Survivin' Acute Myeloid Leukaemia-A Personalised Target for inv(16) Patients.

Despite recent advances in therapies including immunotherapy, patients with acute myeloid leukaemia (AML) still experience relatively poor survival rates. The Inhibition of Apoptosis (IAP) family member, survivin, also known by its gene and protein name, Baculoviral IAP Repeat Containing 5 (BIRC5), remains one of the most frequently expressed antigens across AML subtypes. To better understand its potential to act as a target for immunotherapy and a biomarker for AML survival, we examined the protein and pathways that BIRC5 interacts with using the Kyoto Encyclopedia of Genes and Genomes (KEGG), search tool for recurring instances of neighbouring genes (STRING), WEB-based Gene Set Analysis Toolkit, Bloodspot and performed a comprehensive literature review. We then analysed data from gene expression studies. These included 312 AML samples in the Microarray Innovations In Leukemia (MILE) dataset. We found a trend between above median levels of BIRC5 being associated with improved overall survival (OS) but this did not reach statistical significance (p = 0.077, Log-Rank). There was some evidence of a beneficial effect in adjusted analyses where above median levels of BIRC5 were shown to be associated with improved OS (p = 0.001) including in Core Binding Factor (CBF) patients (p = 0.03). Above median levels of BIRC5 transcript were associated with improved relapse free survival (p < 0.0001). Utilisation of a second large cDNA microarray dataset including 306 AML cases, again showed no correlation between BIRC5 levels and OS, but high expression levels of BIRC5 correlated with worse survival in inv(16) patients (p = 0.077) which was highly significant when datasets A and B were combined (p = 0.001). In addition, decreased BIRC5 expression was associated with better clinical outcome (p = 0.004) in AML patients exhibiting CBF mainly due to patients with inv(16) (p = 0.007). This study has shown that BIRC5 expression plays a role in the survival of AML patients, this association is not apparent when we examine CBF patients as a cohort, but when those with inv(16) independently indicating that those patients with inv(16) would provide interesting candidates for immunotherapies that target BIRC5.

Novel small-molecule LG1836 inhibits the in vivo growth of castration-resistant prostate cancer.

BACKGROUND: Androgen deprivation therapy (ADT) is the mainstay of treatment for castration-resistant prostate cancer (CRPC). Unfortunately, although ADT initially prolongs survival, most patients relapse and develop resistance. Clinical failure of these treatments in CRPC highlights the urgent need to develop novel strategies to more effectively block androgen receptor (AR) signaling and target other oncogenic factors responsible for ADT resistance. METHODS: We developed a small-molecule compound LG1836 and investigated the in vitro and in vivo activity of LG1836 against CRPC in cellular and animal models. RESULTS: LG1836 exhibits potent in vitro cytotoxicity in CRPC cells. Mechanistic studies demonstrated that LG1836 inhibits the expression of AR and AR variant 7, partially mediated via proteasome-dependent protein degradation. LG1836 also suppresses survivin expression and effectively induces apoptosis in CRPC cells. Significantly, as a single agent, LG1836 is therapeutically efficacious in suppressing the in vivo growth of CRPC in the subcutaneous and intraosseous models and extends the survival of tumor-bearing mice. CONCLUSIONS: These preclinical studies indicate that LG1836 is a promising lead compound for the treatment of CRPC.

The SMAC mimetic LCL161 is a direct ABCB1/MDR1-ATPase activity modulator and BIRC5/Survivin expression down-regulator in cancer cells.

Upregulation of ABCB1/MDR1 (P-gp) and BIRC5/Survivin promotes multidrug resistance in a variety of human cancers. LCL161 is an anti-cancer DIABLO/SMAC mimetic currently being tested in patients with solid tumors, but the molecular mechanism of action of LCL161 in cancer cells is still incompletely understood. It is still unclear whether LCL161 is therapeutically applicable for patients with ABCB1-overexpressing multidrug resistant tumors. In this study, we found that the potency of LCL161 is not affected by the expression of ABCB1 in KB-TAX50, KB-VIN10, and NTU0.017 cancer cells. Besides, LCL161 is equally potent towards the parental MCF7 breast cancer cells and its BIRC5 overexpressing, hormone therapy resistance subline MCF7-TamC3 in vitro. Mechanistically, we found that LCL161 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multi-drug efflux activity at low cytotoxic concentrations (i.e. 0.5xIC50 or less). Further analysis revealed that LCL161 also decreases intracellular ATP levels in part through BIRC5 downregulation. Therapeutically, co-treatment with LCL161 at low cytotoxic concentrations restored the sensitivity to the known ABCB1 substrate, paclitaxel, in ABCB1-expressing cancer cells and increased the sensitivity to tamoxifen in MCF7-TamC3 cells. In conclusion, LCL161 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide important information to physicians for designing a more "patient-specific" LCL161 clinical trial program in the future.

