Atorvastatin's Therapeutic Potential in Atherosclerosis: Inhibiting TGF-ß-Induced Proteoglycan Glycosaminoglycan Chain Elongation through ROS-ERK1/2-Smad2L Signaling Pathway Modulation in Vascular Smooth Muscle Cells.

OBJECTIVE: According to the response-to-retention hypothesis, the inception of atherosclerosis is attributed to the deposition and retention of lipoprotein in the arterial intima, facilitated by altered proteoglycans with hyperelongated glycosaminoglycan (GAG) chains. Recent studies have elucidated a signaling pathway whereby transforming growth factor-ß (TGF-ß) promotes the expression of genes linked to proteoglycan GAG chain elongation (CHSY1 and CHST11) via reactive oxygen species (ROS) and the downstream phosphorylation of ERK1/2 and Smad2L. Atorvastatin is known to exhibit pleiotropic effects, including antioxidant and anti-inflammatory. The purpose of the present research was to ascertain the influence of atorvastatin on TGF-ß-stimulated expression of CHSY1 and CHST11 and associated signaling pathways using an in vitro model. MATERIALS AND METHODS: In this experimental study, vascular smooth muscle cells (VSMCs) were pre-incubated with atorvastatin (0.1-10 µM) prior to being stimulated with TGF-ß (2 ng/ml). The experiment aimed to evaluate the phosphorylation levels of Smad2C, Smad2L, ERK1/2, the NOX p47phox subunit, ROS production, and the mRNA expression of CHST11 and CHSY1. RESULTS: Our research results indicated that atorvastatin inhibited TGF-ß-stimulated CHSY1 and CHST11 mRNA expression. Further experiments showed that atorvastatin diminished TGF-ß-stimulated ROS production and weakened TGF-ß-stimulated phosphorylation of p47phox, ERK1/2, and Smad2L; however, we observed no effect on the TGF-ß- Smad2C pathway. CONCLUSION: These data suggest that atorvastatin demonstrates anti-atherogenic properties through the modulation of the ROS-ERK1/2-Smad2L signaling pathway. This provides valuable insight into the potential mechanisms by which atorvastatin exerts its pleiotropic effects against atherosclerosis.

24-Hydroxycholesterol Moderates the Effects of Amyloid-ß on Expression of HMG-CoA Reductase and ABCA1 Proteins in Mouse Astrocytes.

Background: Elevated brain cholesterol increases the risk of Alzheimer's disease. Production of 24-hydroxycholesterol (24s-OHC) by neurons prevents cholesterol accumulation in the brain. In this study, we investigated the effect of 24s-OHC on the HMG-COA reductase and ABCA1 which are involved in the brain cholesterol homeostasis with or without ß-amyloid in astrocytes. Methods and Materials: Astrocytes were treated with 24s-OHC with or without Aß. Western blot and real-time polymerase chain reaction were done to detect protein and gene expression of ß-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) and ABCA1, respectively. Cholesterol release was determined using a quantitation kit. Results: Protein levels of HMGCR and ABCA1 were significantly increased by Aß; however, the 24s-OHC was able to restore their levels and diminish the effect of amyloid-ß. Aß did not have a significant effect on HMGCR expression, while 24s-OHC reduced it by 68%. Aß-induced ABCA1 expression did not increase cholesterol efflux as the lower levels of cholesterol in conditioned medium of Aß-treated cells were found. Conclusion: Our novel findings show that Aß affects two key elements in the brain cholesterol homeostasis, HMGCR and ABCA1, which are crucial in cholesterol synthesis and efflux. Since 24s-OHC could suppress the Aß effects on enhancement of HMGCR and ABCA1, therefore the cytochrome P450 46A1 (Cyp46A1), which is exclusively expressed in the central nervous system and responsible for producing of 24s-OHC, could consider as a therapeutic target in the cholesterol-related neurodegenerative diseases such as Alzheimer's disease.

Comparison of the effects of cholesterol, palmitic acid, and glucose on activation of human hepatic stellate cells to induce liver fibrosis.

Background: In hepatic damage, Hepatic stellate cells (HSCs) become active, proliferate, and change to myofibroblasts. Increasing the fibrogenic genes, such as Transforming growth factor-ß (TGF-ß), Alpha Smooth Muscle Actin (α-SMA), and Collagen1 α (COL 1α) show that the activation of HSCs can lead to hepatic fibrosis. Purpose: These days people consume much cholesterol, palmitic acid, and glucose which can have adverse effects on an individuals' health, but their influences on activating human HSCs and inducing liver fibrosis have not been assessed. Our purpose is to investigate the effects of these three main and abundant ingredients in the diet on the activation of human HSCs and inducing liver fibrosis. Methods: To measure cholesterol, palmitic acid, and glucose cytotoxic effects on the viability of the cells, the MTT technique was used. Then the treated cells were incubated in media containing cholesterol, palmitic acid, and glucose with different concentrations for 24 h. At last, the α-SMA, COL 1α, and TGF-ß, genes mRNA expression were measured by real-time PCR. Results and Conclusions: Our results demonstrated that high concentrations of cholesterol and palmitic acid can activate human HSCs that lead to an increase in the mRNA expressions of fibrogenic genes. Thus, controlling fat intaking and knowing its mechanism is crucial to prevent and attenuate hepatic fibrosis.

Methylation-mediated silencing of miR-125a-5p facilitates breast cancer progression by inducing autophagy.

BACKGROUND: microRNA-125a-5p (miR-125a) is a tumor suppressor gene whose role in autophagy remains poorly understood. In the current study, we aimed to investigate the methylation status of miR-125a, its transfection into SK-BR3 cells, and its effects on autophagy. METHODS: Sixty samples of tumor and non-tumor adjacent tissue were collected and the methylation status of miR-125a was evaluated by methylation-specific PCR (MSP). The effect of 5-Aza-dC on miR-125a expression was investigated in the SK-BR3 cells. Cells were also transfected with miR-125a mimic/antimiR. The expression of miR-125a and its target genes was evaluated by Real-Time PCR. Protein levels of ATG5 and LC3 were assessed by Western blotting. HER2 expression was investigated by immunocytochemistry (ICC). RESULTS: The data showed that the miR-125a promoter CpG Island was significantly hypermethylated in breast cancer tissues (p < 0.01) and in SK-BR3 cells. The 5-Aza-dC could significantly increase miR-125a expression by decreasing its methylation (p < 0.05). In addition, Western blot analysis indicated the expression of ATG5 and LC3 II/ LC3I, as autophagy biomarkers, was significantly reduced in SK-BR3 cells transfected with miR-125a (p < 0.05). CONCLUSIONS: Our data showed miR-125a expression was significantly decreased in tumor tissues due to its promoter hypermethylation. Overexpression of miR-125a was associated with a reduction in autophagy, which could provide a new therapeutic avenue for advanced-stage breast cancer treatment.

Quercetin potentiates the chemosensitivity of MCF-7 breast cancer cells to 5-fluorouracil.

BACKGROUND: Breast cancer is one of the leading causes of cancer mortality worldwide. 5-fluorouracil (5-FU) is one of the chemotherapy drugs to treat breast cancer, but it is associated with several side effects. Combination therapy is a way to increase the effectiveness of chemo drugs and decrease their usage dose. Quercetin (Quer) is one of the natural polyphenols with anti-cancer properties. This study investigated the apoptotic effect of 5-FU in combination with Quer compared with 5-FU alone on MCF-7 breast cancer cells. METHOD AND RESULTS: Different single and combined concentrations of 5-FU and Quer were applied to MCF 7 cells for 48 h. Cell viability, apoptosis, gene expression of Bax, Bcl2, and p53, caspase activity, and colony number were assessed using MTT assay, flow cytometry, quantitative real-time PCR, enzyme-linked immunosorbent (ELISA), and Colony formation assay, respectively. The combination of 5-FU and Quer compared to 5-FU alone improved apoptosis by increasing the gene expression of Bax and p53 and caspase-9 activity and decreasing the Bcl2 gene expression. Colony formation in MCF-7 cells significantly decreased in the combined state compared to 5-FU alone. CONCLUSION: Quer potentiates the sensitivity of breast cancer to 5-FU so that this combination may be proposed as a treatment for breast cancer. Therefore, this combination can be suggested for future in vivo studies.

Improvement of Targeted Chemotherapy of HER2-positive Ovarian Malignant Cell Line by ZHER2-Idarubicin Conjugate: An in vitro Study.

BACKGROUND & OBJECTIVE: Overexpression of human epidermal growth factor receptor 2 (HER2) causes cell transformation and development of various types of malignancies. Idarubicin is an effective anti-neoplastic drug but its specific delivery to the targeted cells is still a great challenge. Affibody as a cost-effective peptide molecule with low molecular weight has a high affinity for HER2 receptors. Breast and ovarian cancers as wide speared types of malignancies are associated with high expression of HER2. In the current study, we assessed the cytotoxic effects of idarubicin-ZHER2 affibody conjugate on the positive-HER2 cancer cell lines. METHODS: The cytotoxic effects of constructed idarubicin-ZHER2 affibody conjugate on the SK-BR-3, SK-OV-3, and MCF-7 cells with various levels of HER2 expression were evaluated by MTT assay following 48 hours of incubation. RESULTS: Idarubicin showed a potent and dose-dependent cytotoxic effect against all treated cell lines while the SK-OV-3 cells were significantly more sensitive. The dimeric form of the ZHER2 affibody molecule showed a mild effect on the cell viability of all treated cells at its optimum concentration. The constructed Idarubicin-ZHER2 affibody conjugate decreased the viability of SK-OV-3 cells at its optimal concentration, more efficiently and specifically than other treated cells. CONCLUSION: The ZHER2-affibody conjugate of idarubicin has a more specific cytotoxic effect compared with idarubicin alone against HER2-overexpressing ovarian cancerous cells. It appears the ZHER2-affibody conjugate of idarubicin has great potential to be implicated as an innovative anti-cancer agent in future clinical trials in patients with HER2-overexpressing ovarian cancer.

The association between mRNA expression of resistin, TNF- α, IL-6, IL-8, and ER-α in peripheral blood mononuclear cells and breast cancer.

BACKGROUND: Adipocytokines, adipose tissue-derived proteins, were demonstrated to be involved in the pathogenesis of breast cancer. We assessed the mRNA expression of resistin, tumor necrosis factor-alpha (TNF-α), interleukins 6 and 8 (IL-6, and IL-8), and estrogen receptor alpha (ER-α) in peripheral blood mononuclear cells (PBMCs) of women with and without breast cancer. METHODS: The PBMCs were isolated from the whole blood of 32 women with breast cancer and 18 women without breast cancer using density gradient centrifugation. The mRNA expression of the target genes was measured by reverse-transcription polymerase chain reaction (RT-PCR). Body mass index was calculated, additionally, clinicopathological characteristics of the breast cancer patients were determined by histopathological examination. RESULTS: The mRNA expression of resistin (3.5-fold) and IL-6 (15-fold) in PBMCs of breast cancer patients significantly increased in comparison to healthy controls. Resistin expression was significantly associated with inflammatory markers including TNF-α, IL-6, IL-8, but not with anthropometric indices. Logistic regression analysis revealed the studied adipokines were not associated with breast cancer. Based on the ROC curve analysis the diagnostic performance of IL-6 was significant (0.825, 95% CI: 0.549-0.94, p = 0.030), thus, it might be considered as a breast cancer biomarker that reflecting an early and inflammatory stage of the disease. DISCUSSION: Breast cancer is not associated with increased expression of inflammatory cytokines in PBMCs. Our results suggested that a PBMC-based gene expression test may be developed to detect breast cancer early.

Metformin synergistically increases the anticancer effects of lapatinib through induction of apoptosis and modulation of Akt/AMPK pathway in SK-BR3 breast cancer cell line.

Objectives: Combination chemotherapy is a beneficial intervention for breast cancer, versus single therapy. We investigated the effect of Metformin (Met) on Lapatinib (Lap)-induced apoptosis in SK-BR3 cells. Materials and Methods: Toxic effect of Met and Lap on SK-BR3 cells was measured using MTT assay. Flow cytometry was used to measure the co-treatment effect of Met on lapatinib-induced apoptosis. The relative expression of Bax, Bcl2, and P21 was measured using a real-time PCR. The activity of caspase 3 and 9 was measured using an ELISA kit. The protein level of AMPK and Akt was determined using Western blot analysis. Results: Metformin and lapatinib alone and combined form showed significant time- and dose-dependent toxic effects on SK-BR3 cell viability. The greatest synergistic inhibitory effect on the cell viability [combination index (CI) = 0.51] was remarkable at Met 100 mM combined with Lap 100 nM. The combination has a stronger apoptotic death (46%) versus lapatinib alone. The combination considerably increased the mRNA expression of Bax and P21, and caspase 3 and 9 activity, while, decreasing the mRNA expression of Bcl2. Additionally, the combination significantly up-regulated and down-regulated the protein levels of AMPK and Akt, respectively. Conclusion: The metformin-lapatinib combination can induce more potent apoptotic death versus each compound individually. The combination may be suggested as a valuable therapeutic intervention in patients with breast cancer. However, additional in vivo studies are necessary to evaluate the clinical use of the combination for induction of apoptosis and its antitumor effects.

Synergistic anti-cancer effects of silibinin-etoposide combination against human breast carcinoma MCF-7 and MDA-MB-231 cell lines.

OBJECTIVES: Recently, there is a significant focus on combination chemotherapy for cancer using a cytotoxic drug and a phytochemical compound. We investigated the effect of silibinin on etoposide-induced apoptosis in MCF-7 and MDA-MB-231 breast carcinoma cell lines. MATERIALS AND METHODS: The cytotoxic effects of silibinin and etoposide were determined using MTT assay after 24 and 48 hr incubation with these drugs individually and combined. The mRNA expression of Bax and Bcl2, and protein levels of P53, phosphorylated p53 (P-P53), and P21 were determined using real-time PCR and western blot analysis, respectively. The caspase 9 activity was measured using an ELISA kit. RESULTS: Silibinin and etoposide alone and combined significantly inhibit cell growth in a dose and time-dependent manner in both cell lines. The strongest synergistic effects in terms of MCF-7 cell growth inhibition [combination index (CI) = 0.066] were evident. The silibinin-etoposide combinations cause a much powerful apoptotic death (47% and 40%) compared with each compound individually in MCF-7 and MDA-MB 231 cells, respectively. Additionally, the silibinin-etoposide combinations significantly increased the expression of P53, P-P53, and P21 in MCF-7 cells. Neither silibinin nor etoposide individually increased the level of P53 and P-P53 in MDA-MB-231 cells, but both of them individually and combined increased the level of P21. CONCLUSION: Since the silibinin-etoposide combination induces apoptosis in both cell lines with and without expression of p53, thus, it is suggested that this combination may be a successful therapeutic strategy for breast cancer regardless of P53 status.

Combination of Silibinin and Curcumin Reduced Leptin Receptor Expression in MCF-7 Human Breast Cancer Cell Line.

BACKGROUND: Leptin and leptin receptor (Ob-R) are associated with worse prognosis, distant metastasis, and poor survival of breast cancer. We investigated the cytotoxic effect of silibinin and curcumin, individually and combined, on Ob-R expression in MCF-7 cells. METHODS: This study was performed from October 2017 to April 2018 at the Department of Clinical Biochemistry, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. The cytotoxic effect of silibinin and curcumin, individually and combined, and their corresponding half-maximal inhibitory concentration (IC50) values were determined using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The cells were treated with different concentrations of silibinin (50-400 µM), curcumin (10-35 µM), and their combinations for 24 and 48 hours. The expression of Ob-R was measured using the Western blot analysis by treating the cells with different concentrations of curcumin (10-25 µM), silibinin (50-250 µM), and their respective combinations. The difference in mean cell viability between the groups was calculated using one-way ANOVA followed by Tukey's post hoc test. RESULTS: Silibinin and curcumin exerted time- and dose-dependent cytotoxic effect on MCF-7 cells. After treatment with silibinin, the IC50 values were about 250 and 50 µM at 24 and 48 hours, respectively. In terms of treatment with curcumin, the IC50 values were about 25 and 15 µM at 24 and 48 hours, respectively. Following treatment with silibinin, the Western blot analysis showed that Ob-R expression significantly decreased at 150 µM (P=0.031) and 200 µM (P=0.023) concentrations. Curcumin did not significantly decrease the Ob-R expression, however, the expression significantly decreased (P=0.004) when it was combined with silibinin. CONCLUSION: The combination of silibinin and curcumin significantly reduced Ob-R expression in MCF-7 cells compared with their individual effects.

Specific targeting of HER2-positive human breast carcinoma SK-BR-3 cells by amygdaline-ZHER2 affibody conjugate.

Amygdalin induces apoptotic death in several carcinoma cells. Affibody is an engineered protein with a high affinity for human epidermal receptor 2 (HER2). We assessed the cytotoxic effects of the amygdalin-ZHER2 affibody conjugate on two breast carcinoma cell lines. The ZHER2 affibody gene was synthesized and transferred into E. coli BL21 as an expression host. After purification, the ZHER2 affibody was conjugated to amygdalin. The cytotoxic effects of amygdalin and its ZHER2 affibody conjugate on the SK-BR-3, with overexpression of HER2, and MCF-7 cells were evaluated by MTT assay. The effects of amygdalin and its conjugate on apoptotic death and expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were measured. Amygdalin individually showed a potent cytotoxic effect against both MCF-7 (IC50 = 14.2 mg ml-1) and SK-BR-3 cells (IC50 = 13.7 mg ml-1). However, the amygdalin-ZHER2 affibody conjugate had a more cytotoxic effect on SK-BR-3 (IC50 = 8.27 mg ml-1) than MCF-7 cells (IC50 = 19.8 mg ml-1). Amygdalin had a significant apoptotic effect on both cell lines and the effect of its conjugate on SK-BR-3 cells was significantly more potent than MCF-7 cells. Amygdalin increased Bax and decreased Bcl-2 expression in both cell lines. However, the effect of its conjugate on the Bax and Bcl-2 expression in SK-BR-3 was more potent than MCF-7 cells. In conclusion, the amygdalin-ZHER2 affibody conjugate may be considered as a valuable candidate for specific treatment of breast cancer patients with overexpression of HER2. However, further in vivo studies are required to explain the antitumoral effects of constructed amygdalin-ZHER2 affibody conjugate.

In vitro and in silico anticancer activity of amygdalin on the SK-BR-3 human breast cancer cell line.

In spite of several studies that have shown the cytotoxic effects of amygdalin on the different cancer cell lines, however, the chemopreventive potential of amygdalin on the breast cancer cell line is not completely understood. We investigated the effect of amygdalin on the cell death and the level of pro-apoptotic Bax protein and anti-apoptotic Bcl-2 protein in SK-BR-3 human breast cancer cell line. The cell viability of SK-BR-3 cells was evaluated by MTT assay in different concentration of amygdalin. The level of Bax and Bcl-2 in SK-BR-3 cells were measured by western blot analysis. For statistical analysis, One-way ANOVA was used for the comparison of Bax and Bcl-2 protein level and percent of cell viability between groups. The molecular docking studies of amygdalin within the Bcl-2 (PDB ID: 4LVT) and HER2 (PDB ID: 3RCD) active site, were performed using AutoDock 4.2.5. Amygdalin induced a significant reduction of cell viability in SK-BR-3 after 24-h treatment in a dose-dependent manner. Also, amygdalin causes an increase in pro-apoptotic Bax protein and a decrease in anti-apoptotic Bcl-2 protein expression in the SK-BR-3 cells. Molecular docking studies showed that amygdalin interacts with the active site amino acids of Bcl-2 and HER2 through hydrogen bonding and some hydrophobic interactions. Amygdalin can induce apoptotic death in SK-BR-3 cells by increasing pro-apoptotic Bax protein and decreasing anti-apoptotic Bcl-2 protein expression. The results suggest that amygdalin may be a valuable candidate for the treatment of breast cancer, especially in HER2 positive cells.

Effect of Hydroalcoholic Ginger Extract on Brain HMG-CoA Reductase and CYP46A1 Levels in Streptozotocin-induced Diabetic Rats.

BACKGROUND: Patients with diabetes present with lipid disorders, including hypercholesterolemia, which can be a high-risk factor for atherosclerosis. Recently, increasing interest has been focused on anti-lipidemic function of herbal medicines, especially Zingiber officinale (known as ginger), in diabetes. However, the mechanism underlying the effect of ginger on some players involved in cholesterol homeostasis of Central Nervous System (CNS) among diabetic patients remains unclear. To our knowledge, this is the first study to investigate the effect of ginger on brain regulation of Hydroxymethylglutaryl-CoA Reductase (HMG-CoA reductase) and Cholesterol 24-hydroxylase (CYP46A1), which provides a rational model for understanding brain dyslipidemia mechanisms associated with diabetes. METHODS: Brains of rats were isolated from four groups: control, non-treated diabetic, and treated diabetic groups receiving 200 or 400 mg/kg of hydroalcoholic extracts of ginger for eight weeks. HMG-CoA reductase and CYP46A1 levels in brain homogenates were determined by western-blot technique. RESULTS: Ginger root extract caused a significant decrease in HMG-CoA reductase and an increase in CYP46A1 levels in treated diabetic groups compared to diabetic control. In comparison to diabetic group, these effects were more remarkable with 400 mg/kg concentration of ginger extract. CONCLUSION: The findings showed that ginger extract has a regulatory effect on proteins involved in cholesterol homeostasis in CNS by a significant down- and up-regulation of HMG-CoA reductase and CYP46A1 levels, respectively. It can be suggested that adding ginger to daily diet of diabetic patients has useful effects and may ameliorate diabetes complications.

Immunological Responses against HER2-targeted Idarubicin-ZHER2 Conjugate in BALB/c Mice.

Targeting of cancerous cells with a high level of human epidermal growth factor receptor 2 (HER2) expressions by drug immunoconjugates is a new approach for specific delivery of chemotherapeutic agents. Our previous work indicated that idarubicin-ZHER2 affibody conjugate has a great potential for the treatment of HER2-overexpressing malignant cell lines but possible induced immune response against constructed conjugate was not addressed. In the current study, the possibility of induction of humoral and cellular immune responses against idarubicin-ZHER2 affibody conjugate in BALB/c mice was investigated. For assessment of the induced immune response, prepared and qualified idarubicin-ZHER2 affibody conjugate was administrated intravenously to BALB/c mice and the induced cellular immune response was evaluated by measuring secretion levels of interferon gamma (IFN-γ) and interleukin 10 (IL-10) cytokines by the splenocytes. Humoral response of treated mice was also assessed by measuring total immunoglobulin G (IgG) titer in mice sera. The obtained results showed that idarubicin-ZHER2 affibody conjugate at any examined concentrations could not induce secretion of IFN-γ as a pro-inflammatory cytokine. A mild increase in the level of regulatory IL-10 cytokine was seen in the treated mice although no dose dependency in the level of IL-10 production was observed. Furthermore, results showed that idarubicin-ZHER2 conjugate could not induce IgG production in the treated mice. Based on these findings, the idarubicin-ZHER2 conjugate can be considered as a candidate for the development of new therapeutics against HER2-overexpressing cancers although further in vivo studies are needed.

Effects of the Hydroalcoholic Extract of Zingiber officinale on Arginase I Activity and Expression in the Retina of Streptozotocin-Induced Diabetic Rats.

BACKGROUND: Emerging evidence suggests that an increased arginase activity is involved in vascular dysfunction in experimental animals. Zingiber officinale Roscoe, commonly known as ginger, has been widely used in the traditional medicine for treatment of diabetes. OBJECTIVES: This study aimed at investigating the effects of the hydroalcoholic extract of Z. officinale on arginase I activity and expression in the retina of streptozotocin (STZ)-induced diabetic rats. METHODS: In this experimental study, 16 male Wistar rats weighing 200 - 250 g were assessed. Diabetes was induced via a single intraperitoneal injection of STZ (60 mg/kg body weight). The rats were randomly allocated into four experimental groups. Untreated healthy and diabetic controls received 1.5 mL/kg distilled water. Treated diabetic rats received 200, and 400 mg/kg of the Z. officinale extract dissolved in distilled water (1.5 mL/kg). Body weight, blood glucose and insulin concentration were measured by standard methods. The arginase I activity and expression were determined by spectrophotometric and western blot analysis, respectively. RESULTS: Our results showed that blood glucose concentration was significantly decreased in diabetic rats treated with the extract compared to untreated diabetic controls (P < 0.01). Treatment with 400 mg/kg of the extract reduced arginase I activity and expression (P < 0.05). A significant elevation in body weight was observed in diabetic rats treated with the extract. Serum insulin was significantly increased in diabetic rats treated with 400 mg/kg of the extract compared to diabetic controls (P < 0.05). CONCLUSIONS: Our results suggest that the Z. officinale hydroalcoholic extract may potentially be a promising therapeutic option for treating diabetes-induced vascular disorders, possibly through reducing arginase I activity and expression in the retina.

Effect of Silibinin on Maspin and ERα Gene Expression in MCF-7 Human Breast Cancer Cell Line.

BACKGROUND AND OBJECTIVE: According to reports, a serine protease inhibitor (Maspin) suppresses metastasis, invasion and angiogenesis in breast and prostate cancers. Silibinin is a natural polyphenolic flavonoid with anti-cancer activity. We assessed the effects of silibinin on cell viability, maspin and ERα gene expression in MCF-7 cell line. METHODS: The human MCF-7 breast cancer cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with different concentrations of silibinin (100-600 µg/mL) for 24, 48 and 72 hours. The cytotoxic effect of silibinin on MCF-7 viability was determined using Methyl-Thiazolyl-Tetrazolium (MTT) assay by IC50 determination. The fold changes of Maspin and ERα expression were determined by reverse-transcription real-time Polymerase Chain Reaction (PCR). All experiments on the cells were performed in triplicates. RESULTS: The maximum inhibitory effect of silibinin on cell viability was observed at 600 µg/mL after 72-hour incubation (p = 0.001). Incubation of the cells with silibinin for 48 and 72 hours significantly decreased IC50 values to 250 and 207 µg/mL (p = 0.005 and p= 0.006), respectively. The expression of maspin and ERα in the treated cells compared to controls was significantly decreased following treatment with different concentrations of silibinin during a 24-hour period. CONCLUSIONS: Silibinin reduces both maspin and ERα gene expression in MCF-7 cell line. The therapeutic effect of silibinin on the treatment of breast cancer may be mediated by the reduction of ERα expression. For verifying this hypothesis and the possible therapeutic implication of silibinin on breast cancer, further studies in this direction are necessary.

Association of I405V polymorphism of colesteryl ester transfer protein gene with coronary artery disease in men with type 2 diabetes.

BACKGROUND: Colesteryl ester transfer protein (CETP) plays a key role in the metabolism of lipoproteins; therefore, polymorphisms of its gene can affect susceptibility to coronary artery disease (CAD) in diabetes mellitus. The aim of the present study was to investigate association between I405V polymorphism of CETP gene and risk of CAD in patients with type 2 diabetes mellitus. METHODS: The current case-control study was conducted on 143 patients with type 2 diabetes and angiographically diagnosed CAD and 150 patients with type 2 diabetes and without CAD. Genotyping was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The presence of CAD was defined as higher than 50% reduction in coronary artery diameter. RESULTS: The genotype frequencies of I405V polymorphism were II (27.3% vs. 23.2%), IV (61.5% vs. 67.5%), and VV (11.2% vs. 9.3%) in diabetic with CAD compared to diabetic without CAD (χ2 = 1.164) (P = 0.55). The I and V alleles were found at frequencies of 63.6% and 61.6% in the diabetic with CAD group and 36.4% and 38.4% in the diabetic without CAD group (χ2 = 0.263) (P = 0.60). No significant difference was observed between two groups in terms of genotype and allele frequency. Moreover, no significant association was observed between II, IV, and VV genotypes and lipid profiles in both groups. However, a significant difference was observed between genotype distributions of I405V polymorphism in men according to the severity of CAD. CONCLUSION: It is speculated that I405V polymorphism may be associated with the severity of coronary artery stenosis only in men with type 2 diabetes mellitus.

Effects of Two Common Promoter Polymorphisms of Transforming Growth Factor-ß1 on Breast Cancer Risks in Ahvaz, West South of Iran.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) has a critical role in breast cancer initiation and progression. OBJECTIVES: We have investigated the possible differences in two promoter polymorphisms (-509C/T and -800G/A) of TGF-ß1 gene between breast cancer cases and controls. PATIENTS AND METHODS: A total of 100 patients with confirmed breast cancer and 100 subjects without breast cancer was selected. Two promoter polymorphisms (-509C/T and -800G/A) of TGF-ß1 gene were genotyped using PCR-based restriction fragment length polymorphism (RFLP) method. RESULTS: The allele frequencies were 63% for C allele and 37% for T allele of SNP -509C/T and 66% for G allele and 34% for A allele of SNP -800G/A. Although no significant difference has observed between two groups, according to the genotype distribution, However, the TT genotype of -509 and AA genotype of -800 was significantly associated with breast cancer risk [odds ratio (OR) = 2.409; 95% confidence interval (CI) = 1.087 - 5.337, P = 0.030; and OR = 2.383; CI = 1.039 - 5.40, P = 0.040, respectively]. In addition, a multinomial logistic regression model shown, homozygous of -800 G/A (OR = 0.570; 95% CI = 0.362 - 0.896, P = 0.015); and HDL-C (OR = 0.935; 95% CI = 0.906 - 0.965, P < 0.001) were the selected variables associated with the presence of breast cancer. Haplotype analysis has shown no significant association between TGF-ß1 haplotypes and breast cancer risk. CONCLUSIONS: Our results indicated that among two promoter polymorphisms of the TGF-ß1gene, -800G/A compared to -509C/T is more associated with breast cancer.

Lack of association between MTHFR C677T polymorphism and breast cancer risk in Ahvaz, west south-Iran.

BACKGROUND: Association between C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR), a key enzyme involved in folate metabolism and DNA methylation, and breast cancer risk are inconsistent. We investigated in a case-control study, possible effect of the common MTHFR C677T polymorphism on breast cancer risk in a sample of Iranian patients. MATERIALS AND METHODS: The study subjects comprised of 123 breast cancer cases and 110 cancer-free control, who were matched for age and body mass index (BMI). C677T genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Lipid profile was measured in all subjects by standard method. RESULTS: The genotypes distributions (CC, CT, and TT) were 55.3, 39, and 5.7% in breast cancer cases and 51.8, 44.5, and 3.6% in controls. Chi square analysis revealed that there was no significant association between breast cancer risk and MTHFR genotypes and alleles. Additionally, no significant association was observed between C677T genotypes and biochemistry parameters. A multinomial logistic regression model with MTHFR genotypes, lipid profiles, BMI and age as covariates revealed that there is no significant association between MTHFR genotypes and risk of breast cancer, but higher values of LDL and HDL significantly increase risk of breast cancer. CONCLUSIONS: Our findings do not support the hypothesis that genetic variation in the MTHFR C677T polymorphism is implicated in the breast cancer risk in a sample of Iranian patients.

Association between two common polymorphisms (single nucleotide polymorphism -250G/A and -514C/T) of the hepatic lipase gene and coronary artery disease in type 2 diabetic patients.

BACKGROUND: Variations in the hepatic lipase (HL) gene are the potential candidate for coronary artery disease (CAD) especially in type 2 diabetes mellitus (T2DM) in diverse populations. We assessed the association of -514C/T and -250G/A polymorphisms in HL (LIPC) gene with CAD risk in Iranian population with type 2 diabetes. MATERIALS AND METHODS: We evaluated 322 type 2 diabetic patients, 166 patients with normal angiograms as controls and 156 patients those identified with CAD undergoing their first coronary angiography as CAD cases. Genotyping of -514C/T and -250G/A polymorphisms in the promoter of the LIPC gene were studied by polymerase chain reaction (PCR)-restriction fragment length polymorphism technique. RESULTS: Genotype distributions in CAD cases (73.7%, 20.5%, and 5.8% for -250G/A) and (62.2%, 32.7%, and 5.1% for -514C/T) were significantly different from those in controls (60.8%, 37.4%, and 1.8% for -250G/A) and (51.2%, 48.2%, and 0.6% for -514C/T). CAD cases had lower A-allele frequency than controls (0.131 vs. 0.196, P = 0.028). The odds ratio for the presence of -250 (GG + GA) genotype and A allele in CAD cases were 2.206 (95% confidence interval [CI] =1.33-3.65, P = 0.002) and 1.609 (95% CI = 1.051 -2.463, P = 0.029) respectively. Haplotype analysis demonstrated a significant association between especially LIPC double mutant (-250 A/-514 T) haplotype and presence of CAD. CONCLUSION: Our findings indicated that -250 G/A polymorphism rather than -514 C/T polymorphism of LIPC gene is more associated with the increased risk of CAD particularly in women with T2DM.