Cytotoxicity of alkaloids isolated from Peganum harmala seeds on HCT116 human colon cancer cells.

BACKGROUND: The present study aimed to elucidate the potential anticancer activity and mechanism of P. harmala's alkaloid extract, harmine (HAR), and harmaline (HAL) in HCT-116 colorectal cancer cells. METHODS AND RESULTS: P. harmala's alkaloid was extracted from harmala seeds. HCT-116 cells were treated with P. harmala's alkaloid extract, HAR and HAL. Cytotoxicity was determined by MTT assay, apoptotic activity detected via flow cytometry and acridine orange (AO)/ethidium bromide (EB) dual staining, and cell cycle distribution analyzed with flow cytometry. The mRNA expression of Bcl-2-associated X protein (Bax) and glycogen synthase kinase-3 beta (GSK3ß) was measured by real-time PCR. Furthermore, the expression of Bax, Bcl-2, GSK3ß and p53 proteins, were determined by western blotting. The findings indicated that, P. harmala's alkaloids extract, HAR and HAL were significantly cytotoxic toward HCT116 cells after 24 and 48 h of treatment. We showed that P. harmala's alkaloid extract induce apoptosis and cell cycle arrest at G2 phase in the HCT116 cell line. Downregulation of GSK3ß and Bcl-2 and upregulation of Bax and p53 were observed. CONCLUSION: The findings of this study indicate that the P. harmala's alkaloid extract has anticancer activity and may be further investigated to develop future anticancer chemotherapeutic agents.

Evaluation of t-DARPP Expression Alteration in Association with DDR1 Expression in Non-Small Cell Lung Cancer.

Background: Discoidin domain receptor 1 (DDR1) signaling plays a critical role in various cellular functions. Increased DDR1 expression has been shown in different human cancers. t-DARPP is a truncated isoform of DARPP-32, and its upregulation promotes cell survival and migration. Most lung cancer patients have non-small cell lung cancer (NSCLC), and their survival rate is low. Therefore, it is necessary to study new and effective targeted therapies. Increased t-DARPP expression in NSCLC patients is associated with patient survival and can act as a prognostic marker correlated with increasing stages of NSCLC. The current study aimed to evaluate alteration in DDR1 expression and its effects on t-DARPP expression in NSCLC. Methods: Two human lung adenocarcinoma cell lines, A549 and Calu-3, were treated with collagen type I and transfected with DDR1 siRNA. The relative expression of DDR1 and t-DARPP was evaluated using qRT-PCR. Results: The results indicated that collagen type I could stimulate DDR1 expression in NSCLC cells. Also, DDR1 upregulation resulted in a significant increase in t-DARPP expression. In contrast, suppression of DDR1 expression significantly decreased t-DARPP expression. Conclusion: Our findings propose that modification in the expression of DDR1, caused by collagen type I and siRNA, might influence the expression of t-DARPP in NSCLC that is linked to NSCLC progression. Moreover, this alteration could potentially serve as an innovative target for therapeutic intervention.

LRIG1 expression and colorectal cancer prognosis.

BACKGROUND: To make the right treatment decisions about colorectal cancer (CRC) patients reliable predictive and prognostic data are needed. However, in many cases this data is not enough. Some studies suggest that LRIG1 gene (leucine-rich repeats and immunoglobulin-like domains1) has prognostic implications in different kinds of cancers. METHODS: One hundred and two patients with colorectal cancer were retrospectively analyzed for LRIG1 expression at both mRNA and protein levels. SYBR Green Real-Time RT-PCR technique was used for mRNA expression analyses and Glyceraldehyde-3-Phosphate Dehydrogenase gene (GAPDH) was considered as a reference gene for data normalization. LRIG1 protein expression was analyzed using Immunohistochemistry. Additionally, appropriate statistic analyses were used to assess the expression of LRIG1 in test and control groups. The prognostic significance of LRIG1 expression was analyzed using the univariate and multivariate analyses. RESULTS: The data revealed that the expression of LRIG1 in both mRNA and protein levels was down regulated in colorectal tumor tissues (P < 0.01) but is not clinically relevant prognostic indicator in CRC. CONCLUSIONS: Therefore, it is suggested that LRIG1 expression analyses may not be considered as an important issue when making informed and individualized clinical decisions regarding the management of colorectal cancer patients.

Aberrant DNA Methylation of Two Tumor Suppressor Genes, p14ARF and p15INK4b, after Chronic Occupational Exposure to Low Level of Benzene.

BACKGROUND: Exposure to benzene would be associated with many diseases including leukemia. Epigenetic alterations seem to be among the main mechanisms involved. OBJECTIVE: To determine if chronic occupational exposure to low level of benzene would be associated with DNA methylation. METHODS: Global DNA methylation and promoter-specific methylation of the two tumor suppressor genes, p14ARF and p15INK4b, were assessed employing methylation-specific PCR using the DNA extracted from 40 petrochemical workers exposed to ambient benzene levels of <1 ppm, and 31 office workers not exposed to benzene or its derivatives. RESULTS: While an increase in global DNA methylation of 5% in p14ARF (p=0.501) and 28% in p15INK4b (p=0.02) genes was observed in the exposed group, no hypermethylation in either of the studied genes was observed in the unexposed group. No significant association was found between the frequency of aberrant methylation and either of age, work experience, and smoking habit in the exposed group. CONCLUSION: Chronic occupational exposure to lower than the permissible exposure limit of benzene may still result in DNA methylation of tumor suppressor genes that may ultimately lead to development of cancer.