Does survivin overexpression enhance the efficiency of fibroblast cell-based wound therapy?

Cell-based wound therapy is faced with some limiting factors that decrease the therapeutic efficacy of transplanted cells. In this study, we aimed to genetically modify fibroblast cells with anti-apoptotic Survivin gene (Birc5) before cell transplantation. In vitro, pIRES2-eGFP-Survivin plasmid was transfected into the fibroblast cells and the growth curve was evaluated for transfected and normal cells performing MTT assay. In vivo, two 6-diameter cutaneous wounds were created at mice dorsal skin. Fibrin clot was used as a delivery vehicle to transfer cells into the wound bed. The effects of four treatment groups including (a) Cell-SVV-Clot (b) Cell-GFP-Clot, (c) Normal cell-Clot and, (d) Clot alone were evaluated. After 1,2,3,7 and 14 days post-transplantation, the wounds were photographed for evaluating the wound closure rate and wound samples were obtained. Angiogenesis and formation of granulated tissue were assessed via H&E staining for wound samples. The expression levels of Survivin, VEGF, and bFGF genes were also determined using qRT-PCR. The MTT assay showed similar proliferation potential of transfected cells with normal cells verifying that Survivin had no detrimental effect. Compared to the Normal cell-Clot group, the Survivin overexpression was seen for 3 days in the Cell-SVV-Clot group verifying the cell survival during the early stage of wound healing. The Survivin further upregulated VEGF and bFGF expressions resulting in more angiogenesis and formation of granulated tissue by day 3 and 14. The treated wounds with Cell-SVV-Clot were regenerated with a higher wound closure rate by day 7 compared to Normal cell-Clot and Clot groups. Survivin enhanced wound healing through induction of VEGF and bFGF at particular times post-wounding that led to a more structured-epidermis with higher angiogenesis and granulation tissue formation rate.

An engineered mouse embryonic stem cell model with survivin as a molecular marker and EGFP as the reporter for high throughput screening of embryotoxic chemicals in vitro.

Embryonic stem cell test (EST) is the only generally accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of EST for regulatory embryotoxicity screening are impeded by its technical complexity, long testing period, and limited endpoint data. In this study, a high throughput embryotoxicity screening based on mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by a human survivin promoter and a human cytomegalovirus promoter, respectively, was developed. These EGFP expressing mESCs were cultured in three-dimensional (3D) fibrous scaffolds in microbioreactors on a multiwell plate with EGFP fluorescence signals as cell responses to chemicals monitored noninvasively in a high throughput manner. Nine chemicals with known developmental toxicity were used to validate the survivin-based embryotoxicity assay, which showed that strongly embryotoxic compounds such as 5-fluorouracil, retinoic acid, and methotrexate downregulated survivin expression by more than 50% in 3 days, while weakly embryotoxic compounds such as boric acid, methoxyacetic acid, and tetracyclin showed modest downregulation effect and nonembryotoxic saccharin, penicillin G, and acrylamide had negligible downregulation effect on survivin expression, confirming that survivin can be used as a molecular endpoint for high throughput screening of embryotoxicants. The potential developmental toxicity of three Chinese herbal medicines were also evaluated using this assay, demonstrating its application in in vitro developmental toxicity test for drug safety assessment.

Evaluation of immunohistochemical expression of survivin and its correlation with -31G/C gene polymorphism in colorectal cancer.

