Low-dose chemotherapeutic drugs induce reactive oxygen species and initiate apoptosis-mediated genomic instability

Abstract

Prolonged cancer cell survival, acquiring drug resistance, and secondary cancer development despite chemotherapy are the major challenges during cancer treatment, whose underlying mechanism still remains elusive. In this study, low-doses of chemotherapeutic drugs (LDCD) - doxorubicin (DOX), etoposide (ETOP), and busulfan (BUS) were used to ascertain the effect of residual concentrations of drugs on breast cancer cells. Our results showed that exposure to LDCD caused significant induction of ROS, early signs of apoptosis and accumulation of cells in S and G₂-M phases of the cell cycle in MCF-7 and MDA-MB-231 cell lines. Under drug-free recovery conditions, a decrease in the number of apoptotic cells and an increase in the number of colonies formed were observed. Analysis of the molecular mechanism showed lower expression of cleaved products of caspase 3, 9, PARP and occurrence of DNA strand breaks in recovered cells compared to LDCD-treated cells, suggesting incomplete cell death activation and survival of cells with genomic damage after therapeutic insult. Thus, LDCD induces defective apoptosis in cancer cells allowing a small population of cells to escape from cell cycle check points and survive with accumulated genetic damage that could eventually result in secondary cancers that warrants further studies for better therapeutic strategies.

Fig. 1. Response of MCF-7 and MDA-MB-231 cells to a range of low-dose chemotherapeutic drugs after 12 and 24 h. (A) Doxorubicin – 0, 1, 2, 3, 4, 5, and 6 μM; (B) etoposide – 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 μM and (C) busulfan – 0, 20, 40, 60, 80, and 100 μM. Data expressed as mean ± S.D. of three independent experiments.

Fig. 2. Hoechst 33258 and propidium iodide double staining of MCF-7 and MDA-MB-231 cells at 12 h incubation (A) with LDCD and after 24 h recovery (B). Apoptotic bodies (yellow arrow) and swollen enlarged cells typical for necrosis (purple) were observed in experimental cells (magnification – 400×).

Fig. 3. Influence of LDCD on the colony formation of MCF-7 (A) and MDA-MB-231 (B) cells. Data expressed as mean ± S.D. of three independent experiments. *p < 0.05, control versus drug-treated cells.

Fig. 4. Intracellular ROS generation of MCF-7 (A) and MDA-MB-231 (B) cells after LDCD treatment for 12 h. data expressed as mean ± S.D. of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, control versus drug-treated cells.

Fig. 5. Effect of LDCD on the expression of apoptotic proteins in MCF-7 (A & B) and MDA-MB-231 (E & F) cells. Total protein from the cells treated with LDCD (A & E) and post recovery in drug-free medium (C & F), assessed for expression of apoptotic proteins. β-Actin – endogenous control. Densitometry analysis of cleaved caspase-3, 9 and PARP with full-length procaspase 3, 9 and PARP and relative changes are indicated (B, D, G, H & I). *p < 0.05, control versus drug-treated cells.

Fig. 6. Differences in the cell cycle distribution of MCF-7 (A & C) and MDA-MB-231 (B & D) cells exposed to LDCD. Cell cycle analysis by flow cytometry 12 h after LDCD treatment and percentage distribution of cells in the sub-G1, G1, S and G₂-M phases from the resulting histograms and the mean values are shown.

Fig. 7. Frequencies of the mitotic index in MCF-7 (A) and MDA-MB-231 cells (B) treated with LDCD. Data expressed as mean ± S.D. of three independent experiments. *p < 0.05, vehicle control versus drug-treated cells.