Inhibitor Of Apoptosis Protein Family Research Articles

Canine squamous cell carcinoma cell lines with high expression of survivin are sensitive to survivin inhibitor YM155

Treatment of unresectable canine squamous cell carcinoma (SCC) remains challenging and new therapeutic strategies are needed. Survivin is a member of the inhibitor of apoptosis protein family and its inhibitor, YM155, is a potential anti-tumour agent. In the present study, 10 canine tumour cell lines (representing eight different tumour types) were screened for sensitivity to YM155; the drug potently inhibited the growth of the HAPPY SCC cell line. The growth inhibitory properties of YM155 were then examined in more detail using a panel of seven SCC cell lines. YM155 inhibited the growth of the cell lines HAPPY and SQ4; in contrast to the other lines in the panel, these two cell lines had high levels of expression of survivin. In HAPPY cells, YM155 inhibited expression of the survivin gene at the transcriptional level. In contrast, YM155 down-regulated survivin at the post-transcriptional level in SQ4 cells. YM155 suppressed cell growth in HAPPY cells, mostly via induction of apoptosis, but this was not the case in SQ4 cells. Two canine SCC cell lines with high cellular expression of survivin were sensitive to YM155. The possible underlying mechanisms of the cytotoxic effect of YM155 in these cell lines were different. One cell line had down-regulation of survivin mRNA and protein expression, associated with induction of apoptotic cell death. The other cell line had post-transcriptional down-regulation of survivin expression and subsequent induction of non-apoptotic cell death. Targeting survivin with YM155 is a potential approach for the treatment of canine SCCs with high expression of survivin.

Survivin Ser81 Plays An Important Role in PI3K/Akt/mTOR Signaling Pathway

Background: Survivin, a member of the inhibitor of apoptosis protein family, has been associated with protection from cell apoptosis and regulation of mitosis. Phosphorylated-Survivin at Ser81 was reported to provide cytoprotection against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in L929 cells by inducing a backloop activation of phosphatidylinositol 3-kinase (PI3K). Therefore Akt as a possible substrate of PI3K was investigated.Methods: L929 cells were pretreated with/without 50 μM LY294002 or 10 μM Perifosine, and infected with viral particle of Survivin, anti sense of Survivin, Ser81Ala mutated Survivin or vector only. Cells were then harvested, lysed and subjected to immunoblot assay to detect Akt, phosphorylated Akt (Ser473), mammalian target of rapamycin (mTOR), phosphorylated-mTOR (Ser2448).Results: Survivin induced Akt and mTOR phosphorylations in a viral particle concentration dependent manner. Pretreatment of LY294002 or Perifosine prior to Survivin infection, attenuated Akt or mTOR phosphorylations, respectively. Low Akt or mTOR phosphorylations were observed when L929 cells were infected with Ser81Ala mutated Survivin.Conclusion: Ser81 phosphorylation site of Survivin played an important role in activating Survivin/PKA/PI3K/Akt/mTOR signaling pathway.Keywords: survivin, Ser81, Akt, mTOR, LY294002, perifosine

Evaluation of survivin gene expression as a prognostic biomarker in pediatric B-acute lymphoblastic leukemia

Objective The aim of this study was to evaluate the level of survivin mRNA expression in Egyptian patients with acute pediatric lymphoblastic leukemia and to outline any significant relation between the level of survivin expression and the clinical and hematological findings in those patients. Background Survivin, a unique member of the inhibitor of apoptosis protein family, plays an important role in regulating both apoptosis and cell division and was found to be highly expressed in various kinds of tumors. Patients and methods Survivin expression was quantitatively determined by real-time (RT) PCR in 30 newly diagnosed acute lymphoblastic leukemia (B-ALL) pediatric patients before and after induction therapy and in 15 age-matched and sex-matched immune thrombocytopenic purpura individuals serving as a control group. Results A highly significant elevation (P Conclusion Survivin is overexpressed in B-ALL at presentation and may be used as a prognostic marker for ALL in pediatric patients.