Colorectal cancer (CRC) placed among the most common neoplasm. Survivin is a member of the inhibitor apoptosis gene family. This gene could be associated with aggressive behavior in numerous types of cancers. The aim of the present study was to evaluate the immunohistochemical expression of survivin gene and its correlation with -31G/C polymorphism in CRC patients. This case-control study was performed on 90 cases: 30 adenocarcinoma, 30 adenomatous polyp, and 30 normal colon. Immunohistochemical expression of survivin evaluated on formalin-fixed paraffin-embedded tissue and -31G/C polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis. Results showed that the subjects carrying C/C genotype with 43.3% (p = 0.002' OR = 12.188, CI = 2.530-58.720) and G/C genotype with 43.3% (p = 0.032' OR = 4.432, CI = 1.133-17.341) significantly had increased risk of CRC compared with subjects carrying GG genotype. Allelic frequencies showed statistically significant difference (p = 0.001) among adenocarcinoma (G = 35%, C = 65%), adenomatous (G = 43.3, C = 56.7), and normal group (G = 68.3, C = 31.7). Immunohistological evaluation showed nuclear survivin protein expression in patients with the CC genotype higher than in patient with the GG and GC genotypes (p = 0.002). The results suggest that C allele of - 31G/C polymorphism in survivin might be cooperative in CRC development.

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

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

NY-ESO-1- and survivin-specific T-cell responses in the peripheral blood from patients with glioma.

The prognosis for patients with glioblastoma is grim. Ex vivo expanded tumor-associated antigen (TAA)-reactive T-cells from patients with glioma may represent a viable source for anticancer-directed cellular therapies. Immunohistochemistry was used to test the survivin (n = 40 samples) and NY-ESO-1 (n = 38 samples) protein expression in tumor specimens. T-cells from peripheral blood were stimulated with TAAs (synthetic peptides) in IL-2 and IL-7, or using a combination of IL-2, IL-15 and IL-21. CD4+ and CD8+ T-cells were tested for antigen-specific proliferation by flow cytometry, and IFN-γ production was tested by ELISA. Twenty-eight out of 38 cancer specimens exhibited NY-ESO-1 protein expression, 2/38 showed a strong universal (4+) NY-ESO-1 staining, and 9/40 cancer lesions exhibited a strong (4+) staining for survivin. We could detect antigen-specific IFN-γ responses in 25% blood samples for NY-ESO-1 and 30% for survivin. NY-ESO-1-expanded T-cells recognized naturally processed and presented epitopes. NY-ESO-1 or survivin expression in glioma represents viable targets for anticancer-directed T-cells for the biological therapy of patients with glioma.

Role of Survivin and p53 Expression in Response of Primary Culture of Ovarian Cancer Cells to Treatment With Chemotherapeutic Agents.

BACKGROUND: Ovarian cancer is associated with a high relapse rate and is the fifth leading cause of cancer deaths in women. The genetic profile of a tumor is responsible for deciding response to chemotherapeutic agents. In this study, we investigate the relation between survivin and p53 expression and response to chemotherapeutic agents of primary cultures of ovarian cancer cells established from ascitic fluid. MATERIALS AND METHOD: Ascitic fluid and Dulbecco's modified Eagle medium was mixed in equal proportion in culture flasks and incubated to establish primary culture. The cells were treated with different combinations of carboplatin and paclitaxel with and without survivin small interfering RNA transfection. Cell survival was estimated by MTT assay. Survivin and p53 expression was quantified by real-time polymerase chain reaction. RESULTS: Out of 19 ascitic fluid samples, 13 primary cultures of ovarian cancer cells were established. The half maximal inhibitory concentration doses of carboplatin (≥70 µg/mL) and paclitaxel (≥18 µg/mL) were high for 10/13 and 5/13 patients, respectively. Survivin messenger RNA expression was significantly downregulated on treatment with carboplatin (100 µg/mL), paclitaxel (12.5 µg/mL), and a combination of carboplatin (50 µg/mL) and paclitaxel (6.25 µg/mL). Only paclitaxel-treated ovarian cancer cells showed decrease in expression of p53. Survivin small interfering RNA increased sensitivity of the primary cultures to chemotherapeutic agents. CONCLUSIONS: The present study highlights the fact that establishing primary cultures from ascitic fluid may help to develop personalized treatment regime for individual patients based on their molecular profile. Our study also shows that supplementing taxols drugs with survivin inhibitors may prove to be beneficial in the treatment of ovarian cancer patients.

