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M1639 Guggulsterone Induces Apoptosis in Colon Cancer Cells and Inhibits Tumor Growth in a Murine Xenograft Model: Therapeutic Potential in the Treatment of Colorectal Cancer
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celecoxib treated-AGS cells in dose dependent manner (0, 5, 10, 25, or 50 uM) (P < 0.05). The expression of FKHR and phosphorylated GSK3 also decreased in dose dependent manner in AGS cells. Procaspase 9 (47 kDa) has been cleaved into 37, 35, and 17 kDa fragments in the celecoxib-treatment group. However, these changes of cell signal transduction were not observed in the indometacin treated cells. In Vivo study, only one rat of 29 rats treated with MNNG had carcinoma at squamo-columnar junction, and microscopic glandular distension was frequently found in the MNNG treated rats than in control (P = 0.042). The expression of phosphorylated Akt was lower in the celecoxib-treated rats than control (P < 0.05). Conclusion: Anti-cancer effects of celecoxib on gastric cancer cells might be mediated by down-regulation of Akt, fork head transcriptional factor and phosphorylated GSK3 and up-regulation of caspase-9, In Vitro. The mediation of Akt-pathway was also supported by In Vivo experiment.
AGA Abstracts
Guggulsterone Induces Apoptosis in Colon Cancer Cells and Inhibits Tumor Growth in a Murine Xenograft Model: Therapeutic Potential in the Treatment of Colorectal Cancer Min Ji An, Jae Hee Cheon, TAE IL KIM, Won Ho Kim Background & Aims: The plant sterol guggulsterone (GGS) has been shown to exert antiinflammatory effects through NF-κB inhibition. Furthermore, it has been recently reported to have anti-tumor effects by enhancing apoptosis in some cancer cell types. However, it is unknown whether GGS is effective in the treatment of colorecal cancer. Moreover, the precise nature of their anti-tumor effects and their modes of action vis-à-vis mechanisms remain unknown. Therefore, we investigated the anti-tumor effects of GGS on colon cancer cells and elucidated its molecular mechanisms in terms of apoptosis and determined its effect of on the tumor growth of murine xenograft model. Methods: Human HT-29 cells and IEC-18 cells were treated with GGS for various times. The apoptotic effects of GGS were examined by cell survival assay including Hoechst 33258 staining and fluorescenceactivated cell sorting analysis (FACS). Western blot analyses were used to determine the levels of various down-stream intracellular proteins involved in apoptosis In Vitro. For In Vivo study, male nude mice were assigned to tumor control, GGS 20mg/kg group, or GGS 40mg/Kg group. Tumor xenograft was induced by subcutaneous injection with HT-29 cells. After the tumors reached an approximate volume of 100-150 mm3, the mice received daily intraperitoneal injections of GGS. Weight changes and tumor size were assessed daily. At day 15 after GGS injection, mice were sacrificed. Results: GGS significantly decreased cell viability and increased the numbers of apoptotic cells in colon cancer cells in a dose dependent manner but in nontransformed intestinal epithelial cells. Furthermore, it provoked chromatin condensation in colon cancer cells. GGS increased the activation of caspases-3, 8 and the levels of truncated Bid, Fas, p-JNK, and p-c-Jun, but decreased those of bcl-2 and IAPs. Xenograft tumors were significantly smaller in GGS-treated mice (both GGS 20 and GGS 40 groups) than in mice treated with the vehicle. Conclusion: Our data demonstrate that GGS induces apoptosis in colon cancer cells and inhibits growth of HT-29 xenografts In Vivo, which suggests that it has a therapeutic potential in the treatment of colorectal cancer.
