Expression Of Drug Resistance Gene ABCG2 In Esophageal Carcinoma And Resistance-reversal Effect Of Artesunate

Objectives:Esophageal carcinoma is one of the most common malignant gastrointestinal tumors. The incidence of esophageal carcinoma is high in China, which is the second in gastrointestinal cancers, and it is a great threaten to human health. In the present, chemotherapy is one of the esophageal carcinoma combined modality therapies, but the mutidrug resistance (MDR) in the course of chemotherapy is a major barrier to the success of chemotherapy, especially to recurrent tumors. Multidrug resistance is a kind of resistance of cancer cells to multiple classes of chemotherapic drugs that can be structurally and mechanistically unrelated. When tumor cells develop drug resistance, they become resistant not only to the treated drug, but also to a variety of structurally and functionally unrelated drugs. Because the therapeutic effect is determined by multidrug resistance, multidrug resistance is generally studied lately. There is accumulating evidence that active export of anticancer drugs from cells is one of the major mechanisms of mutidrug resistance. Tumor cells often gain drug resistance through the overexpression of membrane trasport proteins that effectively efflux anticancer drugs. It has been convincingly documented that several ATP-dependent drug transporters can cause drug resistance in cancer cells by actively extruding the clinically administered chemotherapeutic drugs. Increased transmembrane efflux of antitumor drugs is one of the best-characterized mechanisms of MDR and is mediated through the overexpression of ATP-binding cassette (ABC) transporter superfamily members. By far the well-known major drug transporters, i.e., ABCB1 (P-glycoprotein or MDR1) and ABCG2 (BCRP/MXR/ABCP), have been characterized in details with respect to their structure and function. These drug trasporters belong to the human ABC transporter gene family that consists of 48 gene members at least. The best-characerized mechanism of MDR involves P-gp. P-gp overexpression in tumor cells results in broad resistance to a variety of anticancer drugs with different chemical structures and mechanism of action, so we call it to"typical multidrug resistance". Recently, it's reported that overexpression of ABCG2 confered drug resistance upon malignant cells to various chemotherapeutic drugs. Expression of ABCG2 in various tumors was high and participated in the development of multidrug resistance.In our study, the gene and protein expression of ABCB1 and ABCG2 was detected by various experimental methods, to study the relationship between the expression of ABCB1, ABCG2 and the resistance of esophageal carcinoma. Multidrug resistance cell Eca109/ADM induced by ADM was established. Expression of ABCB1 and ABCG2 in Eca109/ADM was studied. In order to study the relationship between the ABCG2 and esophageal carcinoma MDR, the ABCG2 gene was transfected into Eca109 cells.Artesunate is a remarkable antimalarial agent, espacially to severe and drug-resistant cases. In the present investigation, artesunate is not only an antimalarial agent but also an anticancer agent. Artesunate could inhibit the growth of varietal tumor cells. We have studied that artesunate could inhibit the growth of esophageal cancer cells. Artesunate could also have anticancer effect to drug-resistant cells which hint that artesunate could reverse the drug resistance of cancer cells. Artesunate has low adverse effect, so it can be developed to multidrug resistance reversing drugs. In our research, the drug reversal effect of artesunate to esophageal carcinoma was studied in cellular and animal levels.Therefore, the relationship between ABCB1, ABCG2 expression in esophageal carcinoma and the esophageal carcinoma multidrug resistance, and drug reversal effect of artesunate were investigated