Induction of cell apoptosis by biliverdin reductase inhibitor in MCF-7 and MDA-MB-468 breast cancer cell lines: Experimental and in silico studies.

Biliverdin reductase, biliverdin and bilirubin are known as important components of cellular signaling pathways that play major roles in cell proliferation and apoptosis, although their physiological relevance is still under evaluation. This study was designed to investigate the expression and activity of BVR-A and its apoptotic effect in the breast cancer cell lines, MCF-7 and MDA-MB-468. The expression of BVR-A was examined by real-time PCR and western blot analysis. Bilirubin concentration was measured by HPLC and molecular docking was performed to identify an appropriate inhibitor for BVR-A. To detect cell apoptosis, annexin V-PI staining, caspase-3, -8, and -9 activities were evaluated. Cell viability was reduced by biliverdin, in a dose-dependent manner, and an intrinsic apoptotic response occurred which was evidenced by caspase-3 and -9 activities. The intra- and extracellular concentrations of bilirubin were higher in MCF-7 cells than those of MDA-MB-468 cells. The expression of BVR-A, at mRNA and protein levels, in MCF-7 was also higher than that of MDA-MB-468 cells. Treatment of both cell lines with biliverdin plus DTNB, a BVR-A inhibitor, increased the cell death significantly when compared with biliverdin alone. Using annexin V-PI staining and assessment of caspase-3 activity, it was confirmed that biliverdin together with DTNB increases apoptosis in breast cancer cells. In conclusion, biliverdin has an important role in cell apoptosis and inhibition of biliverdin reductase increases the apoptotic effect of biliverdin.

Expression of SR-B1 receptor in breast cancer cell lines, MDAMB-468 and MCF-7: Effect on cell proliferation and apoptosis.

OBJECTIVES: High-density lipoprotein (HDL) is necessary for proliferation of several cells. The growth of many kinds of cells, such as breast cancer cells (BCC) is motivated by HDL. Cellular uptake of cholesterol from HDL which increases cell growth is facilitated by scavenger receptors of the B class (SR-BI). The proliferative effect of HDL might be mediated by this receptor. It is also believed that HDL has an anti-apoptotic effect on various cell types and promotes cell growth. This study was designed to investigate SR-BI expression, proliferation and apoptotic effect of HDL on human BCC lines, MCF-7 and MDA-MB-468. MATERIALS AND METHODS: Real-time-PCR method was used to evaluate expression of SR-BI, and cholesterol concentration was measured using a cholesterol assay kits (Pars AZ moon, Karaj, Iran). Cell viability was assessed using the MTT test. To identify cell apoptosis, the annexin V-FITC staining test and caspase-9 activity assay were applied. RESULTS: Treatment of both cell lines (MCF-7, MDA-MB-468) with HDL results in augmentation of SR-BI mRNA expression and also elevation of the intracellular cholesterol (P<0.01). HDL induced cell proliferation, cell cycle progression, and prevented activation of caspase-9 (P<0.05). We also demonstrated that inhibition of SR-B1 by BLT-1 could reduce cell proliferation, and induction of SR-B1 receptor by quercetin increased HDL-induced proliferation in both cell lines (P<0.05). CONCLUSION: It can be concluded that alteration in HDL levels by SR-B1 activator (Quercetin) or inhibitor (BLT-1) may affect BCC growth and apoptosis induction.

Differential expression of miR-1297, miR-3191-5p, miR-4435, and miR-4465 in malignant and benign breast tumors.

OBJECTIVES: MicroRNAs (miRs) are a class of small non-coding RNAs which are associated with tumor growth and progression. In the present study, we assessed the expression of selected miRs in malignant, benign, and adjacent normal breast tissues. MATERIALS AND METHODS: The expression of miR-1297, miR-3191-5P, miR-4435, and miR-4465 were evaluated in malignant (n=50), benign (n=35), and adjacent normal breast tissues (n=20) using qRT-PCR. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were generated for evaluating the diagnostic values of miRs. To evaluate diagnostic efficacy, miRs-based score was obtained using the logistic regression model. RESULTS: Among malignant tumors, the expression of miR-1297, miR-3191-5p, and miR-4435 was significantly lower (P=0.024, P<0.001 and P=0.031), respectively. The expression of miR-4465 was higher (P=0.023) than that of normal tissue. The expression of these miRs was lower than those of benign tumors (P<0.01, P<0.001, P<0.0001, and P<0.01, respectively). We observed a positive correlation between miR-4465 expression levels and tumor stage (P=0.042) and a negative correlation with grade and Ki-67 score (P<0.05). The AUCs for miR-1297, miR-3191-5p, miR-4435, and miR-4465 in malignant tumors versus normal tissues were 0.784, 0.700, 0.976, and 0.865 and versus benign tumors they were 0.938, 0.857, 0.981, and 0.785, respectively. The optimal logit(P) value of 0.262 distinguished malignant from normal subjects with a sensitivity of 0.91, specificity of 0.85, and an overall accuracy of 0.89. CONCLUSION: The panel of these miRs are suggested as possible onco-miRs(miR-4465) or tumor suppressor-miRs (miR-3191-5P, miR-1297, miR-4435). Overall, our results indicated that these miRs could be introduced as diagnostic biomarkers in breast cancer patients.