Cloning, Expression, and Purification of the Human Synthetic Survivin Protein in Escherichia coli Using Response Surface Methodology (RSM).

Survivin is one of the novel members of the apoptosis inhibitor protein family in humans. The main activity of the Survivin protein is to suppress caspases activity resulting in negative regulation of apoptosis. Survivin protein can be a potential target for the treatment of cancers between cancerous and normal cells. In the present research, the synthetic Survivin gene with PelB secretion signal peptide was cloned into a prokaryotic expression vector pET21a. The recombinant plasmid pET21a-PelB-Surv was expressed in Escherichia coli (E.coli) BL21, and the relative molecular mass of expressed protein was calculated 34,000 g/mol, approximately. The recombinant protein was purified through chromatography column and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Response surface methodology (RSM) was used to design 20 experiments for optimization of IPTG concentration, post-induction period, and cell density of induction (OD600). The optimum levels of the selected parameters were successfully determined to be 0.28 mM for IPTG concentration, 10 h for post-induction period, and 3.40768 for cell density (OD600). These findings resulted in 4.14-fold increases in the Survivin production rate of optimum expression conditions (93.6363 mg/ml).

CircANKRD52 Promotes the Tumorigenesis of Hepatocellular Carcinoma by Sponging miR-497-5p and Upregulating BIRC5 Expression.

CircRNAs participate in the pathogenesis of a variety of cancers. Previous studies showed that baculoviral IAP repeat containing 5 (BIRC5) can promote tumor progression. But, the mechanisms by which circRNAs regulate BIRC5 expression in hepatocellular carcinoma (HCC) remain unknown. The clinical prognosis of BIRC5 or miR-497-5p expression in patients with HCC was assessed by TCGA RNA-seq dataset. hsa_circ_0026939 (circANKRD52) or BIRC5 was identified to bind with miR-497-5p by luciferase gene report, RIP and circRIP assays. MTT, colony formation, Transwell assays and a xenograft tumor model were used to estimate the role of miR-497-5p or circANKRD52 in HCC cells. As a result, we found that elevated expression of BIRC5 or decreased expression of miR-497-5p was linked to poor survival in HCC. Restored expression of miR-497-5p repressed cell proliferation, colony formation and invasiveness by targeting BIRC5, but its inhibitor showed the opposite results. Furthermore, circANKRD52 possessed a tumor-promoting effect by acting as a sponge of miR-497-5p and thereby upregulated BIRC5 in HCC cells. In conclusion, our findings demonstrated that circANKRD52 enhances the tumorigenesis of HCC by sponging miR-497-5p and upregulating BIRC5 expression.

Survivin expression is essential for early activation of hepatic stellate cells and fibrosis progression in chronic liver injury.

AIM: Hepatic fibrosis in injured liver is characterized by the activation of hepatic stellate cells (HSCs) from their quiescent state. Survivin (BIRC5) is one of the key genes that are upregulated during activation of HSCs but their role in HSC activation and fibrosis progression is unknown. Here, we have investigated the role of survivin protein in early fibrogenic activation of HSCs and fibrosis progression in chronic liver injury. MATERIALS & METHODS: Primary quiescent HSCs were isolated from healthy mice liver through perfusion and cultured for fibrogenic activation. Survivin expression was suppressed by its pharmacological suppressant, YM155. We developed chronic liver injury induced fibrotic mice model through administrating repeated dose of CCl4 for 2 weeks and 4 weeks. Mice were pre-treated with YM155 a week before CCl4 administration till 2nd week of dosing and then discontinued. Hepatic parameters were characterized and underlying mechanisms were investigated. KEY FINDINGS: Survivin expression gradually increased along with the expression of αSMA, collagen I activation maker in HSCs during their activation from quiescent state. Survivin suppression through YM155 downregulated αSMA, collagen I. Pre-treatment of YM155 in mice ceased the early activation of HSCs and onset of fibrosis in injured liver. However, discontinuation of YM155 initiated the activation of HSCs and fibrosis progression that shows survivin expression in HSCs is essential for their early activation and onset of liver fibrosis. SIGNIFICANCE: Survivin expression induces with activation of HSCs and drives onset of liver fibrosis in injured liver. Targeting survivin protein in activated HSCs could be a potential anti-fibrotic therapeutic approach in chronic liver injury.

