New perspectives of quercetin and vitamin C effects on fibronectin-binding integrins and chemokine receptors in prostate cancer cell lines.

OBJECTIVES: The aim of this study is to investigate the effect of two abundant dietary supplements, quercetin and vitamin C on some factors involved in metastasis and proliferation of prostate cancer, which are resistant to conventional chemotherapies in late stages. BACKGROUND: Bone and brain are two common sites of metastases in prostate cancer, nevertheless the factors involved in their metastatic pathways are not well understood. METHODS: The effect of quercetin (75µM) and vitamin C (100 µM) on CXCR4, CXCR7 chemokine receptors, α4, α5 and ß1 integrins, ki-67 proliferation marker and Vascular endothelial growth factor, VEGF was evaluated using Quantitative Reverse Transcription PCR (RT-qPCR). RESULTS: The effect of quercetin and vitamin C alone was different on PC3 and DU145 prostate cancer cell lines, but sequential combination reduced significantly the expression of CXCR and CXCR7 chemokine receptors, α4, α5 and ß1 integrin subunits, VEGF and Ki-67 proliferation markers in PC3 and DU145 cell lines. CONCLUSION: Our results indicated the beneficial effect of quercetin and vitamin C on prostate cancer cells with different metastatic sites and their differential response to the treatment which in turn may lead us to reach suitable therapeutic outcomes to combat cancer (Fig. 3, Ref. 36).

Antioxidant ameliorating effects against H2O2-induced cytotoxicity in primary endometrial cells.

Oxidative stress and a disrupted antioxidant system are involved in a variety of pregnancy complications. In the present study, the role of vitamin E (Vit E) and folate as radical scavengers on the GSH homeostasis in stress oxidative induced in rat endometrial cells was investigated. Primary endometrial stromal cell cultures treated with 50 and 200 µM of H2O2 and evaluated the cytoprotective effects of Vit E (5 µM) and folate (0.01 µM) in H2O2-treated cells for 24 h. Following the exposure of endometrial cells to H2O2 alone and in the presence of Vit E and/or folate, cell survival, glutathione peroxidase (GPx) and glutathione reductase activities and the level of reduced glutathione (GSH) were measured. Cell adhesions comprise of cell attachment and spreading on collagen were determined. Flow cytometric analysis using annexin V was used to measure apoptosis. H2O2 treatment showed a marked decrease in cell viability, GPx and GR activities and the level of GSH. Although Vit E or folate had some protective effect, combination therapy with Vit E and folate attenuated all the changes due to H2O2 toxicity. An increasing number of alive cells was showed in the cells exposed to H2O2 (50 µM) accompanied by co-treatment with Vit E and folic acid. The present findings indicate that co-administration of Vit E and folate before and during pregnancy may maintain a viable pregnancy and contribute to its clinical efficacy for the treatment of some idiopathic infertility.

Adaptive Response Induced by Pre-Exposure to 915 MHz Radiofrequency: A Possible Role for Antioxidant Enzyme Activity.

BACKGROUND: Over the past few years, the rapid use of high frequency electromagnetic fields like mobile phones has raised global concerns about the negative health effects of its use. Adaptive response is the ability of a cell or tissue to better resist stress damage by prior exposure to a lesser amount of stress. This study aimed to assess whether radiofrequency radiation can induce adaptive response by changing the antioxidant balance. MATERIALS AND METHODS: In order to assess RF-induced adaptive response in tissues, we evaluated the level of GSH and the activity of GR in liver. 50 rats were divided into 5 groups. Three groups were pre-exposed to 915 MHz RF radiation, 4 hours per day for one week at different powers, as low, medium and high. 24 hours after the last exposure to radiation, they were exposed to 4 Gy sublethal dose of gamma radiation and then sacrificed after 5 hours. Their livers were removed, washed and were kept at -80o C until used. RESULTS: Our finding showed that pre-exposure to 915 MHz radiofrequency radiation with specific power could induce adaptive response in liver by inducing changes in the activity and level of antioxidant enzymes. CONCLUSION: It can be concluded that pre-exposure to microwave radiation could increase the level of GSH and the activity of GR enzyme, although these increases were seen just in low power group, and the GR activity was indicated in medium power group. This increase protects tissue from oxidative damage induced by sublethal dose of gamma radiation.

Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells.

BACKGROUND: Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. OBJECTIVE: Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. METHOD: 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. RESULTS: Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. CONCLUSION: The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated.

Methylation of Integrin α4 and E-Cadherin Genes in Human Prostate Cancer.

