Showing papers by "Fazlul H. Sarkar published in 2002"

Mechanisms of cancer chemoprevention by soy isoflavone genistein.

Abstract: Diet has been implicated to play an important role in cancers. Epidemiological studies have revealed that Asians, who consume a traditional diet high in soy products, have relatively low incidences of breast and prostate cancers, while the incidences are much higher in the Western world. Asians who immigrate to the United States and adopt a Western diet are at higher risks of breast and prostate cancers. Soy isoflavones have received much attention as dietary components having an important role in reducing breast and prostate cancers. Genistein, one of the predominant soy isoflavones, has been shown to inhibit the growth of cancer cells through the modulation of genes that are related to the homeostatic control of cell cycle and apoptosis. It has been found that genistein inhibits the activation of the nuclear transcription factor, NF-kappaB and Akt signaling pathway, both of which are known to maintain a balance between cell survival and programmed cell death (apoptosis). Genistein is known to have anti-oxidant property, and commonly known as phytoestrogen, which targets estrogen and androgen-mediated signaling pathway in the processes of carcinogenesis. Moreover, genistein is also found to be a potent inhibitor of angiogenesis and metastasis. Hence, significant advances have been made, both by in vitro and in vivo studies showing that genistein is a promising agent for cancer chemoprevention and/or treatment.

Inhibition of Nuclear Factor κB Activation in PC3 Cells by Genistein Is Mediated via Akt Signaling Pathway

Abstract: Prostate cancer is the second leading cause of cancer-related deaths in menin the United States. Genistein is a prominent isoflavonoid found in soy products and has been proposed to be responsible for lowering the rate of prostate cancer in Asians. However, the molecular mechanism(s) by which genistein elicits its effects on prostate cancer cells has not been fully elucidated. We have previously shown that genistein induces apoptosis and inhibits the activation of nuclear factor kappaB (NF-kappaB) pathway, which could be mediated via Akt signaling pathway, the most important survival pathway in cellular signaling. In this study, we investigated whether there is any cross-talk between Akt and NF-kappaB during genistein-induced apoptosis in PC3 prostate cancer cells. We found that genistein inhibits cell growth and induces apoptotic processes in PC3 prostate cancer cells but not in nontumorigenic CRL-2221 human prostate epithelial cells. Immunoprecipitation, kinase assays, and Western blot analysis showed that genistein specifically inhibits Akt kinase activity and abrogates the epidermal growth factor-induced activation of Akt in prostate cancer cells. NF-kappaB DNA-binding analysis and transfection studies with Akt cDNA and NF-kappaB-Luc constructs revealed that Akt transfection results in the induction of NF-kappaB activation and this is completely inhibited by genistein treatment. Moreover, genistein abrogated the epidermal growth factor-induced activation of NF-kappaB, which was mediated via Akt signaling pathway. From these results, we conclude that the inhibition of Akt and NF-kappaB activity and their cross-talk provide a novel mechanism by which genistein inhibits cell growth and induces apoptotic processes in tumorigenic but not in nontumorigenic prostate epithelial cells.

Effects of Lycopene Supplementation in Patients with Localized Prostate Cancer

Abstract: Epidemiological studies have shown an inverse association between dietary intake of lycopene and prostate cancer risk. We conducted a clinical trial to investigate the biological and clinical effects of lycopene supplementation in patients with localized prostate cancer. Twenty-six men with newly diagnosed prostate cancer were randomly assigned to receive a tomato oleoresin extract containing 30 mg of lycopene (n = 15) or no supplementation (n = 11) for 3 weeks before radical prostatectomy. Biomarkers of cell proliferation and apoptosis were assessed by Western blot analysis in benign and cancerous prostate tissues. Oxidative stress was assessed by measuring the peripheral blood lymphocyte DNA oxidation product 5-hydroxymethyl-deoxyuridine (5-OH-mdU). Usual dietary intake of nutrients was assessed by a food frequency questionnaire at baseline. Prostatectomy specimens were evaluated for pathologic stage, Gleason score, volume of cancer, and extent of high-grade prostatic intraepithelial neoplasia. Plasma levels of lycopene, insulin-like growth factor-1, insulin-like growth factor binding protein-3, and prostate-specific antigen were measured at baseline and after 3 weeks of supplementation or observation. After intervention, subjects in the intervention group had smaller tumors (80% vs 45%, less than 4 ml), less involvement of surgical margins and/or extra-prostatic tissues with cancer (73% vs 18%, organ-confined disease), and less diffuse involvement of the prostate by high-grade prostatic intraepithelial neoplasia (33% vs 0%, focal involvement) compared with subjects in the control group. Mean plasma prostate-specific antigen levels were lower in the intervention group compared with the control group. This pilot study suggests that lycopene may have beneficial effects in prostate cancer. Larger clinical trials are warranted to investigate the potential preventive and/or therapeutic role of lycopene in prostate cancer.

Akt Inactivation Is a Key Event in Indole-3-carbinol-induced Apoptosis in PC-3 Cells

Abstract: Indole-3-carbinol (I3C) is a bioactive compound present in Brassica vegetables that shows an antitumor activity in experimental animals and inhibits the growth of human cancer cells in vitro. In recent years, studies on prostate cancer (PCa) chemoprevention have been intensified, because there is a long latency for the development of clinical PCa, which makes the PCa a better target for chemoprevention. We have shown previously that I3C induces cell growth inhibition by G(1) cell cycle arrest and induces apoptosis in a dose- and time-dependent manner in PC-3 PCa cells; however, the mechanism(s) by which I3C induces apoptosis in PC-3 cells is still not clear. A cell survival pathway involving phosphatidylinositol 3'-kinase (PI3K) and Akt is known to play an important role in inhibiting apoptosis in response to growth factor signaling, which prompted us to investigate whether this pathway plays any role in I3C-induced apoptosis in PCa cells. Here we report that I3C inhibits the phosphorylation and subsequent activation of Akt kinase. In addition, I3C abrogated epidermal growth factor (EGF)-induced activation of Akt in PC-3 cells. Western blot analyses of EGF receptor showed that I3C down-regulates the EGF receptor levels and its autophosphorylation. This was also accompanied by the inhibition of EGF-induced phosphorylation of PI3K by I3C treatment. Furthermore, the known downstream modulators of the Akt/PI3K cell survival pathway, Bcl-x(L), and BAD proteins showed decreased expression after I3C treatment. From these results, we conclude that I3C-induced apoptosis is partly mediated by the inhibition of Akt activation, resulting in the alterations in the downstream regulatory molecules of Akt activation in PC-3 cells. However, further in-depth investigation is needed to establish a cause-and-effect relationship between Akt pathway and I3C effect.

Gene Expression Profiles of Genistein-Treated PC3 Prostate Cancer Cells

Abstract: Our previous studies have shown that genistein inhibits the growth of PC3 prostate cancer cells and induces apoptosis by inhibiting nuclear factor kappaB (NF-kappaB) and Akt signaling pathways. To better understand the precise molecular mechanism(s) by which genistein exerts its effects on PC3 cells, we utilized cDNA microarray to interrogate 12,558 known genes to determine the gene expression profiles altered by genistein treatment. We found a total of 832 genes that showed a greater than twofold change after genistein treatment from two independent experiments with a high degree of concordance. Among these genes, 774 genes were down-regulated and 58 genes were up-regulated with genistein treatment. Cluster analysis showed nine different types of expression alternations. These genes were also subjected to cluster analysis according to their biological functions. We found that genistein regulated the expression of genes that are critically involved in the regulation of cell growth, cell cycle, apoptosis, cell signaling transduction, angiogenesis, tumor cell invasion and metastasis. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to confirm the results of cDNA microarray, and the results of RT-PCR were consistent with the microarray data. We conclude that genistein affected the expression of a large number of genes that are related to the control of cell survival and physiologic behaviors. The gene expression profiles provide comprehensive molecular mechanism(s) by which genistein exerts its pleiotropic effects on cancer cells. Genistein-induced regulation of these genes may be further exploited for devising chemopreventive and/or therapeutic strategies for prostate cancer.

Expression of prostate-specific antigen is transcriptionally regulated by genistein in prostate cancer cells

Abstract: Prostate cancer is the second-leading cause of cancer-related deaths in men in the United States. Unfortunately, there is no effective therapy when prostate cancer becomes metastatic and refractory to conventional treatments. For this reason, the identification and exploration of new agents that reduce prostate cancer cell growth are of paramount importance. High consumption of plant-derived phytoestrogens is inversely associated with the incidence and mortality rate of prostate cancer. Previous studies, including our own, have shown that the phytoestrogen genistein inhibits prostate cancer cell growth in vitro and in vivo and decreases secreted and intracellular levels of the androgen-regulated protein prostate-specific antigen (PSA), but the role of genistein as an agonist/antagonist for hormone receptors remains unclear. To elucidate the mechanism by which genistein modulates PSA protein expression in prostate cancer cells, we investigated the effects of genistein on androgen-mediated and estrogen-mediated transcriptional regulation of PSA, androgen receptor (AR) mRNA and protein expression, and the ability of nuclear proteins to bind to androgen-response elements (AREs) in LNCaP cells. We showed that genistein decreased the transcriptional activation of PSA by both androgen-dependent and androgen-independent methods in LNCaP cells. The reduction of androgen-mediated transcriptional activation of PSA was correlated with decreased AR protein and mRNA levels and decreased binding to AREs. In contrast, genistein had differential effects on 17beta-estradiol-mediated PSA expressions. Low concentrations of genistein enhanced 17beta-estradiol-mediated PSA expressions, whereas high concentrations of genistein inhibited estrogen-mediated PSA expression in LNCaP cells. Genistein did not inhibit AR protein expression in the presence of 17beta-estradiol. These results suggest that ligand-dependent differences in the ability to activate PSA expression may contribute to the agonistic/antagonistic responses observed with genistein in prostate cancer cells.

Ciprofloxacin inhibits cell growth and synergises the effect of etoposide in hormone resistant prostate cancer cells.

Abstract: Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer related deaths in men in the United States. Ciprofloxacin is a relatively non-toxic antibiotic that can be easily administered orally with large volume of distribution and good tissue penetration. Studies from others and our laboratory have recently reported its anti-tumor activity in a variety of human tumor cells. In our current experiment, we studied the effect of ciprofloxacin on a hormone resistant prostate cancer (HRPC) cell line, PC-3. Our study shows significant in vitro cell growth inhibition of PC-3 cell line (p=0.0001) and also shows that there is a synergistic increase in the antiproliferative effect of etoposide when these cells are pretreated with ciprofloxacin for 24 h, prior to etoposide exposure (p=0.0001). Western blot analysis of the protein extracts from these cells showed down-regulation of Bcl-2, altering the ratio of Bax:Bcl-2 favoring apoptosis. In our study no significant effect was seen on p21WAF1 expression by the combination of ciprofloxacin and etoposide but there was down-regulation of p21WAF1 gene by ciprofloxacin alone. Ciprofloxacin also inhibited NF-kappaB binding to DNA. Further studies in this area are warranted as the roles of p21WAF1, Bax/Bcl-2 and NF-kappaB may be important molecular events in mediating the antiproliferative and apoptosis inducing effect of etoposide in combination with ciprofloxacin in HRPC cells.

