Mohammad R. Salabat

Increased ACE 2 and Decreased ACE Protein in Renal Tubules From Diabetic Mice: A Renoprotective Combination?

Abstract—Unlike the ubiquitous angiotensin-converting enzyme (ACE), the ACE-related carboxypeptidase 2 (ACE 2) is predominantly expressed in the heart, kidney, and testis. ACE 2 degrades angiotensin (Ang) II to Ang (1–7) and Ang I to Ang (1–9). We investigated the expression of ACE and ACE 2 in a rodent model of type 2 diabetes. ACE and ACE 2 were measured in kidney and heart from 8-week-old no diabetic control (db/m) mice and diabetic (db/db) mice, which at this young age have obesity and hyperglycemia without nephropathy. In renal cortical tissue, ACE mRNA was reduced (db/db 0.31±0.06 versus db/m 0.99±0.05; P <0.005), whereas ACE 2 mRNA was not (db/db 0.94±0.05 versus db/m 1.03±0.11, NS). ACE protein was markedly reduced in kidney cortex of db/db mice (db/db 0.24±0.13 versus db/m 1.02±0.12; P <0.005), and this was associated with a corresponding decrease in renal ACE activity (db/db 12.7±3.7 versus db/m 61.6±4.4 mIU/mg protein; P <0.001). ACE 2 protein, by contrast, was increased in kidneys from diabetic mice (db/db 1.39±0.14 versus db/m 0.53±0.04; P <0.005). An increase in ACE 2 protein and a decrease in ACE protein, respectively, were also seen by immunostaining of renal cortical tubules from the db/db mice. In heart tissue, there were no significant differences between db/db and db/m mice in either ACE mRNA and protein or ACE 2 mRNA and protein. We conclude that in young db/db mice, ACE 2 protein in renal cortical tubules is increased, whereas ACE protein is decreased. We propose that the pattern of low ACE protein coupled with increased ACE 2 protein expression may be renoprotective in early stages of diabetes.

Overexpression of 5-Lipoxygenase in Colon Polyps and Cancer and the Effect of 5-LOX Inhibitors In vitro and in a Murine Model

Purpose: Arachidonic acid metabolism via the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways modulates cell growth and apoptosis. Many studies have examined the effects of COX inhibitors on human colorectal cancer, but the role of 5-LOX in colonic cancer development has not been well studied. The purpose of this study was to evaluate the expression of 5-LOX in colonic polyps and cancer and the effect of 5-LOX inhibition on colon cancer cell proliferation. Experimental Design: Colonic polyps, cancer, and normal mucosa were evaluated for 5-LOX expression by immunohistochemistry. Reverse transcription-PCR was used to establish 5-LOX expression in colon cancer cells. Thymidine incorporation and cell counts were used to determine the effect of the nonspecific LOX inhibitor Nordihydroguaiaretic Acid and the 5-LOX inhibitor Rev5901 on DNA synthesis. A heterotopic xenograft model in athymic mice using HT29 and LoVo human colon cancer cells was used to evaluate the effect of the 5-LOX inhibitor zileuton on tumor growth. Results: 5-LOX is overexpressed in adenomatous polyps and cancer compared with that of normal colonic mucosa. LOX inhibition and 5-LOX inhibition decreased DNA synthesis in a concentration- and time-dependent manner in the Lovo cell line (P < 0.05). Inhibition of 5-LOX in an in vivo colon cancer xenograft model inhibited tumor growth compared with that of controls (P < 0.05). Conclusions: This study showed that 5-LOX is up-regulated in adenomatous colon polyps and cancer compared with normal colonic mucosa. The blockade of 5-LOX inhibits colon cancer cell proliferation both in vitro and in vivo and may prove a beneficial chemopreventive therapy in colon cancer.

Crosstalk between Mast Cells and Pancreatic Cancer Cells Contributes to Pancreatic Tumor Progression

Purpose: To assess the clinical and pathologic significance of mast cell infiltration in human pancreatic cancer and evaluate crosstalk between mast cells and cancer cells in vitro. Experimental Design: Immunohistochemistry for tryptase was done on 53 pancreatic cancer specimens. Mast cell counts were correlated with clinical variables and survival. Serum tryptase activity from patients with cancer was compared with patients with benign pancreatic disease. In vitro, the effect of pancreatic cancer–conditioned medium on mast cell migration was assessed. The effect of conditioned medium from the human mast cell line, LAD-2, on cancer and normal ductal cell proliferation was assessed by thymidine incorporation. Matrigel invasion assays were used to evaluate the effect of mast cell–conditioned medium on cancer cell invasion in the presence and absence of a matrix metalloproteinase inhibitor, GM6001. Results: Mast cell infiltration was significantly increased in pancreatic cancer compared with normal pancreatic tissue (11.4 ± 6.7 versus 2.0 ± 1.4, P < 0.001). Increased infiltrating mast cells correlated with higher grade tumors (P < 0.0001) and worse survival. Patients with pancreatic cancer had elevated serum tryptase activity (P < 0.05). In vitro, AsPC1 and PANC-1 cells induced mast cell migration. Mast cell–conditioned medium induced pancreatic cancer cell migration, proliferation, and invasion but had no effect on normal ductal cells. Furthermore, the effect of mast cells on cancer cell invasion was, in large part, matrix metalloproteinase–dependent. Conclusions: Tumor-infiltrating mast cells are associated with worse prognosis in pancreatic cancer. In vitro, the interaction between mast cells and pancreatic cancer cells promotes tumor growth and invasion. Clin Cancer Res; 16(8); 2257–65. ©2010 AACR.

