Role Of ABCC2 In Cisplatin-resistance For Nasopharyngeal Carcinoma

BACKGROUND & OBJECTIVENasopharyngeal carcinoma (NPC) is a common malignant epithelial tumor in Southern China. Because the primary anatomical site of tumor growth is located in a cryptic area, with slightly early symptoms, the NPC patients tend to present a more advanced stage of disease in clinic with higher metastatic potential, which makes chemotherapy a necessary treatment for these NPC patients. But multidrug-resistance (MDR) results in the failure of chemotherapy. It's important to elucidate the mechanism of MDR.Of all the chemotherapy drugs, cisplatin (DDP) is the most commonly used one which is more efficient than other drugs. The mechanism of resistance to DDP is very complex. Among the mechanisms, the most commonly focused one is dealing with adenosine triphosphate binding cassette (ABC) transporter families which is located on the cell membrane and supposed to exclude the cytotoxic drugs out of the cells.There are 48 genes in ABC transporter families, and is divided into 7 subfamilies according to the transmembrane region. Among these genes, ten genes are reported to be correlated with multidrug resistance, including ABCA2, ABCB1 (also named as MDR1 or P-gp), ABCC1 (also named as MRP1), ABCC2 (also named as MRP2 or cMOAT), ABCC3, ABCC4, ABCC5, ABCC6, ABCC11, and ABCG2. Of all the ABC transporters, ABCC2 was identified to confer cellular resistance of tumor cells to various anticancer drugs including DDP. It has been found that accompanied by the reduction of ABCC2 mRNA, some tumor cells resistant to DDP showed resistance level against DDP decreased, while cells with overexpressed ABCC2 mRNA showed resistance to DDP enhanced. Interestingly, ABCC2 was found to be located not only in cellular membrane, but also in the nuclear membrane. Giving that DNA is the primary target of DDP, these findings strongly indicate that there is a close relationship between ABCC2 and DDP. But up till now, no evidence has shown a relationship between ABCC2 and DDP in NPC. In this research, the relationship between ABCC2 and DDP resistance in NPC was to be studied in order to provide guidlines for chemotherapeutic regime in NPC patients.METHODS1. Paraffin-embedded NPC specimens with clinical data were collected, and divided into sensitive or resistant group according to the DDP treatment effect. Immunohistochemistry (IHC) method was used to detect the ABCC2 expression. The relationship between ABCC2 expression and sex, age, tumor stage, metastasis and chemotherapeutic effect were analyzed. According to the chemotherapy design, these cases were divided into 4 groups: PF (DDP+5-Fu), PFB (DDP+5-Fu+ bleomycin), PFD (DDP+5-Fu+doctacxol) and PV (DDP+Vincristine), and the relationship between ABCC2 expression and chemotherapy design was analyzed.2. CNE2 cell line was induced by DDP, DDP with 5-fluorouracil (5-Fu) individually for one year. All of these cells were used in the following experiments. The ability of cellular proliferation was detected by MTT method, and cell cycle was detected by flow cytometry method. MTT method was used to detect the inhibition rate of DDP in each cell line with or without the action of PSC833, followed by IC50 and resistance index calculation. The modulations of mRNA expression of ten ABC transporters related to drug-resistance in these cells were detected by quantitative real time PCR. Immunocytochemistry (ICC) method was used to detect the protein expression of ABCC2. The modulation of intracellular accumulation of DDP in these cells with or without the action of PSC833 was detected by high performance liquid chromatography (HPLC).3. CNE2 cell line was treated with lentiviral vector containing specific anti-ABCC2 short hairpin RNA. The cell clones stably expressed the shRNA targeting ABCC2 were picked out and expanded. Quantitative real time PCR was used to detect the efficiency of interference of each cell clone, followed by western blot and immunocytochemistry method to detect the modulation of ABCC2 protein. The ability of cellular proliferation was detected by MTT method, and cell cycle was detected by flow cytometry. Inhibition rate of each cell clone proliferation by DDP was detected by MTT method, followed by IC50 calculation. The modulation of intracellular accumulation of DDP in these cells was detected by HPLC. The cell clone with higher sensitivity to DDP was used to transplant subcutaneously in nude mice. ABCC2 expression in tumor tissues was detected by IHC. Relative tumor size and rates of relative tumor growth were calculated and analyzed in statistics.RESULTS1. The expression of ABCC2 in NPC specimens and its clinical significance(1) 84 cases with clinical data were collected. The results of IHC showed that ABCC2 mainly located in cellular membrane and cytoplasm in NPC cases. ABCC2 can also be found to be located in nucleus in a few cases. No correlation was found between ABCC2 expression and sex, age or tumor stage (P>0.05).