Benzyl Isothiocyanate Suppresses Pancreatic Tumor Angiogenesis and Invasion by Inhibiting HIF-α/VEGF/Rho-GTPases: Pivotal Role of STAT-3

Our previous studies have shown that benzyl isothiocyanate (BITC) suppresses pancreatic tumor growth by inhibiting STAT-3; however, the exact mechanism of tumor growth suppression was not clear. Here we evaluated the effects and mechanism of BITC on pancreatic tumor angiogenesis. Our results reveal that BITC significantly inhibits neovasularization on rat aorta and Chicken-Chorioallantoic membrane. Furthermore, BITC blocks the migration and invasion of BxPC-3 and PanC-1 pancreatic cancer cells in a dose dependant manner. Moreover, secretion of VEGF and MMP-2 in normoxic and hypoxic BxPC-3 and PanC-1 cells was significantly suppressed by BITC. Both VEGF and MMP-2 play a critical role in angiogenesis and metastasis. Our results reveal that BITC significantly suppresses the phosphorylation of VEGFR-2 (Tyr-1175), and expression of HIF-α. Rho-GTPases, which are regulated by VEGF play a crucial role in pancreatic cancer progression. BITC treatment reduced the expression of RhoC whereas up-regulated the expression of tumor suppressor RhoB. STAT-3 over-expression or IL-6 treatment significantly induced HIF-1α and VEGF expression; however, BITC substantially suppressed STAT-3 as well as STAT-3-induced HIF-1α and VEGF expression. Finally, in vivo tumor growth and matrigel-plug assay show reduced tumor growth and substantial reduction of hemoglobin content in the matrigel plugs and tumors of mice treated orally with 12 µmol BITC, indicating reduced tumor angiogenesis. Immunoblotting of BITC treated tumors show reduced expression of STAT-3 phosphorylation (Tyr-705), HIF-α, VEGFR-2, VEGF, MMP-2, CD31 and RhoC. Taken together, our results suggest that BITC suppresses pancreatic tumor growth by inhibiting tumor angiogenesis through STAT-3-dependant pathway

Role of Mitochondrial Electron Transport Chain Complexes in Capsaicin Mediated Oxidative Stress Leading to Apoptosis in Pancreatic Cancer Cells

We evaluated the mechanism of capsaicin-mediated ROS generation in pancreatic cancer cells. The generation of ROS was about 4–6 fold more as compared to control and as early as 1 h after capsaicin treatment in BxPC-3 and AsPC-1 cells but not in normal HPDE-6 cells. The generation of ROS was inhibited by catalase and EUK-134. To delineate the mechanism of ROS generation, enzymatic activities of mitochondrial complex-I and complex-III were determined in the pure mitochondria. Our results shows that capsaicin inhibits about 2.5–9% and 5–20% of complex-I activity and 8–75% of complex-III activity in BxPC-3 and AsPC-1 cells respectively, which was attenuable by SOD, catalase and EUK-134. On the other hand, capsaicin treatment failed to inhibit complex-I or complex-III activities in normal HPDE-6 cells. The ATP levels were drastically suppressed by capsaicin treatment in both BxPC-3 and AsPC-1 cells and attenuated by catalase or EUK-134. Oxidation of mitochondria-specific cardiolipin was substantially higher in capsaicin treated cells. BxPC-3 derived ρ0 cells, which lack mitochondrial DNA, were completely resistant to capsaicin mediated ROS generation and apoptosis. Our results reveal that the release of cytochrome c and cleavage of both caspase-9 and caspase-3 due to disruption of mitochondrial membrane potential were significantly blocked by catalase and EUK-134 in BxPC-3 cells. Our results further demonstrate that capsaicin treatment not only inhibit the enzymatic activity and expression of SOD, catalase and glutathione peroxidase but also reduce glutathione level. Over-expression of catalase by transient transfection protected the cells from capsaicin-mediated ROS generation and apoptosis. Furthermore, tumors from mice orally fed with 2.5 mg/kg capsaicin show decreased SOD activity and an increase in GSSG/GSH levels as compared to controls. Taken together, our results suggest the involvement of mitochondrial complex-I and III in capsaicin-mediated ROS generation and decrease in antioxidant levels resulting in severe mitochondrial damage leading to apoptosis in pancreatic cancer cells

Tumor aerobic glycolysis: new insights into therapeutic strategies with targeted delivery

Introduction: Cancer cells acclimatize to the harsh tumor microenvironment by altering cellular metabolism in favor of aerobic glycolysis. This process provides a source of energy and also generates essential components for macromolecular biosynthesis, which enables cellular survival. As the dependence of cancer cells on glycolysis affects tumorigenesis, it has become an attractive target for therapeutic intervention. Several preclinical studies have shown the effectiveness of using biological targets from the glycolytic pathway for anticancer therapy.Areas covered: This review provides an insight into the glycolytic pathway, highlighting potential targets for glycolytic inhibition. We then discuss recent advancement in delivery strategies that have the potential to circumvent some of the problems posed by current glycolytic inhibitors, enabling resurrection of abandoned therapeutic agents.Expert opinion: Targeting the glycolysis pathway is a tactical approach for cancer therapy. However, the current nonspecific therapeutic strategies have several drawbacks such as poor bioavailability, unfavorable pharmacokinetic profile and associated nonspecific toxicity, thereby limiting preclinical investigation. In recent years, nanoparticle systems have received recognition for the delivery of therapeutic agents directly to the tumor tissue. Thus, it is envisaged that this strategy can be expanded for the delivery of current glycolytic inhibitors specifically to tumor tissues providing improved anticancer activity

BITC inhibits migration and invasion of pancreatic cancer cells.