MGMT hypermethylation and BCL-2 overexpression associated with superficial bladder cancer and recurrence.

BACKGROUND: Urinary bladder carcinoma is one of the leading causes of death among men, and its high recurrence rates make it one of the most solid tumors to treat. The silencing of the tumor suppressor gene by hypermethylation of the CpG islands and overexpression of proto-oncogene proteins are the main mechanisms in cancers. Here, we investigate methylation status of O6-methylguanine-DNA-methyltransferase (MGMT), a tumor suppressor gene and expression level of BCL-2 a proto-oncogene protein that is frequently observed in bladder carcinoma and its recurrences. MATERIALS AND METHODS: We analyzed the methylation of MGMT in 80 tissue samples of patients suffering from bladder cancer and 80 urine samples of cancer-free individuals by MS-PCR. Additionally, BCL-2 protein expression level was analyzed on these 80 tissue samples by immunohistochemistry. RESULTS: 45% of patients had MGMT methylation, of which this hypermethylation does not have significant association with an increase in grade, but there was significant association in cases with recurrence tumors and metastasis tumors. Among patients with recurrence tumor, 92.5% patients showed MGMT hypermethylation; 66% of these showed BCL-2 overexpression. CONCLUSION: Our data indicate that MGMT hypermethylation and BCL-2 overexpression may have an intense role in superficial bladder cancer recurrences.

Interleukin-12 and interleukin-6 gene polymorphisms and risk of bladder cancer in the Iranian population.

Interleukin-12 (IL-12) as an antitumor and interleukin-6 (IL-6) as an inflammatory cytokine, are immunomodulatory products that play important roles in responses in cancers and inflammation. We tested the association between two polymorphisms of IL-12(1188A>C; rs3212227) and IL-6 (-174 C>G) and the risk of bladder cancer in 261 patients and 251 healthy individuals. We also investigated the possible association of these SNPs in patients with high-risk jobs and smoking habits with the incidence of bladder cancer. The genotype distributions of IL-6 (-174 C/G) genotype were similar between the cases and the control groups; however, among patients with smoking habits, the association between IL-6 gene polymorphism and incidence of bladder cancer was significant. After a control adjustment for age and sex, the following results were recorded: CC genotype (OR= 2.11, 95%CI=1.56-2.87, p=0.007), GC genotype (OR=2.18, 95%CI=1.16-4.12, p=0.014) and GC+ CC (OR=2.6, 95%CI=1.43-4.47, p=0.011). A significant risk of bladder cancer was observed for the heterozygous genotype (AC) of IL-12 (OR=1.47, 95%CI=1.01-2.14, p=0.045) in all cases, and among smokers (AC) (OR=3.13, 95%CI=1.82-5.37, p=0.00014), combined AC+CC (OR=3.05, 95%CI=1.8-5.18, p=0.000015). Moreover among high risk job patients, there was more than a 3-fold increased risk of cancer in the carriers of IL-12 beta heterozygous (OR=3.7, 95%CI=2.04-6.57, p=0.000056) and combined AC+CC(OR=3.29, 95%CI=1.58-5.86, p=0.00002) genotypes as compared with the AA genotype with low-risk jobs. As a conclusion, this study suggests that IL-12(3'UTR A>C) and IL-6 (-174 C>G) genotypes are significantly associated with an increased risk of bladder cancer in the Iranian population with smoking habits and/or performing high-risk jobs.

Neural differentiation of mouse embryonic and mesenchymal stem cells in a simple medium containing synthetic serum replacement.

Neural differentiation of embryonic and adult stem cells has been reported previously. Several studies have used different proportions of serum or a cocktail of growth and differentiation factors for this purpose. In the present study, we examined neural differentiation of mouse embryonic stem (ES) cells in KoSR-containing media. We also investigated neural differentiation of mouse adipose tissue-derived stem cells (ADSCs) in a medium containing KoSR, a synthetic serum replacement, and compared it with neural differentiation in low-serum condition. Meanwhile, effect of ß-ME on neural differentiation was investigated in both conditions. As revealed by RT-PCR and immunocytochemistry analyses, KoSR-containing medium induced neural differentiation of mouse ES cells. Moreover, under the culture conditions we tested, ADSCs were differentiated to neuron-like cells and expressed some neuronal markers. Low concentration of ß-ME improved neuron-like differentiation of the ADSCs in the 4% FBS-supplemented medium, while addition of ß-ME in KoSR condition decreased neural differentiation. KoSR-containing medium without any additional factor improved generation of neuron-like cells, upregulated the expression of mature neuronal markers and led to the formation of cytoplasmic processes. In summary, our findings are indicating that mouse embryonic and mesenchymal stem cells are capable of neural development in KoSR-containing media.