than exposure to each agent individually in both cell lines. Combined treatment with PXD101 and SN38 exerts time and dose dependent Formononetin eVects on the expression of XIAP protein, especially in HCT116 cells To determine the eVect of single and combined treatment on the expression of acetyl H3, H3, p21, and XIAP over time and with diVerent doses of each agent , we performed Western blotting in both cancer cells. The time and dose dependent eVect of PXD101 on acetylated histone H3 levels was evident in both cancer cells. However, eVect is more sensitive in the HCT116 cells. HDACIs or irinotecan are known to be associated with induction of p21 . Therefore, to explore whether PXD101 and SN38 combine to exert a synergistic eVect on p21 expression, we performed Western blotting for p21.
Both PXD101 Maraviroc molecular weight and SN38 alone induced a time and dosedependent slightly increase in p21 levels in HCT116 and HT29 cells; however, there was no evidence for a synergistic eVect of the two agents in combination. We analyzed the expression of XIAP as a representative antiapoptotic protein . After treating HCT116 cells with combined treatment for 8 h, XIAP expression was reduced compared to treatment with either agent alone. In HT29 cells, combined treatment for 8 h did not signiWcantly reduce XIAP levels. However, at higher concentrations, combined treatment reduced XIAP expression in HT29 cells. These results indicate that combined treatment exerts a time and dose dependent eVect on XIAP expression in colon cancer cells, an eVect that is more prominent in the HCT116 cell line.
To determine whether the combination of PXD101 with irinotecan also acts synergistically in vivo, we evaluated the antitumor eVect of combined treatment against HCT116 and HT29 xenografts. Combined treatment was signiWcantly more eVective in suppressing the growth of HCT116 tumors than either agent alone . In HCT116 xenografts, tumor growth was inhibited by 62.9, 75.6, and 88.2% at day Imatinib price 21 after treatment with PXD101 alone, irinotecan alone, and combined PXD101/irinotecan, respectively . In HT29 xenografts, there was a tendency toward increased eVectiveness of combined treatment, although this diVerence did not reach statistical signiWcance . Thus, consistent with the in vitro Wndings, HT29 xenografts were less sensitive to Fulvestrant ic50 these agents than HCT116 xenografts.
Body weight was not signiWcantly diVerent among colon cancer cell xenograft recipients in the diVerent treatment groups . Combined treatment with PXD101 and irinotecan showed enhanced eVects on HCT116 xenografts in psychological examination TUNEL and colony forming assays To evaluate the apoptotic eVects of agents on xenograft tumors, the percentage of apoptotic cells was determined . There was a modest increase in apoptosis with time after treatment with either agent alone. However, the combination treatment induced a clear increase in apoptosis above this level, indicating that combined treatment was more eVective by this measure than either agent alone. The antiproliferative activity of combined treatment and treatment with either agent alone in HCT116 xenograft tumors was assessed using a soft agar colony formation assay . Combined treatment, and treatment with PXD101 alone, inhibited colony formation on day 16 by 87 and 50%, respectively.