Apigenin inhibits the GLUT-1 glucose transporter and the phosphoinositide 3-kinase/Akt pathway in human pancreatic cancer cells.

Objectives: The antiproliferative mechanisms of flavonoid drugs inpancreatic cancer cells remain unclear. In this study, we evaluated the effects of the flavonoid apigenin on glucose uptake, on the expression of the glucose transporter 1 (GLUT-1), and on the phosphoinositide 3-kinase (PI3K)/Akt pathway in human pancreatic cancer cells. Methods: Human pancreatic cancer cells were treated with apigenin and then underwent glucose uptake assays. Real-time reverse transcription-polymerase chain reaction and Western blot analysis were conducted to evaluate GLUT-1 and pAkt expression in CD18 and S2-013 human pancreatic cancer cells after treatment with apigenin or PI3K inhibitors (LY294002 and wortmannin). Results: Apigenin (0-100 &mgr;M) significantly inhibited, in a dose-dependent fashion, glucose uptake in CD18 and S2-013 human pancreatic cancer cell lines. Apigenin inhibited both GLUT-1 mRNA and protein expression in a concentration- and time-dependent fashion. The PI3K inhibitors, like apigenin, downregulated both GLUT-1 mRNA and protein expression. Conclusions: Our results demonstrate that the flavonoid apigenin decreases glucose uptake and downregulates the GLUT-1 glucose transporter in human pancreatic cancer cells. In addition, the inhibitory effects of apigenin and the PI3K inhibitors on GLUT-1 are similar, indicating that the PI3K/Akt pathway is involved in mediating apigenins effects on downstream targets such as GLUT-1.

Apigenin down-regulates the hypoxia response genes: HIF-1α, GLUT-1, and VEGF in human pancreatic cancer cells.

BACKGROUND The flavonoid apigenin exhibits anti-proliferative and anti-angiogenic activities. Our objective was to evaluate the effect of apigenin on hypoxia responsive genes important in pancreatic cancer cell proliferation. MATERIALS AND METHODS Immunohistochemistry for GLUT-1 expression was conducted on human pancreatic cancer samples and adjacent controls. Real-time RT-PCR, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA) were conducted on CD18 and S2-013 human pancreatic cancer cells treated with apigenin (0-50 μM) in normoxic and hypoxic conditions to evaluate HIF-1α, GLUT-1, and VEGF mRNA and protein expression and secretion. RESULTS GLUT-1 expression was significantly increased in pancreatic adenocarcinoma samples versus adjacent controls (P < 0.001). Hypoxic conditions induced HIF-1α, GLUT-1, and VEGF protein expression in both CD18 and S2-013 pancreatic cancer cells. Apigenin (50 μM) blocked hypoxia induced up-regulation of all three proteins in both cell lines. Apigenin also impeded hypoxia-mediated induction of GLUT-1 and VEGF mRNA in both cell lines (P < 0.05). CONCLUSIONS Apigenin inhibits HIF-1α, GLUT-1, and VEGF mRNA and protein expression in pancreatic cancer cells in both normoxic and hypoxic conditions. This may account for the mechanism of apigenins anti-proliferative and anti-angiogenic effects and further supports the potential of apigenin as a future chemopreventive agent for pancreatic cancer.

A high omega-3 fatty acid diet mitigates murine pancreatic precancer development.

BACKGROUND Diets containing omega-3 (ω-3) fat have been associated with decreased tumor development in the colon, breast, and prostate. We assessed the effects of a diet rich in ω-3 fat on the development of pancreatic precancer in elastase (EL)-Kras transgenic mice and examined the effect of an ω-3 fatty acid on pancreatic cancer cells in vitro. MATERIALS AND METHODS Two cohorts of EL-Kras mice were fed a high ω-3 fat diet (23% menhaden oil) for 8 and 11 mo and compared with age-matched EL-Kras mice fed standard chow (5% fat). Pancreata from all mice were scored for incidence and frequency of precancerous lesions. Immunohistochemistry was performed for proliferating cell nuclear antigen (PCNA) to assess proliferative index in lesions of mice fed either a high ω-3 or standard diet. In vitro, the effect of the ω-3 fatty acid, docosahexaenoic acid (DHA), on two pancreatic cancer cell lines was assessed. Cancer cell proliferation was assessed with an MTT assay; cell cycle analysis was performed by flow cytometry; and apoptosis was assessed with annexin/PI staining. RESULTS The incidence, frequency, and proliferative index of pancreatic precancer in EL-Kras mice was reduced in mice fed a high ω-3 fat diet compared with mice fed a standard chow. In vitro, DHA treatment resulted in a concentration-dependent decrease in proliferation through both G1/G0 cell cycle arrest and induction of apoptosis. CONCLUSIONS A high ω-3 fat diet mitigates pancreatic precancer by inhibition of cellular proliferation through induction of cell cycle arrest and apoptosis.