YAP promotes sorafenib resistance in hepatocellular carcinoma by upregulating survivin.

BACKGROUND: Sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), but its use is hampered by secondary drug resistance. Yes-associated protein (YAP) is a downstream effector of the Hippo signaling pathway, which is crucial for liver tumorigenesis. As yet, however, the mechanism underlying sorafenib resistance and the role of YAP therein is not fully understood and needs to be explored further. METHODS: Western blotting, flow cytometry and CCK-8 assays were used to assess the role of YAP in HCC sorafenib resistance. Next, qRT-PCR and Western blotting were performed to identify survivin as a YAP downstream effector, and rescue experiments were performed to confirm that YAP induces sorafenib resistance via survivin. Additionally, Western blotting, flow cytometry and in vivo xenograft models were used to evaluate the effect of verteporfin in combination with sorafenib on HCC. RESULTS: We found that sorafenib enhances YAP nuclear accumulation and activation, thereby promoting sorafenib resistance through inhibiting apoptosis in HCC cells. In addition, we found that survivin acts as a downstream mediator of YAP to resist sorafenib-induced apoptosis. Pharmacological inhibition of YAP by verteporfin increased the sensitivity of HCC cells to sorafenib and reversed sorafenib resistance. Moreover, verteporfin in combination with sorafenib significantly suppressed in vivo HCC tumor growth. CONCLUSIONS: Our data indicate that YAP promotes sorafenib resistance through upregulation of survivin expression in HCC cells. Targeting YAP may be a therapeutic strategy to improve the antitumor effects of sorafenib in HCC.

The Evaluation of Survivin and Bcl-2 Expression on the Medical Radiation Doses for Neural Tube Defect Development.

AIM: To investigate the effects of different radiation doses on the development of the neural tube defect in chick embryos using computed tomography (CT), and assess its correlation with survivin and Bcl-2 expressions. MATERIAL AND METHODS: A total of 150 chicken eggs were used and grouped into five categories. In Group 1 (n=30), the embryos were not exposed to radiation. In Group 2 (n=30), the embryos were irradiated using lung cancer screening chest CT protocol. In Groups 3 and 4 (n=30 each), the abdominopelvic and adult routine head CT protocols, respectively, were used to irradiate the embryos. In Group 5 (n=30), the embryos were irradiated using adult brain perfusion CT protocol. Subsequently, the embryos were examined under a stereomicroscope to assess the presence of neural tube developmental abnormalities. Moreover, immunohistochemical staining was performed to determine the survivin and Bcl-2 expression levels. RESULTS: The risk of developing neural tube defect increased with the amount of exposed radiation. Moreover, no significant correlation was observed between the survivin and Bcl-2 expression levels and the radiation dose. CONCLUSION: Overall, the results of this study indicate that the radiation from CT may cause neural tube defect in chicken embryos.

Inhibition of Sp1-mediated survivin and MCL1 expression cooperates with SLC35F2 and myeloperoxidase to modulate YM155 cytotoxicity to human leukemia cells.

Although YM155 is reported to suppress survivin (also known as BIRC5) expression in cancer cells, its cytotoxic mechanism in human acute myeloid leukemia (AML) cells has not been clearly resolved. In this study, we analyzed the mechanistic pathways that modulate the sensitivity of human AML U937 and HL-60 cells to YM155. YM155 induced apoptosis in AML cells, which was characterized by p38 MAPK phosphorylation and downregulation of survivin and MCL1 expression. Phosphorylated p38 MAPK causes autophagy-mediated Sp1 degradation, thereby inhibiting the transcription of survivin and MCL1. The reduction of survivin and MCL1 levels further facilitated Sp1 protein degradation through autophagy. The restoration of Sp1, survivin, or MCL1 expression protected U937 and HL-60 cells from YM155-mediated cytotoxicity. U937 and HL-60 cells were continuously exposed to hydroquinone (HQ) to generate U937/HQ and HL-60/HQ cells, which showed increased SLC35F2 expression. The increase in SLC35F2 expression led to an increase in the sensitivity of U937/HQ cells to YM155-mediated cytotoxicity, whereas no such effect was observed in HL-60/HQ cells. Of note, myeloperoxidase (MPO) activity in HL-60 and HL-60/HQ cells enhanced YM155 cytotoxicity in these cells, and the enforced expression of MPO also increased the sensitivity of U937 cells to YM155. Taken together, we conclude that p38 MAPK-modulated autophagy inhibits Sp1-mediated survivin and MCL1 expression, which, in turn, leads to the death of U937 and HL-60 cells following YM155 treatment. In addition, our data indicate that SLC35F2 increases the sensitivity of U937 cells to YM155-mediated cytotoxicity, whereas MPO enhances YM155 cytotoxicity in U937 and HL-60 cells.