M1642 Simvastatin Potentiates Sensitivity to Apoptosis in Microsatellite-Unstable Colon Cancer Cells Yeon Sil Jang, Eun Suk Choi, Dong Kyung Chang, Young-Ho Kim, Poong-lyul Rhee, Jae J. Kim, Jong Chul Rhee Background/Aim: Genetic or epigenetic inactivation of DNA mismatch repair (MMR) genes results in a strong mutator phenotype, known as the microsatellite instability (MSI). The defective MMR system occurs in about 15% of colorectal cancer and has been shown to confer resistance to various chemotherapeutic reagents. Numerous recent reports suggest that statins (hydroxyl-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. This study aims to compare the anticancer efficacy of simvastatin in between MMR-profient and -deficient colon cancer cells. Methods: MMR-deficient colon cell lines HCT116, HCT116/p53-/-, HCT116/p21-/and its MMR-proficient colon cell lines HCT116+ch3, HCT116+ch3/E6 were treated with simvastatin dose-and time-dependently. Results: MMR-deficient HCT116 cells were more sensitive to simvastatin than to MMR-proficient HCT116+ch3 as cytotoxicity was determined by MTT assay and apoptotic cell death was measured by annexin-V/PI staining. Apoptosis occurred time- and dose-dependently in both cells as indicated by Bcl-xL down-regulation, caspase-3 activation and PARP cleavage. However, their levels of changes were not different based on the existence or nonexistence of MLH1 and p53. On the other hand, simvastatin stimulated phosphorylation of c-jun which was completely abolished by the c-jun NH2terminal kinase (JNK) inhibitor SP600125, which also reduced the cytotoxic effect of simvastatin in both MMR-deficient and -proficient colon cells to the similar degree. Conclusion: Simvastatin seems to induce apotosis more in MMR-deficient cells. Its apoptotic effect involves JNK in HCT116 colon cancer cell series independent of their MLH1 and p53 expression status. Some other mechanisms may contribute to the higher level of apoptosis in MMR-deficient cells. Our findings suggest a potential of simvastatin for the therapies of microsatellite-unstable colon cancers resistant to currently used chemotherapeutic drugs.
M1640 CK2 Inhibition Sensitizes Chemopreventive-Induced Apoptosis of Colon Carcinoma Cells Serge Dionne, Denise Levesque, Ernest G. Seidman Background: Colon cancer is often resistant to apoptosis induced by single agents. Targeting several apoptosis pathways with combined therapy represents a promising therapeutic strategy. CK2 is a highly pleiotropic kinase whose activity is aberrantly elevated in diverse tumor types including colorectal carcinomas (CRC). Specific CK2 inhibitors were recently developed allowing study of affected signaling pathways and their role in proliferation and apoptosis. Aim: To investigate the effect of 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) in combination with other anticancer drugs on apoptosis of CRC cells. Methods: HT-29 and Caco-2 cells were cultured with DMAT +/- suboptimal doses of TRAIL, TWEAK, 15dPGJ2, rosiglitazone, GW9662, butyrate and sulindac sulfide. Apoptosis was measured with the M30 assay which specifically recognizes caspase cleaved cytokeratin 18. ROS were measured using H2DCFDA and loss of mitochondrial potential (MP) was measured with the cationic dye JC-1 by flow cytometry. Results: DMAT dose-dependently decreased CRC cell viability. This effect was partially antagonized in the presence of FCS. DMAT (40 µM) did not affect viability in presence of 10% FCS, as assessed by MTT assay. CRC cell viability after 72 h incubation with TRAIL 100 ng/ml (93%), TWEAK 100 ng/ml (91%), 15d-PGJ2 9 µM (67%), rosiglitazone 15 µM (105%), GW9662 15 µM (109%) and sulindac sulfide 35 µM (116%) was significantly decreased in the presence of 40 µM DMAT (80%, 81%, 41%, 82%, 89%, and 85%, respectively). Sensitization with combined treatment was less marked with butyrate 2.5 mM (79% vs 68%). Apoptosis rate (48 h) was enhanced by 1.6 to 4 fold in the presence of DMAT. Combined treatment resulted in enhanced caspase-3 and 8 activation, dissipation of MP and ROS production. Modulation of ERK phosphorylation, as well as prolonged JNK phosphorylation were also observed. Apoptosis was decreased by over 60% by pre-treatment with the caspase inhibitor zVAD and 30-50% by the antioxidant NAC. Notably, combined treatment resulted in decreased levels of the β-catenin/LCF target survivin and NF-kB target c-FLIP. Conclusions: We hypothesized that CK2 inhibition facilitates apoptotic commitment by decreasing NF-kB and β-catenin/LCF activities in cancer cells which resulted in decreased expression of c-FLIP and survivin. This in turn favors increased ROS production, JNK and caspases activation leading to cell death. Our results shows that sensitization of CRC cells to low doses of chemo preventive agents is achievable in the presence of suboptimal CK2 inhibition. Supported by the Dairy Foundation of Canada and the Canadian Institutes of Health Research.
