Bystander response triggered by doxorubicin-killed dead cells contributes to acquire drug resistance but increasing radiosensitivity In vitro

Abstract

Introduction: A bystander effect typically refers to the death, altered growth or damage of cells that have not directly received chemotherapy or irradiation. Chemotherapeutic drugs like doxorubicin cause a drastic increase in the number of dead cells more towards the periphery and low towards the centre of the tumor prompting us to test for the existence of a bystander effect in view of the tumor microenvironment. Materials and Methods: HeLa cervical cancer cells were acutely exposed to doxorubicin to trigger cell death. Bystander HeLa cells in varying amounts were co cultured with fix amount of dead cells. The surviving mutant clones were isolated by serial culturing and checked for morphology, growth pattern and resistance to doxorubicin or radiation. Results: Co-culture results showed, growth arrest, SA-γ-galactosidase activity, an enlarged cell size, collectively indicating a premature senescent state. Up regulation of p53 and γH2AX indicated a DNA damage response pathway. Co-culturing of a fixed number of dead cells with increasing number of bystander cells showed highest number of clones formed in least number of bystander cells. The individual clones obtained were morphologically altered, reduced proliferation and resistant to doxorubicin. Conversely, clones were sensitive to γ radiation compared to control HeLa cells. Conclusion: The results suggest that dead cells conferred significant resistance towards drug but not radiation in cloned bystander tumor cells. This point to possible mechanism of drug resistance in vitro, which might explain the success of radiation therapy and cause of frequent tumor recurrence observed in patients undergoing chemotherapy.