Investigation of the Effect of Zebularine in Comparison to and in Combination with Trichostatin A on p21Cip1/Waf1/ Sdi1, p27Kip1, p57Kip2, DNA Methyltransferases and Histone Deacetylases in Colon Cancer LS 180 Cell Line

Background: The heart of the cell cycle regulatory machine is a group of enzymes named cyclin-dependent kinases (Cdks). The active form of these enzymes includes a kinase and its partner, a cyclin. The regulation of cyclin-Cdk complexes is provided by Cdk inhibitors (CKIs) such as Cip/Kip family comprising p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2. The hypermethylation and deacetylation of Cip/Kip gene family seem to be frequent in numerous cancers. It has been indicated that increased expression of DNMTs and HDACs contributes to cancer induction. Previously, we reported the effect of DNA demethylating agents and histone deacetylase inhibitors on histone deacetylase 1, DNA methyltransferase 1, and CIP/KIP family in colon cancer. The current study was designed to evaluate the effect of zebularine in comparison to and in combination with trichostatin A (TSA) on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, DNA methyltransferases (DNMT1, 3a and 3b) and histone deacetylases (HDAC1, 2, and 3) genes expression, cell growth inhibition and apoptosis induction in colon cancer LS 180 cell line. Materials and Methods: The colon cancer LS 180 cell line was cultured and treated with zebularine and TSA. To determine cell viability, apoptosis, and the relative expression level of the genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively. Results: Both compounds significantly inhibited cell growth, and induced apoptosis. Furthermore, both compounds increased p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 significantly. Additionally, zebularine and TSA decreased DNMTs and HDACs gene expression respectively. Conclusion: The zebularine and TSA can reactivate the CIP/KIP family through inhibition of DNMTs and HDACs genes activity.     

Effect of Sodium Butyrate on p16INK4a,p14ARF,p15INK4b,Class I HDACs (HDACs 1, 2, 3) Class II HDACs (HDACs 4, 5, 6), Cell Growth Inhibition and Apoptosis Induction in Pancreatic Cancer AsPC-1 and Colon Cancer HCT-116 Cell Lines

Background: In higher eukaryotes, cell-cycle transitions are regulated by different cyclin-dependent kinases (Cdks) and Cdk inhibitors (CKIs). CKIs include two groups, the Ink4 (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the Cip/Kip (p21Cip1, p27Kip1, and p57Kip2) families. The hyperactivity of histone deacetylases (HDACs) is associated with cancer induction. Histone deacetylase inhibitors (HDACIs) such as sodium butyrate (NaBT) can inhibit HDAC activity resulting in apoptosis induction. The present study was designed to investigate the effect of sodium butyrate on p16INK4a, p14ARF, p15INK4b, class I HDACs (HDACs 1, 2, 3), and class II HDACs (HDACs 4, 5, 6), cell growth inhibition, and apoptosis induction in pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines. In fact, we want to know whether sodium butyrate can reactivate Ink4 and Cip/Kip families by HDACs inhibition. Materials and Methods: The AsPC-1 and HCT-116 cells were treated with sodium butyrate at different periods. Then, the MTT assay, cell apoptosis assay, and qRT-PCR were done to determine viability, apoptosis, and the relative expression level of the genes respectively. Results: The sodium butyrate increased p16INK4a, p14ARF, and p15INK4b and decreased class I and II HDACs significantly. Besides, HCT-116 cell was more sensitive to sodium butyrate in comparison to AsPC-1 cell. Conclusion: The sodium butyrate can reactivate the p16INK4a, p14ARF, and p15INK4b through inhibition of HDACs in AsPC-1 and HCT-116 cell lines.     

Antitumour effect of cyclin-dependent kinase inhibitors (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)) on malignant glioma cells

Cyclin-dependent kinase inhibitors (CDKIs) are considered as novel anticancer agents because of their ability to induce growth arrest or apoptosis in tumour cells. It has not yet been fully determined, however, which CDKI is the best candidate for the treatment of malignant gliomas and whether normal brain tissues are affected by CDKI expression. Using recombinant adenoviral vectors that express CDKIs (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)), we compared the antitumour effect of CDKIs on malignant glioma cell lines (A172, GB-1, T98G, U87-MG, U251-MG and U373-MG). p27(KIP1) showed higher ability to suppress the growth of all tumour cells tested than other CDKIs. Interestingly, overexpression of p27(KIP1) induced autophagic cell death, but not apoptosis in tumour cells. On the other hand, p27(KIP1) overexpression did not inhibit the viability of cultured astrocytes (RNB) nor induced autophagy. Overall, our findings suggest that gene transfer of p27(KIP1) may be a promising approach for the therapy of malignant gliomas.     