in this study, which could be beneficial to the chemotherapy of esophageal carcinaoma in clinic. Methods:1 Expression of ABCB1 and ABCG2 in esophageal carcinoma and related biology significance Esophageal carcinoma tissues, paired adjacent mucosa (2~5cm from margin of esophageal carcinoma), and paired normal mucosa (at least 5cm from margin of esophageal carcinoma) were obtained from 150 resected surgical specimens of esophageal squamous cell carcinoma (ESCC). All the specimens were verified by pathologic diagnosis, 80 cases of esophageal squamous cell carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were selected from 150 specimens. 80 ESCC tissues included well and moderately differentiated ESCC(n=59), poorly differentiated ESCC(n=21) ; fibrous membrane invasion(n=54), fibrous membrane untouched(n=26); lymph node metastasis positive(n=27), lymph node metastasis negative(n=53). ABCB1, ABCG2 gene and protein expression in 80 resected surgical specimens of esophageal carcinoma, dysplasia of esophageal squamous epithelium and normal esophageal mucosa were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry(IHC) and flow cytometry(FCM). Relationship between their expressions and clinical pathological features was analyzed.2 Expression of ABCG2 in adriamycin-resistant human esophageal cancer cellsDrug-resistant cells of esophageal cancer was established by gradually increasing the ADM concentration in the culture medium of human esophageal cancer cell line (Eca109) from 0.002μg/ml ADM to 0.02μg/ml ADM for 8 months. The drug-resistant cell was named Eca109/ADM cell.Cell inhibitory rate of ADM on the growth of Eca109 and Eca109/ADM cells was investigated by MTT and IC50, resistance index (RI) (RI=Eca109/ADM IC50/ Eca109 IC50) was calculated. ABCG2 gene and protein expression in Eca109 and Eca109/ADM cells were investigated by RT-PCR, Western-blot as well as FCM. ABCB1 protein was detected by FCM. Location and expression of ABCG2 in Eca109/ADM cells were investigated by Laser scan confocal microscope (LSCM). Drug excretion of Eca109/ADM cells was detected by FCM. The apoptosis, ABCB1 and ABCG2 expression of Eca109, Eca109/ADM cells after treatment of Art and ADM were investigated by FCM.3 Establishment of Eca109/ABCG2, a drug resistant esophageal cancer cell line, and its biological profileWe clone the whole length of ABCG2 gene from gene bank and ligate the whole length of ABCG2 gene to the PCDNA3.1 vector to construct the custom-crafted plasmid PCDNA3.1-ABCG2. The positive clones PCDNA3.1-ABCG2 was transfected to Eca109 cells and positive cell clones were selected with G418 after transfected 72h. Eca109 cell transfected with PCDNA3.1 as control group. Eca109 cell transfected with PCDNA3.1-ABCG2 and PCDNA3.1 was named Eca109/ABCG2 and Eca109/PCDNA3.1 cell respectively.Transfected rate was investigated by FCM and the inhibitory effect of ADM, DNR and MIT on the growth of Eca109/ABCG2, Eca109/PCDNA3.1 as well as Eca109 cells was detected by MTT. The content of ADM in cells after treatment of ADM was investigated by FCM. Gene and protein expression of ABCG2 were investigated by RT-PCR, FCM as well as Western-blot. Location of ABCG2 in cells was measured by immunocytochemistry (ICH).4 The ADM resistance reversal effect of Art on Eca109/ABCG2 cells and related mechanismThe inhibitory effect of Art, ADM and Art+ADM on the growth of Eca109/ABCG2 cells was investigated by MTT. Apoptosis of Eca109/ABCG2 cells and the content of ADM in Eca109/ABCG2 cells after treatment of Art, ADM as well as Art+ADM were detected by FCM. Gene and protein expression of ABCG2 in Eca109/ABCG2 cells after Art, ADM and Art+ADM interference were detected by RT-PCR and FCM.5 The resistance-reversal effect of Art on human esophageal cacer transplanted in nude mice60 (30 of male and 30 of female) nude mice (BALB/c, nu/nu, 4 wk old, 17~20g) were randomly divided into 10 groups (6 in each group) by weight. Each of nude mice in 7 groups was inoculated with 200μl Eca109/ABCG2 cells (6×106 each mouse) subcutaneously on the left subscapularis. Each of nude mice in 3 groups was inoculated with 200μl Eca109 cells (6×106 each mouse) subcutaneously on the left subscapularis. On the seventh day after inoculation, all of the nude mice were formed tumor, starting to use drugs.Modality and time of drug injection: Art, peritoneal injection per a day for 2 courses, each course contained 7 days and there was a seven-day interval between two courses. ADM was treated with peritoneal injection per three days for 7 times.Nude mice inoculated with Eca109/ABCG2 cells were divided into 7 groups as follows: 2 groups received Art (25, 50mg/kg/d, respectively). 2 groups received ADM (1, 4mg/kg/3d, respectively). 2 groups received ADM (1mg/kg/3d) + Art (25, 50mg/kg/d) respectively. The negative control group received saline.Nude mice inoculated with Eca109 cells were divided into 3 groups as follows: the experimental groups received ADM (1, 4mg/kg/3d, respectively). The negative control group received saline.The weight and the shortest, longest diameters of the tumors were measured with vernier caliper each 2 days.Mice were sacrificed after experiments course and all tumor tissues were weighed. Cell apoptosis, the ADM content and ABCG2 protein of tumor cells were examined by FCM. Expression and location of ABCG2 protein of tumor cells were detected by IHC. ABCG2 mRNA expression of tumor cells was investigated by RT-PCR.Results:1 Expression of ABCB1 and ABCG2 in esophageal carcinoma and related biology significanceRT-PCR and FCM results show: mRNA and protein expression of ABCB1, ABCG2 in esophageal cancer tissues were significantly higher than normal tissues (P<0.01). In esophageal cancer tissues, mRNA and protein expression of ABCB1, ABCG2 were no difference between age and gender (P>0.05), but the expression of ABCB1, ABCG2 in poorly differentiated, fibrous membrane invasion and lymph node metastasis positive ESCC were significantly higher than the expression in well and moderately differentiated, fibrous membrane untouched and lymph node metastasis negative (P<0.05). There was no significant association between ABCG2 mRNA, protein expression and ABCB1 mRNA, protein expression (rs =0.077,P=0.499; rs =-0.087,P=0.444, respectively).IHC results show: ABCB1 and ABCG2 positive protein were located in cell membrane and cytolymph as buffy granular and diffused distribution. The positive rates of ABCB1 and ABCG2 protein were significantly higher in ESCC (77.50% and 86.25%) than those in squamous dysplasia (31.25% and 26.25%) and normal squamous epithelium (6.25% and 3.75%) (P<0.01).2 Expression of ABCG2 in adriamycin-resistant human esophageal cancer cellsDrug resistant cell Eca109/ADM was established successfully for 8 monthes. Comparing Eca109/ADM cell with Eca109 cell, Eca109/ADM cell became bigger volume and more irregularity morphology. The IC50 of Eca109/ADM and Eca109 cells to ADM was 15.45±1.15,4.69±0.88 after the treatment of ADM for 24h. The RI of Eca109/ADM was 3.29.ABCG2 mRNA and protein expression of Eca109/ADM cells were significantly higher than Eca109 cells (P<0.05). ABCG2 protein of Eca109/ADM was located in cell membrane and cytolymph and the protein expression was higher than Eca109 cells. ABCB1 protein expression of Eca109/ADM cells was significantly higher than the expression of Eca109 cells (P<0.05). Drug excretion of Eca109/ADM cells was higher than Eca109 cells.The cell apoptosis rate of Art+ADM group on Eca109/ADM cells was significantly higher than ADM group (P<0.05). ABCG2 protein expression of Eca109/ADM cells was significantly decreased after the treatment of Art for 48h (P<0.05), but ABCB1 protein expression of Eca109/ADM cells was no difference (P>0.05).3 Establishment of Eca109/ABCG2, a drug