Raf-1 kinase inhibitor protein modulation of the cellular response to chemotherapeutic drugs and PDE5 inhibitors.

RKIP was initially discovered as an endogenous inhibitor of the ERK and NF-κB pathways,and was also shown to prolong the activation of GPCRs via inhibition of the GRK2 protein. Now increasing evidence has linked RKIP to a metastases suppressing and chemo-sensitising role in cancer cells.The chemo-sensitising effect of RKIP was investigated in a colon carcinoma cell line using a variety of chemotherapeutic agents from conventional agents to newer targeted therapies. Initial results suggested that role of RKIP in the modulation of chemotherapeutic drug response was at the level of apoptosis; there did not appear to be great observable effects in the cell proliferative response and the cell cycle distribution of the colon carcinoma cells after treatment with selected agents. Apoptosis modulation by RKIP occurred after treatment with doxorubicin, FasL, paclitaxel and TRAIL. TRAIL-treated colon carcinoma cells displayed increased cell death as the levels of RKIP within the cell were increased. In contrast, doxorubicin, FasL and paclitaxel-treated cells displayed a scaffold-like response as the levels of RKIP were increased in the cell; with WT RKIP-expressing cells being more sensitive to doxorubicin, FasL and paclitaxel-induced apoptosis than low or high RKIP-expressing colon carcinoma cells. There was no modulation of 5-FU, cisplatin and etoposide-induced apoptosis by RKIP. Indeed, these three agents did not appear to induce cell death in this colon carcinoma cell line. RKIP modulation of chemo-sensitivity has never been shown before in a colon carcinoma cell line and this is the first time that doxorubicin and FasL-induced apoptosis has been shown to be modulated by RKIP. Further, it is shown here, for the first time, that the modulation of chemotherapy-induced apoptosis by RKIP can change depending upon the cytotoxic drug employed as treatment. TRAIL and FasL, both members of the TNF super-family, were selected for further analysis due to the distinctive cell death responses observed as a consequence of the levels of RKIP within the cell. WT RKIP cells were sensitive to FasL treatment, and high RKIP cells were most sensitive to TRAIL administration. Increased sensitivity of high RKIP-expressing colon cells to TRAIL treatment appeared to involve up-regulation of the DR5 receptor; down-regulation of the anti-apoptotic molecule Bcl-xl; pIKK which activates the NF-κB pathway; and TRAF2 which has been shown to activate the NF-κB pathway. Whether RKIP directly interacts with these molecules is unknown however RKIP has been shown to bind upstream activators of the NF-κB pathway and another TRAF subtype TRAF6. YY1 expression was evident in the TRAIL-treated cells but the expression was unchanged as the levels of RKIP within the cell were altered. The FasL-treated cells also displayed decreased pIKK levels as the levels of RKIP were increased; it is possible that NF-κB was behaving as both pro- and anti-apoptotic within this cell line. Thus RKIP inhibition of the NF-κB pathway may have prevented FasL-induced apoptosis in the high RKIP-expressing colon carcinoma cells. The expression of TRAF6, which has been shown to bind RKIP, displayed a scaffold-like response with WT RKIP-expressing cells having the highest TRAF6 expression. This was also the case for the transcriptional regulator YY1, thus it is possible that both YY1 and TRAF6 were behaving in a pro-apoptotic-like manner in the WT RKIP-expressing cells. TRAF2 was also evident in the FasL-administered cells but the expression did not change regardless of the levels of RKIP within the cell. Overall, it appears that differential expression of TRAF adaptor proteins is responsible for the contrasting responses of TRAIL and FasL-treated cells with low, WT and high RKIP expression. Utilisation of particular TRAF adaptors or TRAF combinations by the TRAIL and Fas receptors may also account for the pro- and anti-apoptotic roles of the NF-κB pathway, and the recruitment or down-regulation of other proteins dependent upon the cell stimulus. How RKIP affects these proteins requires further investigation, however these results are exciting and novel, and strengthen evidence surrounding the role of RKIP in chemosensitivity. On another note, RKIP has been shown to bind the PDE5 inhibitor PF-3717842, therefore investigation of the effects of the PDE5 inhibitors sildenafil citrate and vardenafil citrate on RKIP inhibition of the ERK pathway in a colon carcinoma cell line were examined. The effects of the PDE5 inhibitors were compared to the cell migration inhibitor locostatin that has been shown to bind and inhibit RKIP, and prevent the RKIP-Raf-1 interaction. With TPA and EGF stimulation, locostatin appeared to act in a manner consistent with its known function as an RKIP inhibitor. The PDE5 inhibitors sildenafil citrate and vardenafil citrate displayed a similar trend to that of locostatin, although their effects on the ERK pathway were not as potent. It is possible that after EGF stimulation, the strong activation of B-Raf was over-shadowing the subtle effects of the drug treatments. Under growth conditions, the RKIP inhibitor locostatin did not appear to behave as an inhibitor of RKIP nor did the PDE5 inhibitors sildenafil citrate and vardenafil citrate. It is possible that the strong activation of various growth and proliferative cascades was impinging upon the ERK pathway, were overshadowing the drug effects, or resulting in off-target (RKIP-unrelated) effects of the drugs. In summary, the role of RKIP within the cell is becoming an increasingly exciting avenue of research and is consistently yielding new and interesting roles and interactions within the cell. Understanding and elucidating the roles of this intriguing protein within the cell will not only strengthen our knowledge of signal transduction regulation and modulation, but may also provide a new source of targeted therapy and means of manipulation in the treatment of cancer and chemotherapeutic drug resistance.