M1643 GSK3 Activity Regulates Proliferation and Survival of Human Pancreatic Cancer Cells Benoit Marchand, Marie-Josee Boucher The glycogen synthase kinase-3 (GSK3) was initially described as a key enzyme involved in glycogen metabolism. Since then, it has been found to regulate a diverse range of cell functions including proliferation, differentiation and apoptosis. The role of GSK3 greatly depends on cellular context. Thus, the aim of the study was to clarify the role of GSK3 with respect to pancreatic cancer cell proliferation and survival. Methods. Cell proliferation and cell survival were evaluated in different human pancreatic cancer cell lines (BxPC-3, PANC1, MiaPaCa-2) following inhibition of GSK3 activity by different specific inhibitors (ARA014418, BIO, SB216763). Results. Inhibition of GSK3 activity led to a marked reduction in cell proliferation (evaluated by reduced cell number) of all pancreatic cancer cell lines tested. Further analysis revealed a hypophosphorylated status of pRb suggesting that inhibition of GSK3 activity induced a blockade of cell proliferation in the G1-phase of the cell cycle. Also, reduced cyclinD1 and cyclinE protein expressions were observed while the expression of the cell cycle inhibitor p21 was increased. Reduction in cyclin A protein expression was also observed. Furthermore, prolonged inhibition of GSK3 activity (24h) induced apoptosis, an effect that was visualized by DNA fragmentation, PARP cleavage and induction of caspase-7 activity. Importantly, cell apoptosis induced by GSK3 inhibition could not be rescue by addition of serum. Taking together, our results demonstrated that the activity of GSK3 contributes to the proliferation of human pancreatic cancer cells and is imperative for their survival. Hence, GSK3 activity could be an attractive new therapeutic target in the treatment of unresectable pancreatic cancer.
M1641 M1644 Anti-Gastric Cancer Effect of Celecoxib, a Selective COX-2 Inhibitor, Through Inhibition of AKT Signaling Nayoung Kim, Chung Hyun Kim, Ji Hyun Park, Mi Kyung Lee, Soo-Jeong Cho, Joo Sung Kim, Hyun Chae Jung, In Sung Song
Characterisation of Progenitor Cells in Colorectal Cancer and the Adult Human Colon Jamie Murphy, Sahira Khalaf, Rebecca Hands, Jun Tian, Norman S. Williams, Stephen A. Bustin
Background and aim: Previously we found that the anti-cancer effects of celecoxib on gastric cancer cells appeared to be mediated by cell cycle arrest and apoptosis, and not by antiCOX-2 effect or PGE2 suppression alone. This study was carried out In Vitro and In Vivo to investigate the mechanism of celecoxib associated anti-cancer effect. Methods: In Vitro experiment Western blot analysis of total Akt, phosphorylated Akt, fork head transcriptional factor, phosphorylated GSK3 and caspase-9 was performed in the condition of celecoxib or indomethacin-treatment in AGS cells. In Vivo experiment, vehicle (control) or celecoxib (5 and 10 mg/kg/day) was gavaged for 40 weeks to 45 Wistar rats, which had been treated by MNNG (N-mehtyl-N'-nitro-Nnitrosoguanidine) and 10% NaCl for 6 weeks as a gastric cancer inducer. Western blot analysis of tAkt and pAkt was performed In Vivo experiment. Results: The expression of phosphorylated Akt but not total Akt became lower in the
AGA Abstracts
Background: A subset of mutated stem cells within each colorectal cancer appear to initiate and sustain tumour growth. Prominin-1, which encodes the CD133 cell surface antigen, has been proposed as a marker for these cells. The purpose of this study was to clarify CD133 expression patterns in the normal human bowel, primary colorectal cancer, and colorectal cancer metastases. Methods: Normal intestinal tissue was obtained from 20 patients undergoing surgery for benign disease. Full thickness colorectal cancer samples were obtained from 20 patients, while matched samples of laser micro-dissected non-malignant adjacent epithelium, central and peripheral primary colorectal cancer, vascular invasion, and lymph node and liver metastasises were obtained from a further 13 patients. Assessment of prominin1 mRNA / CD133 protein expression was performed by RT-qPCR, immunohistochemistry,
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celecoxib treated-AGS cells in dose dependent manner (0, 5, 10, 25, or 50 uM) (P < 0.05). The expression of FKHR and phosphorylated GSK3 also decreased in dose dependent manner in AGS cells. Procaspase 9 (47 kDa) has been cleaved into 37, 35, and 17 kDa fragments in the celecoxib-treatment group. However, these changes of cell signal transduction were not observed in the indometacin treated cells. In Vivo study, only one rat of 29 rats treated with MNNG had carcinoma at squamo-columnar junction, and microscopic glandular distension was frequently found in the MNNG treated rats than in control (P = 0.042). The expression of phosphorylated Akt was lower in the celecoxib-treated rats than control (P < 0.05). Conclusion: Anti-cancer effects of celecoxib on gastric cancer cells might be mediated by down-regulation of Akt, fork head transcriptional factor and phosphorylated GSK3 and up-regulation of caspase-9, In Vitro. The mediation of Akt-pathway was also supported by In Vivo experiment.
