Hypoxia induced aggressiveness of prostate cancer cells is linked with deregulated expression of VEGF, IL-6 and miRNAs that are attenuated by CDF

Abstract

Tumor hypoxia with deregulated expression of hypoxia inducing factor (HIF) and its biological consequence leads to poor prognosis of patients diagnosed with solid tumors, resulting in higher mortality, suggesting that understanding of the molecular relationship of hypoxia with other cellular features of tumor aggressiveness would be invaluable for developing newer targeted therapy for solid tumors. Emerging evidence also suggest that hypoxia and HIF signaling pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation, all of which contribute to radiation therapy and chemotherapy resistance. However, the detailed mechanisms by which hypoxia/HIF drive these events are not fully understood. Here, we have shown that hypoxia leads to increased expression of VEGF, IL-6, and CSC marker genes such as Nanog, Oct4 and EZH2, and also increased the expression of miR-21, an oncogenic miRNA, in prostate cancer (PCa) cells (PC-3 and LNCaP). The treatment of PCa cells with CDF, a novel Curcumin-derived synthetic analogue previously showed anti-tumor activity in vivo, inhibited the productions of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 under hypoxic condition. Moreover, CDF treatment of PCa cells led to decreased cell migration under hypoxic condition. Taken together, these results suggest that the anti-tumor effect of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become useful for cancer therapy.

Figure 1. Effect of CDF on cell survival, clonogenicity, and the expression of HIF-1α protein in human prostate cancer (PCa) cells under hypoxic condition.

The panels A, B, and C represent the data of cell survival, clonogenicity, and Western blot analysis, respectively. The bars in the figures indicate standard deviation of n = 4.

Figure 2. Effect of CDF on the productions of VEGF and IL-6 in human PCa cells under hypoxic condition.

The conditioned media were collected from cells cultured under normoxic and hypoxic conditions as described under the methods section. The measurements of VEGF and IL-6 were conducted by ELISA. The bars in the figures indicate standard deviation of n = 3.

Figure 3. Effect of CDF on angiogenesis, cell migration, and invasion of PCa cells under hypoxic condition.

The panels A&B, C&D, and E represent the data of angiogenesis in vitro, cell migration, and invasion. As described under the Methods section, angiogenesis in vitro was evaluated by the tube formation assay; cell migration was evaluated by wound healing assay; invasion was evaluated by chamber invasion assay.

Figure 4. Effect of CDF on the expression of Nanog, Oct4, EZH2, miR-21 and miR-210 in PCa cells cultured under hypoxic conditions.

Real time RT-PCR was conducted as described under the Methods section. The bars in the figures indicate standard deviation of n = 3.

Figure 5. Effect of CDF and anti-miR-21 on the formation of prostaspheres, the expression of CD44 and EpCAM, and the production of VEGF and IL-6 in the CSC-like sphere-forming cells of PCa cells under hypoxic conditions.

The panels A and B represent the data of the formation of prostaspheres, the expression of CD44 and EpCAM, and the production of VEGF and IL-6 in the CSC-like sphere-forming cells of PCa cells, respectively. The sphere formation assay was conducted to examine the self-renewal capacity of CSCs of PCa cells as described under the Methods section. Confocal microscopy was conducted to measure the expression of CSC surface markers CD44 and EpCAM in the sphere-forming cells derived from PCa cells as described under the Methods section. The bars in the figures indicate standard deviation of n = 3.

Figure 6. Effect of CDF and/or anti-miR-21 on miRNA expression and miR-21-mediated luciferase activity in human PCa cells under hypoxic conditions.

The panels A and B represent the data of miRNAs in the CSC-like sphere-forming cells derived from human PCa cells and luciferase activities in PCa cells, respectively. The transfection of miR-21-mediated reporter gene luciferase vector and anti-miR-21 were conducted in PC-3 cells, as described in detail under the Methods section. The luciferase activity was measured by using Promega luciferase assay system kit, following the manufacturer’s manual. The bars in the figures indicate standard deviation of n = 3.