Major mechanisms contributing to RLIP76-mediated anti-cancer effects and chemo-radiotherapy resistance in pancreatic cancer.

<p>RLIP76 depletion leads to predominant inhibition of PI3K/Akt pathway as revealed by the inhibition of PI3K as well as decrease in the levels and phosphorylation of Akt. The associated downstream proteins that regulate differentiation, proliferation and apoptosis like E-cadherin, Bim, Bcl2 and CDK4 are also differentially regulated due to RLIP76 targeted interventions. The collective impact of RLIP76 depletion on the regulation of cellular signaling pathways as well as detoxification of glutathione-conjugates (GS-Es) of lipid peroxidation and chemotherapy drugs represents precious opportunities for the development of novel and effective therapeutic interventions for pancreatic cancers. <i>Green arrow</i>: Up regulation following RLIP76 depletion; <i>Red arrow</i>: down regulation following RLIP76 depletion; <i>Blue arrow</i>: Normal signal transduction.</p

RLIP76 mediated DOX transport and radiation-protection in pancreatic cancer cells.

<p>The crude membrane vesicles (inside-out vesicles, IOV) from BxPC3 cells were separately coated with 40 µg/ml final concentration of either anti-RLIP76 IgG, anti-MRP1 IgG, or anti-Pgp IgG and used to measure the ATP-dependent uptake of ¹⁴C-DOX. ATP-dependent uptake of ¹⁴C-DOX was determined by subtracting the radio-activity (cpm) of the controls without ATP from that of the experimental groups containing ATP (<b>panel A</b>). Approximately, 2.5×10³ BxPC3 cells grown in RPMI-1640 medium, were treated with control and RLIP76-liposomes (50 µg/ml final concentration) for 24 h prior to radiation at 100, 200, 500 and 1000 cGY (6 MeV photons). After 7 days, cells were stained with methylene-blue and the colonies were counted using Alpha Imager HP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Nagaprashantha1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Nagaprashantha2" target="_blank">[33]</a> (<b>panel B</b>) * p<0.05, ** p<0.01 compared to controls. The fold change in the radiation-resistance of RLIP76 <i>vs.</i> control-liposome treated pancreatic cancer cells at each dose of irradiation is represented in the bar diagram (<b>panel C</b>). The results presented are the mean and s.d. from three separate experiments (n = 9) * p<0.05, ** p<0.01 compared to control.</p

RLIP76 protein expression in human normal and pancreatic cancer cells.

<p>Cell lines were cultured in respective medium and homogenate was prepared from 10⁸ cells. RLIP76 was purified from total crude membrane fraction using DNP-SG affinity column chromatography and quantified by ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Awasthi4" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Singhal6" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Singhal7" target="_blank">[31]</a>. Values represent mean ± s.d. from three separate determinations.</p

Treatment with RLIP76 antibody, siRNA or antisense causes regression of established BxPC-3 pancreatic cancer xenografts.

<p>For xenografts studies, we used thirty-six 11-weeks-old Hsd: Athymic nude nu/nu mice (Harlan, Indianapolis, IN) randomized 6 animals each into six groups as follow: 1) pre-immune serum, 2) scrambled siRNA, 3) scrambled anti-sense DNA, 4) RLIP76 antibodies, 5) RLIP76 siRNA and 6) RLIP76 antisense. All 36 animals were injected with 2×10⁶ human pancreatic cancer cells (BxPC-3) suspensions in 100 µl of PBS, subcutaneously into one flank of each nu/nu nude mouse. Animals were examined daily for signs of tumor growth and body weights. When tumors reached a cross-sectional area of ∼42 mm² (47 d later), animals were randomized in to treatment groups as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone-0034582-g004" target="_blank">Figure 4</a>. Treatment consisted of 200 µg of RLIP76 antibodies, siRNA or antisense in 100 µl PBS. Control groups were treated with 200 µg/100 µl of either pre-immune serum, scrambled siRNA or scrambled anti-sense DNA. Tumors were measured in two dimensions using calipers. Tumor measurements are presented with all control groups (pre-immune IgG, scrambled siRNA or antisense) versus all treated groups (anti-RLIP76 IgG, RLIP76 siRNA, or anti-sense) (<b>panel A</b>). Tumor-weight is reduced at day 47 after the treatment start (<b>panel B</b>) and tumor RLIP76 is depleted by antisense (<b>panel B, inset</b>). Tumors were excised 48 h after antisense-treatment, weighed, and homogenized, and aliquots containing 100 µg protein from each, were loaded to SDS-PAGE for Western blotting against anti-RLIP76 IgG (n = 3; lanes 1–3, scrambled antisense treated, and lanes 4–6, RLIP76-antisense treated). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#s3" target="_blank">Results</a> were quantified by scanning densitometry. Membrane was stripped and re-probed with β-actin antibody to verify equal protein loading, * p<0.05 compared with respective controls. Western blot analysis of tumor tissue lysates: Mechanistic basis for the tumor growth inhibition by RLIP76-antisense was determined in the tumor lysates from control and RLIP76-antisense treated mice. Tumor tissues were homogenized, and ∼70 µg protein was resolved on SDS-PAGE and probed for pPI3K, PI3K, pAkt, Akt, PCNA, Bim, E-cadherin, Cyclin B1 and CDK4. The blots were stripped and reprobed for GAPDH to ensure equal protein loading (<b>panel C</b>). Bar diagram represents densitometry analyses. Dotted line represents no significant change as observed with scrambled antisense (<b>panel D</b>).</p

RLIP76 transport activity in human normal and pancreatic cancer cells.

<p>For transport studies, in-side out vesicles (IOVs) prepared from 20×10⁶ cells was enriched for IOVs by wheat germ agglutinin affinity chromatography <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Awasthi4" target="_blank">[26]</a>. Transport activity was calculated from measurements of uptake of ¹⁴C-DOX (sp. activity, 8.5×10⁴ cpm/nmol) and ³H-DNP-SG (sp. activity, 3.6×10³ cpm/nmol) into the IOVs (20 µg/30 µl reaction mixture) in the absence or presence of 4 mM ATP as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Awasthi4" target="_blank">[26]</a>. Each transport experiment was done in triplicates in three separate experiments (n = 9).</p

Comparison of RLIP76 levels in pancreatic cancer cells <i>vs</i> non-malignant cells.

<p>Aliquots of crude membrane fractions of pancreatic cancer cells (BxPC-3 and Panc-1) and normal control cells (HUVEC) containing 100 µg protein were used for SDS-PAGE and Western blotting. Intensity of the full-length RLIP76 protein (∼95 kDa) band was quantified by scanning densitometry using Innotech Alpha Imager HP. β-actin was used as an internal control (<b>panel A</b>). Impact of anti-RLIP76 IgG, RLIP76 siRNA and RLIP76 antisense on normal and pancreatic cancer cells: Effect of anti-RLIP76 IgG (40 µg/ml final concentration) on the cell survival was determined by MTT assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Singhal6" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Stuckler1" target="_blank">[30]</a>. Depletion of RLIP76 expression by RLIP76 siRNA and RLIP76 antisense (each 10 µg/ml final concentration) was done, using Transmessenger Transfection-Reagent-kit (Qiagen), and Maxfect Transfection-Reagent (Molecula, Inc.), respectively, according to the manufacturer's instructions. Cell survival was measured by MTT cytotoxicity assay 48 h after treatment. The values are presented as mean ± SD from two separate determinations with eight-replicates each (n = 16), <b>black bars</b>, normal HUVEC cells; <b>gray bars</b>, BxPC-3 pancreatic cancer cells (<b>panel B</b>) * p<0.05, ** p<0.01 compared to respective controls. Effect of RLIP76 antisense on PI3K and Akt signaling in pancreatic cancer cells: RLIP76 antisense caused inhibition of PI3K/Akt pathway in BxPC-3 and Panc-1 cells. Cells were treated with 10 µg/ml of RLIP76 antisense for 24 h and immune-blotted for pPI3K, PI3K, pAkt, Akt, Bim, Bcl2, cyclin B1 and CDK4. The same blot was stripped and reprobed for GAPDH to ensure equal protein loading (<b>panel C</b>). Bar diagram shows the quantitation of respective Western blots. Dotted line represents no significant change as observed with scrambled antisense (<b>panel D</b>).</p

Proteomic Analysis of Signaling Network Regulation in Renal Cell Carcinomas with Differential Hypoxia-Inducible Factor-2α Expression

<div><p>Background</p><p>The loss of von Hippel–Lindau (<i>VHL</i>) protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of <i>VHL</i> in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α) instead of hypoxia-inducible factor-1α (HIF1α).</p><p>Methods and Principal Findings</p><p>We used renal carcinoma cell lines that recapitulate the differences between mutant <i>VHL</i> and wild-type <i>VHL</i> genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography–mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in <i>VHL</i>-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in <i>VHL</i>-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335.</p><p>Conclusions and Significance</p><p>Pathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct <i>VHL</i> genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced <i>VHL</i>-mutant, HIF2α-over-expressing tumors.</p></div