PKCδ activation mediates angiogenesis via NADPH oxidase activity in PC-3 prostate cancer cells - PubMed (original) (raw)

. 2011 Jun 15;71(9):946-54.

doi: 10.1002/pros.21310. Epub 2010 Nov 23.

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PKCδ activation mediates angiogenesis via NADPH oxidase activity in PC-3 prostate cancer cells

Jeewon Kim et al. Prostate. 2011.

Abstract

Background: PKCδ is generally known as a pro-apoptotic and anti-proliferative enzyme in human prostate cancer cells.

Methods: Here, we investigated the role of PKCδ on the growth of PC-3 human prostate cancer cells in vivo and in vitro.

Results: We found that sustained treatment with a specific PKCδ activator (ψδ receptor for active C kinase, ψδRACK) increased growth of PC-3 xenografts. There was increased levels of HIF-1α, vascular endothelial growth factor and CD31-positive cells in PC-3 xenografts, representative of increased tumor angiogenesis. Mechanistically, PKCδ activation increased the levels of reactive oxygen species (ROS) by binding to and phosphorylating NADPH oxidase, which induced its activity. Also, PKCδ-induced activation of NADPH oxidase increased the level of HIF-1α.

Conclusions: Our results using tumors from the PC-3 xenograft model suggest that PKCδ activation increases angiogenic activity in androgen-independent PC-3 prostate cancer cells by increasing NADPH oxidase activity and HIF-1α levels and thus may partly be responsible for increased angiogenesis in advanced prostate cancer.

Copyright © 2010 Wiley-Liss, Inc.

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Figures

Fig. 1

Fig. 1. PKCδ activation increases PC-3 prostate tumor growth

(A) The level of the active form of PKCδ was determined by Western blot analyses of cytosolic (C) and particulate (P) fractions from primary normal human prostate epithelial cells (hPEC) and PC-3 cells grown in culture. Cells were fractionated into cytosolic and particulate fractions as described in Methods. Quantification of the active forms of PKCδ (translocation; expressed as percentage of PKC isozyme in the particulate fraction over sum of cytosolic and particulate fraction enzymes, i.e., total cellular enzyme) is provided in the graph below (n=3, *; p<0.05). A 2-tailed Student’s t test was used to determine significance. Loading controls for cytosolic and particulate fractions (GAPDH and Gαi) are shown in the lower bands. IB; immunoblot. (B) PC-3 xenografts were grown s.c. on nude mice for up to 8 weeks and tumors were obtained at weeks 3-8. PKCδ translocation was analyzed using Western blot and quantification is shown on the graph below (n=3, *; p<0.05) vs. week 3. (C) One week after PC-3 cell injection, mice were implanted with osmotic pumps with control peptide (TAT) at 24 mg/kg/day (30mM) or δV1-1 conjugated to TAT at 1.4 mg/kg/day (1mM) or ψδRACK at 3.8 or 38 mg/kg/day (3mM or 30mM). The peptides were dissolved in saline and administered at a constant rate (0.5μl/hr) for 2 weeks and were replaced once for the next 2 weeks. Tumor volume was measured weekly. Tumors were excised and weighed at week 5. (White squares, TAT; small gray circles, δV1-1; small black circles, 3mM ψδRACK and large black ovals, 30mM ψδRACK, *; p<0.05, repeated ANOVA, n=5-8 each, TAT vs. 30mM ψδRACK-treated group). An inserted graph shows final tumor volumes of each treatment group.

Fig. 2

Fig. 2. PKCδ activation increases tumor angiogenesis

(A) The level of angiogenesis at the end of the 5-week growth was measured with immunofluorescence by staining blood vessels with anti-CD31 monoclonal antibodies conjugated with FITC (n=3 each, *; p<0.05, Scale bar: 10μm). (B) One week after PC-3 cell injection, mice were implanted with osmotic pumps with control peptide (TAT) or ψδRACK at 38 mg/kg/day (30mM) for 4 weeks. HIF-1α levels were measured by Western blot analyses using whole cell lysates from tumors (Figure 2B left, *; p<0.05 t test, n=3 each). GAPDH was used as a loading control. (C) Next, human VEGF concentration was measured in the serum from mice treated with control peptide (TAT) or ψδRACK by ELISA (Figure 2C left, *; p<0.05, n=4 each). Mouse VEGF concentration was measured in the serum from mice treated with control peptide (TAT) or ψδRACK by ELISA (Figure 2C, right).

