Moussa Alkhalaf

Resveratrol-Induced Apoptosis Is Associated with Activation of p53 and Inhibition of Protein Translation in T47D Human Breast Cancer Cells

Background and Purpose:Trans-resveratrol (RSVL; 3,4′,5-trihydroxystilbene), a natural compound found in grapes, berries, peanuts and red wine exerts certain anticancer roles in different human cancer types. However, the exact molecular mechanism(s) behind such a role remains to be elucidated, thus the aim of this study. Experimental Approach: T47D human breast cancer cells were treated with RSVL and cell proliferation was measured by cell counting. Apoptosis was analyzed by Giemsa staining, poly(ADP-ribose) polymerase (PARP) fragmentation analysis and annexin V assay. Regulation of p53 tumor suppressor protein, p70S6K, and pS6 ribosomal protein was measured by detecting their phosphorylated active forms using ECL-immunoblot analysis. Results: The present results show that RSVL-induced growth inhibition in T47D cells is caused by apoptosis as demonstrated by morphological changes and PARP fragmentation. RSVL-induced apoptosis is associated with the activation of the p53 in a dose- and a time-dependent manner. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002 abolished the effect of RSVL on p53 activation. Interestingly, RSVL inhibits the expression of p70S6K and the phosphorylation of pS6RP. Conclusions and Implications: These findings demonstrate that RSVL affects multiple intracellular signaling transduction pathways such as p53 activation/protein translation inhibition/apoptosis, and strongly support a contemplated use of this natural compound as a preventive and/or an adjuvant therapeutic drug for breast cancer. The data indicate that these proteins may be used as predictive biomarkers to evaluate the treatment efficacy of RSVL in clinical trials.

Resveratrol-induced Apoptosis in Human Breast Cancer Cells Is Mediated Primarily through the Caspase-3-dependent Pathway

BACKGROUND Resveratrol (RSVL), a nontoxic natural compound found in a wide variety of plants with known antioxidant and anti-inflammatory properties, is emerging as a potent chemopreventive and anticancer drug. Recently, we demonstrated that RSVL-induced apoptosis in several human cancer cell lines was associated with cleavage of the proapoptotic 116 kDa poly(ADP-ribose) polymerase protein (PARP) into its 89-kDa fragment. METHODS Western blotting was used to check the levels of caspase-3 and PARP proteins. The caspase activity was analyzed with the caspase-3 colorimetric substrate DEVD-pNA. Apoptotic cells were quantified by annexin V-FITC-propidium iodide double staining. RESULTS We show that RSVL cleaved the immature caspase-3 (35 kDa) into the active fragments (p12, p17, p20) in a dose- and time-dependent manner. In addition, RSVL markedly increased caspase-3 activity (5-fold) in cells. Interestingly, RSVL-induced PARP cleavage and apoptosis was blocked specifically by inhibiting caspase-3. CONCLUSIONS Collectively, the data suggest that caspase-3 activation by RSVL is required for PARP degradation and induction of apoptosis in MDA-MB-231 cells and provide additional insights into the action of RSVL, thus substantiating the chemopreventive potential of RSVL against human breast cancer.

Resveratrol reverses hydrogen peroxide-induced proliferative effects in human coronary smooth muscle cells: a novel signaling mechanism.

BACKGROUND In human coronary smooth muscle cells (HCSMCs), we tested the proatherogenic/proliferative potential of the reactive oxygen species (ROS), hydrogen peroxide (HP), and the ability of the polyphenol stilbene resveratrol (RSVL) to protect against such effects. METHODS Activity for ERK1/2 and the kinase-G cascade were determined and correlated with HCSMC count before and after treatment with HP and/or RSVL. RESULTS HP evoked concentration-dependent cell proliferation and stimulated ERK1/2 phosphorylation at active sites. Pretreatment with the MEK-ERK inhibitor (PD98059) reversed these effects of HP. RSVL (1-100 microM) elicited more prominent inhibition of HP-evoked cell proliferation and ERK1/2 activation. In addition, RSVL markedly enhanced cGMP formation, a response that was insensitive to the soluble guanylyl-cyclase (sGC) inhibitor (ODQ, 10 microM) but was obliterated with the phorbol ester, (PMA, 0.1 microM), a desensitizer of the pGC enzyme. Likewise, the RSVL-evoked cytostatic and ERK inhibitory effects were significantly reversed by the kinase-G-inhibitor, KT-5823 (10 microM). CONCLUSIONS Collectively, RSVL activates the kinase-G system to counteract HP-induced ERK1/2 activation and coronary arterial proliferation. These effects for RSVL remain functional in endothelium-disrupted arteries, scenarios that commonly occur in advanced coronary heart disease.

