Background: Our previous work has demonstrated the role of cancer stem cells (CSCs) in the initiation and propagation of human liver cancer, and the effect of CD44 on CSC survival. The present study was designed to characterize the role of CD44 in chemoresistance of liver CSCs. Materials and Methods: In vitro, CD90+ and CD90- cells were sorted human HCC cell lines, PLC and MHCC97L. Cell response to chemocytotoxic agents in the presence or absence of anti-CD44 antibody was determined by Annexin V and PI labeling. Quantitative RT-PCR and western blot were used to detect genetic and proteomic changes with treatment. In vivo, HCC tumor xenografts in nude mice were treated with chemocytotoxic agents alone, or in combination with different time-course of anti-CD44 antibody. Tumor growth kinetics was monitored for 4 weeks after treatment. Results: In vitro,IC50 of cisplatin or doxorubicin in the sorted CD90+ cells from both cell lines were significantly higher than that in CD90- cells. When treated with cisplatin or doxorubicin alone, less CD90+ cells were undergoing apoptosis than CD90- cells. In both PLC and MHCC97L cells, lethal doses of cisplatin or doxorubicin could not completely eradicate tumor cells, but enriched CD90+ cells in the remaining population. Administration of anti-CD44 antibody increased the number of apoptotic cells in the sorted CD90+ cells in both cell lines. In the total cell population, a sequential treatment protocol using cytotoxic agents and anti-CD44 antibody significantly increased the number of apoptotic cells, compared with cytotoxic agents alone. Both anti-CD44 antibody alone and combination treatment downregulated the expression of heat shock protein 90 (Hsp90) and Bcl-xL. Inhibition of Hsp90 activity attenuated the pro-apoptotic effects of anti-CD44 antibody. In vivo, cisplatin, doxorubicin, and anti-CD44 antibody treatment alone retarded tumor growth, whereas combined treatment exhibited a more prominent inhibitory effect on tumor growth. Tumor regression was observed in one mouse with the combined treatment. Histological studies showed extensive areas of necrosis in the tumor xenografts treated with cisplatin or doxorubicin in the presence of anti-CD44 antibody. Immunohistochemical staining detected an increased proportion of CD90+ cells in the cisplatin or doxorubicin treated tumor xenografts. TUNEL assay identified an increased number of apoptotic cells in the tumor xenografts treated with cisplatin or doxorubicin alone, compared with non-treatment control, and an enhancement of cell apoptosis was detected in the group treated with anti-CD44 antibody and cisplatin or doxorubicin. Conclusion: The present study reveals that targeting CSCs can enhance chemosensitivity of human liver cancer.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1067.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO