The non-responsiveness of cancer cells to chemotherapeutic regiments has been a major obstacle in the treatment of cancers. The survival rate for HCC patients in advanced stages which could not treated with surgical procedures (including liver transplantation) has not been changed for 30 years
. One of the important issues in multidrug resistance in cancer is the high expression of ABC transporters proteins, including ABCG2.
Due to the inconclusive data on the role of ABCG2 in HCC we assessed the functional and gene expression of this ABC transporter in two different but related systems such as a series of human derived liver cell lines and patients with HCC and cirrhosis. For the in vitro model 4 differently differentiated hepatic cell lines were used. Three derived from HCC while one originates from a normal adult liver. The ABCG2 mRNA expression in tumoral lines was higher than in immortalized normal hepatocyte (IHH) and this was paralleled by the protein contents. Of notice was the effect observed when the cells were exposed to 5 μM Dox. This treatment induced an up-regulation of both gene expression and protein content in all cell lines, but the up-regulation was inversely proportional to the basal level of expression. This behavior suggests that in the presence of toxic drug, ABCG2 is induced to export the drug from the cell and to prevent cytotoxicity as one of the major defense mechanisms and that this effect is maximal when the basal level of the expression is low. The data we observed in HepG2 cells agree with a previous report in which ABCG2 mRNA was up-regulated in Dox-resistant HepG2
To assess the activity of ABCG2 in cancer cell lines, we performed a functional assay using the Hoechst 33342 test. The efflux of Hoechst 33342 is accounted by both ABCG2 and ABCB1 transporters, and verapamil had been shown to effectively inhibit ABCB1 but not ABCG2
. The addition of verapamil resulted in the increase of intracellular Hoechst 33342 in the well differentiated HepG2 and HuH-7 cells but not in the poorly differentiated cells (JHH-6). This data points to the conclusion that in the poorly differentiated HCC cell line, the efflux is mainly depend on the ABCG2 activity while in the more differentiated ones, drug export is also accounted by the activity of ABCB1. This conclusion is in line with very high ABCB1 expression in both HepG2 and HuH-7 cells. This finding indicates that a high expression of ABCG2 transcription is associated with functional role of the transporter which results in the protection of the cell from doxorubicin-induced cytotoxicity.
To compare the data obtained in vitro, we studied the ABCG2 mRNA expression in samples of adult normal liver and tissues obtained from HCC patients. There are several limitations in this type of studies such as the effect of the different treatments to which each patient undergoes before being considered for hepatic resection or transplantation. Our data in human tissues samples show a rather high variation in ABCG2 expression, as expected. The large variations of intra- and inter-groups ABCG2 expression observed in this study had also been reported in acute leukemia
, breast cancer
 and lung cancer
. We believe that these variations are likely linked to several factors such as age, sex, type of the drugs, duration of the treatments, severity of the diseases, and many others. In line with this conclusion is the observation that when we compared the expressions of normal adult liver with that obtained from children, ABCG2 expression was higher in the adults (data not shown), suggesting that ABCG2 expression is age-related and dependent to drug exposure which obviously increases with age.
The overall distribution showed that the expression of the ABCG2 mRNA was slightly higher and more dispersed in HCC and cirrhotic tissues as compared to normal tissues, in line with a previous report
. Interestingly, the ABCG2 mRNA expression was higher in the HCC than in the surrounding cirrhotic tissue in about 60% of HCC patients. Of notice was the observation that this up-regulation was consistently observed in poorly differentiated pathological grade G3/G4 than in G1/G2 HCC tissue. This is different from what described by Gupta et al. who reported in 3 specimens of liver cancers that both the mRNA and protein level of ABCG2 were decreased
. The discrepancy suggests that the ABCG2 expression is variable and dependent by the tissue type, even in the single individual due to variability of HCC tissues profile. A relationship between cell differentiation and ABCG2 expression had been previously reported in other cell lines. A high level of functional ABCG2 was detected in undifferentiated human embryonic cells and decreased during cellular differentiation
. In hematopoietic system, the ABCG2 expression is confined to the most immature progenitor cells and down-regulated at the committed progenitor level
. Recent data showed that knock-down of ABCG2 inhibited breast cancer and lung cancer cells proliferation, suggesting the role of ABCG2 in maintenance the cancer cells
Our finding reemphasized the importance of intrinsic factors like age, differentiation, and disease status in the expression of drug-transporters in human tissues. Our data expand the previous report showing that ABCG2 protein and mRNA were higher in HCC
 and that in hepatoblastoma patients the transporter was up-regulated following chemotherapy
. For the ABCG2 and pathological grade of cancer, our data in HCC is concordant with those observed in glioma