Resveratrol reverses ET-1-evoked mitogenic effects in human coronary arterial cells by activating the kinase-G to inhibit ERK-enzymes.

In human coronary smooth muscle cells (HCSMC), treatment with the vascular mitogen; endothelin-1 (ET-1), induced cell proliferation and stimulated ERK-1/2 phosphorylation at active sites. Pretreatment with the MEK-ERK inhibitor (PD98059) appreciably reversed the mitogenic effects of ET-1. On the other hand, pretreatment with the polyphenolic stilbene resveratrol (RSVL, 1-100 microM) triggered more prominent inhibition of ET-1-evoked cell proliferation and ERK1/2 activation. Besides, RSVL also markedly (2-3 fold) and rapidly enhanced cGMP formation, but had no effect on cAMP levels. This RSVL-evoked upregulation of cGMP was insensitive to pretreatment with the soluble guanylyl cyclase (sGC)-inhibitor (ODQ, 10 microM), but was ablated with an inhibitor of pGC (PMA, 0.1 microM). Further, pretreatment with the specific cGMP-phosphodiesterase inhibitor, zaprinast (10 microM) appreciably augmented RSVL-evoked cGMP formation, ERK inhibition, and cytostatic response. Moreover, the RSVL-induced ERK-inhibitory effects were significantly reversed by the kinase-G inhibitor, KT-5823 (10 microM; 69%), but not by the kinase-A inhibitor (KT-5720). These results demonstrate a novel signaling pathway for RSVL that leads from activation of the pGC/kinase-G system to inhibition of ERK1/2 and their downstream nuclear targets. This pathway functions to counteract the atherogenic signaling induced by vascular mitogens.

Apolipoprotein E, CI and B Gene Polymorphisms in a Sample of Patients with Coronary Heart Disease in the Kuwaiti Population

Objectives: The objective of this study was to investigate the possible association of clinical variables and apolipoprotein (APOE, APOCI and APOB) polymorphisms with the development of myocardial infraction (MI) and coronary heart disease (CHD) in Kuwaitis. Subjects and Methods: APOE, APOCI and APOB genotypes were determined by polymerase chain reaction followed by restriction fragment length polymorphism in 143 Kuwaiti CHD patients with (n = 88) and without (n = 55) MI and in 122 controls matched for gender and age. Statistical and genetic analyses of the genotype, allele and haplotype frequencies, as well as regression analyses of genetic and clinical variables were done. Results: There was a statistically significant association between CHD and medical history of diabetes mellitus (p < 0.001), hypertension (p < 0.01), high cholesterol (p < 0.05) and family history of CHD (p < 0.001). A highly significant association (p < 0.001) was found, with an adjusted odds ratio of 9.32, for family history and the development of MI. No significant differences were found for allele or genotype frequencies between CHD patients and controls. Conclusion: The strong effect of family history suggests a major genetic component for the development of CHD in Kuwaitis, but this association does not appear to be related to the APO genes studied here. The results in this study encourages future research into these and other polymorphisms and their potential association with MI and CHD in the Kuwaiti population.

Consumption of green tea alters glial fibriliary acidic protein immunoreactivity in the spinal cord astrocytes of STZ-diabetic rats

Abstract We examined the effect of green tea consumption on glial fibriliary acidic protein (GFAP) expression in spinal cord of streptozotocin (STZ) treated rats. Three groups (n = 10) were used in this study: (i) controls; (ii) STZ-induced diabetic rats given tap water; and (iii) an STZ-induced diabetic group given green tea. Immunohistochemistry showed a significant (P < 0.001) decrease in the number of GFAP immunoreactive astrocytes in spinal cord sections of diabetic rats compared to non-diabetic controls. Diabetic rats treated with green tea showed a significant (P < 0.01) increase in the number GFAP-immunoreactive astrocytes in all the spinal cord gray areas as compared to water-drinking diabetic rats. Immunoblotting confirmed that the diabetic spinal cord tissue expressed 71.0 ± 7.0% less GFAP compared to non-diabetic controls and that the GFAP content in diabetic rats increased up to 86.34 ± 18.74% compared to non-diabetic controls after 12 weeks of green tea consumption. In conclusion, consumption of green tea may represent an achievable adjunct therapy for improving changes seen in diabetic spinal cord.