Expression of SIRT1 and survivin correlates with poor prognosis in esophageal squamous cell carcinoma.

This study assessed the association of sirtuin type 1 (SIRT1) and survivin expression with the clinicopathological features and survival of esophageal squamous cell carcinoma (ESCC) patients after concurrent chemoradiotherapy.SIRT1 and survivin proteins were immunohistochemically stained in 93 ESCC tissue specimens.SIRT1 was expressed in ESCC (80.6% vs 25.8% in normal mucosae) and survivin was expressed in 67.7% of ESCC vs 19.4% normal tissues (P < .01), and SIRT1 expression was associated with survivin expression (r = 0.39, P < .05). Furthermore, expression of both SIRT1 and survivin was associated with tumor size, depth of tumor invasion, tumor differentiation, lymph node metastasis, advanced clinical stage, and chemoradiotherapy (P < .05) as well as poor progression-free survival (PFS; P < .05) of ESCC patients after concurrent chemoradiotherapy (P < .05). Patient age, chemotherapy, tumor size, clinical stage, lymph node metastasis, and SIRT1 and survivin expression were independent PFS predictors (P < .05).Expression of both SIRT1 and survivin was associated with poor ESCC PFS.

The prognostic significance of Caspase-3 and survivin expression in colorectal cancer patients.

PURPOSE: To investigate the expressions of caspase-3 and survivin in colorectal cancer patients and their possible associations with clinicopathological parameters and the oncological outcome. METHODS: Between January 2008 and December 2011, 85 patients with sporadic colorectal cancer were submitted to colectomy with curative intent. Postoperatively, all patients were followed every three months up to the 36th month. Immunohistochemical detection of the apoptosis-related proteins was carried out on 4-µm-thick deparaffinized sections from all primary tumors. Univariate and multivariate analyses were performed by using the R software for Windows, version 3.3.2. RESULTS: Setting the cut-off point for caspase-3 positivity at 5%, 48% of the patients were characterized as caspase-3(+). Caspase-3 positivity was not found related either to any clinicopathological parameter or to the oncological outcome. Choosing simple survivin positivity as the cut-off point for its expression, 78% of the patients were considered as survivin(+). Survivin inexpression predisposed to poorly differentiated tumors of advanced T stage. However, neither a dismal nor a favorable prognostic role for survivin expression or inexpression was disclosed. By dividing all enrolled patients in four different groups, a trend for worse 3-year overall survival rate in the caspase-3(-)/survivin(-) subgroup of patients was noticed (p=0.067). CONCLUSION: Caspase-3 expression was unrelated to the oncological outcome in colorectal cancer patients. The proposed favorable prognostic role for survivin inexpression was not confirmed. On the contrary, survivin(-) tumors were mainly of poor differentiation and advanced T stage. An inverse relationship between caspase-3 and survivin expressions was also not confirmed. Future studies focusing on specific survivin isoforms expression or inexpression may give answers on apoptotic-antiapoptotic interactions on cancer cell death.

Selected protein expression in a new prognostic model for patients with non-muscle-invasive bladder cancer.