AGA Abstracts
Guggulsterone Induces Apoptosis in Colon Cancer Cells and Inhibits Tumor Growth in a Murine Xenograft Model: Therapeutic Potential in the Treatment of Colorectal Cancer Min Ji An, Jae Hee Cheon, TAE IL KIM, Won Ho Kim Background & Aims: The plant sterol guggulsterone (GGS) has been shown to exert antiinflammatory effects through NF-κB inhibition. Furthermore, it has been recently reported to have anti-tumor effects by enhancing apoptosis in some cancer cell types. However, it is unknown whether GGS is effective in the treatment of colorecal cancer. Moreover, the precise nature of their anti-tumor effects and their modes of action vis-à-vis mechanisms remain unknown. Therefore, we investigated the anti-tumor effects of GGS on colon cancer cells and elucidated its molecular mechanisms in terms of apoptosis and determined its effect of on the tumor growth of murine xenograft model. Methods: Human HT-29 cells and IEC-18 cells were treated with GGS for various times. The apoptotic effects of GGS were examined by cell survival assay including Hoechst 33258 staining and fluorescenceactivated cell sorting analysis (FACS). Western blot analyses were used to determine the levels of various down-stream intracellular proteins involved in apoptosis In Vitro. For In Vivo study, male nude mice were assigned to tumor control, GGS 20mg/kg group, or GGS 40mg/Kg group. Tumor xenograft was induced by subcutaneous injection with HT-29 cells. After the tumors reached an approximate volume of 100-150 mm3, the mice received daily intraperitoneal injections of GGS. Weight changes and tumor size were assessed daily. At day 15 after GGS injection, mice were sacrificed. Results: GGS significantly decreased cell viability and increased the numbers of apoptotic cells in colon cancer cells in a dose dependent manner but in nontransformed intestinal epithelial cells. Furthermore, it provoked chromatin condensation in colon cancer cells. GGS increased the activation of caspases-3, 8 and the levels of truncated Bid, Fas, p-JNK, and p-c-Jun, but decreased those of bcl-2 and IAPs. Xenograft tumors were significantly smaller in GGS-treated mice (both GGS 20 and GGS 40 groups) than in mice treated with the vehicle. Conclusion: Our data demonstrate that GGS induces apoptosis in colon cancer cells and inhibits growth of HT-29 xenografts In Vivo, which suggests that it has a therapeutic potential in the treatment of colorectal cancer.
