat is a promising treatment of HNSCC because of its antiproliferative, chemosensitizing, and radiosensitizing effects. © 2017 Wiley Periodicals, Inc. Head Neck 39: 900-907, 2017.

Chlorella sorokiniana induces mitochondrial-mediated apoptosis in human non-small cell lung cancer cells and inhibits xenograft tumor growth in vivo

BackgroundLung cancer is one of the leading causes of cancer related deaths worldwide. Marine microalgae are a source of biologically active compounds and are widely consumed as a nutritional supplement in East Asian countries. It has been reported that Chlorella or Chlorella extracts have various beneficial pharmacological compounds that modulate immune responses; however, no studies have investigated the anti-cancer effects of Chlorella sorokiniana (CS) on non-small cell lung cancer (NSCLC).MethodsIn this study, we evaluated the anti-cancer effects of CS in two human NSCLC cell lines (A549 and CL1-5 human lung adenocarcinoma cells), and its effects on tumor growth in a subcutaneous xenograft tumor model. We also investigated the possible molecular mechanisms governing the pharmacological function of CS.ResultsOur results showed that exposure of the two cell lines to CS resulted in a concentration-dependent reduction in cell viability. In addition, the percentage of apoptotic cells increased in a dose-dependent manner, suggesting that CS might induce apoptosis in human NSCLC cells. Western blot analysis revealed that exposure to CS resulted in increased protein expression of the cleaved/activated forms of caspase-3, caspase-9, and PARP, except caspase-8. ZDEVD (caspase-3 inhibitor) and Z-LEHD (caspase-9 inhibitor) were sufficient at preventing apoptosis in both A549 and CL1-5 cells, proving that CS induced cell death via the mitochondria-mediated apoptotic pathway. Exposure of A549 and CL1-5 cells to CS for 24 h resulted in decreased expression of Bcl-2 protein and increased expression of Bax protein as well as decreased expression of two IAP family proteins, survivin and XIAP.ConclusionsWe demonstrated that CS induces mitochondrial-mediated apoptosis in NSCLC cells via downregulation of Bcl-2, XIAP and survivin. In addition, we also found that the tumors growth of subcutaneous xenograft in vivo was markedly inhibited after oral intake of CS.

The natural flavonoid apigenin sensitizes human CD44+ prostate cancer stem cells to cisplatin therapy.

Prostate cancer (PCa) is the second most common type of cancer and the fifth leading cause of cancer-related death among men. Development of chemoresistance, tumor relapse and metastasis remain major barriers to effective treatment and all been identified to be associated with cancer stem cells (CSCs). Natural flavonoids such as apigenin have been shown to have the ability to improve the therapeutic efficacy of common chemotherapy agents through CSCs sensitization. Thus, the aim of this study was to evaluate the combination of apigenin with cisplatin on CD44+ PCa stem cell growth and migration. Platinum-based anti-neoplastic drugs have been used to treat a number of malignancies including PCa. However, acquired resistance and side effects unfortunately have limited cisplatin's use. A CD44+ subpopulation was isolated from human androgen-independent PC3 PCa cells by using human CD44-PE antibody. IC50 values were determined by MTT test. RT-qPCR, Western blot analyses and image-based cytometer were used to investigate apoptosis, cell cycle and their underlying molecular mechanisms. Cell migration was evaluated by wound healing test. The combination of the IC50 doses of apigenin (15μM) and cisplatin (7.5μM) for 48h significantly enhanced cisplatin's cytotoxic and apoptotic effects through downregulation of Bcl-2, sharpin and survivin; and upregulation of caspase-8, Apaf-1 and p53 mRNA expression. The combined therapy suppressed the phosphorylation of p-PI3K and p-Akt, inhibited the protein expression of NF-κB, and downregulated the cell cycle by upregulating p21, as well as cyclin dependent kinases CDK-2, -4, and -6. Apigenin significantly increased the inhibitory effects of cisplatin on cell migration via downregulation of Snail expression. In conclusion, our study showed the possible therapeutic approach of using apigenin to potentially increase the effects of cisplatin by targeting CSCs subset in prostate cancer.

