Investigation of the apoptotic and anti-proliferative effects of receptor tyrosine kinase and JAK/STAT pathway inhibitors in colon cancer cell lines

Colon cancer is the fourth most common cause of cancer death in men and women in the world. The Epithelial Growth Factor Receptor (EGFR) is a tyrosine kinase (TK) receptor that is frequently overexpressed in colorectal cancer. Gefitinib (Gef) is an orally active inhibitor of TK that targets the ATP binding site of the intracellular TK domain of EGFR. The EGFR-associated signaling pathways, such as JAK-STAT, play an important role in cancer development. Cucurbitacin B (CuB) is a triterpenoid compound, which is a potent and selective inhibitor of JAK-STAT signaling pathway with potent antitumor activity. In this study, we aimed to investigate the apoptotic and anti-proliferative effects of CuB as a single agent and in combination with Gef on HT-29 ve HCT-116 colon cancer cell lines.In our study, cell viability was identified in the p53 mutant HT-29 and p53 wild type HCT-116 human colon adenocancer cell lines by MTT assay method and AO/EtBr staining and BrdU ELISA methods, for different dosages and incubation times, following CuB alone and in combination with Gef treatments. Apoptotic ratio was assessed using LDH, DNA Fragmentation and Caspase-3 ELISA methods. Quantitative Real-Time PCR and Western Blot methods were used for analysis of mRNA and protein expression levels of Bcl-2, Bax, Bak, Bad, cyclin D and p27kip1 genes which take part in apoptotic and proliferative signal pathways.Our results showed that treatment with CuB alone and in combination with Gef resulted in an apoptotic response in a dose dependent manner in both cell lines. We also found that the apoptotic effects of CuB increased in the presence of Gef. These results were also confirmed with appropriate changes in the mRNA and protein expression levels of apoptotic and anti-proliferative genes and proteins, in comparison to the control group. Our data suggest that CuB could be useful as a potential anti-neoplastic agent in the management of colon cancer. In conclusion, our study draws attention to the possible clinical effectiveness of use of CuB with chemotherapeutic agents with the opportunity of attaining convenient apoptotic and anti-proliferative effect with reduced dose and time in cancer treatment targeting JAK/STAT pathway. In addition to this, determination of the exact molecular effects of cucurbitacins would allow us to identify new molecular targets for the treatment of the colon cancer.