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.

Soluble guanylate cyclase isoenzymes: The expression of α1, α2, ß1, and ß2 subunits in the benign and malignant breast tumors.

Soluble guanylate cyclase (sGC) encompasses α and ß subunits. This study examined the expression of α1, α2, ß1, and ß2 subunits in the malignant and benign breast tumors using the Western blot analysis. Both benign and malignant tumors showed a significantly higher expression of the α1 subunit in comparison with normal tissues (p < 0.0001). In contrast, the expression of α2 and ß2 sGC were significantly lower in these tumors than normal tissues (p < .0015 and p < .001, p < .007 and p < .0001, respectively). The expression level of α1 sGC was significantly correlated with ER + PR+ (p < .0001). A significant correlation was also detected for sGC-α1 and -α2 expression with c-erbB2-negative status (p < .01). However, the expression level of sGC was not associated with tumor stage, tumor grade, or other clinicopathological features. In conclusion, as the expression of α1 sGC is upregulated and α2 and ß2 sGC are downregulated in malignant breast tumors. Variations in the expression of sGC isoenzymes may be suggested as an indicator to confirm the enzyme antitumor activity.

Differential expression of alternative transcripts of soluble guanylyl cyclase, GYCY1a3 and GUCY1b3 genes, in the malignant and benign breast tumors.

Extensive alterations in splicing is one of the molecular indicator for human cancers. Soluble guanylyl cyclase (sGC), an obligatory heterodimer, is composed of α1 and ß1 subunits. Each subunit is encoded by a separate gene, GUCY1a3 and GUCY1b3, correspondingly. sGC activity has been regulated by an alternative splicing and it has an important effect on the breast cancer. sGC alternative splicing has been evaluated in the 55 malignant, 25 benign and 30 normal breast tissues using qRT-PCR and RT-PCR. The differences between groups were analyzed by Mann-Whitney U. The expression of six different splice forms have been detected, three for α1 and three for ß1 sGC. Expressions of Tr1, Tr2 ß1 sGC and Tr7, Tr6 α1 sGC mRNA in the malignant breast tumors were significantly lower than those of benign and normal breast tissues. However, the expression of Tr3 α1 sGC mRNA was significantly higher than that of benign and normal tissues. Present data have provided some evidences for an alteration in the expression of α1 and ß1 sGC alternative splicing forms which may contribute to the loss of sGC functions in the breast cancer. The observed information might be discussed by the cGMP status.

Targeting Cell Necroptosis and Apoptosis Induced by Shikonin via Receptor Interacting Protein Kinases in Estrogen Receptor Positive Breast Cancer Cell Line, MCF-7.

BACKGROUND: Recognition of a new therapeutic agent may activate an alternative programmed cell death for the treatment of breast cancer. OBJECTIVE: Here, it has been tried to evaluate the effects of Shikonin, a naphthoquinone derivative of Lithospermum erythrorhizon, on the induction of necroptosis and apoptosis mediated by RIPK1-RIPK3 in the ER+ breast cancer cell line, MCF-7. METHODS: In the current study, cell death modalities, cell cycle patterns, RIPK1 and RIPK3 expressions, caspase-3 and caspase-8 activities, reactive oxygen species and mitochondrial membrane potential have been evaluated in the Shikonin-treated MCF-7 cells. RESULTS: Necroptosis and apoptosis have been occurred by Shikonin, with a significant increase in RIPK1 and RIPK3 expressions, although necroptosis was the major rout in MCF-7 cells. Shikonin significantly increased the percentage of the cells in sub-G1 and also those in the later stages of cell cycle, which represents an increase in necroptosis and apoptosis. Under caspase inhibition by Z-VAD-FMK, Shikonin has stimulated necroptosis, which could be arrested by Nec-1. An increase in ROS levels and a decrease in the mitochondrial membrane potential have also been observed. CONCLUSION: On the basis of present findings, Shikonin has been suggested as a good candidate for the induction of cell death in ER+ breast cancer, although further investigations, experimental and clinical, are required.

Evaluation of RIP1K and RIP3K expressions in the malignant and benign breast tumors.

