Synergistic antitumor effect of TRAIL and doxorubicin on colon cancer cell line SW480.
OBJECTIVE
TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent. Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms.


METHODS
SW480 cells were cultured in the regular condition and incubated with different levels of agents. Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT) assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detect apoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay.


RESULTS
(1) SW480 cells were not sensitive to TRAIL, with IC(50)>1 mg/L(-1) and dose-independent cytotoxicity. (2) SW480 cells were sensitive to doxorubicin at a certain degree, with dose-dependent cytotoxicity and IC(50)=65.25+/-3.48 micromol/L(-1). (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC(50) of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 microg/L(-1)), combined with subtoxic doxorubicin (0.86 micromol/L(-1)), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12+/-2.67 %, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82+/-1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (P>0.05).


CONCLUSIONS
SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentration of doxorubicin to induce apoptosis effectively. The status of p53 protein is not involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubicin against colon cancers.
Xu
Li-Hong
LH
Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Donghu Road 169, Wuhan 430071, Hubei Province China. hqy222@yahoo.com.cn
Deng
Chang-Sheng
CS
Zhu
You-Qing
YQ
Liu
Shi-Quan
SQ
Liu
Dong-Zhou
DZ
D000970:Antineoplastic Agents / Q000494:pharmacology*; D051017:Apoptosis Regulatory Proteins; D003110:Colonic Neoplasms / Q000473:pathology*; D004317:Doxorubicin / Q000494:pharmacology*; D004357:Drug Synergism; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D053221:TNF-Related Apoptosis-Inducing Ligand; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D004317:Doxorubicin