Low-dose 5-fluorouracil sensitizes HepG2 cells to TRAIL through TRAIL receptor DR5 and survivin-dependent mechanisms

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer treatment due to its highly selective apoptosis-inducing action on tumour cells without harming normal cells. However, because of TRAIL resistance by some cancer cells, combined treatment with sensitizing agents is required to enhance the anticancer potential of TRAIL. In the present study, we investigated the sensitizing effect of 5-fluorouracil (5-FU) on TRAIL-induced apoptosis in TRAIL-resistant HepG2 hepatocarcinoma cells. The results show that 5-FU pretreatment could sensitize HepG2 cells to TRAIL-mediated apoptosis. The enhanced induction of cell death by the 5-FU/TRAIL combination was mediated by DR5 up-regulation and survivin down-regulation. Furthermore, this combination treatment significantly inhibited the growth of human xenografts in vivo. In conclusion, this study demonstrates that the combination of a sensitizing agent and TRAIL may be a novel and effective therapeutic regimen for treating human hepatocellular carcinoma.

Abstract A04: Novel indol-based tambjamine analogs down-regulate survivin in lung cancer

Abstract Introduction: Lung cancer is the most frequent cause of cancer death worldwide, with an increasing incidence and a poor prognosis that depends on tumor stage at presentation. Characterization of molecular abnormalities that play a crucial role on lung cancer will allow the development of targeted therapies for this illness. Our aim is to test novel indol-based compounds chemically related to tambjamines and to characterize the alterations in signaling pathways caused by their treatment in order to identify their molecular targets. Materials and Methods: Cell lines representative of the four major groups of lung cancer types were selected to perform this study: A549 (adenocarcinoma), SW900 (squamous cell carcinoma), H460 (large cell carcinoma) and DMS53 (small cell lung carcinoma). Cell viability evaluation was performed by the standard MTT assay. In order to characterize the main transcriptional changes produced by the selected compounds, a QIAGEN array of genes related to human cancer drug targets was used. Transcriptional alteration of a set of genes was further corroborated by TaqMan gene expression assays in order to validate the array. Additionally, the protein expression levels of several apoptotic-related proteins were assessed by immunoblot in the A549 cell line exposed to IC25, IC50 and IC75 values of the selected compounds. Moreover, Z-VAD-FMK (a pan-caspase inhibitor) treatment was performed and the outcome analyzed using the MUSE cell count and viability assay kit in order to assess the reversion in viability upon compounds treatment. Lastly, the p38 specific inhibitor SB202190 was used to assess its potential reversion of survivin levels and apoptosis by Western blot. Results and Discussion: Cell viability was significantly reduced in a dose-dependent manner by tambjamine analogs in all the lung cancer cell lines studied. Our results, using gene expression arrays, showed the impact of our compounds in several targets from apoptosis (e.g. BCL2 and BIRC5/survivin) and cell cycle (e.g. AURKs, CDKs and PLKs), among others. The down-regulation of the anti-apoptotic proteins survivin and Bcl2, as well as the activation of several pro-apoptotic proteins (initiator caspases, effector caspases and final executor PARP protein) was analyzed by immunoblot. Furthermore, the inhibition of caspase activity significantly reversed the cytotoxic effect of the selected compounds. Finally, the p38 stress kinase was activated after compound treatment and experiments using a specific inhibitor showed to partially recover the down-regulation of survivin levels and the activation of apoptosis. All these results hint that after compounds treatment cell death process is triggered mainly by apoptosis and that survivin down-regulation might be the responsible for the induction of this process. Conclusion: Altogether, our selected compounds show more potent cytotoxic properties than the first-line treatment cisplatinum, altering the expression of several key signaling proteins in lung cancer cells, especially apoptotic related proteins. These compounds strongly down-regulate the survivin cellular pool thereby triggering apoptotic cell death via caspase activation. Acknowledgements: This work was supported by the grant from the Spanish government and the EU (FIS PI13/00089) and a grant from La Marató de TV3 Foundation (20132730). LKG holds a FCT (SFRH/BPD/91766/2012) fellowship. Citation Format: Pilar Manuel-Manresa, Luis Korrodi-Gregório, Ananda Marina Rodilla, Roberto Quesada, Vanessa Soto-Cerrato, Ricardo Pérez-Tomás. Novel indol-based tambjamine analogs down-regulate survivin in lung cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A04.