Prostate cancer is the second most common malignancy in men worldwide. Abnormal epigenetic alterations such as DNA methylation and histone modification play an important role in tumor initiation, progression and regulation of cancer-related genes such as integrin α4 and E-cadherin. Expression of these genes was determined by semi-quantitative reverse transcriptase-PCR in prostate cancer cell lines, DU145 and PC3, before and after treatment with 5-aza-2-deoxycytidine and trichostatin A. Laser capture microdissection microscopy was used to obtain exclusively affected epithelial cells from prostate gland biopsies of 30 patients with prostate cancer and 40 with benign prostate hyperplasia. DNA bisulfite modifications followed by methylation-specific PCR were used to evaluate the promoter methylation status of E-cadherin and α4 integrin genes in extracted DNA from patients and aforementioned cell lines. The integrin α4 promoter in DU145 was fully methylated, whereas in PC3 cells, partial methylation was detected. E-cadherin was expressed in both cell lines; trichostatin A and 5-aza-2-deoxycytidine treatment had no effect on E-cadherin expression, however the combined treatment of both drugs or 5-aza-2-deoxycytidine alone increased integrin α4 expression. Integrin α4 and E-cadherin were hypermethylated in 66.6 % and 6.6 % of prostate cancer cases, respectively; no hypermethylation was observed in patients with benign prostate hyperplasia. These results together suggest that aberrant DNA methylation is one of the mechanisms involved in integrin α4 expression and may play an important role in human prostate carcinogenesis. In addition, the higher rate of integrin α4 gene methylation in prostate cancer patients elects it as a potential molecular tumor marker.

Microsatellite instability typing in serum and tissue of patients with colorectal cancer: comparing real time PCR with hybridization probe and high-performance liquid chromatography.

Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI+). MSI is detected in about 15% of all CRCs; 3% are of these are associated with Lynch syndrome and the other 12% are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI+ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100% in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.

Expression and Purification of Recombinant Mycobacterium Tuberculosis (TB) Antigens, ESAT-6, CFP-10 and ESAT- 6/CFP-10 and Their Diagnosis Potential for Detection of TB Patients.

BACKGROUND: One of the most widely used methods to detect tuberculosis (TB) infection is the tuberculin skin test (TST). The completion of Mycobacterium tuberculosis (M. tuberculosis) genome sequence has led to identification of several antigens that can be utilized for accurate diagnosis and control of TB. The aim of this study was to purify the recombinant M. tuberculosis antigens for the evaluation of their potential in TB diagnosis. METHODS: The recombinant secretory antigens, ESAT-6, CFP-10 and ESAT-6/CFP-10 were produced by PCR and cloning methods. To investigate antigen specific responses of these recombinant antigens in detection of TB, ex vivo enzyme linked immunospot (ELISPOT) test in 30 clinically diagnosed TB patients was evaluated. RESULTS: The selected M. tuberculosis antigens were cloned, expressed and purified in Escherichia coli (BL21). ELISPOT assay for detection of TB showed the sensitivity of 93, 90 and 100% for recombinant ESAT-6, CFP-10 and ESAT-6/CFP-10 proteins respectively, which is significantly higher than conventional TST. CONCLUSION: The recombinant antigens of ESAT-6, CFP-10 and ESAT-6/CFP-10 can be used as an accurate means of detecting TB in Iran.

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.

Age and sex dependence of the effects of an aqueous extract of Physalis alkekengi fruits on rat hepatic glucose 6-P dehydrogenase activity.

Intraperitoneal injections of an aqueous extract of winter cherry fruits (Physalis alkekengi) to new-born, weanling and adult female rats and to weanling and adult male rats had no effect on body weight, liver weight and liver cytosol protein content. The specific activities of hepatic glucose 6-P dehydrogenase (an estrogen induced protein) in rats of different age and sex groups in terms of mU/mg protein were: treated new-born females, 15.9 +/- 0.5; control, 29.1 +/- 0.6; treated weanling females, 14.9 +/- 0.3; control, 24.8 +/- 0.7; treated adult females, 25.7 +/- 0.5; control, 26.1 +/- 0.5; treated weanling males, 7.9 +/- 0.2; control, 7.9 +/- 0.1; treated adult males, 9.6 +/- 0.4; and control, 9.7 +/- 0.3. Treatment of new-born and weanling female rats with the extract resulted in 40-45% reduction in hepatic G6PD activity. However, treatment of adult females, and weanling and adult males produced no significant change in the activity of this enzyme. The data are discussed both in terms of the increase in the capacity of rodent liver to metabolize steroidal compounds with age and the presence of low levels of circulating estradiol necessary for enzyme induction in male rats.