Receptor-interacting protein kinase 1 (RIP1K) and RIP3K belong to RIPK family, which regulate cell survival and cell death. In the present investigation, the expression levels of RIP1K and RIP3K were evaluated in the 30 malignant, 15 benign, and 20 normal breast tissues, and their correlation with clinicopathological characteristics was also studied. The expression levels of RIP1K and RIP3K were determined, by western blot analysis. The relative RIP1K expression was significantly higher in the malignant and benign tumors when compared to those of normal tissues (P < 0.0001 and P < 0.001, respectively). However, the expression level of RIP3K was significantly lower in the malignant tumors than those of normal and benign values (P < 0.001 and P < 0.01, respectively). Positive significant correlation was found for RIP1K expression with tumor size (P < 0.001), grades (P < 0.0001), and c-erbB2 (P < 0.001), but negative significant correlation was detected with patient's age (P < 0.001), estrogen receptor (ER) (P < 0.001), progesterone receptor (PR) (P < 0.01), and P53 (P<0.01) status. RIP3K expression was significantly lower in the pre-menopauses (P < 0.01), grade III (P < 0.05), ER-negative (P < 0.05), and c-erbB2-negative malignant tumors, but no correlation was detected with tumor size, PR, and P53 status. No significant correlation was observed for RIP1K and RIP3K expressions with Ki67 and Her2. Based on the present results, it is concluded that reduction of RIP3K expression in the malignant breast tumor might be an important evidence to support the antitumor activity of this enzyme in vivo. However, RIP1K expression was shown to be higher in the malignant breast tumors than those of normal and benign breast tissues, which probably designates as a poor prognostic factor.

RIP1K and RIP3K provoked by shikonin induce cell cycle arrest in the triple negative breast cancer cell line, MDA-MB-468: necroptosis as a desperate programmed suicide pathway.

Resistance to cell death and reprogramming of metabolism are important in neoplastic cells. Increased resistance to apoptosis and recurrence of tumors are the major roadblocks to effective treatment of triple negative breast cancer. It has been thought that execution of necroptosis involves ROS generation and mitochondrial dysfunction in malignant cells. In this study, the effect of shikonin, an active substance from the dried root of Lithospermum erythrorhizon, on the induction of necroptosis or apoptosis, via RIP1K-RIP3K expressions has been examined in the triple negative breast cancer cell line. The expression levels of RIP1K and RIP3K, caspase-3 and caspase-8 activities, the levels of ROS, and mitochondrial membrane potential have been studied in the shikonin-treated MDA-MB-468 cell line. An increase in the ROS levels and a reduction in mitochondrial membrane potential have been observed in the shikonin-treated cells. Cell death has mainly occurred through necroptosis with a significant increase in the RIP1K and RIP3K expressions, and characteristic morphological changes have been observed. In the presence of Nec-1, caspase-3 mediating apoptosis has occurred in the shikonin-treated cells. The current findings have revealed that shikonin provoked mitochondrial ROS production in the triple negative breast cancer cell line, which works as a double-edged executioner's ax in the execution of necroptosis or apoptosis. The main route of cell death induced by shikonin is RIP1K-RIP3K-mediated necroptosis, but in the presence of Nec-1, apoptosis has prevailed. The present results shed a new light on the possible treatment of drug-resistant triple negative breast cancer.

Shikonin Induced Necroptosis via Reactive Oxygen Species in the T-47D Breast Cancer Cell Line.

Breast cancer, the most common cancer in the women, is the leading cause of death. Necrotic signaling pathways will enable targeted therapeutic agents to eliminate apoptosis-resistant cancer cells. In the present study, the effect of shikonin on the induction of cell necroptosis or apoptosis was evaluated using the T-47D breast cancer cell line. The cell death modes, caspase-3 and 8 activities and the levels of reactive oxygen species (ROS) were assessed. Cell death mainly occurred through necroptosis. In the presence of Nec-1, caspase-3 mediated apoptosis was apparent in the shikonin treated cells. Shikonin stimulates ROS generation in the mitochondria of T-47D cells, which causes necroptosis or apoptosis. Induction of necroptosis, as a backup-programmed cell death pathway via ROS stimulation, offers a new strategy for the treatment of breast cancer.

Targeting prostate cancer cell metabolism: impact of hexokinase and CPT-1 enzymes.

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.

Apoptosis Induced by 13-S-hydroxyoctadecadienoic acid in the Breast Cancer Cell Lines, MCF-7 and MDA-MB-231.