(2) 17 cases showed high expression of ABCC2 in 50 sensitive group (34%), while in resistant group, 22 cases showed high expression of ABCC2 (65%), which was much higher than in sensitive group with statistically significance (Z=3.71, P<0.01). This result indicates that the expression of ABCC2 is correlated with chemotherapeutic effect. A further study showed that no correlation was found between the expression of ABCC2 and PFB, PFD or NP treatment, but a significant correlation was found between the expression of ABCC2 and PF treatment (Z=1.5, P<0.05).(3) 36 cases showed high expression of ABCC2 in 74 cases of metastasis group (50%), 3 cases showed high expression of ABCC2 in 10 cases of non-metastasis group, which was significantly lower than that in metastasis group (Z=2.05, P <0.01). We also found that in metastases group, ABCC2 expression showed higher expression in resistant group than in sensitive group significantly (Z=3.52, P<0.01) .2. Establishment of resistance cell lines and detection of ABCC2 expression(1) After CNE2 cell line was induced by DDP (named as CNE2/DDP), DDP with 5-fluorouracil (named as CNE2/DDP+5Fu) for one year, we found that compared to CNE2, both CNE2/DDP and CNE2/DDP+5Fu cell lines showed more resistance to DDP in terms of IC50 values, and can be reversed by pSC833 partially. Resistance index (RI) was 2.63 for CNE2/DDP and decreased to 1.63 after reversed by PSC833, while RI was 5.35 for CNE2/DDP+5Fu and decreased to 4.62 after reversed by PSC833, respectively.(2) Both results of growth ability by MTT method and cell cycle by flow cytometry showed no significant difference (P>0.5).(3) The results of quantitative real time PCR showed that in ten ABC transporters, only ABCC2 mRNA were increased in both resistant cells with statistically significance (F=37.24, P<0.05), with 1.73 folds in CNE2/DDP and 3.96 folds in CNE2/DDP+5Fu respectively, compared to CNE2.(4) The results of ICC showed that expression of ABCC2 protein enhanced in resistant cells obviously. Interestingly, ABCC2 is found to be located in nucleus of CNE2/DDP+5Fu but not in CNE2 or CNE2/DDP.(5) The results of HPLC showed that the intracellular accumulation of DDP decreased in resistant cells, with 2.07 folds in CNE2/DDP and 2.82 folds in CNE2/DDP+5Fu respectively, compared to CNE2. After reversed by PSC833, intracellular accumulation of DDP decreased to 1.35 folds for CNE2/DDP and 1.36 for CNE2/DDP+5Fu.3. The targeting ABCC2 effect of RNAi technique on CNE2 cell line(1) The lentiviral vector containing specific anti-ABCC2 shRNA and negative control were transfected into 293FT cell line to produce lentivirus particles, followed by transduced into CNE2 cell line. After selected by blasticidin for 14 days, 12 resistant cell clones were picked out, expanded for about 3 months and analyzed separately. Quantitative real time PCR was used to detect the efficiency of RNAi targeting ABCC2 for each clone. 2 clones with higher efficiency (66.1 %,66.8%) of RNAi were chosen for the following study. Western blot and ICC method were used to detect the modulation of ABCC2 protein. The results showed that ABCC2 expression was similar in both CNE2 and negative control, while both selected clones showed expression of ABCC2 decreased obviously.(2) The results of growth ability by MTT method and cell cycle by flow cytometry showed no significant difference in all of these cells (P>0.5).(3) MTT results showed that both selected clones, compared to CNE2, displayed increased sensitivity to DDP in terms of IC50 values with statistically significant difference (F=4.08, P<0.05), while no significant modulation for negative control was observed. The sensitivity to DDP for both selected clones increased about 3.96 folds and 3.42 folds respectively.(4) The results of HPLC showed that the intracellular accumulation of DDP increased in selected clones with statistically significant difference (F=97.23, P<0.01), with 2.66 folds and 3.11 folds respectively, compared to CNE2, while no significant modulation was found for negative control.(5) The cell clone with higher sensitivity to DDP was used to transplant subcutaneously in nude mice. IHC method was used to detect the ABCC2 expression in tumor tissues, and the results showed that the expression of ABCC2 in the cell clone was decreased significantly compared to CNE2 and negative control. Tumor relative size and rates of tumor relative growth were calculated, and both of the results showed that after treated by DDP, the growth of the cell clone was much slower than other groups including CNE2 and negative control.CONCLUSION1. The expression of ABCC2 was correlated with metastasis and chemotherapy effect, especially with PF treatment, which suggests that we can take the expression of ABCC2 as one of the index to predict the effect of chemotherapy treatment and prognosis.2. Two cell lines resistant to DDP and DDP+5Fu repectively were established successfully, with up-regulated expression of ABCC2, decreased intracellular accumulation of DDP, and more resistant to DDP.3. Tow clones with downregulated expression of ABCC2 were selected by RNAi technique targeting ABCC2. Both clones showed more intracellular accumulation of DDP, and more sensitivity to DDP compared to CNE2 and negative control.4. This study shows that the expression of ABCC2 is correlated with NPC resistant to DDP.