Exploration On The Relationship Between ABCG2 And Drug Resistance In Renal Cell Carcinoma

Background and Purposes:Renal Cell Carcinoma(RCC) is one of the most common malignant tumor in urology system with the high morbidity, ranked 2nd place. Patients diagnosed with RCC in the first encounter often show a high prevalence(30%) of metastases and patients will suffer from the recurrence and/or metastases even after receiving the surgery. RCC is kind of solid tumor which approved to be insensitive to radiotherapy and chemotherapy and the etiology and pathologic mechanism is still unclear. Recently, with intervention of the target therapy, the efficiency is promising for the advanced/metastatic RCC patients and the disease control rate is about 80%. However, a part of patients still have to face to the recurrence and disease progress even administered with target therapy and finally they decease with short term survival. The TKI(Tyrosine Kinase Inhibitors)Sunitinib malate(SU11248; Sutent) is an orally active, small molecule ATP-competitive multi-targeted inhibitor of vascular endothelial growth factor receptors type 1 and 2, the platelet-derived growth factor receptors a and b, the stem cell factor receptor c-KIT, FMS-like TK-3receptor and the glial cell-line derived neurotrophic factor receptor. Sunitinib is approved by the Food and Drug Administration for the treatment of advanced or metastatic RCC.The failure of tumor treatment is mostly considered to be due to the multidrug resistance(MDR). Therefore, researchers are focusing on the topic about how to reversethe MDR and improve the sensitivity of tumor cell to the drug and surmount the RCC in the future.ABCG2 was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone by extruding the compound out of the cell. ABCG2 is the second member of the G subfamily of the large ATP-binding cassette(ABC) transporter superfamily, which is located at the cell membrane. ABCG2 is a semi-transport protein in nature and is capable of transporting variable drugs as “drug pump”, playing an important role in the drug accumulation in the cell. Sunitinib is also the substrate of ABCG2. It was reported that high expression of ABCG2 protein in RCC cell was associated with the survival of RCC patients and may participate, to some extent, in the formation of multidrug resistance in RCC, so it can be regarded as a reliable factor of multidrug resistance to guide the RCC treatment in clinic.Fumitremorgin C(FTC) has been approved to be the ABCG2-specific inhibitor.Firstly, in our study, we try to explore the expression of ABCG2 in renal cell carcinoma in sporadic RCC patients and then to study the treatment efficiency by inhibiting the ABCG2 function with FTC in vitro and in vivo.Materials and Methods:1 120 cases of RCC specimens confirmed as clear cell carcinoma in histology were collected and 10 cases of non-tumor renal hydronephrosis specimens were reviewed as control group. Immunohistochemical stained standard procedure was used to detect the expressions of ABCG2. The relationship between ABCG2 expression and clinicopathological factors was analyzed by SSPS 20.0 software.2 Western Blot method was used to investigate the expression of ABCG2 protein and three lines of RCC with high expression of ABCG2 was sorted. Sunitinib IC50(half maximal inhibitory concentration) was detected by microplate reader method. Growth inhibitive effect of ABCG2 in combination with FTC and Sunitinib on the selective 3RCC cell lines was detected by CCK-8 assay. Effect of cell apoptosis was detected by flow cytometry(FCM). Effects of the accumulation and efflux rate of Sunitinib in cell lines was also detected by FCM. Cell proliferation was observed and compared in the cell lines with and without FTC intervention by the Clone forming test. The migration of RCC cell lines with and without FTC intervention was detected by transwell assay.3 A total of 18 786-O renal cancer-bearing nude male mice(BLAB/c nu), 5 week-old,were divided into three groups randomly, as follows: control group(C group),Sunitinib group(S group)and Sunitinib+FTC(F group). Xenografts models of 786-O human RCC were established by cell suspension inoculation. The mice were treated with the designed process of management: mice in S group and F group were