The flavonoid apigenin potentiates the growth inhibitory effects of gemcitabine and abrogates gemcitabine resistance in human pancreatic cancer cells.

Objectives: The aim of the study was to evaluate the effect of combination therapy of apigenin and gemcitabine on cell proliferation, the cell cycle, and gemcitabine resistance in human pancreatic cancer cells. Methods: Cell counting was used to assess the effect of single-agent and combination treatment on the proliferation of CD18 and AsPC-1 pancreatic cancer cells. Flow cytometry was performed to assess the effect of combination treatment on cell cycle progression and induction of apoptosis. Western blot analysis was used to evaluate phosporylated AKT (pAkt) and cell cycle proteins. The effect of apigenin on gemcitabine-resistant AsPC-1 cells was assessed via thymidine incorporation. Results: Apigenin in combination with gemcitabine inhibited pancreatic cancer cell proliferation more than either agent alone. Combination treatment induced both S and G2/M phase arrest and increased apoptosis. Apigenin down-regulated pAkt expression and abrogated gemcitabine-mediated pAkt induction. In gemcitabine-resistant AsPC-1 cells, apigenin significantly inhibited cell proliferation in a dose-dependent manner. Conclusion: Combination treatment with apigenin and gemcitabine inhibited pancreatic cancer cell growth via cell cycle arrest, down-regulation of the prosurvival factor pAkt, and induction of apoptosis. Combination therapy may prove useful for the treatment of pancreatic cancer.

Geminin is overexpressed in human pancreatic cancer and downregulated by the bioflavanoid apigenin in pancreatic cancer cell lines.

Pancreatic adeniocarcinoma is among the deadliest of human cancers. Apigenin, an antitumor flavonoid, inhibits pancreatic cancer cell proliferation in vitro. Geminin is a recently identified novel protein that plays a critical role in preventing abnormal DNA replication by binding to and inhibiting the essential replication factor Cdt1. Microarray analysis identified geminin to be downregulated in pancreatic cancer cells treated with apigenin. Therefore, we investigated the effects of apigenin on geminin expression and other proteins involved in replication (Cdc6, Cdt1, and MCM7) in pancreatic cancer cell lines CD18 and S2013. Real time RT‐PCR and western blotting analysis showed that geminin expression is downregulated by apigenin at both mRNA and protein levels. Furthermore, treatment of cells with proteosome inhibitor MG132 reversed the downregulation of geminin by apigenin, supporting our hypothesis that the degradation pathway is another mechanism by which apigenin affects geminin expression. Apigenin treatment also resulted in downregulation of Cdc6 at both mRNA and protein levels. However, Cdt1 and MCM7 expression was not affected in apigenin‐treated cells. The effect of apigenin treatment on geminin promoter activity was measured by transient transfection of Hela cells with a reporter gene, demonstrating that apigenin inhibited geminin promoter activity. Geminin expression was also evaluated in human pancreatic tissue (n = 15) by immunohistochemistry and showed that geminin is overexpressed in human pancreatic cancer compared to normal adjacent pancreatic tissue. In conclusion, our studies demonstrated that geminin is overexpressed in human pancreatic cancer and downregulated by apigenin which may contribute to the antitumor effect of this natural flavonoid.

On the mechanisms of 12-O-tetradecanoylphorbol-13-acetate-induced growth arrest in pancreatic cancer cells.

Objectives: Protein kinase C (PKC) is involved in cell growth, differentiation, and apoptosis. We investigated the effects of the PKC activator, the tetradecanylphorbol acetate (TPA), in human pancreatic cancer cells. Methods: Cell proliferation was measured by thymidine incorporation. Expression of cell cycle proteins was investigated by Western blot. Real-time reverse transcriptase-polymerase chain reaction was used to measure p21WAF1 messenger RNA expression, whereas knockdown of its expression was accomplished with a specific small interferring RNA. Cell cycle phases were determined by flow cytometry. Results: TPA time and concentration dependently inhibited thymidine incorporation in Panc-1 and CD18 cells and induced G2/M cell cycle arrest. The TPA decreased cyclin A and B expression, increased cyclin E, and markedly increased the expression of p21WAF1 at both the messenger RNA and protein levels. TPA-induced p21WAF1 expression and growth inhibition were blocked by the PKC inhibitor, bisindoylmaleimide. TPA induced extracellular signal-regulated kinase1/2 phosphorylation, whereas the MEK inhibitor, PD98059, blocked the TPA-induced p21WAF1 expression. Small interferring RNA targeted to p21WAF1 blocked TPA-induced p21WAF1 protein expression but not TPA-induced cell growth arrest. Conclusions: TPA-induced p21WAF1 expression is mediated by the MEK/ERK pathway but is not involved in TPA-induced growth inhibition. In contrast, cyclin A and cyclin B are likely involved in TPA-induced G2/M arrest because both proteins are involved in S phase and G2/M transition during cell proliferation.