UNLABELLED: Objective(s) : The 15-Lipoxygenase-1(15-LOX-1) pathway has become of considerable interest as a promising molecular approach for the modulation of cancer cell growth. 13-S-hydroxyoctadecadienoic acid (13(S)-HODE) is a main metabolite of 15-LOX-1 which is proposed to influence the cancer cell's growth. This study aims to investigate the role of 13(S)-HODE in the regulation of cell growth and apoptosis in the breast cancer cell lines. Materials and Methods : MTT assay was used to examine the cytotoxic effect of 13(S)-HODE in the breast cancer cells, MCF-7 and MDA-MB-231.Annexin-V-FITC staining and cell cycle analysis were performed using flow cytometry. The effect of 13(S)-HODE on the expression level of Peroxisome proliferator-activated receptors-δ (PPAR-δ) was also evaluated. Results : The results demonstrated that 13(S)-HODE inhibited cell growth in a dose and time dependant manner in MCF-7 and MDA-MB-231 cell lines. The reduction of cell growth was associated with the induction of cell cycle arrest and apoptosis in the breast cancer cell lines. Moreover, PPAR-δ was down-regulated in response to 13(S)-HODE administration. CONCLUSION: This study conducted evidences in to the stimulatory effect of 13(S)-HODE on the inhibition of cell growth and induction of apoptosis in the breast cancer cell lines.

Apoptosis Induced by 13-S-hydroxyoctadecadienoic acid in the Breast Cancer Cell Lines, MCF-7 and MDA-MB-231.

UNLABELLED: Objective(s) : The 15-Lipoxygenase-1(15-LOX-1) pathway has become of considerable interest as a promising molecular approach for the modulation of cancer cell growth. 13-S-hydroxyoctadecadienoic acid (13(S)-HODE) is a main metabolite of 15-LOX-1 which is proposed to influence the cancer cell's growth. This study aims to investigate the role of 13(S)-HODE in the regulation of cell growth and apoptosis in the breast cancer cell lines. Materials and Methods : MTT assay was used to examine the cytotoxic effect of 13(S)-HODE in the breast cancer cells, MCF-7 and MDA-MB-231.Annexin-V-FITC staining and cell cycle analysis were performed using flow cytometry. The effect of 13(S)-HODE on the expression level of Peroxisome proliferator-activated receptors-δ (PPAR-δ) was also evaluated. Results : The results demonstrated that 13(S)-HODE inhibited cell growth in a dose and time dependant manner in MCF-7 and MDA-MB-231 cell lines. The reduction of cell growth was associated with the induction of cell cycle arrest and apoptosis in the breast cancer cell lines. Moreover, PPAR-δ was down-regulated in response to 13(S)-HODE administration. CONCLUSION: This study conducted evidences in to the stimulatory effect of 13(S)-HODE on the inhibition of cell growth and induction of apoptosis in the breast cancer cell lines.

Induction of apoptosis by Trichostatin A in human breast cancer cell lines: involvement of 15-Lox-1.

15-Lipoxygenase-1 (15-Lox-1) is a key enzyme mediating oxidative metabolism of polyunsaturated fatty acids and has attracted considerable interest as a potential target for the induction of apoptosis in cancer cells. Knowledge of relationship between 15-Lox-1 and histone deacetylase inhibitors is lacking in the breast cancer. This study is aimed to investigate the role of Trichostatin A (TSA) and 13(S)-HODE, as a metabolite of 15-Lox-1, in the regulation of breast cancer cell growth. The cytotoxic effect of TSA, as a potent HDAC inhibitor, was measured using MTT assay. Annexin V-FITC and PI staining were performed to detect apoptosis and cell cycle distribution using Flow cytometry. The role of 15-Lox-1 in the regulation of cell growth was assessed by 15-Lox-1 inhibitor and the level of 15-Lox-1 metabolite was measured to determine 15-Lox activity after treatment by TSA. The results demonstrated that TSA induced cell growth inhibition via 15-Lox-1, in a dose- and time-dependent manner, and subsequently accompanied by the cell cycle arrest and induction of apoptosis. Moreover, growth inhibitory effect of TSA was associated with the elevation of 15-Lox-1 metabolite (13(S)-HODE). This study provided evidences that the inhibitory effect of TSA on the breast cancer cell growth occurs via the induction of 15-Lox-1 activity and 13(S)-HODE production. Our findings underline the possible role of 15-Lox-1/13(S)-HODE pathway as a promising molecular approach for the induction of apoptosis in breast cancer cells.