M1642 Simvastatin Potentiates Sensitivity to Apoptosis in Microsatellite-Unstable Colon Cancer Cells Yeon Sil Jang, Eun Suk Choi, Dong Kyung Chang, Young-Ho Kim, Poong-lyul Rhee, Jae J. Kim, Jong Chul Rhee Background/Aim: Genetic or epigenetic inactivation of DNA mismatch repair (MMR) genes results in a strong mutator phenotype, known as the microsatellite instability (MSI). The defective MMR system occurs in about 15% of colorectal cancer and has been shown to confer resistance to various chemotherapeutic reagents. Numerous recent reports suggest that statins (hydroxyl-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. This study aims to compare the anticancer efficacy of simvastatin in between MMR-profient and -deficient colon cancer cells. Methods: MMR-deficient colon cell lines HCT116, HCT116/p53-/-, HCT116/p21-/and its MMR-proficient colon cell lines HCT116+ch3, HCT116+ch3/E6 were treated with simvastatin dose-and time-dependently. Results: MMR-deficient HCT116 cells were more sensitive to simvastatin than to MMR-proficient HCT116+ch3 as cytotoxicity was determined by MTT assay and apoptotic cell death was measured by annexin-V/PI staining. Apoptosis occurred time- and dose-dependently in both cells as indicated by Bcl-xL down-regulation, caspase-3 activation and PARP cleavage. However, their levels of changes were not different based on the existence or nonexistence of MLH1 and p53. On the other hand, simvastatin stimulated phosphorylation of c-jun which was completely abolished by the c-jun NH2terminal kinase (JNK) inhibitor SP600125, which also reduced the cytotoxic effect of simvastatin in both MMR-deficient and -proficient colon cells to the similar degree. Conclusion: Simvastatin seems to induce apotosis more in MMR-deficient cells. Its apoptotic effect involves JNK in HCT116 colon cancer cell series independent of their MLH1 and p53 expression status. Some other mechanisms may contribute to the higher level of apoptosis in MMR-deficient cells. Our findings suggest a potential of simvastatin for the therapies of microsatellite-unstable colon cancers resistant to currently used chemotherapeutic drugs.
M1640 CK2 Inhibition Sensitizes Chemopreventive-Induced Apoptosis of Colon Carcinoma Cells Serge Dionne, Denise Levesque, Ernest G. Seidman Background: Colon cancer is often resistant to apoptosis induced by single agents. Targeting several apoptosis pathways with combined therapy represents a promising therapeutic strategy. CK2 is a highly pleiotropic kinase whose activity is aberrantly elevated in diverse tumor types including colorectal carcinomas (CRC). Specific CK2 inhibitors were recently developed allowing study of affected signaling pathways and their role in proliferation and apoptosis. Aim: To investigate the effect of 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) in combination with other anticancer drugs on apoptosis of CRC cells. Methods: HT-29 and Caco-2 cells were cultured with DMAT +/- suboptimal doses of TRAIL, TWEAK, 15dPGJ2, rosiglitazone, GW9662, butyrate and sulindac sulfide. Apoptosis was measured with the M30 assay which specifically recognizes caspase cleaved cytokeratin 18. ROS were measured using H2DCFDA and loss of mitochondrial potential (MP) was measured with the cationic dye JC-1 by flow cytometry. Results: DMAT dose-dependently decreased CRC cell viability. This effect was partially antagonized in the presence of FCS. DMAT (40 µM) did not affect viability in presence of 10% FCS, as assessed by MTT assay. CRC cell viability after 72 h incubation with TRAIL 100 ng/ml (93%), TWEAK 100 ng/ml (91%), 15d-PGJ2 9 µM (67%), rosiglitazone 15 µM (105%), GW9662 15 µM (109%) and sulindac sulfide 35 µM (116%) was significantly decreased in the presence of 40 µM DMAT (80%, 81%, 41%, 82%, 89%, and 85%, respectively). Sensitization with combined treatment was less marked with butyrate 2.5 mM (79% vs 68%). Apoptosis rate (48 h) was enhanced by 1.6 to 4 fold in the presence of DMAT. Combined treatment resulted in enhanced caspase-3 and 8 activation, dissipation of MP and ROS production. Modulation of ERK phosphorylation, as well as prolonged JNK phosphorylation were also observed. Apoptosis was decreased by over 60% by pre-treatment with the caspase inhibitor zVAD and 30-50% by the antioxidant NAC. Notably, combined treatment resulted in decreased levels of the β-catenin/LCF target survivin and NF-kB target c-FLIP. Conclusions: We hypothesized that CK2 inhibition facilitates apoptotic commitment by decreasing NF-kB and β-catenin/LCF activities in cancer cells which resulted in decreased expression of c-FLIP and survivin. This in turn favors increased ROS production, JNK and caspases activation leading to cell death. Our results shows that sensitization of CRC cells to low doses of chemo preventive agents is achievable in the presence of suboptimal CK2 inhibition. Supported by the Dairy Foundation of Canada and the Canadian Institutes of Health Research.