Theoretical study of the intermolecular recognition mechanism between Survivin and substrate based on conserved binding mode analysis

Survivin is the smallest member of IAP (inhibitor of apoptosis protein) family, which plays important roles in both mitosis and apoptosis. It has become an attractive drug target due to its overexpression in many human cancers. Survivin has been proven to bind to Smac/DIABLO protein that indirectly inhibits apoptosis. Meanwhile, it is the key subunit of chromosome passenger complex (CPC) which bind to the N-terminal tail of phosphorylated histone H3T3ph during mitosis. Up to now, Survivin directly targeting inhibitor has yet to merge since the difficulty of disrupting the protein-protein interactions (PPIs) between Survivin and its substrate proteins. Nevertheless, currently known binding partners of Survivin provide crucial information about conserved recognition mechanism, which can assist in the detection of some uncharted substrates and also the Survivin inhibitors. Herein, we adopted a method that using four substrates to analyze the common binding mode of Survivin. To accomplish this, conventional molecular dynamics (MD) simulations, molecular mechanics/generalized born surface area (MM-GBSA) binding free energy calculations and energy decomposition were carried out to assess the binding affinity and per-residue contributions. We found that there are two anchor sites of Survivin responsible for maintaining the binding conformation and one sub-pocket for intermolecular recognition. The results of this study synthetically describe the binding mechanism and provide valuable guidance for rational drug design of PPI inhibitor.

IRF8 Regulates Transcription of Naips for NLRC4 Inflammasome Activation

Inflammasome activation is critical for host defenses against various microbial infections. Activation of theNLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection invivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.

XIAP impairs mitochondrial function during apoptosis by regulating the Bcl-2 family in renal cell carcinoma.

Efficient apoptosis requires Bcl-2 family-mediated mitochondrial outer membrane permeabilization (MOMP), which releases pro-apoptotic proteins to the cytosol, activating apoptosis and inhibiting X-linked inhibitor of apoptosis protein (XIAP). XIAP is a member of the inhibitors of apoptosis protein family whose expression is elevated in many cancer types and participates in the release of pro-apoptotic proteins. To explore the association between XIAP and the Bcl-2 family, and the influence of XIAP on mitochondria, RNA interference of XIAP was performed in Caki-1 cells and the dynamic change in the levels of related proteins was compared with the original Caki-1 cells upon induction of apoptosis. Upon knockdown of XIAP, the release of cytochrome c (Cyt-c), second mitochondria-derived activator of caspase (Smac) and apoptotic protease activating factor 1 (Apaf-1) from mitochondria proceeded normally, whereas in Caki-1 cells, the release of these pro-apoptotic proteins was significantly prolonged, and incomplete. Downregulation of XIAP through small interfering RNA resulted in an increase of apoptosis and a marked decrease in Bcl-2 and Bcl-xl levels at 3 h. Additionally, the regulation of the level of XIAP protein affected the specific ratios of Bcl-2/Bax and Bcl-xl/Bax, which play decisive roles in cell death. In the present study, it was revealed that XIAP can feed back to mitochondria, delaying Cyt-c and Apaf-1 release. Furthermore, XIAP can limit the release of its inhibitor Smac with the involvement of Bcl-2 family proteins.

Silencing Livin improved the sensitivity of colon cancer cells to 5-fluorouracil by regulating crosstalk between apoptosis and autophagy.

Colorectal cancer (CRC) is the third most common cause of cancer-associated mortality worldwide. Currently, 5-fluorouracil (5-FU) remains a widely used chemotherapeutic drug in the treatment of CRC; however, 5-FU resistance during treatment has become a common problem. Livin, a member of the inhibitor of apoptosis protein family, is considered to be associated with tumor resistance to chemotherapy. In the present study, Livin-silenced cells were generated by introducing a lentivirus into HCT116 and SW620 colon cancer cell lines. Acridine orange/ethidium bromide staining was used as an indicator of cell death. Western blot analysis was performed to detect protein expression levels, and transmission electron microscopy was used to assess autophagy. The half-maximal inhibitory concentration of 5-FU in colon cancer cells was evaluated using a Cell Counting Kit-8 assay. The results of the present study confirmed that silencing Livin significantly enhanced colon cancer cell death in the presence of 5-FU, increased expression levels of various apoptosis- and autophagy-associated proteins and augmented chemotherapeutic sensitivity to 5-FU. Furthermore, the present study demonstrated that this effect may be reversed when autophagy or apoptosis was inhibited, indicating that apoptosis and autophagy were involved in this process. The protein kinase B signaling pathway and B-cell lymphoma-2 expression levels significantly decreased following Livin knockdown, suggesting they may contribute to the regulation of apoptosis and autophagy crosstalk, which caused the Livin knockdown-induced cell death observed.

