Induction of Paraptosis: A novel strategy to overcome radiation resistance in cancer cells

2018 Fiscal Year Research-status Report

• Project/Area Number: 18K15585
• Research Institution: University of Toyama
• Principal Investigator: REHMAN MATIUR 富山大学, 大学院医学薬学研究部(医学), 特命助教 (10810921)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Keywords: Radiation / Paraptosis / Oxidative stress

Outline of Annual Research Achievements

The objective was to determine the effects of chemical agents that modify intracellular redox balance and ER stress mediated induction of distinct cell death types in alone or combined with radiation.It was found that CDDO-Me decreases cell viability in HCT-116 colorectal cancer cells. CDDO-Me induced extensive cytoplasmic vacuolation, which was triggered by CDDO-Me induced ROS generation. As pre-treatment with an antioxidant NAC alleviated vacuolation-mediated death. Mechanistic investigation revealed that cytoplasmic vacuolation was brought out by lack of caspase activation or PARP cleavage, and DNA fragmentation. In addition, CDDO-Me persistently induces the expression of autophagy marker LC3-II, along with endoplasmic (ER) stress markers, Bip and CHOP. However, in contrast failure of both apoptosis- and autophagy- inhibitors to reversed cytoplasmic vacuolation, suggest that vacuolation-mediated cell death is different from classical apoptotic and autophagic cell death. Cycloheximide treatment prevent CDDO-Me vacuolation and suggest the induction of paraptosis. Intrestingly, it was also found that these effects are concentration and cell death type dependent, as CDDO-Me induces apoptosis not paraptosis in HCT-15 colorectal cancer cells. In addition, in HeLa cells it was confirmed that combined used of CDDO-Me and glycolysis inhibitors enhanced cell death. Similarly, it was also confirmed that the combined used of romidepsin (FK228), a novel histone deacytylase inhibitor and hyperthermia also enhanced cell death in U937 and MOLT-4 cells.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Both the apoptosis and autophagy inhibitors fail to reverse the cytoplasmic vacuolation and it was found that cycloheximide prevents the cytoplasmic vacuolation, which confirms the involvement of paraptosis. NAC pre-treatment also protects the vacuolation mediated death and the expression of ER stress markers Bip and CHOP showed that intracellular redox modification and ER stress was involved in paraptosis by CDDO-Me. Research is progressing smoothly, in line with the purpose of research.

Strategy for Future Research Activity

In continuation of last year research, the expression of specific markers associated with paraptosis cell death will be examined by western blot and immunofluorescence imaging methods. Further, its confirmation will be done using positive marker for paraptosis. The involvement of calcium, ER stress and unfolded protein response in paraptosis will be investigated by using specific siRNAS and inhibitors. Mainly the effects will be observed using human colorectal cancer HCT-116 cells but the comparative examination will be done using other colon cancer cell lines. Next, the enhancement of radiation-induced cancer cell death combined with drugs will be investigated. The aim is to elucidate the molecular mechanism for the enhancement of radiation-induced cancer cell death by modifying intracellular redox balance and ER stress. The results will be summarized and published as a research paper.

Causes of Carryover

(Reason)Because several specific siRNAS are required for confirmation of ER stress mediated paraptosis this year. Therefore, I changed to buy siRNAS this year.
(Usage Plan)I plan to purchase these specific siRNAS immediately in this year and perform accurate detection of paraptosis.