Depletion of intrinsic expression of interleukin-8 in prostate cancer cells causes cell cycle arrest, spontaneous apoptosis and increases the efficacy of chemotherapeutic drugs
The progression of all cancers characterized by increased cell proliferation and decreased apoptosis. Many chemokines and cytokines are suspects to cause this increased tumor cell survival that ultimately leads to resistance to therapy and demise of the host.
Castration-resistant prostate tumors and cells constitutively express abundant amount of the pro-inflammatory chemokine, Interleukine-8 (IL-8). The contribution of IL-8 up-regulation in androgen-independent prostate cancer cells is incomplete at present.
This report is to show the pervasive role of IL-8 in malignant progression of androgen-independent prostate cancer (AIPC) and to provide a potential new therapeutic avenue, using RNA interference.
Methods: The functional consequence of IL-8 depletion by RNA interference in two metastatic prostate cancer cell lines, PC-3 and DU145 was investigated. Cells were transfected with RISC-free siRNA (control) or validated-pool of IL-8 siRNA (Smartpool siRNA, Dharmacon Inc, USA).
Consequences of siRNA induced intrinsic IL-8 depletion on tumor cell physiology were evaluated using colorimetric assay, flow cytometry, immunoblotting, Matrigel invasion assay, real-time q-PCR and densitometry.
Results: Transfection of PC-3 and DU145 cells with 50 nM IL-8 siRNA caused >95% depletion of IL-8 mRNA and >92% decrease in IL-8 protein. This reduction in IL-8 led to cell cycle arrest at G1/S boundary and decreases in cell cycle-regulated proteins: cyclin D1 and cyclin B1 (all decreased >50%) and inhibition of MAP-kinase activity by >50%.
Further, the spontaneous apoptosis was increased by >43% in IL-8 depleted cells, evidenced by increases in caspase-9 activation and cleaved-PARP. In addition, IL-8 depletion caused significant decreases in anti-apoptotic proteins, BCL-2, BCL-xL due to decrease in both mRNA and post-translational stability, but increased levels of pro-apoptotic BAX and BAD proteins.
More significantly, depletion of intracellular IL-8 sensitized the tumor cells to cytotoxic activity of several chemotherapeutic drugs. Specifically, the cytotoxicity of Docetaxel, Staurosporine and Rapamycin increased significantly (>40% at IC50 dose) in IL-8 depleted cells when compared to control-siRNA transfected cells.
Conclusion: These results show the pervasive role of IL-8 in promoting tumor cell survival, and resistance to cytotoxic drugs, regardless of the cytotoxic mechanism of antiproliferative drugs, and point to potential therapeutic significance of IL-8 depletion in men with AIPC.
Author: Rajendra SinghBal Lokeshwar
Credits/Source: Molecular Cancer 2009, 8:57
Published on: 2009-07-31
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
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