Ras Puts the Brake on Doxorubicin-mediated Cell Death in p53-expressing Cells

Sunil K Manna,Charitha Gangadharan,Damodar Edupalli,Nune Raviprakash,Thota Navneetha,Sidharth Mahali,Maikho Thoh

doi:10.1074/jbc.m110.191916

Sunil K Manna, Charitha Gangadharan + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.191916

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Abstract

Doxorubicin is one of the most effective molecules used in the treatment of various tumors. Contradictory reports often open windows to understand the role of p53 tumor suppressor in doxorubicin-mediated cell death. In this report, we provide evidences that doxorubicin induced more cell death in p53-negative tumor cells. Several cells, having p53 basal expression, showed increase in p53 DNA binding upon doxorubicin treatment. Doxorubicin induced cell death in p53-positive cells through expression of p53-dependent genes and activation of caspases and caspase-mediated cleavage of cellular proteins. Surprisingly, in p53-negative cells, doxorubicin-mediated cell death was more aggressive (faster and intense). Doxorubicin increased the amount of Fas ligand (FasL) by enhancing activator protein (AP) 1 DNA binding in both p53-positive and p53-negative cells, but the basal expression of Fas was higher in p53-negative cells. Anti-FasL antibody considerably protected doxorubicin-mediated cell death in both types of cells. Activation of caspases was faster in p53-negative cells upon doxorubicin treatment. In contrast, the basal expression of Ras oncoprotein was higher in p53-positive cells, which might increase the basal expression of Fas in these cells. Overexpression of Ras decreased the amount of Fas in p53-negative cells, thereby decreasing doxorubicin-mediated aggressive cell death. Overall, this study will help to understand the much studied chemotherapeutic drug, doxorubicin-mediated cell signaling cascade, that leads to cell death in p53-positive and -negative cells. High basal expression of Fas might be an important determinant in doxorubicin-mediated cell death in p53-negative cells.

Highlights

Doxorubicin is one of the most effective molecules used in the treatment of various tumors
Doxorubicin treatment often causes DNA damage and oxidative stress that leads to change in mitochondrial membrane potential followed by cell death [3, 4]
MCF-7 cells have wild type p53 that might be a target of doxorubicin, p53-mutated breast tumor cells such as SKBr3 or MDA-MB-231 are targeted by doxorubicin more aggressively for cell death

Summary

EXPERIMENTAL PROCEDURES

Materials—Doxorubicin, EDTA, EGTA, PMSF, propidium iodide, Ac-DVED-pNA, Ac-ITED-pNA, 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT), glycine, oleandrin, and anti-tubulin antibody were obtained from Sigma. The cells were extracted with lysis buffer (part of the luciferase assay kit from Promega), and the extracts were incubated with the firefly luciferin (substrate from Promega). Determination of DNA Fragmentation—MCF-7 and HeLa cells were treated with doxorubicin and harvested, and DNA was extracted following the method described earlier [20]. After different treatments, 1 ϫ 105 cells were stained with the LIVE/DEAD cell assay reagent. JNK complex from whole-cell extract (300 ␮g) was precipitated with anti-JNK1 antibody (1 ␮g) followed by incubation with protein A/G-Sepharose beads (Pierce). After a 2-h incubation, the beads were washed with lysis buffer and assayed for activity of JNK using 5 ␮g of GST-Jun (amino acids 1–100) substrate protein. The p Ͻ 0.05 was considered to be significant

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS