An Effective Concentration of 5-Aza-CdR to Induce Cell Death and Apoptosis in Human Pancreatic Cancer Cell Line through Reactivating RASSF1A and Up-Regulation of Bax Genes

Background: Promoter hyper-methylation of tumor suppressor genes is a common event that occurs in cancer. As methylation is a reversible modification, agents capable of reversing an abnormal methylation status should help to combat cancer. 5-Aza-CdR is a DNA methyl-transferase inhibitor. The present study aimed to evaluate the effect of 5-Aza-CdR on the proliferation of human pancreatic cancer cell line (PANC-1) and the expression of RASSF1A and Bax genes. Methods: PANC-1 cells were cultured and treated with 5 and 10 µM/L of 5-Aza-CdR for 24, 48, 72, and 96 hours and the percentages of cell viability and apoptosis were measured by MTT and flow cytometry. RASSF1A gene promoter methylation was assessed by methyl-specific primer-PCR (MSP-PCR) and the expression of RASSF1A and Bax genes was measured using quantitative real-time PCR (qPCR). All quantitative data are presented as mean±SD (standard deviation). The one-way analysis of variance (ANOVA) with the LSD post hoc test was performed for statistical analysis using the SPSS software package, version 16.0. Results: 3-[4,5-dimethythiaziazol-2yl]-2,5-diphenyl tetrazoliumbr omide (MTT) assay revealed that 5-Aza-CdR significantly inhibit the growth and proliferation of PANC-1. The flow cytometry results showed over 40% and 70% of early and late apoptotic cells after treatment with 5 and 10 µm/L of 5-Aza-CdR, respectively. MSP-PCR data indicated that the treatment of cells with 10 µm/L 5-Aza-CdR resulted in partial demethylation of RASSF1A gene promoter. qPCR results showed significant re-expression of RASSF1A and up-regulation of Bax genes after 96 hours treatment of cells with 10 µm/L 5-Aza-CdR versus control cells (P<0.01). Conclusion: The result demonstrated that 5 and 10 µM of 5-Aza-CdR induce cell death and apoptosis by epigenetic reactivation of RASSF1A and up-regulation of Bax genes

Association of TP53 gene codon 72 polymorphism with endometriosis risk in Isfahan

Background: Endometriosis is a female health disorder that occurs when cells from the lining of the uterus grow in other areas of the body. The cause of endometriosis is unknown. Objective: The purpose of this study was to investigate TP53 gene codon 72 polymorphism in women with endometriosis and compared it with healthy samples in Isfahan. Materials and Methods: We undertook a case-control study to examine the possible association of the TP53 gene codon 72 polymorphism with the risk of endometriosis in Isfahan. Ninety whole blood specimens from normal people as controls and ninety endometriosis specimens were analyzed. p53 codon 72 genotypes were identified using allele-specific polymerase chain reaction. Results: Frequency of genotype Arg/Arg (Arginine/Arginine) in the samples of endometriosis was 28.9% and in healthy samples 42.2%. Frequency of genotype Pro/Pro (Proline/Proline) in the samples of endometriosis was 15.6% and in healthy ones. Frequency of heterozygote's Arg/Pro was 55.6% in endometriosis samples and 54.45% in healthy ones 3.3%. By comparing statistical genotype Pro/Pro with two other genotypes in both groups there was a statistical meaningful difference between control group and endometriosis group. [p=0.009, CI=95%, OR=5.34 (1047-19.29)]. Conclusion: Recent research shows that genotype Pro/Pro codon72 exon4 TP53 gene may be one predisposing genetic factor for endometriosis in Isfahan

All-trans retinoic acid and genistein induce cell apoptosis in OVCAR-3 cells by increasing the P14 tumor suppressor gene

In this study, we evaluated the effects of all-trans retinoic acid (ATRA) alone or in combination with genistein (GEN) in p14 tumor suppressor gene and subsequent apoptosis of human ovarian carcinoma cells (OVCAR-3). The cells were treated with ATRA or GEN at concentrations of 50 and 25 μM respectively, either alone or in combination for 24 and 48 h. The cell viability was evaluated using 3-(4, 5- dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The percentage of cell apoptosis was determined using flow cytometry and p14 gene expression was measured using real time PCR. The MTT results showed that in both ATRA and GEN treated groups, the cell viabilityviability in group treated for 48 h was significantly lower than group treated for 24 h. The flow cytometry results showed that the percentage of apoptotic cells in groups that treated with ATRA and GEN in combination for 24 h and 48 h was significantly more than all other tested groups. The real time results showed that the mRNA level of p14 in cells treated with both drugs for 48 h was significantly higher than all other groups. In conclusion, we confirm that GEN in combination with ATRA is an effective strategy to up regulate the p14 tumor suppressor gene and induce cell apoptosis in OVCAR-3 cell line

The effect of CTB on P53 protein acetylation and consequence apoptosis on MCF-7 and MRC-5 cell lines