Survivin as a novel target protein for reducing the proliferation of cancer cells.

Survivin, also known as baculoviral inhibitor of apoptosis repeat-containing 5, is a novel member of the inhibitor of apoptosis protein family. Survivin is highly expressed in tumors and embryonic tissues and is associated with tumor cell differentiation, proliferation, invasion and metastasis; however, survivin is expressed at low levels in normal terminally differentiated adult tissues. Meanwhile, the expression level of survivin is also a negative prognostic factor for patients with cancer. These unique characteristics of survivin make it an exciting potential therapeutic target for cancer treatment. This review will discuss the biological characteristics of survivin and its potential use as a treatment target to reduce cancer cell proliferation.

Knockdown of survivin results in inhibition of epithelial to mesenchymal transition in retinal pigment epithelial cells by attenuating the TGFβ pathway

Proliferative vitreoretinopathy (PVR) is a common complication of open globe injury and the most common cause of failed retinal detachment surgery. The response by retinal pigment epithelial (RPE) cells liberated into the vitreous includes proliferation and migration; most importantly, epithelial to mesenchymal transition (EMT) of RPE plays a central role in the development and progress of PVR. For the first time, we show that knockdown of BIRC5, a member of the inhibitor of apoptosis protein family, using either lentiviral vector based CRISPR/Cas9 nickase gene editing or inhibition of survivin using the small-molecule inhibitor YM155, results in the suppression of EMT in RPE cells. Knockdown of survivin or inhibition of survivin significantly reduced TGFβ-induced cell proliferation and migration. We further demonstrated that knockdown or inhibition of survivin attenuated the TGFβ signaling by showing reduced phospho-SMAD2 in BIRC5 knockdown or YM155-treated cells compared to controls. Inhibition of the TGFβ pathway using TGFβ receptor inhibitor also suppressed survivin expression in RPE cells. Our studies demonstrate that survivin contributes to EMT by cross-talking with the TGFβ pathway in RPE cells. Targeting survivin using small-molecule inhibitors may provide a novel approach to treat PVR disease.

Clinicopathological and functional implications of the inhibitor of apoptosis proteins survivin and XIAP in esophageal cancer.

Based on their overexpression and important roles in progression and therapy-resistance in malignant diseases, the inhibitor of apoptosis protein family (IAP) members, survivin and X-linked inhibitor of apoptosis protein (XIAP), represent attractive candidates for targeted therapy. The present study investigated the prognostic and biological relevance of survivin and XIAP in esophageal squamous-cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Survivin and XIAP expression was analyzed by immunohistochemistry using tissue microarrays containing 120 ESCC and 90 EAC samples as well as the corresponding non-neoplastic esophageal mucosa samples. IAP expression levels were then correlated to clinicopathological parameters and overall survival to identify any associations. In addition, esophageal cancer cell lines were treated with the survivin inhibitor YM155, and the XIAP inhibitors Birinapant and GDC-0152 in vitro. Survivin and XIAP expression were significantly increased in EAC and ESCC when compared with tumor-adjacent mucosa. In patients with ESCC XIAP expression was associated with female gender and advanced tumor stages, and nuclear survivin expression was associated with poor grading. High XIAP expression was identified as an independent negative prognostic marker in ESCC. By contrast, XIAP inhibitors did not affect cancer cell viability in vitro, and the small molecule survivin inhibitor YM155 significantly reduced cell viability and proliferation in esophageal cancer cell lines. Western blot analysis revealed a dose dependent decrease of survivin accompanied by an increased poly (adenosine diphosphate-ribose) polymerase cleavage following YM155 treatment. These findings underline the potential role of survivin and XIAP in the oncogenesis of esophageal cancer and provide a rationale for future clinical studies investigating the therapeutic efficacy of IAP directed therapies in patients with esophageal cancer.

