Glycolysis has been shown to be required for the cell growth and proliferation in several cancer cells. However, prostate cancer cells were accused of using more fatty acid than glucose to meet their bioenergetic demands. The present study was designed to evaluate the involvement of hexokinase and CPT-1 in the cell growth and proliferation of human prostate cancer cell lines, PC3, and LNCaP-FGC-10. Hexokinase and CPT-1 activities were examined in the presence of different concentrations of their inhibitors, lonidamine and etomoxir, to find the concentration of maximum inhibition ([I max]). To assess cell viability and proliferation, dimethylthiazol (MTT) assay was carried out using [I max] for 24, 48, and 72 h on PC3 and LNCaP cells. Apoptosis was determined using annexin-V, caspase-3 activity assay, Hoechst 33258 staining, and evaluation of mitochondrial membrane potential (MMP). Moreover, ATP levels were measured following lonidamine and etomoxir exposure. In addition, to define the impact of exogenous fatty acid on the cell growth and proliferation, CPT-1 activity was evaluated in the presence of palmitate (50 μM). Hexokinase and CPT-1 activities were significantly inhibited by lonidamine [600 μM] and etomoxir [100 μM] in both cell lines. Treatment of the cells with lonidamine [600 μM] resulted in a significant ATP reduction, cell viability and apoptosis, caspase-3 activity elevation, MMP reduction, and appearance of apoptosis-related morphological changes in the cells. In contrast, etomoxir [100 μM] just decreased ATP levels in both cell lines without significant cell death and apoptosis. Compared with glucose (2 g/L), palmitate intensified CPT-1 activity in both cell lines, especially in LNCaP cells. In addition, activity of CPT-1 was higher in LNCaP than PC3 cells. Our results suggest that prostate cancer cells may metabolize glucose as a source of bioenergetic pathways. ATP could also be produced by long-chain fatty acid oxidation. In addition, these data might suggest that LNCaP is more compatible with palmitate.
Background: p53 alterations have been implicated in the development of many cancers, such as gastric cancer, but there is no evidence of p53 intron alterations in gastritis lesions. The aim of this study was to investigate the p53 intron alterations in gastritis along with p53 and mismatch repair protein expression and microsatellite status. Materials and Methods: PCR-sequencing was conducted for introns 2-7 on DNA extracted from 97 paired samples of gastritis lesions and normal adjacent tissue. Abnormal accumulation of p53 and mismatch repair proteins was investigated using immunohistochemistry. In addition, microsatellite status was evaluated with reference to five mononucleotide markers. Results: Gastritis cases included 41 males and 56 females in the age range of 15-83 years, 87.6% being H.pylori positive. IVS2+38, IVS3ins16 and IVS7+72 were the most polymorphic sites. Their minor allele frequency values were as follows: 0.38, 0.21 and 0.06, respectively. Samples with GG genotype at IVS2+38 and CT at IVS7+72 had no insertion. Moreover, most of the stable samples (91.9 %) had a G allele at IVS2+38. All of the samples were IHC negative for p53 protein, microsatellite stable and expressed mismatch repair proteins. p53 alterations were prominent in the H. Pylori+ group, but without statistical significance. Conclusions: According to our results, some p53 polymorphisms such as IVS2+38, IVS3ins16 and IVS7+72, because of their correlations together or with microsatellite status may contribute to gastritis development. However, so far effects on p53 expression and function remain unclear. Therefore, a comprehensive survey is needed to delineate their biological significance.
