Evaluation of the antiproliferative effects of calcitriol and menadione in colon cancer cells

Abstract

Colon cancer is one of the leading causes of death worldwide, highlighting the need for new pharmacological strategies, especially in resistant variants. In previous studies, we have shown that drugs such as menadione (MEN), D,L-buthionine-S,R-sulfoximine or calcitriol (D), alone or in combination, enhance the sensitivity of breast and colon tumor cell lines by modifying the oxidative status. The present work aimed to evaluate the mechanisms underlying the antiproliferative effects of the combination of MEN and D in Caco-2 cells, a human colon adenocarcinoma cell line.

We used MEN (20 µM), D (200 nM), both or vehicle (ethanol) in Caco-2 cells. We quantified cell proliferation by crystal violet staining in the presence/absence of autophagy inhibitors and antioxidants; cell migration by wound healing assay (0-9h); morphological changes using May-Grünwald Giemsa and ethidium bromide/acridine orange stains; superoxide anion (SA) levels and catalase (CAT) activity by spectrophotometry. We determined apoptosis, cell cycle phases, and quantified reactive oxygen species (ROS, with dichlorofluorescein diacetate probe) by flow cytometry. Statistical analysis was performed using one-way ANOVA and Bonferroni tests.

The antiproliferative effects of MEN+D began at 24 h showing time and dose dependence. The combination reduced cell proliferation by 50% at 48 h at the selected doses (p<0.05 vs control). At the same time, it delayed cell migration and produced morphological changes compatible with necrosis and apoptosis. Significant alterations in oxidative status were observed: SA level increased by 80% (p<0.05 vs control), CAT by 150% (p<0.05 vs control), and total ROS content by 60% (p<0.05 vs control) at 48 h. Cell cycle arrest occurred in G2/M concomitant with a x12 increase in the level of apoptosis and x4 in necrosis (control vs MEN+D).

This study demonstrates that the combination of MEN+D has antiproliferative actions in Caco-2 cells, and suggests that it could be an additional strategy to treat conventional therapies resistant tumors.