Effects of novel targeted anticancer drugs on cytotoxicity, apoptosis, angiogenesis, EMT, drug resistance and autophagic mechanism

Abstract

Abstract Background Metastatic colon cancer has a survival rate less than %5 despite the use of effective chemotherapeutic regimen. Prognostic and predictive importance of EGFR is still contradictive in colon cancer. Tyrosine kinase inhibitor canertinib was used in this study to block the EGFR receptor. The effect of Palladium (Pd) (II) compound [PdCl(terpy)](sac).2H2O] and canertinib combination treatment was investigated in this study. 5-fluorouracil (5-FU), a chemotherapeutic drug was used as positive control. Methods The cytotoxic effects of the combination of Pd (II)+canertinib and the 5-FU+canertinib combination on colon cancer cell line (HCT-15, HT-29) were determined by the SRB test. Apoptosis, the acidic vesiculations, mitochondrial depolarization were determined by flow cytometry, Annexin-V-Cy3/SYTOX, acridine orange, JC-1 staining. Alterations in proteins related to EGFR-associated pathway, EMT, apoptosis/autophagy and drug carrier were evaluated by western blot. Invasion, wound healing and colony forming ability were determined. Tube forming in HUVEC and vessel forming ability was assessed by matrigel and CAM test respectively. Results A significant decrease in p-ERK, p-P38, p-EGFR protein levels was observed with EGFR inhibition, while the cell viability was decreased. Cell death was determined as mitochondrial apoptosis. LC3-II, Beclin-1, Atg5 protein levels, and increased acidic vesicles and decreased p-MTOR protein levels were associated with increased autophagy in combination therapy. In addition, increased E-cadherin expression and decreased N-cadherin, fibronectin and vimentin expression related to decrease in invasion, migration, and colony forming ability. ROS expression and DNA damage increased. Significant differences in MDR-1 and MRP-1 protein levels were observed with EGFR inhibition. In addition, the ability to create tubes and vessels has decreased significantly. Conclusions EGFR inhibition along with Pd(II) has been shown to increased cytotoxicity, decreased invasion, migration and vessel formation abilities. Furthermore, induced ROS levels and increased autophagic signaling are found to be necessary for mitochondria-dependent apoptosis in colon cancer cells. Legal entity responsible for the study Uludag University. Funding Scientific Research Projects Foundation (BAP) of Uludag University of Turkey [Project No. (DDP(F)-2017)]. Disclosure All authors have declared no conflicts of interest.