Expression level of VLDL receptor and VLDL-c levels in the malignant and benign breast tumors: The correlation with miRNA-4465 and miRNA-1297.

Breast cancer as one of the most prevalent cancers has high morbidity and mortality. Very low-density lipoprotein receptor (VLDLR) is a multifunctional receptor which plays a principal role in the tumor development through affecting cell metastasis and proliferation. The VLDLR as a target for miRNA-4465 and miRNA-1297 was predicted using bioinformatics analysis. Tissue specimens of malignant (n = 50), benign (n = 35) and corresponding normal breast (n = 20) were considered to evaluate the expression of VLDLR using RT-qPCR and western blotting. The VLDL cholesterol (VLDL-C) levels were quantified using a colorimetric assay. The relative VLDLR expression was found in the malignant tumors, which was significantly lower than that in the normal tissues (P<0.05). The expression levels of VLDLR had no significant difference between malignant and benign tissues (P>0.05). Correlation analysis revealed that the VLDLR expression level had a direct correlation with miRNA-1297 (R=0.566, P<0.05), but a reverse one with miRNA-4465 (R = -0.663, P<0.0001). The VLDL-C level in the malignant and normal tissues was lower than that in the benign tumors, which was not significant (P>0.05). The expression levels of VLDLR in E+P-H- (ER+,PR-,HER2-) tumors were higher than those in other subtypes (P<0.05). Furthermore, a negative correlation was found between the VLDLR expression level and the Ki 67% score. These data revealed that the lower expression of VLDLR mediated by the high expression levels of miRNA-4465 may be involved in the development of breast cancer. These results might provide some evidence for the effect of VLDLR on the breast cancer.

Evaluation of LDL receptor and Scavenger Receptor, Class B, Type 1 in the malignant and benign breast tumors: The correlation with the expression of miR-199a-5p, miR-199b-5p and miR-455-5p.

AIMS: The purpose of present study was to examine the correlations of LDL (LDLR) and HDL (SR-B1) receptors with lipoproteins, miR-199a-5p, miR-199b-5p, miR-455-5p in the malignant and benign breast tumors. METHODS: Total cholesterol-rich-lipoproteins and the receptors were determined using enzymatic-homogeneous and ELISA methods. The expression levels of miRNAs were detected by qRT-PCR. RESULTS: Receptor expressions and lipoproteins concentration were significantly higher in the malignant tumors (p < 0.05). Positive correlation was found for LDLR with Ki67% and Her2+. HDL-C content of TNBC tumors was higher than those of Non-TNBC (p < 0.05). The expression level of miR-199a-5p was found to be downregulated significantly in the malignant tumors of <2 cm, TNBC, HER2- or stage3. The expression of miR-199b-5p was downregulated in the malignant tumors and was negatively associated with TNBC, stage and Her2+. The expression of miR-455-5p was significantly correlated with Her2- (p < 0.05). A positive correlation was observed for SR-B1 or LDLR with HDL-C or LDL-C and also for SR-B1 with LDLR, although a reverse association was detected for the expression of miR-199b-5p with LDLR in the malignant tumors (p < 0.05). No significant correlations were found for miR-199a-5p or miR-455-5p with LDLR or SR-B1 expressions and also for LDL-C and SR-B1 with clinicopathological features (p ≥ 0.05). CONCLUSIONS: Mechanisms potentially involved in the present findings may be due to the lipid internalization and lipoprotein consumption through LDLR and SR-B1 over expression. It is noteworthy that the expression of miR-199b-5p is negatively correlated with LDLR which may suggest it as a suppressor for LDLR expression in the breast cancer.