Purpose: Emerging evidence implies that electromagnetic fields (EMFs) can negatively affect angiogenesis. In this regard, the effects of extremely low frequency pulsed electromagnetic field (ELF–PEMF) exposure on the relative expression level of angiogenic factors involved in the pathogenesis of ocular disorders were evaluated in human retinal pigment epithelial (hRPE) cells in order to investigate a noninvasive therapeutic method for patients with several ocular diseases associated with neovascularization. Methods: After separating hRPE cells from globes, hRPE cells were exposed to 15 mT of ELF–PEMF (120 Hz) at 5, 10, and 15 min for seven days. Cell proliferation and apoptosis of treated cells were evaluated via ELISA assay. Moreover, relative expression changes of HIF-1α, CTGF, VEGFA, MMP-2, cathepsin D, and E2F3 were performed using real-time RT-PCR. Results: ELF–PEMF exposure had no significant effects on the apoptosis and proliferation rate of hRPE cells. Expression level of HIF-1α, CTGF, VEGFA, MMP- 2, cathepsin D, and E2F3 was downregulated following 5 min of ELF–PEMF exposure. Conclusion: As ELF–PEMF showed inhibitory effects on the expression of angiogenic genes in hRPE cells with no cytotoxic or proliferative side effects, it can be introduced as a useful procedure for managing angiogenesis induced by retinal pathogenesis, although more studies with adequate follow-up in animal models are needed.
Background: It has been frequently shown that p53 alterations have an important role in the development of gastric cancers but there is no data on p53 alteration in gastric cancer and its precancerous lesions from Iran although this country experiences one of the highest gastric cancer incidence and mortality rates in the world. The purpose of this study was to do a comprehensive assessment of p53 alterations in the Iranian population of gastritis patients and to evaluate the association between p53 alterations, microsatellite status and clinicopathological aspects. Methods: After DNA extraction, PCR sequencing was done for exons 2–7. Also microsatellite status was evaluated using five microsatellite markers: NR-27, NR-21, NR-24, BAT-25 and BAT-26. Results: The highest rate of alteration was seen in codons 72 (85.6%, SNP) and 248 (30.9%, mutation). Also, we found 2 new mutations in codons 9 and 146. In contrast with previous work, transition at the CpG codons was relatively rare. Nucleotide alterations were more prevalent in the Helicobacter pylori-positive group but not significantly. Neither nuclear staining for p53 protein nor microsatellite instability was seen in gastritis lesions. Conclusion: p53 alterations might contribute to the pathogenesis of gastritis and perhaps gastric cancer in Iran. However, the different spectrum seen here implies other mechanism(s) in gastritis and gastric cancer development in the Iranian population.
Global genome hypomethylation as an epigenetic phenomenon may induce (pre)neoplastic transformation through inducing chromosomal and genomic instability and activating oncogenes. Global genome hypomethylation has a fundamental role in early stages of tumorigenesis but little is known about this epigenetic event in gastric precancerous lesions such as gastritis. Therefore, we decided to evaluate this issue in gastritis lesion for obtaining new insight toward molecular biology of gastric cancer. Here we used a technique composed of restriction enzyme digestion and pyrosequencing known as luminometric methylation assay to evaluate this issue. DNA obtained from normal and gastritis lesions was digested with HpaII (sensitive to methylation in its cut site) and MspI (insensitive). Overhangs resulting from these enzymes then fill in by polymerase extension assay using pyrosequencing instrument. Nucleotide incorporation during polymerase extension generates light, which expresses as pick in the pyrogram. By comparing the height of picks obtained form both enzymes it can be possible to evaluate and compare global genome methylation level of gastritis and normal tissues. If the target site is fully methylated, the HpaII/MspI (their pick height) will approach zero. If not, this ratio will be around 1. In the other conditions this ratio varies between 0 and 1. Comparing the ratio of normal and gastritis sample, it can be inferred whether or not gastritis is hypomethylated. This study was performed on 83 gastritis and normal adjacent tissues. The patients included 34 male and 49 female and were 15 to 83 years old. According to our study, gastritis tissue was hypomethylated more than the normal tissue (p = 0.028). Global genome methylation has no significant correlation with MSI, pathological findings, age, and gender. We conclude that global genome hypomethylation occurs in the gastritis level. This reduction probably continues in the next steps toward gastric cancer and may induce other epigenetic and/or genetic changes (such as MSI) that promote carcinogenesis.
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