A cytogenetic study of Kuwaiti couples with infertility and reproductive disorders: short arm deletion of chromosome 21 is associated with male infertility

Chromosomal anomalies may be a reason for both male and female infertility. The aim of this study was to investigate the contribution of chromosomal abnormalities in sterile couples from Kuwait. A total of 118 patients with clinical diagnosis of infertility was analyzed using cytogenetic banding techniques. Common chromosomal abnormalities were detected in 12 patients. We describe here one new case of an infertile male with the karyotype 46,XY, del(21)(pter;q11.2). The overall incidence of 11% abnormality indicates that routine chromosome analysis of infertile couples in Kuwait should be considered before the planning of intracytoplasmic sperm injection.

Role of Na+/H+ exchanger in resveratrol-induced growth inhibition of human breast cancer cells.

Cancer cells maintain low intracellular pH [pHi]; therefore, it is likely that resveratrol [RSVL] inhibits cell growth through interference with regulation of pHi. Na–H exchanger [NHE] regulates pHi and NaCl uptake. In this study, we investigated a putative role of NHE-1 and NHE-3 isoforms in the RSVL-induced cell death using MDA-MB-231 estrogen receptor-negative [ER−] and MCF-7 [ER+] human breast cancer cell lines. ECL Western blot analysis and fluorescence morphometeric analysis were used. Cell viability and counting were performed using standard procedures. RSVL caused a dose- and time-dependent induction of NHE-1 and NHE-3 proteins in both cancer cell lines as shown by ECL Western blot analysis and fluorescence measurement. Interestingly, the level of actin, an internal control, remains unaltered. Thus, it is concluded that RSVL-inhibited cell growth and viability, increased cell size, and volume along with an increased apoptotic activity are due to the induction of NHE-1 and NHE-3 isoforms in the present breast cancer cell lines. Induction of NHE will increase the uptake of NaCl and decrease pHi leading to disturbance in Ca2+ homeostasis, which is responsible for cell death.

Cytogenetic and Immunohistochemical Characterization of Fragile X Syndrome in a Kuwaiti Family: Rapid Antibody Test for the Diagnosis of Mental Retardation Patients

Objectives: To study a Kuwaiti family with fragile X mental retardation syndrome and present a rapid, noninvasive antibody test to be used for fragile X syndrome diagnosis on all mentally retarded patients in Kuwait. Methods: For cytogenetic analysis, the fragile site was induced by culturing blood lymphocytes in a medium containing a low concentration of folate (M199) or by adding 0.1 µM of fluorodeoxyuridine to the culture media. For the immunohistochemical test on blood smears, monoclonal antibodies against fragile X mental retardation (FMR1) gene product were used. The test is based on the presence of the protein in lymphocytes from normal individuals and its absence in lymphocytes from fragile X patients. Results: A Kuwaiti family with fragile X syndrome in 4 affected males is described. The immunohistochemical study with the monoclonal antibodies against FMR protein gave conclusive results on the same day for the affected males. Conclusion: To our knowledge, this is the first report describing a large Kuwaiti family with the fragile X syndrome. These data suggest that the immunohistochemical test used is an ideal method for the diagnosis of fragile X syndrome in mentally retarded patients in Kuwait.

Progesterone inhibition of MDM2 p90 protein in MCF-7 human breast cancer cell line is dependent on p53 levels

The mdm2 gene encodes several protein isoforms with different molecular weights (p90, p80, p76 and p57). MDM2 p90 (usually considered to be the major MDM2 protein) binds to and inactivates P53. We have recently shown that growth inhibition of MCF-7 human breast cancer cells by progesterone is associated with P53 down-regulation. In this work, we analyzed the expression pattern of MDM2 proteins in three human breast cancer cell lines by western blotting with anti-MDM2 antibodies. We found a prominent expression of MDM2 p57 protein in cell lines which have non-functional P53 protein (T47D and MDA-MB-231) as compared to the p90, p80 isoforms, whereas p90 was the major protein isoform in MCF-7 cells that contain functional P53 protein. When MCF-7 cells were treated with 100 nM of progesterone, MDM2 p90 was inhibited but the highly expressed MDM2 p57 isoform was not. The inhibition of MDM2 p90 protein by progesterone was abrogated in MCF-7 cells transfected with a P53 expressing vector. To our knowledge, this is the first report linking progesterone-induced growth inhibition with down-regulation of the MDM2 protein. We present evidence that reestablishing of P53 expression by transient transfection of P53 cDNA in these cells enhances the expression level of MDM2 p90 isoform. The data indicate that expression of MDM2 p90 is regulated through a P53-dependent pathway in response to progesterone.