Expression of Inhibitor of Apoptosis Gene Family Members in Bladder Cancer Tissues and the 5637 Tumor Cell Line

Background:Apoptosis is suppressed in cancer tissues and tumor cell lines because anti-apoptosis genes are over- expressed. The inhibitor of apoptosis proteins (IAP) gene family contributes to control of apoptosis. The expression profile of eight genes of the IAP family in biopsies from patients with a history of bladder cancer and normal bladder tissues, as well as a bladder tumor cell line (5637), was assessed in the present study.Methods:Cancer tissue samples were obtained at surgery and the 5637 tumor cell line was cultured in RPMI1640 medium. Beyond tumor margins were selected as normal tissue. Expressional profile of interested genes was obtained by using specific primers and the real-time PCR method.Results:The results showed that expression of seven of the studied genes was up-regulated in cancer tissues and the cell line whereas BIRC4 (XIAP) was down-regulated in both.Conclusions:The results showed that these genes were expressed to a greater extent in cancer tissue and cancer cells than in normal tissues. The data suggested that over-expression of anti-apoptotic genes such as IAP family members, can trigger cells to escape from apoptosis.

XIAP underlies apoptosis resistance of renal cell carcinoma cells.

X-linked inhibitor of apoptosis (XIAP), a key member of the inhibitors of apoptosis protein family, can inhibit apoptosis by directly binding to the initiator caspase-9, −3 and −7, thereby promoting tumor cell survival during tumor progression. In the present study, XIAP basal expression levels were investigated and its contribution to the resistance to apoptosis was evaluated, in the RCC cell lines exposed to apoptosis-inducing drugs. This was investigated by histological methods and western blot analysis. Using RNA interference, elimination of XIAP in Caki-1 cells was also studied, and its contribution to the sensitivity to apoptosis induced through the intrinsic pathway was observed. Differences in XIAP expression were detected between ClearCa-2 and ClearCa-6 cell lines. ClearCa-6 cells with lower expression of XIAP were more sensitive to apoptosis-inducing drugs, compared with ClearCa-2 cells. However, the levels of XIAP expression in both cell lines were stable during apoptosis. Furthermore, a Caki-1 cell line with no XIAP expression was used, and was demonstrated to be more sensitive to the apoptosis induced by the mitochondrial pathway. These results suggested that downregulation of XIAP expression could enhance the sensitivity of RCC cells to apoptosis, and the basal expression of XIAP during apoptosis is stable. This may provide novel insight for targeted gene therapy against XIAP, in the clinic.

Livin serves as a prognostic marker for mid-distal rectal cancer and a target of mid-distal rectal cancer treatment.

Livin is a novel member of the inhibitor of apoptosis protein family, which has been identified to be expressed in various malignancies and is suggested to be associated with poor prognostic significance. However, no data are available concerning the significance of livin in mid-distal rectal cancer. In the present study, livin expression, and its association with clinicopathological characteristics and prognosis was examined in patients with mid-distal rectal cancer. Apoptotic susceptibility, invasion capacity and chemosensitivity of LoVo cells were investigated using small interfering RNA (siRNA)-mediated knockdown of livin. It was revealed that livin was highly expressed in mid-distal rectal cancer tissues compared with the normal rectal mucosal tissues. Livin expression was associated with pathological grade, extent of invasion (T stage) and extent of lymph node metastasis (N stage) of tumor, contributing to poor prognosis of mid-distal rectal cancer following surgery. The data suggest that aggressive surgery should be applied in patients with mid-distal rectal cancer with high expression of livin. It was also revealed that knockdown of livin by siRNA increased the apoptotic rate, suppressed invasion of LoVo cells, and decreased the half-maximal inhibitory concentration of oxaliplatin and 5-fluorouracil by ~50% in LoVo cells significantly compared with control groups. The data suggested that a combination of downregulation of livin and anticancer drugs may significantly decrease the toxicity of anticancer drugs. Taken together, the present study indicated that livin may be a promising target in clinical therapy of mid-distal rectal cancer.