<p><b>A</b>. BITC inhibits migration of BxPC-3 cells. BxPC-3 cells were plated, scratched with pipette tip, and incubated in the absence or presence of 5 µM BITC. Photomicrographs were made at regular intervals using inverted microscope. <b>B</b>. Quantitative representation of migration assay. Wound area in BITC-treated and control cells were quantified by Image J software and presented as mean ± SD of triplicates. p<0.01, statistically significant when compared to corresponding time points in controls cells. <b>C–D</b>. BITC inhibits the invasion of BxPC-3 and PanC-1 cells. Invasion assay was performed using Boyden's chamber. Results are presented as mean ± SD of triplicates. p<0.05, statistically significant when compared controls.</p

BITC inhibits angiogenesis <i>ex vivo</i>.

<p><b>A</b>. BITC inhibits VEGF-induced vessel sprouting <i>ex vivo</i>. Aortic rings (1 mm) were harvested from Sprague-Dawley rats, immerged in matrigel, and treated with VEGF (20 ng/mL) in the absence or presence of BITC (0, 2.5 and 5 and 10 µM) for 4 days and photographed under microscope (4X). Representative photographs are presented. <b>B</b>. Quantitative analysis of aortic ring assay. Aortic ring sprouting was quantified by Image J software and presented as mean ± SD of triplicates. <b>C</b>. Inhibition of CAM angiogenesis by BITC. Eggs were incubated at 37°C for 3 days. A Whatman filter disc containing the test compound (BITC 5 µmol) was placed on the CAM of eggs (n = 10) through pre-opened window and further incubated. On day 9–12 of incubation, photographs were made after removing the filter discs. A representative photograph is presented. <b>D</b>. Blood vessels density was quantified by Image J software and represented as a bar diagram.</p

BITC inhibits angiogenesis in HUVECs.

<p><b>A</b>. BITC inhibits the secretion of proangiogenic factors from HUVECs. Cells were plated, stimulated with VEGF, and treated with BITC for 24 h. Media was collected and assayed for MMP-2 and VEGF by ELISA kit. *p<0.01 statistically significant when compared with controls. #p<0.01 statistically significant when compared with VEGF-stimulated controls. <b>B</b>. Regulation of VEGF mediated signaling by BITC. HUVECs were treated with various concentrations of BITC and whole cell lysates were analyzed by western blot. <b>C</b>. BITC down-regulates VEGFR2 and MMP-2 mRNA in HUVECs. Total RNA from BITC-treated HUVECs was isolated with Trizol and analyzed for the expression levels of VEGFR-2 and MMP-2 by RT-PCR. GAPDH was used as internal control. mRNA expression levels were quantified by Image J software and presented as bar diagram (lower panel). <b>D</b>. BITC inhibits STAT-3 DNA binding activity in HUVECs. HUVECs were treated with BITC and nuclear fraction was collected. Around 5 µg of nuclear protein subjected to STAT-3 DNA binding activity by Universal EZ-TFA transcription factor assay colorimetric kit according to the manufacturer's protocol. #p<0.01 statistically significant when compared with controls. <b>E</b>. BITC inhibits invasion of HUVECs. Invasion assay was performed using Boyden's chamber. *p<0.01 statistically significant when compared with controls. #p<0.01 statistically significant when compared with VEGF-stimulated controls</p

BITC inhibits secretion of proangiogenic factors from BxPC-3 and PanC-1 cells under normoxic and hypoxic conditions.

<p>Serum-starved BxPC-3 or PanC-1 cells were treated with various concentrations of BITC in a 96-well plate and incubated for 24 h. For hypoxia treatment, cells were exposed to 1% pO₂ for 24 h. Culture supernatants were collected and assayed for MMP-2 or VEGF by ELISA kit -. <b>A–B</b>. BITC suppresses secretion of VEGF from BxPC-3 and PanC-1 cells. <b>C–D</b>. BITC blocks secretion of MMP-2. Values are mean ± SD of triplicates. *p<0.01 statistically significant when compared with normoxic controls. #p<0.01, statistically significant when compared with hypoxic controls.</p

BITC inhibits <i>in vivo</i> tumor growth by inhibiting proangiogenic proteins.

<p><b>A</b>. BITC suppresses tumor growth <i>in vivo</i>. BxPC-3 xenografts bearing mice (n = 10) were orally fed with 12 µmol BITC daily for 40 days. Right side panel shows photographs of isolated tumors from control and BITC-treated mice. <b>B</b>. BITC inhibits tumor angiogenesis. BxPC-3 xenografts or matrigel plug-bearing mice were fed with 12 µmol BITC daily for 40 or 7 days, respectively. Tumors and plugs were collected and 50 mg of tumor or plugs were analyzed for hemoglobin content by Drabkin's reagent. <b>C</b>. BITC down-regulates pro-angiogenic proteins in tumor xenografts. Cell lysates were prepared from isolated tumor xenografts, subjected to western blot and analyzed for VEGFR-2, MMP-2, HIF-α, and Rho-GTPases <b>D</b>. Quantitative analysis of tumor western blots. *p<0.01 statistically significant when compared with controls.</p