INTRODUCTION: After transurethral resection of a bladder tumor, patients frequently have a recurrence of the disease, thereby requiring adjuvant therapy. PURPOSE: The study aimed to determine the prognostic value of expression levels of p53, Ki-67, and survivin, and to develop a new prognostic model for patients with non-muscle-invasive bladder cancer (NMIBC) after transurethral resection of a bladder tumor. METHODS: The study group consisted of 101 patients with primary NMIBC. Univariate followed by multivariate Cox proportional hazard regression analysis was performed to obtain a model including the smallest possible number of descriptive variables with the highest statistical significance and impact on risk. RESULTS: The RECINT model (RECurrence In Not Treated) including factors independently associated with cancer recurrence (tumor size [HR 1.148; p = 0.034], intensity of the color reaction for p53 [HR 1.716; p = 0.008], Ki-67 [HR 3.001; p = 0.022], and survivin [HR 1.461; p = 0.021]) adequately stratified recurrence free-survival (R2 = 0.341, p < 0.001) in patients with primary NMIBC. Patients with the lowest RECINT score (0-6) had the lowest probability of cancer recurrence (1- and 5-year recurrence of 16%) in comparison with other groups (p < 0.001). CONCLUSIONS: The RECINT model may be useful for stratifying the risk of recurrence in patients with non-muscle-invasive bladder cancer and may allow for identification of those who may benefit the most from adjuvant BCG immunotherapy.

Assessment of Relationship Between Expression of Survivin Protein and Histopathology Diagnosis and Malignancy Severity in Colon Specimen.

BACKGROUND: Survivin is a member of the inhibitor of an apoptosis protein family that has been shown to inhibit apoptosis, promote cell proliferation and enhance angiogenesis. In this study, the survivin protein expression in normal, colon polyp, and adenocarcinoma tissues was investigated. METHODS: Immunohistochemical staining for nuclear survivin was carried out on 45 normal colon tissue samples, 38 samples of a colonic polyp, and 37 cases of colon adenocarcinoma operated by colonoscopy or colectomy. The percentages of cells that expressed survivin were classified qualitatively into four categories (0, 1+, 2+, and 3+) based on the intensity of staining and the percentage of cells. An area of samples with colon polyp diagnosis or colon adenocarcinoma that had no microscopic pathology was considered as normal tissues. RESULTS: Survivin protein expression was negative in all cases of normal colon tissue samples while it was expressed in 31 out of 38 colon polyp specimens (81.5%) and in 35 out of 37 (94.5%) colon adenocarcinoma samples. Amount of expression in the colon adenocarcinoma (p < 0.001) was significantly higher than the amount of expression in the colon polyp. There was not a significant correlation between the survivin protein expression and the low and high grade adenocarcinoma (p = 0.874). CONCLUSIONS: Survivin protein was not expressed in normal colon tissues and its amount was higher in the colonic adenocarcinoma compared to the colon polyp. Due to the variations in the intensity of expression in colon polyp (changing from negative to + 3), this marker cannot be used for differentiating the polyp from the adenocarcinoma.

Survivin modulation in the antimelanoma activity of prodiginines.

Melanoma is a type of skin cancer with an elevated incidence of metastasis and chemoresistance. Such features hamper treatment success of these neoplasms, demanding the search for new therapeutic options. Using a two-step resin-based approach, we recently demonstrated that cytotoxic prodiginines bind to the inhibitor of apoptosis protein, survivin. Herein, we explore the role of survivin in melanoma and whether its modulation is related to the antimelanoma properties of three cytotoxic prodiginines (prodigiosin, cyclononylprodigiosin, and nonylprodigiosin) isolated from marine bacteria. In melanoma patients and cell lines, survivin is overexpressed, and higher levels negatively impact survival. All three prodiginines caused a decrease in cell growth with reduced cytotoxicity after 24 h compared to 72 h treatment, suggesting that low concentrations promote cytostatic effects in SK-Mel-19 (BRAF mutant) and SK-Mel-28 (BRAF mutant), but not in SK-Mel-147 (NRAS mutant). An increase in G1 population was observed after 24 h treatment with prodigiosin and cyclononylprodigiosin in SK-Mel-19. Further studies indicate that prodigiosin induced apoptosis and DNA damage, as detected by increased caspase-3 cleavage and histone H2AX phosphorylation, further arguing for the downregulation of survivin. Computer simulations suggest that prodigiosin and cyclononylprodigiosin bind to the BIR domain of survivin. Moreover, knockdown of survivin increased long-term toxicity of prodigiosin, as observed by reduced clonogenic capacity, but did not alter short-term cytotoxicity. In summary, prodiginine treatment provoked cytostatic rather than cytotoxic effects, cell cycle arrest at G0/G1 phase, induction of apoptosis and DNA damage, downregulation of survivin, and decreased clonogenic capacity in survivin knockdown cells.