Background: P300 is a member of the mammalian histone acetyl transferase (HAT) family, an enzyme that acetylates histones and several non-histone proteins including P53 (the most important tumor suppressor gene) during stress, which plays an important role in the apoptosis of tumor cells. Hereby, this study describes the potency of CTB (Cholera Toxin B subunit) as a P300 activator to induce apoptosis in a breast cancer cell line (MCF-7) and a lung fibroblast cell line (MRC-5) as a non-tumorigenic control sample. Materials and Methods: MCF-7 and MRC-5 were cultured in RPMI-1640 and treated with or without CTB at a concentration of 85.43 μmol/L, based on half-maximal inhibitory concentration (IC50) index at different times (24, 48 and 72 h). The percentage of apoptotic cells were measured by flow cytometry. Real-time quantitative RT-PCR was performed to estimate the mRNA expression of P300 in MCF-7 and MRC-5 with CTB at different times. ELISA and Bradford protein techniques were used to detect levels of total and acetylated P53 protein generated in MCF-7 and MRC-5. Results: Our findings indicated that CTB could effectively induce apoptosis in MCF-7 significantly higher than MRC-5. We showed that expression of P300 was up-regulated by increasing time of CTB treatment in MCF-7 but not in MRC-5 and the acetylated and total P53 protein levels were increased more in MCF-7 cells than MRC-5. Conclusion: CTB could induce acetylation of P53 protein through increasing expression of P300 and consequently induce the significant cell death in MCF-7 but it could be well tolerated in MRC-5. Therefore, CTB could be used as an anti-cancer agent

Evidence for an association of TP53 codon 72 polymorphism with sporadic colorectal cancer risk in Isfahan

&lt;ul&gt; &lt;li&gt;&lt;strong&gt;BACKGROUND&lt;/strong&gt;: A common polymorphism at codon 72 of TP53 gene has been associated with increased risk for many human cancers. We studied this TP53 polymorphism in colorectal adenocarcinomas in small population selected from Isfahan city.&lt;/li&gt; &lt;li&gt;&lt;strong&gt;METHODS&lt;/strong&gt;: Samples: We undertook a case-control study on 180 controls and 180 paraffin block specimens of sporadic colorectal adenocarcinomas. PCR amplification of TP53 codon 72 polymorphism: TP53 codon 72 genotypes were detected by PCR using specific primer pairs for amplifying the Proline or the Arginine alleles.&lt;/li&gt; &lt;li&gt;&lt;strong&gt;STATISTICAL ANALYSES:&lt;/strong&gt; The 42-test was used to assess the significance of any difference in the prevalence of TP53 codon 72 polymorphism between colorectal cancer patients and controls.&lt;/li&gt; &lt;li&gt;&lt;strong&gt;RESULTS&lt;/strong&gt;: In control samples, the genotype distribution for TP53 polymorphism showed 28.3%, 48.9% and 22.8% for the Arginine/Arginine, Arginine/Proline and Proline/Proline genotypes, respectively. In the cancer group 40% of the cases were Arginine/Arginine, 42.2% were Arginine/Proline and 17.8% were Proline/Proline. A significant difference between cases and controls was found for the Arginine/Arginine genotype compared with (grouped) Arginine/Proline and Proline/Proline genotypes (Odds Ratio = 1.686 (1.085-2.620), P = 0.02).&lt;/li&gt; &lt;li&gt;&lt;strong&gt;CONCLUSIONS&lt;/strong&gt;: TP53 codon 72 polymorphism may be a genetic predisposing factor for colorectal adenocarcinomas in Isfahan city.&lt;/li&gt; &lt;li&gt;&lt;strong&gt;KEYWORDS&lt;/strong&gt;: Colorectal adenocarcinoma, TP53, Arginine, Proline, Polymorphism.&lt;/li&gt; &lt;/ul&gt

Inhibitory effect of genistein on PLC/PRF5 hepatocellular carcinoma cell line

Background: Natural compounds including flavonoids like genistein (GE) are able to inhibit cell proliferation and induce apoptosis. GE is the main representative of these groups. GE inhibits carcinogenic tumors such as colon, stomach, lung, and pancreas tumors. The aim of the present study was to analyze the apoptotic effect of GE in the hepatocellular carcinoma (HCC) PLC/PRF5 cell line. Methods: Cells were treated with various doses of GE (1, 5, 10, 25, 50, 75, and 100 μM/L) at different times (24, 48, and 72 h) and the MTT assay was commonly used. Furthermore, cells were treated with single dose of GE (25 μM) at different times and flow cytometry was performed. Results: GE inhibited the growth of liver cancer cells significantly with a time- and dose-dependent manner. The percentage of living cells in GE treatment groups with a concentration of 25 μM at different times were 53, 48 and 47%, respectively (P < 0.001). Result of flow cytometry demonstrated that GE at a 25 μM concentration induces apoptosis significantly in a time-dependent manner. The percentage of apoptotic cells at different times were 44, 56, and 60%, respectively (P < 0.001). Conclusions: GE can significantly inhibit the growth of HCC cells and plays a significant role in apoptosis of this cell line