Results Treatment with IL 6 enhances phosphorylated RKIP levels IL 6 has been shown to lead to STAT3 activation in colon cancer. HCT116 cells were treated for 1, 3 and 6 h with 40 ng ml IL 6 and examined for STAT3 and RKIP phosphorylation. As expected, we observed Tipifarnib solubility an increase in pY705STAT3 but were surprised to also note an increase in pRKIP. To our knowledge this is the first report to show cytokine mediated phosphorylation of RKIP. Oxaliplatin inhibits IL 6 signaling Previous studies have shown that treating CRC CT26 cells with 300 uM OXP for 24 h leads to about 50% of the cells showing signs of apoptosis. In our experiment treatment with OXP induced approximately 32% of the cells to undergo apoptosis, which was lowered to 19% after co treatment with IL 6.
Western blot analysis showed that co treatment of HCT116 cells with IL 6 and 300 uM OXP for 18 hours inhibited the increase in pY705 STAT3 and pRKIP caused by IL 6. OXP induced apoptosis was confirmed with Western blot analysis by measuring PARP cleavage and DNA damage by H2AX phosphorylation. CPT reduces IL 6 induced RKIP phosphorylation and STAT3 transcription Camptothecin is frontline therapy for metastatic CRC. Therefore, we investigated if CPT could affect STAT3 phosphorylation. Western blot analysis revealed a dose dependent decrease of STAT3 pY705 phosphorylation when cells were treated with 40 ng ml IL 6 in the presence of 250 750 nM CPT for 12 h. The same experiment was repeated and the cells were treated with 250 nM CPT and 40 ng ml IL 6. We observed a reduction of pRKIP when the cells were treated with both compounds.
We measured apop tosis in the samples via Annexin staining from Figure 2B and found that treatment with 250 nM CPT led to approximately 17% of the cells to undergo apoptosis, which was reduced to 7% after co treatment with IL 6. STAT3 selleck kinase inhibitor luciferase reporter assay confirmed a significant decrease in STAT3 transcription when cells were treated with IL 6 and CPT. We found that these effects were also recapitulated in HT29 colon cancer cells. In addition to inhibiting TOP I, this CPT analogs can also interfere with cytokine mediating signaling events that lead to RKIP and STAT3 phosphorylation. STAT3 overexpression increases pRKIP IL 6 treatment enhances STAT3 phosphorylation, tran scription and pRKIP. We examined if STAT3 overexpression could directly affect pRKIP and Western blot analysis showed that the expression levels of phosphorylated RKIP increased upon transfection with STAT3. In the presence of CPT, the levels of pRKIP were reduced after STAT3 overexpression when compared to STAT3 alone. This indicates, similar to our IL 6 results that CPT interferes with the kinase activity mediated by STAT3 that results in RKIP phosphorylation.