given Sunitinib through intragastric administration,5 days on and 2 days off every week, lasting for 2weeks, with the drug concentration of 100 mg/kg. On the 15 th day of the experiment,the mice of F group received the FTC through the intratumoral injection, with the FTC dosage of 20 nmol. On the 37 th day, all mice were sacrificed. The tumors were ablated and the volume and weight of tumor were calculated.Results:1 The ABCG2 positive expression rate in 120 samples of sporadic RCC was 52.5% and those in hydronephrosis tissue were 0%, respectively(P=0.001). Microscopically ABCG2 was mostly located at the cell membrane, pale brown in color, stripped shape.ABCG2 expression in tumor tissue was correlated with metastasis and 5-year overall survival(P <0.05), but not with gender, age, tumor size, lymph node metastasis and Fuhrman grade(P>0.05). The rate of 5-year survival was 80.8% in 120 RCC patients.According to the subgroup analysis, the rate of 5-year survival was 94.7% in the ABCG2(-) group and was 68.3% in the ABCG2(+~+++), respectively(P<0.001).The overall survival(OS) in the ABCG2(-) group and ABCG2(+ ~ +++) was86.3±22.5 months and 61.4±33.0 months, respectively, P<0.001.2 There was relative high expression of ABCG2 protein in three lines of RCC(786-O、ACHN、OS-RC-2) which was sorted for further test. Sunitinib IC50 was about 2 μM detected by microplate reader method. The average growth inhibitive rate in combination with FTC and Sunitinib on the above selective 3 RCC cell lines was63.8%、46.3%、45.3% detected by CCK-8 assay. By comparison between combination group and Sunitinib group, the average growth inhibitive rate was significantly different among 786-O and OS-RC-2 lines(P<0.05). The average rate of cell apoptosis was 13.5%、25.8%、17.4% detected by FCM. By comparison between combination group and Sunitinib group, the average rate of cell apoptosis was significantly different among three RCC lines(P<0.05). After intervention of FTC the average rate of Sunitinib accumulation in cell was 142.3%、151.7%、148.0% among three RCC lines respectively and the average rate of Sunitinib efflux which was detected by FCM was 74.9%、61.5%、69.7%, respectively. There was significantly difference in Sunitinib accumulation and efflux comparison with control group without intervention of FTC, P<0.05. In the FTC intervention group, the clone of cell lines was significantly decreased comparison with control group without FTC intervention(P<0.05). We found that there was no significant change in the migration of RCC cell lines with and without FTC intervention(P>0.05).3 The 786-O renal cancer-bearing nude male mice(BLAB/c nu) was successfully established. The mice were treated with the designed process of management. The mice in S group and F group were given Sunitinib through intragastric administration.On the 15 th day of the experiment, the mice of F group received the FTC through the intratumoral injection. On the 37 th day, all mice sacrificed. The volume of tumor was320.3mm3, 582.0mm3 and 825.3mm3 in F group, S group and C group, respectively(P<0.01). The weight of tumor was 249.5mg, 339.7mg and 421.3mg in F group, S group and C group, respectively(P<0.01). The inhibiting rate(IR) base on tumor weight was 26.6% comparison between the F group and S group.Conclusions:1 The positive expression of ABCG2 exists in the sporadic RCC(52.5%). ABCG2 may play an important role in the metastasis and overall survival of RCC patients(P<0.05).2 FTC(ABCG2-specific inhibitor) in combination with Sunitinib has the function of inhibiting the growth and inducing the apoptosis in the 786-O, ACHN and OS-RC-2RCC cell lines in vitro, however has less effect on the migration of cancer cell. We deduce that FTC enhances the drug concentration of Sunitinib in cancer cell by inhibiting the “drug-pump” function of ABCG2. In this way, Sunitinib could kill and suppress cancer cell easily with high concentration.3 Sunitinib can inhibit tumor growth in the 786-O renal cancer-bearing nude male mice.FTC, as ABCG2-specific inhibitor, could enhance this effect and inhibit cancer cell proliferation.4 This research is intended to improve targeted drug sensitivity and overcome drug targeted therapy for RCC in clinical treatment, which provides preliminary experimental basis.