Survivin and XIAP expression in distinct tumor compartments of surgically resected gastric cancer: XIAP as a prognostic marker in diffuse and mixed type adenocarcinomas.

There is considerable evidence that the inhibitor of apoptosis protein (IAP) family serves a role in tumorigenesis. The most studied IAP family members, survivin and X-linked inhibitor of apoptosis (XIAP), have been demonstrated to serve as biomarkers in distinct tumor entities. Thus, the present study aimed to investigate the expression levels of both IAPs in the tumor center, invasion front and lymph node metastases of surgically resected gastric cancer (GC) specimens. Tissue microarrays containing samples from 201 primary GCs were analyzed. IAP expression was detected using immunohistochemistry in different tumor compartments, normal mucosa and lymph node metastases. In addition, the association between the expression levels of these proteins, and clinicopathological parameters and overall survival was investigated. High levels of survivin and XIAP were evident in GC, when compared with normal mucosa, and were correlated with intestinal-type and well-differentiated GC, as well as low International Union Against Cancer stages. Increased XIAP expression was detected in lymph node metastases as compared with corresponding primary tumors. XIAP overexpression was identified to be an independent negative prognostic marker in diffuse and mixed type GC. These results suggest a potential role of survivin and XIAP in the early phase of gastric carcinogenesis. In addition, increased XIAP expression in lymph node metastases supports the observation that IAPs serve an essential role in metastatic tumor disease. Since XIAP expression was identified to be associated with poor survival in diffuse and mixed type GC, XIAP may serve as a novel therapeutic target in these types of GC.

Difference in the Inhibitory Effect of Temozolomide on TJ905 Glioma Cells and Stem Cells.

This study aims to determine the difference in the inhibitory effect of temozolomide (TMZ) on TJ905 glioma cells and stem cells. TJ905 cancer stem cells were isolated. Livin is a member of the inhibitor of apoptosis protein family. The TJ905 cells and cancer stem cells were transfected with a Livin-shRNA and negative-shRNA, respectively, and then treated with TMZ. At 48 h post-transfection, a cell counting kit 8 assay, flow cytometry, and real-time qPCR were performed to detect cell proliferation, the cell cycle, and the expression of the Caspase-3, -7, and -9 mRNAs, respectively. As a result, the suppressive effect of TMZ on TJ905 cells was more significant than its effect on TJ905 cancer stem cells. TMZ exerted an inhibitory effect on the growth of TJ905 glioma cells by arresting them at G0/G1 phase and arresting cancer stem cells at S phase in a dose-dependent manner. TMZ inhibited Livin mRNA expression and increased the expression of the Caspase-3, -7, and -9 mRNAs. Low Livin mRNA expression induced high levels of Caspase-3, -7, and -9 expressions, thus promoting the apoptosis of both TJ905 cells and cancer stem cells in response to TMZ treatment. The TJ905 cells transfected with the Livin-shRNA were more sensitive to TMZ, whereas the TJ905 glioma stem cells transfected with the Livin-shRNA showed no significant changes in their sensitivity to TMZ. In conclusion, the Livin gene may play an important role in the resistance mechanisms of TJ905 glioma cells and cancer stem cells. However, Livin had a more distinct role in TMZ resistance, cell proliferation, and the cell cycle in TJ905 glioma cells than in cancer stem cells.

Characterization of inflammasome-related genes in urine sediments of patients receiving intravesical BCG therapy

BackgroundNowadays, the intravesical Bacillus Calmette-Guérin (BCG) instillation is the method of choice for the postsurgical treatment of high-grade nonmuscle-invasive bladder cancer , to reduce both recurrence rate and risk of progression. BCG is hypothesized to correct the immune system disequilibrium occurring during carcinogenesis, through an immunostimulation with detrimental effects for tumoral cells. Inflammation plays a crucial role in tumor progression. The deregulation of inflammasomes upon carcinogenesis underlines its importance both in physiologic and pathologic human conditions. Nucleotide oligomerization domain-like receptors (NLRs) are key components of this molecular platform and the increase in expression of some members of nucleotide oligomerization domain-like receptors family (NLRP3, NLRP4, NLRP9, and NLR family apoptosis inhibitory protein [NAIP]) in urothelial carcinoma was already demonstrated in our previous work.The first aim of the present work was to estimate whether these inflammasome-related genes show alterations during BCG instillations. The expression levels of NLRP3, NLRP4, NLRP9, and NAIP were assessed in the urine sediments from patients, which underwent surgery for superficial high-grade bladder cancer and further subjected to serial BCG instillations. The eventual association between NLR expression and recurrence was also evaluated. The expression of CK20 mRNA as confirmed marker of bladder cancer was also assayed. MethodsUrine were sampled from patients harboring high-grade superficial bladder cancer and treated postsurgically with weekly BCG instillations for 6 weeks (induction cycle, I). Urine sediments were processed and resulting RNA was reverse transcribed and used for amplification by real-time PCR. ResultsAfter surgery, CK20 levels decreased significantly whereas NLRP4 and NLRP9 genes showed an increase. NLRP3 and NAIP remained substantially unmodified. CK20 mRNA decreased at the end of the induction cycle. NLRP3 did not show relevant modifications. The expression levels of NLRP4 and NLRP9 decreased significantly after 2 BCG administrations and remained substantially downregulated during the whole induction cycle. CK20 was higher in recurrence cases before BCG administration compared to the recurrence-free group, while no significant difference after BCG therapy was recorded. NLRP4 and NLRP9 were higher in patients with recurrence before BCG administration. ConclusionsThe study underlines the importance of NLRP4 and NLRP9 in urothelial carcinoma and if these preliminary data will be confirmed in larger cohort studies, the assessment of NLRP4 and NLRP9 expression levels could help to predict the BCG failure, playing a relevant role in decision making for early radical surgery.

Abstract 12: Evaluation of metformin and clotam combination for medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children. Standard treatment, which includes surgery, chemotherapy, and radiation, is successful for most patients, but survivors often suffer from significant long-term side effects affecting their neurocognitive and growth potential. Therefore, there is a critical need to understand the molecular processes that regulate MB growth and find less toxic therapies. Survivin (BIRC5) was identified as a protein belonging to the Inhibitor of Apoptosis Protein family that inhibits apoptosis by inhibiting caspase activation. Survivin expression is also associated with increased resistance of tumor cells to radiation and chemotherapy. In MB, several studies suggest that increased expression of survivin serves as a marker of tumor morphology and is associated with poor prognosis. Specificity protein 1 (Sp1) is one of the transcription factors that regulates the expression of survivin gene. Sp1 is also shown to be overexpressed in some cancer cells and is associated with poor prognosis. In this study, we are testing a strategy of targeting survivin in MB using anti-diabetic drug Metformin, which targets Sp1, and the NSAID Clotam (Tolfenamic acid, TA), which targets Sp1 and survivin. Dose response curves were established by treating MB cell lines DAOY and D283 with increasing concentrations of metformin or TA and measuring cell viability at 24 and 48h post-treatment using the CellTiter-Glo Cell Viability Assay. A time and dose dependent inhibition of cell proliferation was observed for both the drugs. Metformin dose response was then repeated in the presence of 25 or 50 µM TA. Our results indicate that TA significantly increased the growth inhibitory response of metformin. To further characterize this response, we determined the combination index (CI) using the Chou-Talalay method. We found that the CI values were between 0.85 to 0.61 for various combinations of metformin and TA, strongly suggesting a synergistic effect of the two drugs on cell proliferation. Combination of metformin and TA was also accompanied by a 3-4 fold increase in apoptotic cells at 48h post-treatment, as determined by AnnexinV staining of DAOY cells. Western blot analysis of DAOY cells treated with the two drugs also revealed a decrease in survivin and Bcl-2 protein levels, and an increase in cleaved-PARP. In conclusion, the anticancer activity of metformin in MB cells is enhanced in the presence of TA. Both drugs synergistically combine to inhibit MB cell proliferation and increase apoptosis, which in part may be the result of a decrease in survivin levels. This combination strategy may therefore represent a novel targeted therapy for MB. Studies are underway to determine whether this drug combination can enhance the response of chemotherapy in MB cells, since both TA and metformin are known to sensitize cancer cells to chemotherapy. Citation Format: Umesh T. Sankpal, W Paul Bowman, Jeffrey C. Murray, Riyaz Basha. Evaluation of metformin and clotam combination for medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 12. doi:10.1158/1538-7445.AM2017-12

Abstract 5185: Targeting inhibitors of apoptosis in combination with radiation for the treatment of head and neck squamous cell carcinoma

Abstract Background and purpose: Evasion of apoptosis contributes to radioresistance of head and neck squamous cell carcinoma (HNSCC), calling for novel strategies to overcome apoptotic resistance. Second mitochondria-derived activator of caspase (SMAC) - mimetics are a class of targeted drugs that antagonize selected inhibitor of apoptosis protein (IAP) to block pro-survial signalling, and eventually induce cell apoptosis. The present study was designed to investigate the radiosensitizing effect of one SMAC mimetic, LCL161, in HNSCC and the underlying mechanisms for radiosensitization. Material and methods: We analyzed the correlation between apoptotic molecules and HPV status in HNSCC via mRNA expression using The Cancer Genome Atlas (TCGA) database, as well as protein expression in 6 HNSCC cell lines by immunoblotting. We also examined cIAP1 expression on a tissue microarray (TMA) of HNSCC tumors, and assessed its correlation with HPV status and patient outcome. Clonogenic survival and WST-1 assays were carried out to explore the potential of LCL161 as a radiosensitizer in HNSCC cell lines. Cell cycle analysis, Annexin-V assays and immunoblotting were performed to investigate effects of LCL161 on radiation induced cell apoptosis. Human tumor xenografts were generated to explore the radiosensitization effect of LCL161 on HPV[-] tumors in vivo. Results: TCGA database analysis and immunoblotting from HNSCC cells revealed that expression of cIAP1, Survivin, DIABLO, p65, IK-b, TNF-α, BCL-XL, Caspase-7, and Caspase-8 were elevated in HPV[-] compared to HPV[+] HNSCC tumors. IHC staining showed cIAP1 expression was associated with HPV[-] status (p=0.0239). In addition, cIAP1 expression was significantly higher in HNSCC compared with adjacent normal tissue (p=0.0003). In univariate analysis, cIAP1 was significantly associated with poor overall survival (p=0.0359). LCL161 degraded cIAP1 at nanomolar concentrations but has minimal single-agent cytotoxicity with IC50 range of 32 μM – 95 μM. Interestingly, we found that LCL161 could induce radiosensitization only in HPV[-], but not in HPV[+] HNSCC cells. Mechanistic studies showed LCL161 mediated radiosensitisation was associated with increased cell apoptosis, with enhanced activation of caspases-3, -7, -8, -9, and PARP. Finally, in vivo data showed that LCL161 degraded cIAP1, activated caspases, and profoundly radiosensitized two human HPV[-] HNSCC xenograft models. Conclusion: Taken together, our results demonstrate for the first time that cIAP1 is a potential prognostic and therapeutic biomarker for HNSCC patients. Through targeting cIAP1 and other IAP family members, LCL161, radiosensitizes HNSCC tumors, particularly HPV [-] tumors, via caspase activation and apoptosis induction. Taken together, LCL161 holds promise for future clinical development as a novel radiosensitizer in the treatment of HPV[-] HNSCC tumors. Citation Format: Linlin Yang, Bhavna Kumar, Tianyun Li, Thenodoros N. Teknos, Arnab Chakravarti, Terence M. Williams. Targeting inhibitors of apoptosis in combination with radiation for the treatment of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5185. doi:10.1158/1538-7445.AM2017-5185

