Experimental Study On Drug Efflux Of ABCG2 Positive Side Population Cells In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of most common cancers worldwide, and a large proportion of HCC patients present with late stage cancer at diagnosis. Currently, the treatment is limited for patients with advanced HCC, and the drug resistance to conventional chemotherapy is a major impediment to successful treatment. In HCC, one of leading causes for chemotherapeutic failure has been attributed to the fact that a large proportion of cancer cells express relatively high levels of several ATP-binding cassette (ABC) transporters, which actively "pump" out a broad spectrum of clinical relevant compounds and decrease the intracellular drug accumulation. The ABCG2 transporter (ATP-binding cassette subfamily G, member 2), also known as breast cancer resistance protein (BCRP), was first identified in a doxorubicin selected drug-resistant MCF7 cancer cell line and have been shown to mediate multiple drug resistance (MDR) in various cancers. Previous studies found the ABCG2 expression was upregulated HCCs, suggesting the role of ABCG2 in protecting HCC cells against chemotherapy.Recently, the ABCG2 transporter and its function in Hoechst dye efflux were identified in the "side population" cells in many cancers. The side population, a rare subset of cells distinguished by their low Hoechst dye staining in flow cytometry, was first described by Goodell et al in identifying hematopoietic stem cells in bone marrow. Unlike the common methods by recognizing cell surface markers, side population is a functional phenotype that defined by the cells' performance in "pumping" out DNA binding dye Hoechst 33342. Later studies in leukemia and some solid tumors found this population could be a source of cancer stem cells, though it appeared heterogeneous in terms of cell surface marker profile. Current evidences support ABCG2 is the primary transporter for the Hoechst dye efflux, and the elevated ABCG2 expression on side population was proved to confer intrinsic resistance to chemotherapeutic agents.In HCC cell lines, previous studies had reported the existence of a distinct side population displaying extreme tumorigenicity. However, ABCG2 expression, side population and their relevance to chemotherapy resistance in HCC still need further investigation. In this study, we examined the ABCG2 expression patterns in HCC tissues, and found the diffused ABCG2 expression pattern suggesting the existence of intrinsic drug resistance in HCC. We demonstrated that the ABCG2 expression patterns had great impact on side population fraction and doxorubicin efflux transport in HCC cell lines, especially in the MHCC-97L cell line with intrinsic ABCG2 expression. Further, we found the serum stimulation could elevated the SP proportion in MHCC-97L cells, and the isolated MHCC-97L SP cells exhibited relatively higher Akt signaling activities than the non-SP cells, which prompted us to study the contribution of Akt signaling to the SP fraction. The results showed the Akt signaling blockade by inhibitiors or by siRNA approach could significantly modulate the SP proportion in the MHCC-97L cell line. Further investigation revealed that the Akt signaling inhibition could regulate ABCG2 function by promoting its subcellular redistribution. Moreover, we found inhibiting Akt signaling in MHCC-97L cells could block the doxorubicin efflux and potentiate the drug efficacy on cancer cells. Our findings would improve the existing understanding for ABCG2 and its functional modulation in HCC cells, and provide some clues for clinical regimens targeting HCC cells with inherent drug resistance.