p21~(WAF1/CIP1)在乳腺癌中的表达及意义

目的　探讨p2 1WAF1/CIP1蛋白在乳腺癌中表达的临床意义。方法　运用免疫组化SP法半定量检测p2 1蛋白在癌旁正常乳腺组织、乳腺癌组织中的表达。结果　p2 1蛋白表达位于细胞核 ,呈棕黄色。在 2 0例癌旁正常乳腺组织中 ,无p2 1蛋白表达。在 69例乳腺癌组织中有 3 0例p2 1蛋白阳性表达。在乳腺癌组织中 ,随组织学分级升高 ,p2 1阳性率下降 (P <0 0 5 ) ,随临床分期升高 ,p2 1阳性率下降 (P <0 0 5 )。有淋巴结转移组p2 1阳性率低于无淋巴结转移组 (P <0 0 5 )。p2 1蛋白阳性表达者术后 5年无瘤生存率高于p2 1蛋白阴性者术后 5年无瘤生存率 (P <0 0 5 )。结论　p2 1蛋白可用来评估乳腺癌细胞分化情况及转移潜能 ,可判断乳腺癌患者预后。     

Sensitivity to the non-COX inhibiting celecoxib derivative, OSU03012, is p21(WAF1/CIP1) dependent

OSU03012 is a non-COX inhibiting celecoxib derivative with growth inhibiting and apoptotic activity in many cancer cell lines. To investigate mechanisms related to cell cycle proteins in growth inhibition and apoptosis induced by OSU03012, the primary human oral epithelial cell line, TE1177, was transformed with HPV16 E6 (TE/E6), HPV16 E7 (TE/E7) or empty vector (TE/V). TE/E6 cell lines exhibiting low levels of p53 and undetectable levels of p21(WAF1/CIP1) were sensitized to the growth inhibiting and apoptotic effects of OSU03012. The TE/E7 cell lines expressing low levels of Rb and elevated levels of p53 and p21(WAF1/CIP1) were resistant. OSU03012 reduced the number of cells in the S phase of the TE/E7 and TE/V cell lines with intact p53-p21(WAF1/CIP1) checkpoint, but not in the checkpoint defective TE/E6 cell lines. Treatment with OSU03012 also markedly reduced the levels of cyclin A and Cdk2 in TE/E7 and TE/V, but not in TE/E6 cell lines, which had significantly enhanced basal levels of cyclin A and Cdk2. Consistent with the TE/E6 cell line, p21(WAF1/CIP1)-/- mouse embryo fibroblasts were more sensitive to OSU03012-induced apoptosis as evidenced by PARP and caspase 3 cleavages. These data suggest that p21(WAF1/CIP1) is an important factor in the sensitivity of cells to the growth inhibiting and apoptotic effects of OSU03012.     

HCV C蛋白、p53、Mdm2、p14和p21在原发性肝癌中的表达及相关性

目的:研究原发性肝癌(HCC)组织中核心蛋白、突变p53、Mdm2、p14和p21的表达及其相关性,探讨HCV核心蛋白对突变p53、Mdm2、p14和p21表达影响及临床意义。方法:收集42例HCC石蜡组织,采用免疫组织化学EnVision法检测HCC组织中核心蛋白、突变p53、Mdm2、p14和p21的表达,用统计学方法及临床联系分析它们之间的关系。结果:核心蛋白、突变p53、Mdm2、p14和p21的阳性表达主要定位于细胞核中;HCC组织中核心蛋白、突变p53、Mdm2、p14和p21阳性率分别为40.5%、47.62%、75.57%、45.24%、19.05%;5组间的Kruskal-Wallis检验P=0.000,差异显著,核心蛋白与突变p53、Mdm2、p14和p21间的Mann-WhitneyU秩和检验P值分别为0.043、0.009、1.0、0.023;HCVC蛋白与突变p53、p14、p21阳性强度两者间相关性Spearman检验P值分别为0.000、0.000、0.43,相关系数rs分别为0.67、0.64、-0.29;p53和Mdm2、p21阳性强度间相关性Spearman检验p值为0.000、0.174,rs分别为0.72、-0.45。结论:在HCV感染的HCC中HCVC蛋白可能促进野生p53突变和表达,p14的表达与C蛋白有关联;突变p53和p14很可能促进Mdm2的表达;HCC中p21表达缺陷是十分常见的,HCV相关的HCC主要与p53通路改变有关,C蛋白可能下调p21的表达。因此,HCV相关的HCC中C蛋白、突变p53、Mdm2很可能促进肝细胞增生或恶性生长。     

p185HER-2/neu and p21CIP1/WAF1 Expression in Primary Tumors and Lymph Node Metastases in Non-small Cell Lung Cancer