M1643 GSK3 Activity Regulates Proliferation and Survival of Human Pancreatic Cancer Cells Benoit Marchand, Marie-Josee Boucher The glycogen synthase kinase-3 (GSK3) was initially described as a key enzyme involved in glycogen metabolism. Since then, it has been found to regulate a diverse range of cell functions including proliferation, differentiation and apoptosis. The role of GSK3 greatly depends on cellular context. Thus, the aim of the study was to clarify the role of GSK3 with respect to pancreatic cancer cell proliferation and survival. Methods. Cell proliferation and cell survival were evaluated in different human pancreatic cancer cell lines (BxPC-3, PANC1, MiaPaCa-2) following inhibition of GSK3 activity by different specific inhibitors (ARA014418, BIO, SB216763). Results. Inhibition of GSK3 activity led to a marked reduction in cell proliferation (evaluated by reduced cell number) of all pancreatic cancer cell lines tested. Further analysis revealed a hypophosphorylated status of pRb suggesting that inhibition of GSK3 activity induced a blockade of cell proliferation in the G1-phase of the cell cycle. Also, reduced cyclinD1 and cyclinE protein expressions were observed while the expression of the cell cycle inhibitor p21 was increased. Reduction in cyclin A protein expression was also observed. Furthermore, prolonged inhibition of GSK3 activity (24h) induced apoptosis, an effect that was visualized by DNA fragmentation, PARP cleavage and induction of caspase-7 activity. Importantly, cell apoptosis induced by GSK3 inhibition could not be rescue by addition of serum. Taking together, our results demonstrated that the activity of GSK3 contributes to the proliferation of human pancreatic cancer cells and is imperative for their survival. Hence, GSK3 activity could be an attractive new therapeutic target in the treatment of unresectable pancreatic cancer.
M1641 M1644 Anti-Gastric Cancer Effect of Celecoxib, a Selective COX-2 Inhibitor, Through Inhibition of AKT Signaling Nayoung Kim, Chung Hyun Kim, Ji Hyun Park, Mi Kyung Lee, Soo-Jeong Cho, Joo Sung Kim, Hyun Chae Jung, In Sung Song
Characterisation of Progenitor Cells in Colorectal Cancer and the Adult Human Colon Jamie Murphy, Sahira Khalaf, Rebecca Hands, Jun Tian, Norman S. Williams, Stephen A. Bustin
Background and aim: Previously we found that the anti-cancer effects of celecoxib on gastric cancer cells appeared to be mediated by cell cycle arrest and apoptosis, and not by antiCOX-2 effect or PGE2 suppression alone. This study was carried out In Vitro and In Vivo to investigate the mechanism of celecoxib associated anti-cancer effect. Methods: In Vitro experiment Western blot analysis of total Akt, phosphorylated Akt, fork head transcriptional factor, phosphorylated GSK3 and caspase-9 was performed in the condition of celecoxib or indomethacin-treatment in AGS cells. In Vivo experiment, vehicle (control) or celecoxib (5 and 10 mg/kg/day) was gavaged for 40 weeks to 45 Wistar rats, which had been treated by MNNG (N-mehtyl-N'-nitro-Nnitrosoguanidine) and 10% NaCl for 6 weeks as a gastric cancer inducer. Western blot analysis of tAkt and pAkt was performed In Vivo experiment. Results: The expression of phosphorylated Akt but not total Akt became lower in the
AGA Abstracts
Background: A subset of mutated stem cells within each colorectal cancer appear to initiate and sustain tumour growth. Prominin-1, which encodes the CD133 cell surface antigen, has been proposed as a marker for these cells. The purpose of this study was to clarify CD133 expression patterns in the normal human bowel, primary colorectal cancer, and colorectal cancer metastases. Methods: Normal intestinal tissue was obtained from 20 patients undergoing surgery for benign disease. Full thickness colorectal cancer samples were obtained from 20 patients, while matched samples of laser micro-dissected non-malignant adjacent epithelium, central and peripheral primary colorectal cancer, vascular invasion, and lymph node and liver metastasises were obtained from a further 13 patients. Assessment of prominin1 mRNA / CD133 protein expression was performed by RT-qPCR, immunohistochemistry,
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