Evaluation of PDE5 and PDE9 expression in benign and malignant breast tumors.

BACKGROUND AND AIMS: Phosphodiesterases 5 and 9 (PDE5, PDE9) are enzymes responsible for regulating second messenger signaling by hydrolyzing 3',5' cyclic guanosine monophosphate (cGMP). PDE isoforms are deregulated in some types of human cancer. The present study was carried out to evaluate the expression of phosphodiesterase isoenzymes, PDE5 and PDE9, in benign and malignant breast tumors. METHODS: The expression levels of PDE5 and PDE9 were assayed in malignant and benign breast tumors and corresponding normal breast tissues using quantitative real-time RT-PCR. Moreover, the correlation between PDE5, PDE9 relative expression and clinicopathological characteristics were analyzed. RESULTS: The relative expressions of PDE5 and PDE9 in malignant tumors were significantly higher than those of respective normal breast tissues and benign tumors (5.5-fold, p <0.001 and 6-fold, p <0.001, respectively). Furthermore, a significant positive correlation was found between PDE5 and PDE9 overexpression and tumor grade, stage, and lymph node involvement. However, a negative correlation was observed with age. CONCLUSIONS: Based on the present results, it is concluded that assessment of PDE5 and PDE9 expression may be useful in the differential diagnosis of benign and malignant breast disease and successful treatment of breast cancer. To the best of our knowledge, this is the first study to show that PDE5 and PDE9 expression levels are higher in malignant breast tumors than those of normal and benign breast tissue.

Expression of cGMP-dependent protein kinase, PKGIα, PKGIß, and PKGII in malignant and benign breast tumors.

Cyclic GMP-dependent protein kinases (PKG) constitute a small family of enzymes that are encoded by two genes. Two major forms of PKG have been identified in mammalian cells, PKG I and PKG II. In addition, there are two splice variants of PKG I, which are designated as Iα and Iß. There are increasing evidences that PKG can play an important role in the inhibition of cell proliferation and induction of apoptosis. In our previous studies, the inhibitory effects of cGMP/PKG on the cell growth were indicated using breast cancer cell lines. Accordingly, the present study was designed to compare the expression levels of three PKG isoforms in normal, benign, and malignant breast tissues. The expression level of PKG isoforms was assayed using quantitative real-time RT-PCR. The correlation between relative expression of PKG isoforms and clinicopathological characteristics were also analyzed. Downregulation of PKG isoforms was observed in the malignant and benign tumors when compared to those of respective normal tissues. No significant correlation was found between PKGIα, PKGIß, and PKGII expression and clinicopathological features. The present study is the first to evaluate the expression level of PKG isoforms PKGIα, PKGIß, and PKGII in the malignant and benign breast tumors. Reduction in the PKG expression is an important evidence to support the antitumor activity of this enzyme in vivo.

Adenosine induces cell-cycle arrest and apoptosis in androgen-dependent and -independent prostate cancer cell lines, LNcap-FGC-10, DU-145, and PC3.

BACKGROUND: Adenosine has been shown to inhibit cell growth and induce apoptosis in the several cancer cells via intrinsic and extrinsic pathway. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis in the DU-145, PC3, and LNcap-FGC10 human prostate cancer cells. METHODS: To observe cell viability and proliferation, MTT assay, cell counting, and BrdU assay were carried out in DU-145, PC3, and LNcap-FGC10 cells. Apoptosis was assessed with the analysis of cell cycle, Hoechst 33258 staining, propidium iodide and annexin-V staining, reactive oxygen species (ROS) formation, mitochondrial membrane potential (ΔΨM) measurement, caspase-3 activity assay, Bcl-2 and Bax protein expression. Moreover, the expression of adenosine receptors and the effects of adenosine receptor (A(1) , A(2a) , and A(3) ) antagonists were examined. RESULT: Adenosine significantly reduced cell proliferation in a dose-dependent manner in DU-145, PC3, and LNcap-FGC10 cell lines. Adenosine induced arrest in the cell-cycle progression in G0/G1 phase through Cdk4/cyclinD1-mediated pathway. Adenosine induced apoptosis, which was determined by morphological changes and increased sub-G1 population. Furthermore, increase of ROS, loss of MMP, activation of caspase-3, and down-regulation of Bcl-2 expression was observed. A(1) , A(2a) , A(2b) , and A(3) adenosine receptors mRNA are expressed in the cell lines. Moreover, adenosine-induced apoptosis was inhibited by MRS1220, A(3) adenosine receptor antagonist. CONCLUSION: Our results suggest that adenosine induced apoptosis in prostate cancer cells via the mitochondrial pathway and is related to the adenosine receptors. These data might suggest that adenosine could be used as an agent for the treatment of prostate cancer.