Expression of ERRP in Normal and Neoplastic Pancreata and Its Relationship to Clinicopathologic Parameters in Pancreatic Adenocarcinoma

Abstract: Introduction and Aims: Epidermal growth factor (EGF) and its receptor (EGFR) play crucial roles in cellular signaling in many malignancies, including pancreatic neoplasia. Attenuation of EGFR signaling has been considered novel strategy for the management of human malignancies in several ongoing clinical trials. We recently isolated a novel negative regulator of EGFR, termed EGF receptor related protein (ERRP), whose expression appears to attenuate EGFR activation. In the current study, the expression of ERRP in normal and neoplastic pancreas was investigated and correlated with the clinicopathologic parameters in pancreatic ductal adenocarcinoma (DA). Methodology: Using rabbit polyclonal antibody that specifically interacts with ERRP, immunohistochemical staining was performed on 45 benign pancreata and 106 cases of DA. The intensity and percentage of cells with cytoplasmic and membranous staining were scored as 0, 1, 2, or 3. A combined score was calculated as intensity x percent/3, and for comparative analysis, the data were arbitrarily divided into three groups: <20, 20-49, and ≥50. The expression of ERRP was correlated with patient age, gender, race, tumor size, stage, grade, and survival. Results: ERRP was expressed in most benign ductal epithelium and islet cells, but not in normal acinar cells. In pancreatic ductal adenocarcinoma, ERRP expression frequency decreased progressively from well (WD) to moderate (MD) to poorly differentiated (PD) carcinoma (58%, 43%, and 15% respectively, p < 0.001). ERRP expression was correlated with survival in DA showing decreased median survival with decreased ERRP score (p = 0.0035). Median survival of the lower intensity (0 or 1) group was less than that of the higher intensity (2 or 3) group (8 vs. 14 months, p = 0.002). The higher expressing group (≥50% of cells) had longer median survival (17 months) than the lower expressing (<50% of cells) group (10 months, p = 0.003). Stepwise multiple regression analyses revealed that ERRP expression score and tumor grade are the significant predictors of survival in pancreatic ductal carcinomas (p < 0.03). Conclusion: ERRP is usually expressed in benign ductal epithelium, but not in ductal adenocarcinoma. Its expression decreases with decreasing tumor differentiation. Low levels of ERRP are associated with poor clinical outcome, suggesting that progressive loss of ERRP, a negative regulator of EGFR, may partly stimulate aggressive tumor cell growth in pancreatic adenocarcinoma.

Lycopene in the treatment of prostate cancer

Abstract: Dietary intake of lycopene is associated with reduced risk of prostate cancer (PCa). We conducted a clinical trial in men with prostate cancer to investigate the biological and clinical effects of lycopene supplementation. Twenty-six men with prostate cancer were ran- domly assigned to receive a lycopene supplement or no supplement for three weeks before radical prostatectomy. Subjects in the intervention group (n = 15) were instructed to take a tomato oleoresin extract soft gel capsule (Lyc-O-Mato ® , LycoRed Company, Beer Sheva, Israel) containing 15 mg lycopene, 1.5 mg phytoene, 1.5 mg phytofluene, and 5 mg toco- pherol twice daily with meals. Prostatectomy specimens were evaluated for pathologic stage, Gleason score, volume of cancer, and extent of high-grade prostatic intraepithelial neoplasia (HGPIN). Biomarkers of cell proliferation and apoptosis were assessed by Western blot analysis in benign and cancerous tissue samples obtained from the prostatectomy specimens. Oxidative stress was assessed by measuring the peripheral blood lymphocyte DNA oxidation product 5-hydroxymethyl-deoxyuridine (5-OH-mdU). Plasma levels of lycopene, insulin- like growth factor-1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), and prostate-specific antigen (PSA) were measured at baseline and after three weeks of study period. After the intervention, more men in the intervention group had smaller (<4 cc) tumors, organ-confined disease without involvement of surgical margins or extra-prostatic tissues, and focal involvement of the prostate with HGPIN compared to the control group. Mean plasma PSA levels were lower in the intervention group compared to the control group. This pilot study suggests that a tomato extract containing lycopene and other tomato carotenoids and phytochemicals may have a potential role in the treatment of prostate can- cer. Larger clinical trials are necessary to definitively address potential uses of lycopene or tomato extract in the prevention or treatment of prostate cancer.

Antibodies to a novel EGF-receptor related protein (ERRP)

Abstract: This invention relates to ERRP (EGF-Receptor Related Protein) specific antibodies that could be used 1) to study the functional properties of ERRP and; 2) as a diagnostic and prognostic tool for malignancies. Preferably, the antibody reacts with a segment of ERRP containing 15 amino acids (SEQ ID NO: 4) that possesses the most antigenic property.

Molecular mechanism(s) of actinomycin-D induced sensitization of pancreatic cancer cells to CD95 mediated apoptosis.

Abstract: The death receptor CD95 transduces apoptotic death signaling in many cell types. However, in pancreatic tumor cells CD95 mediated apoptotic machinery is blocked by unknown protein(s). We and others have recently demonstrated that actinomycin-D (ActD) treatment induces sensitization of pancreatic cancer cells as well as other cell types to CD95 mediated apoptosis. In addition, NF-kappaB/Akt system has been implicated in the processes of CD95 mediated apoptosis, however the precise mechanism by which ActD sensitizes pancreatic tumor cells to CD95 mediated apoptosis is still unknown. In the present study, we demonstrated that HPAC and PANC1 pancreatic cancer cells constitutively express high levels of NF-kappaB and phosphorylated form of Akt. ActD at a dose that is known to sensitize pancreatic cancer cells to CD95 mediated apoptosis abrogated the DNA binding activity of NF-kappaB but did not affect expression of phosphorylated Akt. Co-treatment of pancreatic cancer cells with ActD and agonist anti-CD95 antibodies showed no effect on the abrogation on the DNA binding activity of NF-kappaB, but decreased the expression of phosphorylated Akt. Moreover, treatment with PI3-kinase inhibitor LY294002 did not show any sensitization of pancreatic cancer cells to CD95 mediated apoptosis. Our data suggest that ActD sensitizes pancreatic cancer cells to CD95 mediated apoptosis through the abrogation of DNA binding activity of NF-kappaB, rather than PI3-kinase/Akt system. Over-expression of phosphorylated Akt in pancreatic cancer cells is controlled by effective apoptotic signaling from CD95 receptors, but do not protect tumor cells from CD95 induced apoptosis. Thus, our results indicate that modulation of NF-kappaB activity rather then Akt may provide a useful tool to sensitize pancreatic cancer cells to CD95 mediated apoptosis.

Fas and Fas Ligand Expression in Pancreatic Adenocarcinoma

Abstract: Introduction Fas and Fas ligand (FasL) mediate apoptosis of tumor cells in immune surveillance, and expression of FasL by tumors may mediate their counterattack on cytotoxic lymphocytes. Both proteins are expressed in most if not all pancreatic carcinoma cell lines, but their study in primary human tumors has been limited. Aim We performed Fas and FasL immunohistochemical staining on 81 primary pancreaticobiliary or ampullary ductal adenocarcinomas of patients in our institutional database to determine the extent and strength of staining. Methodology The expression of Fas and FasL was compared with regard to clinicopathologic variables, K-ras mutations, and immunoexpression of HER2, p21, p27, and p53. Results and Conclusion Fas was expressed in 19% of patients with strong or intermediate intensity but with variable percentages of tumor cell staining. FasL was expressed in 49% of patients, usually with diffuse expression but variable intensity. Fas expression was more common in women than men, as women had 93% of Fas-positive tumors but only 55% of Fas-negative tumors (p = 0.007), and was associated with strong HER2 expression (67% of Fas-positive versus 18% of Fas-negative patients;p = 0.04). Fas expression tended to be less common in blacks (4% had Fas-positive tumors) than whites (22% had Fas-positive tumors;p = 0.052). FasL expression tended to be associated with stage 4 disease at diagnosis (24% versus 0%;p = 0.07). Neither Fas expression nor FasL expression was associated with survival, a circumstance suggesting that their role, if any, in contributing to the aggressiveness of these tumors is complex.