p185^HER-2/neu, a tyrosine kinase receptor, is one of the target molecules for cancer therapy, and its expression may reduce the sensitivity of tumor cells to anti-cancer drugs. p21^CIP1/WAF1 is a cyclindependent kinase inhibitor, and its expression may also be involved in chemoresistance. Non-small cell lung cancer (NSCLC) is a potentially systemic disease, and systemic therapies play an important role in its treatment. However, there have been no studies comparing the expression of these molecules between primary and metastatic tumors. We investigated the expression of p185^HER-2/neu and p21^CIP1/WAF1 in 57 paired samples of primary NSCLC tumors and corresponding lymph node metastases by immunohistochemistry. Expression of each of p185^HER-2/neu and p21^CIP1/WAF1 was highly correlated between primary tumors and lymph node metastases, and similar correlations were also obtained when adenocarcinoma and squamous cell carcinoma cases were analyzed individually. However we failed to detect any correlation between p185^HER-2/neu and p21^CIP1/WAF1 expression. Our results suggested that expression of both p185^HER-2/neu and p21^CIP1/WAF1 is concordant between primary and metastatic tumors.     

Resistance of primary cultured mouse hepatic tumor cells to cellular senescence despite expression of p16(Ink4a), p19(Arf), p53, and p21(Waf1/Cip1)

Primary cultured mouse hepatic cells become senescent within a short period, although rare cells form colonies from which continuously proliferating cell lines can be established. In contrast, hepatic tumor (HT) cells show little senescence and higher colony-forming capacity. To assess this difference, we investigated p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression in primary normal and HT cells, together with cell lines established from both. In primary normal cells, p16(Ink4a)/p19(Arf) were expressed only in association with senescence and disappeared at later stages of colony formation. In contrast, primary HT cells showed sustained p16(Ink4a)/p19(Arf) expression from the beginning. No p16(Ink4a)/p19(Arf) alterations, such as deletion, mutations, or hypermethylation, were detected in the primary HT cells, although most cell lines derived from either normal or HT cell colonies lost p16(Ink4a) or p19(Arf) expression owing to hypermethylation or homozygous deletion of p16(Ink4a)/p19(Arf). On the other hand, primary normal and HT cells and most cell lines showed constitutively elevated expression of p53/p21(Waf1/Cip1), with a further increment after ultraviolet ir-radiation, indicating a functionally normal p53 pathway. These results indicate that primary HT cells are resistant to senescence despite retaining p16(Ink4a)/p19(Arf)/p53/p21(Waf1/Cip1) expression and that loss of p16(Ink4a)/p19(Arf) function is associated only with establishment of the cell lines.     

Urinary Bladder Lesions after the Chernobyl Accident: Immunohistochemical Assessment of p53, Proliferating Cell Nuclear Antigen, Cyclin D1 and p21WAF1/Cip1

During the 11-year period subsequent to the Chernobyl accident, the incidence of urinary bladder cancer in Ukraine has increased from 26.2 to 36.1 per 100,000 population. Cesium-137 (¹³⁷Cs) accounts for 80–90% of the incorporated radioactivity in this population, which has been exposed to long-term, low-dose ionizing radiation, and 80% of the more labile pool of cesium is excreted via the urine. The present study was performed to evaluate the histopathological features and the immunohistochemical status of p53, p21^WAF1/Cip1, cyclin D1 and PCNA (proliferating cell nuclear antigen) in urinary bladder mucos a of 55 males (49-92 years old) with benign prostatic hyperplasia who underwent surgery in Kiev, Ukraine, in 1995 and 1996. Group I (28 patients) inhabiting radiocontaminated areas of the country, group II (17 patients) from Kiev city with less radiocontamination and a control group III (10 patients) living in so-called "clean" areas of Ukraine were compared. In groups I and II, an increase in multiple areas of moderate or severe dysplasia or carcinoma in situ was seen in 42 (93%) of 45 cases. In addi tion, two small transitional cell carcinomas were found in one patient in each of groups I and II. Nuclear accumulation of p53, PCNA, cyclin D1, and to a lesser extent p21^WAF1/Cip1, was significantly increased in both groups I and II as compared with the control group III, indicating possible transformation events or enhancement of repair activities, that may precede the defect in the regulatory pathway itself, at least in the G1 phase of the cell cycle. Our results suggest that early malignant transformation is taking place in the bladder urothelium of people in the radiocontaminated areas of Ukraine and that this could possibly lead sometime in the future to an increased incidence of urinary bladder cancer.     

Rationale and Design of the IROCAS Study: Multicenter,International, Randomized Phase 3 Trial Comparing Adjuvant Modified (m) FOLFIRINOX to mFOLFOX6 in Patients With High-Risk Stage III (pT4 and/or N2) Colon Cancer—A UNICANCER GI-PRODIGE Trial

Background

According to the IDEA trial, 6-month adjuvant chemotherapy should remain the treatment standard in stage III T4 or N2 colon cancer. The relatively poor survival in this high-risk subgroup—a 3-year disease-free survival (DFS) rate of 65%—and the potential synergistic efficacy of 5-fluorouracil (5-FU), oxaliplatin, and irinotecan suggest that FOLFIRINOX may be a regimen of particular interest in this setting.