Apoptotic signaling is initiated when OXP binds

Apoptotic signaling is initiated when OXP binds selleck catalog to DNA, forming a DNA adduct. Camptothecins are another class of chemotherapeutic compounds used clin ically to treat various malignancies including metastatic CRC. Camptothecin and its congeners target the enzyme topoisomerase 1 by binding to the DNA Top1 complex and preventing the replication of DNA. Camptothecin derivatives can induce RKIP expression and apoptosis in some human cancer cells. One major obstacle in elongating the post treatment survival of patients after conventional therapies, such as radiation and chemotherapeutic drugs like OXP and CPT, is the acquired resistance observed in many patients with colon cancer. One way to understand the mechanism by which this resistance arises is to analyze how the drug modulates proteins involved with survival and apoptosis.

Therefore, it is necessary to find specific gene and protein targets to help improve the outcome of colon cancer treatment. Recent reports indicate that RKIP may serve as a potential biomarker in Dukes B CRC patients and used to identify high risk patients with aggressive CRC and these patients should be considered for adjuvant therapy, which may be dependent on intratumoural heterogeneity. In this study we demonstrate that IL 6 mediated activa tion of STAT3 occurs in conjunction with the phosphoryl ation of RKIP in vitro. OXP and CPT are able to block the IL 6 mediated STAT3 activation and RKIP phosphorylation via the inhibition of the interaction of STAT3 with gp130.

We extended these observations and determined that that STAT3 and nuclear pRKIP are associated with poor patient prognosis in stage II colon cancer patients. Methods Materials The CPT derivative ST2614 was provided by Sigma Tau Inc, Rome, Italy. Recombinant human IL 6 was purchased from BD Pharmingen Biosciences. All other reagents and chemicals were purchased from Sigma Chemical Co. un less otherwise noted. Protein quantification reagents were obtained from Bio Rad Laboratories Inc. and Thermo Scientific. Enhanced chemiluminescence reagents and secondary mouse and rabbit antibodies conjugated to horseradish peroxidase for Western blot analysis were from GE Healthcare. The antibodies to STAT3, pRKIP, gp130 and actin were purchased from Santa Cruz Biotechnology, STAT3 pY705 and PARP from Cell Signaling Technology, RKIP and Histone 2AX from Millipore, Milford, MA.

Cells and plasmid The human colon cancer cell lines, HCT116 and HT29 were purchased from ATCC. The HCT116 cells were grown in McCoys 5A and HT29 cells in RPMI1640 medium supplemented with 10% fetal bovine serum, glutamine, selleck chem non essential amino acids, 100 units ml penicillin, and 100 ug ml streptomycin. They were cultured in a humidified incubator at 37 C containing 5% CO2. Western blot analysis Total cell extracts were prepared as previously reported and the protein concentrations of lysates were determined using either Bradford assay kit or BCA protein assay kit.

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