Fig. 3

Fig. 3. PKCδ regulates NADPH oxidase activities in PC-3 cells

(A) Tumors as described in Figure 1C were analyzed for NADPH oxidase activity. Whole lysates of tumors in PBS with phosphatase and protease inhibitor cocktail were added with lucigenin. Chemiluminescence derived from superoxide and lucigenin was measured using a luminometer (n=5 each). A 2-tailed Student’s t test was used to determine significance. (B) Cells were serum starved for 14 hours and first incubated with PKCδ inhibitor peptide (δV1-1, 1μM) or PKCδ activator peptide (ψδRACK, 1μM) for 15 minutes. After incubation with 1nM PMA or serum for 30 minutes, whole cell lysate was used for the assay. A 2-tailed Student’s t test was used to determine significance (n=5-6 for each treatment). (C) The levels of PKCδ in PC-3 cells were knocked down using siRNA. (D) To test PKCδ regulation of NADPH oxidase activity, NADPH oxidase activities were measured in the cells treated with control siRNA or siRNA of PKCδ. A 2-tailed Student’s t test was used to determine significance (n=3 for each, *; p<0.05, t test).

Fig. 4

Fig. 4. PKCδ regulates NADPH oxidase activity by phosphorylating p47phox and inducing its translocation to the membrane fraction

(A) Western blot analyses were performed with total cell lysates of PC-3 tumors treated with TAT or 38 mg/kg/day of ψδRACK for 4 weeks and immunoblotted with antibodies against phospho-serine/threonine (Ser/Thr) (*; p<0.05, n=3 each, Figure 4A). GADPH was used as a loading control. (B) Tumor lysates were immunoprecipitated with antibodies against p47phox and probed for PKCδ. (*; p<0.05, n=3 each, Figure 4B). p47phox was used as a loading control. (C) Phosphorylation levels of the co-immunoprecipitated p47phox were checked by probing with anti-Ser/Thr antibodies (*; p<0.05, n=3 each, Figure 4C). PKCδ was used as a loading control. (D) Finally, tumor lysates were fractionated (as described in the methods) and the translocation of p47phox from the cytosolic to the membrane (particulate) fraction was determined by probing each fraction with anti-p47phox antibodies (Figure 4D, *; p<0.05, n=3 each). GADPH or Gαi was used as a loading control (IB: immunoblot).

Fig. 5

Fig. 5. PKCδ regulates HIF-1α levels in PC-3 cells via NADPH oxidase

(A) PKCδ regulates HIF-1α levels via NADPH oxidase. PC-3 cells were serum starved for 14 hours and incubated with apocynin (an anti-oxidant and a chemical inhibitor of NADPH oxidase, 1mM) for 5 minutes in the presence of 1% serum (Figure 5A, n=3 each, *; p<0.05). Also, the PC-3 cells were treated with δV1-1 and ψδRACK at 1μM for 4 hours in the presence of 1% serum. The peptides were treated every 1.5 hours and the cells were lysed for Western blot analyses. (B) A schematic diagram summarizes the regulation of tumor-induced angiogenesis via HIF-1α levels by PKCδ and NADPH oxidase in PC-3 cancer cells. p47; p47phox and p67; p67phox.

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References

    1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009;59(4):225–249. - PubMed
    1. Mostaghel EA, Montgomery B, Nelson PS. Castration-resistant prostate cancer: targeting androgen metabolic pathways in recurrent disease. Urol Oncol. 2009;27(3):251–257. - PMC - PubMed
    1. Kumar B, Koul S, Khandrika L, Meacham RB, Koul HK. Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Res. 2008;68(6):1777–1785. - PubMed
    1. Ushio-Fukai M, Nakamura Y. Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy. Cancer Lett. 2008;266(1):37–52. - PMC - PubMed
    1. Yuan G, Nanduri J, Khan S, Semenza GL, Prabhakar NR. Induction of HIF-1alpha expression by intermittent hypoxia: involvement of NADPH oxidase, Ca2+ signaling, prolyl hydroxylases, and mTOR. J Cell Physiol. 2008;217(3):674–685. - PMC - PubMed

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