Evaluation of the Expression of the Inhibitor of Apoptosis Protein Family and Human Telomerase Reverse Transcriptase in Patients With Advanced Colorectal Adenoma

BackgroundIt has been reported that the expression of the inhibitor of apoptosis protein (IAP) family increases in patients with colon cancer. We evaluated the expression of the IAP family and human telomerase reverse transcriptase (hTERT) in normal colon mucosa from patients with advanced colorectal adenoma and investigated their features according to characteristics of advanced colorectal adenoma.MethodsWhile resections of polyps were performed in patients (n = 80) diagnosed with advanced colorectal adenoma or carcinoma in situ, additional normal tissues were obtained from the sigmoid colon. In healthy patients (n = 16), blind biopsies were performed on the sigmoid colon. The expression of the IAP family, including survivin, XIAP, cIAP1, and cIAP2, and hTERT, were analyzed by real-time PCR in both groups.ResultsA total of 80 advanced colorectal adenoma patients (71.3% male, mean age of 60.4 years) and 16 control patients were enrolled in this study. The mean ranking of cIAP2 was higher in the control group (68.88 vs. 44.43, P = 0.001). The expression levels of hTERT, survivin, XIAP, and cIAP from both groups showed no differences. The expression of survivin, XIAP, cIAP1, cIAP2, and hTERT depending on certain factors of advanced adenoma, including the number (two or fewer vs. three or more), size (smaller than 1 cm vs. larger than 1 cm), grade of dysplasia (low grade adenoma vs. high grade adenoma), pathology (tubular adenoma vs. villous adenoma), and presence of endometrial intraepithelial neoplasms, showed no significant correlations in the Mann-Whitney U-test.ConclusionsThe expression of the IAP family and hTERT, except cIAP2, in the normal mucosa of patients with advanced colorectal adenoma were not different from those of the control group. There were no differences in the IAP family and hTERT according to the characteristics of advanced adenoma.

Induction of apoptosis by Dae-Hwang-Mok-Dan-Tang in HCT-116 colon cancer cells through activation of caspases and inactivation of the phosphatidylinositol 3-kinase/Akt signaling

BackgroundDae-Hwang-Mok-Dan-Tang (DHMDT), a traditional Korean medicine, contains five species of medicinal plants and has been used to treat patients with digestive tract cancer for hundreds of years; however, its anticancer mechanism is poorly understood. In the present study, we investigated the proapoptotic effects of DHMDT in human colon cancer HCT-116 cells. MethodsCytotoxicity was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was detected using 4,6-diamidino-2-phenyllindile staining, agarose gel electrophoresis, and flow cytometry. The protein levels were determined using Western blot analysis. Caspase activity was measured using a colorimetric assay. ResultsTreatment with DHMDT resulted in a growth inhibition coupled with apoptosis induction, which was associated with the downregulation of members of IAP (inhibitor of apoptosis protein) family, including XIAP and survivin, and the activation of caspase-9 and -3 accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase and phospholipase C-γ1. DHMDT treatment also showed a correlation with the translocation of proapoptotic Bax to mitochondria, the loss of mitochondrial membrane permeabilization, and the cytochrome c release from the mitochondria to the cytosol. Moreover, DHMDT increased the levels of death receptor-associated ligands and enhanced activation of caspase-8 and cleavage of its substrate, Bid. However, the pan-caspase inhibitor could reverse DHMDT-induced apoptosis. In addition, DHMDT suppressed the phosphoinositide 3-kinase (PI3K)/Akt pathway, and treatment with a potent inhibitor of PI3K further increased the apoptotic activity of DHMDT. ConclusionOur data showed that DHMDT induces HCT-116 cell apoptosis by activating intrinsic and extrinsic apoptosis pathways and by suppressing the PI3K/Akt signal pathway; however, further studies are needed to identify the active compounds.