Role of epigenetic regulation on the induction of apoptosis in Jurkat leukemia cells by white grape pomace rich in phenolic compounds.

Grape pomace is a rich source of phenolic compounds commonly employed for elaboration of dietary supplements. The aim of the present study was to investigate the anticancer effect of a purified white grape pomace extract (PWGPE) in acute lymphoblastic leukemia Jurkat cells and to characterize the underlying mechanism. Apoptosis, mitochondrial membrane potential and reactive oxygen species (ROS) levels were assessed by flow cytometry and protein expression levels were analysed by Western blotting. PWGPE induced apoptosis in Jurkat cells in a time- and concentration-dependent manner. The anticancer effect was associated with an increased expression of p73 and down-regulation of pro-survival factors, including p-Akt, Bcl-2, and survivin. PWGPE reduced the expression of several proteins that block the expression of apoptosis-related genes such as DNMT1, HDAC1/2, UHRF1, and the polycomb group protein members: EZH2, SUZ12, and BMI1. In addition, the extract induced the formation of ROS, whereas pre-treatment with PEG-catalase and N-acetylcysteine prevented the ROS formation and markedly decreased the induction of apoptosis. These findings suggest that PWGPE-induced apoptosis in Jurkat human leukemia cells, is mediated by mitochondrial depolarization and caspase-3 cleavage, and depends on ROS generation and regulation of epigenetic gene silencing. Therefore, PWGPE may play an important role in the treatment of acute lymphoblastic leukemia (ALL).

Rho-Associated Protein Kinase (ROCK) Inhibitors Inhibit Survivin Expression and Sensitize Pancreatic Cancer Stem Cells to Gemcitabine.

Targeting pathways regulating survivin expression, which has been implicated in multidrug resistance of cancer cells, is a promising strategy to overcome cancer chemoresistance. To date, the role of rho-associated protein kinases (ROCKs) in survivin expression remains largely unknown. The effects of ROCK inhibitors Y-27632 and fasudil on survivin expression and cell viability were determined by immunoblot analysis and dye exclusion, respectively, in PANC-1 CSLC, a cancer stem cell line derived from a serum-cultured, gemcitabine-sensitive pancreatic cancer cell line, PANC-1. siRNA-mediated knockdown of survivin revealed that the gemcitabine resistance of PANC-1 CSLC was dependent on survivin expression. Both Y-27632 and fasudil, reduced survivin expression in PANC-1 CSLC cells and sensitized them to gemcitabine. ROCK inhibition also reduced survivin expression in various other human cancer cell lines. Small molecule inhibitor-mediated targeting of ROCK may be a viable strategy to overcome cancer chemoresistance through down-regulation of survivin.

Drug priming enhances radiosensitivity of adamantinomatous craniopharyngioma via downregulation of survivin.

OBJECTIVE In this study, the authors investigated the underlying mechanisms responsible for high tumor recurrence rates of adamantinomatous craniopharyngioma (ACP) after radiotherapy and developed new targeted treatment protocols to minimize recurrence. ACPs are characterized by the activation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR), known to mediate radioresistance in various tumor entities. The impact of tyrosine kinase inhibitors (TKIs) gefitinib or CUDC-101 on radiation-induced cell death and associated regulation of survivin gene expression was evaluated. METHODS The hypothesis that activated EGFR promotes radioresistance in ACP was investigated in vitro using human primary cell cultures of ACP (n = 10). The effects of radiation (12 Gy) and combined radiochemotherapy on radiosensitivity were assessed via cell death analysis using flow cytometry. Changes in target gene expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Survivin, identified in qRT-PCR to be involved in radioresistance of ACP, was manipulated by small interfering RNA (siRNA), followed by proliferation and vitality assays to further clarify its role in ACP biology. Immunohistochemically, survivin expression was assessed in patient tumors used for primary cell cultures. RESULTS In primary human ACP cultures, activation of EGFR resulted in significantly reduced cell death levels after radiotherapy. Treatment with TKIs alone and in combination with radiotherapy increased cell death response remarkably, assessed by flow cytometry. CUDC-101 was significantly more effective than gefitinib. The authors identified regulation of survivin expression after therapeutic intervention as the underlying molecular mechanism of radioresistance in ACP. EGFR activation promoting ACP cell survival and proliferation in vitro is consistent with enhanced survivin gene expression shown by qRT-PCR. TKI treatment, as well as the combination with radiotherapy, reduced survivin levels in vitro. Accordingly, ACP showed reduced cell viability and proliferation after survivin downregulation by siRNA. CONCLUSIONS These results indicate an impact of EGFR signaling on radioresistance in ACP. Inhibition of EGFR activity by means of TKI treatment acts as a radiosensitizer on ACP tumor cells, leading to increased cell death. Additionally, the results emphasize the antiapoptotic and pro-proliferative role of survivin in ACP biology and its regulation by EGFR signaling. The suppression of survivin by treatment with TKI and combined radiotherapy represents a new promising treatment strategy that will be further assessed in in vivo models of ACP.

311P Blockade of vascular endothelial growth factor receptors by tivozanib inhibits growth and restores chemosensitivity in human ovarian carcinoma cells

Background Epithelialovarian cancer(EOC) is themostfatalgynecological malignancyworldwide. Despite initial therapeutic response, the majorityofadvanced- stagepatients relapseandsuccumb tochemoresistantdisease. Therapy-resistantEOC is an incurable malignancy and overcoming drug resistance is the key to successful treatment.Membersofvascularendothelialgrowthfactor(VEGF)familyofligandsand receptorsareover-expressedinEOCandplaykeyrolesinitsmalignantprogression thoughtheexactcontributionofthispathwayindevelopmentofthechemoresistant disease remains largely elusive. Methods Cell proliferation, colony formation and anoikis resistance assays were performedtoevaluatetheeffectsoftivozasniboncellviability.Quantitativereverse transcription-PCRwasconductedtomeasuretheeffectsoftivozanibonexpressionof IL6,IL8,CCNB1,BIRC5,CDK1,CDK2,CHEK1,CHEK2,CDKN1AandSFN.To investigatechangesinexpressionofWEE1,cyclinB1,CDC25C,BCL2,BAX,P21and survivin after tivozanib treatment, Western blot analysis was done. Propium iodide staining was performed to measure cell cycle distribution and a caspase 3 activation assaywasusedtoinvestigatetheeffectsoftivozaniboninductionofapoptosis. Zymoanaylsis, uPA activity assay and cell migration/invasion assays were done to explore the effects of tivozanib treatment on MMP-2 activity, uPA enzymatic levels and motilityofthechemoresistantovariancarcinomacells.Alldatawereevaluatedin triplicate against untreated control cells and collected from three independent experiments. Data were graphed and analysed using GraphPad Prism Software 6.0 usingone-wayANOVAandtheunpairedtwo-tailedStudent's ttest.Alldataare presented as mean 6 standard deviation (SD). Results Inthisreport,weshowedthatexpressionoftheVEGFfamilymembersis higherintherapy-resistantEOCcellscomparedtosensitiveones.Over-expressionof VEGFA, VEGFC and VEGFR2 correlated with dug resistance and tivozanib, a pan- inhibitorofVEGFreceptors,reducedproliferationofthechemoresistantEOCcells through a G2/M cell cycle arrest. Tivozanib decreased adhesive and invasive potential of thesecellsviareductionofintercellularadhesionmolecule-1 (ICAM-1)andenzymatic activity ofurokinase plasminogen activator (uPA) as well as matrix metalloproteinase-2 (MMP-2).Moreover,tivozanibsynergisticallyenhancedanti-tumoreffectsofEGFR- directed therapies including erlotinib, gefitinib and cetuximab via down-regulation of anti-apoptotic proteins survivin and Bcl-2. Conclusions Altogether, these findings suggest that the VEGF/VEGFR pathway may have potential as an attractive therapeutic target in therapy-resistant EOC and VEGFR blockade by tivozanib may yield stronger anti-tumor efficacy and circumvent resistance to EGFR-directed therapies. Legal entity responsible for the study Dr. Majid Momeny Funding Tehran University of Medical Sciences Disclosure All authors have declared no conflicts of interest.

Fish-Oil-Derived DHA-mediated Enhancement of Apoptosis in Acute Lymphoblastic Leukemia Cells is Associated with Accumulation of p53, Downregulation of Survivin, and Caspase-3 Activation

ABSTRACTIn acute lymphoblastic leukemia (ALL), resistance to chemotherapy is associated with inactivation of p53 and upregulation of survivin. Thus, targeting the p53 and survivin expression may provide an attractive strategy for ALL treatment. It has been shown that fish-oil-derived docosahexaenoic acid (DHA) activates several antitumorigenic mechanisms in tumor cells, but little is known regarding the role of DHA on modulating p53 and survivin expression in ALL cells. In this study, we investigated the alterations of the p53 and survivin expression and induction of apoptosis in DHA-treated Molt-4 cells that serve as a model for ALL cells. Molt-4 cells were treated with 50, 100, 150, and 200 μM DHA after which cell proliferation, survivin mRNA and protein levels, p53 protein level, caspase-3 activation, and apoptotic rates were evaluated by different cellular and molecular techniques. After 48- and 72-h treatments with DHA at concentrations ranging from 50 to 200 μM, cell proliferation rates were measured to be 80.5–44.4%, and 73.4–14.4%, respectively, compared to untreated cells. We also found that treatment for 48 h with 200 μM DHA resulted in 10.8- and 3.6-fold increase in p53 protein level and caspase-3 activation followed by 4.7-and 1.6-fold decrease in survivin mRNA and protein levels, respectively, compared to untreated cells. Treatment of cells with different concentrations of DHA dramatically increased the p53/survivin and caspase-3/survivin ratios by 2.8- to 16.9-fold and 3.3 to 5.6-fold increases, respectively, compared to untreated cells. A decrease in the number of cells ranging from 16% to 70% and an increase in the number of apoptotic cells ranging from 9.3% to 93% was also observed with increasing DHA concentrations. In conclusion, p53 and survivin may provide promising targets of DHA in ALL cells and this compound with high proapoptotic capacity represents the possibility of its therapeutic application for ALL treatment.

Combination effects of arsenic trioxide and fibroblast growth factor receptor inhibitor in squamous cell lung carcinoma

ObjectivesLung cancer remains the top cancer killer worldwide, with squamous cell carcinoma (SCC) as the second commonest histologic subtype. Arsenic trioxide (ATO) was previously shown to suppress growth of lung cancer. Fibroblast growth factor receptor (FGFR) amplification was recently demonstrated in lung SCC, with specific FGFR inhibitor (e.g. PD173074) developed as a potential targeted therapy. Therefore the combination effects of ATO and PD173074 in SCC was studied. Materials and methodsThe combination of ATO/PD173074 was studied in a proof-of-principle model using a lung SCC cell line with FGFR1 overexpression: SK-MES-1. The effects of ATO and/or PD173074 on cell viability and protein expression were studied by MTT assay and Western blot respectively. Cell cycle analysis, phosphatidylserine externalization and mitochondrial membrane depolarization were monitored by flow cytometry. FGFR1 knockdown was performed with siRNAs. Proteasome inhibitor (MG-132) was used to study the degradation mechanism. In vivo effect of ATO and/or PD173074 was investigated using a nude mice xenograft model. ResultsCombined ATO/PD173074 reduced cell viability along with increased sub-G1 population, phosphatidylserine externalization and mitochondrial membrane depolarization more significantly than single treatments. Downregulation of FGFR1, p-Akt, Akt, p-Src, Src, p-c-Raf, c-Raf, Erk and survivin as well as upregulation of p-Erk and cleaved PARP were observed upon ATO and/or PD treatment. MG-132 partially reversed the degradation of Akt, Src, c-Raf and Erk induced by ATO/PD, suggestive of ubiquitin-independent proteasome-dependent degradation. However, the mechanism of FGFR1 downregulation remained unknown. Downregulation of FGFR1, Akt, Src, c-Raf and Erk as well as cleaved PARP elevation induced by ATO and/or PD were confirmed in vivo. ConclusionMassive protein degradation (FGFR1, Akt, Src, c-Raf and Erk) was induced by ATO and/or PD173074 treatment mainly mediated by activation of proteasomal degradation in SCC cell line SK-MES-1 in vitro and in vivo.