Induction of apoptosis by type Iß protein kinase G in the human breast cancer cell lines MCF-7 and MDA-MB-468.

Activation of protein kinase G (PKG) by cyclic guanosine 3,5-monophosphate (cGMP) has become of considerable interest as a novel molecular approach for the induction of apoptosis in cancer cells. This study was conducted to investigate the role of PKG isoforms in the regulation of cell growth in human breast cancer cell lines MCF-7 and MDA-MB468. The expression levels of PKG isoforms were also examined using real-time reverse transcriptase polymerase chain reaction. No differences in the gene expression of PKG isoforms were observed between MCF-7 and MDA-MB-468 cells. To investigate the effects of PKG isoforms on the regulation of cell growth, the cGMP analogues 8-APT-cGMP (PKGIα activator), 8-Br-PET-cGMP (PKGIß activator) and 8-pCPT-cGMP (PKGII activator) were employed. Apoptosis was assessed with the Annexin-V-propidium iodide (PI) staining, cell cycle analysis and caspase-3/9 activity assay. Treatment of MCF-7 and MDA-MB-468 cells with 8-Br-PET-cGMP resulted in a concentration-dependent cell growth inhibition and apoptosis, whereas neither PKGIα nor PKGII activators had any effect on the cell growth. The role of PKGIß in the inhibition of cell growth was confirmed using PKGI and PKGII inhibitors. The present study is the first to demonstrate the involvement of PKGIß in the inhibition of cell growth and induction of apoptosis in breast cancer cells.

Molecular mechanisms of A3 adenosine receptor-induced G1 cell cycle arrest and apoptosis in androgen-dependent and independent prostate cancer cell lines: involvement of intrinsic pathway.

PURPOSE: A3 adenosine receptor has shown several physiological and pathological activities, including cell proliferation and apoptosis in various cancer cell lines. This study is designed to investigate molecular mechanism and apoptotic pathway of A3 adenosine receptor in DU-145, PC3 and LNcap-FGC10 human prostate cancer cells. METHODS: The expression level of A3 adenosine receptor was examined using real-time RT-PCR. cAMP concentration was also measured. MTT viability, cell counting and BrdU incorporation tests were used to study the cell proliferation effect of IB-MECA. Cell cycle analysis, Annexin V-FITC staining, Hoechst 33258 staining, mitochondrial membrane potential (ΔΨM), caspase-3 activity, Bcl-2 and Bax protein expression were used to detect apoptosis. RESULT: A3 adenosine receptors mRNAs were detected at different levels. IB-MECA inhibited forskolin-stimulated cAMP. IB-MECA at (1 µM) suppressed cell proliferation and induced G1 cell cycle arrest. Indeed, IB-MECA down-regulated the expression of CDK4, cyclin D1 and up-regulated p53 expression. IB-MECA at (10-100 µM) induced apoptosis. The activity of caspase-3 was also increased. Expression of Bcl-2 was decreased in response to IB-MECA, while the expression of Bax protein was increased. The results showed a significant loss of ΔΨM, in a dose-dependent manner. CONCLUSION: This study introduces a possible mechanism through A3 adenosine receptor activation. IB-MECA inhibited prostate cancer cells proliferation and induced G1 cell cycle arrest through p53, Cdk4/cyclinD1 pathway. Apoptosis determined by characteristic morphological changes and increased in sub-G1 population. Loss of MMP, activation of caspase-3 and down-regulation of Bcl-2 expression indicated mitochondrial signaling pathway that involved in the apoptosis.