Mechanism of Overcoming Oxaliplatin Resistance Using HT29 Subcloning

Abstract Background: Cancer cells are different from normal healthy cells even though they reside within the same tissue and adapt to others. This intra-tumoral heterogeneity is the reason for chemo-resistance, and the emergence of one or more clones, which are resistant to chemotherapeutic drugs, causing clonal outgrowth to form recurrent cancer mass. The need for personalized chemotherapy is increasing because of the tumor heterogeneity and diverse mechanism of chemotherapy resistance. The recent accumulation of extensive evidence for the existence of cancer stem cells strengthens the cancer stem cell hypothesis for chemotherapy resistance and relapse. The development of a primary culture of cancer cells is essential for functional analysis like the sensitivity of chemotherapeutic drugs and the evaluation of the characteristics of cancer stem cells. Methods: We used a clonal cylinder to establish sub-clones originated from a single cell. Twenty-two sub-clones were successfully established, and eleven clones were selected according to their growth rate and analyzed. The sub-clones with low expression of BRAF, MEK2, and ERK, but not EGFR or KRAS, showed a correlation with the doubling time. We grouped the sub-clones as the fast-growing group and the slow-growing group of the HT29 cell line. Three out of five slow-growing sub-clones showed resistance to oxaliplatin treatment. All oxaliplatin-resistant sub-clones overexpressed ABCC2, and no relevance was found with ABCB1 and ABCG2. Active efflux of the drug by ABCC2, but not by ABCB1 or ABCG2, was confirmed with the inhibitor study using each specific inhibitor. The viability of resistant sub-clones decreased after the MK571 treatment, but other clones were not responsive. CD44 expression in oxaliplatin-resistant sub-clones was higher than that of sensitive clones. Conclusions: This study provides definite evidence of heterogeneity using a cancer cell line. Based on our studies, it appears that intra-tumoral heterogeneity of human cancer tissue is responsible for the development of chemotherapy resistance in cancer. These sub-clones are an excellent model for testing efficacy of anti-cancer drug candidates for advanced cancer therapy. The experimental design and concept of this study could be applied to personalized chemotherapy.