resistant esophageal cancer cell line, and its biological profileEca109/ABCG2 and Eca109/PCDNA3.1 cell were established successfully. The IC50 of Eca109/ABCG2, Eca109/PCDNA3.1 and Eca109 cells was 18.61±3.94, 4.18±0.14, 4.69±0.88 after treatment of ADM for 24h. The RI of Eca109/ABCG2 was 3.97. The IC50 of Eca109/ABCG2 cells was significantly higher compared with Eca109 cells (P<0.01). The IC50 of Eca109/ PCDNA3.1 cells was no difference compared with Eca109 cells (P>0.05). The IC50 of Eca109/ABCG2 cells to DNR and MIT was higher than Eca109 cells, The RI was 3.50 and 3.15 respectively.Drug excretion of Eca109/ABCG2 cells was significantly higher than Eca109/ PCDNA3.1 and Eca109 cells (P<0.01).ABCG2 mRNA and protein expression of Eca109/ABCG2 cells was significantly higher than Eca109/PCDNA3.1 and Eca109 cells (P<0.05). ICC results show: ABCG2 protein of Eca109/ABCG2 was located in cell membrane and cytolymph, and the protein expression was higher than the expression of Eca109/PCDNA3.1 and Eca109 cells.4 The ADM resistance reversal effect of Art on Eca109/ABCG2 cell and related mechanism.MTT results show: The inhibitory effect of Art+ADM on the growth of Eca109/ABCG2 cells was significantly higer than Art or ADM (P<0.05). The Eca109/ABCG2 cells apoptosis rate of Art+ADM group was significantly higher than Art or ADM group (P<0.05). ABCG2 mRNA and protein expression of Eca109/ABCG2 cells after the treatment of Art+ADM were significantly lower than ADM (P<0.05).5 The resistance-reversal effect of Art on human esophageal cancer transplanted in nude miceThe model of esophageal cancer xenograft in nude mice was successfully established, the tumorigenic rate was 100%. In the same ADM concentration group, volume and weight of transplanted tumor inoculated with Eca109/ABCG2 cells was significantly higher than transplanted tumor inoculated with Eca109 cells (P<0.05). In the transplanted tumor inoculated with Eca109/ABCG2 cells groups, tumor volume and weight of Art + ADM group was significantly lower than Art or ADM group (P<0.05), and ABCG2 mRNA, protein expression in tumor cells of Art + ADM group were significantly lower than ADM group (P<0.05).Conclusions:1 In the process of carcinogenesis of the esophagus, the expression of ABCB1 and ABCG2 in ESCC were significantly higher than those in squamous dysplasia and normal squamous epithelium. The expression of ABCB1 and ABCG2 in squamous dysplasia were gradually high from gradeⅠto gradeⅢ. High expression of ABCB1 and ABCG2 might be correlated with tumor progression and drug resistance. There was no correlation between ABCB1 expression and ABCG2 expression in ESCC, which suggest that the drug resistance induced by ABCB1 or ABCG2 might have different mechanism. ABCG2 might be new therapy target of esophageal carcinoma drug resistance.2 Drug resistant cell Eca109/ADM was established successfully by gradually increasing the ADM concentration in the culture medium of human esophageal cancer cell line (Eca109). ABCB1 and ABCG2 expression were higher in Eca109/ADM cells than those in Eca109 cells, which suggested that ABCB1 and ABCG2 might participate in the drug resistance of Eca109/ADM cells. Artesunate could significantly increase the inhibitory effect of ADM on the growth of Eca109/ADM cells and decrease the ABCG2 expression of Eca109/ADM cells but not ABCB1, which suggested that artesunate could reverse the drug resistance of Eca109/ADM cells by decreasing ABCG2 expression of Eca109/ADM cells.3 For the first time, multidrug resistance cell (Eca109/ABCG2) of esophageal cancer was successfully established by transfection of liposomes. Eca109/ABCG2 cell possessed ABCG2-resistant biological profile and could stably express the ABCG2 protein, so the Eca109/ABCG2 cell was ideally multidrug resistant cell model which had ABCG2 biological profile.4 For the first time, resistance-reversal effect of artesunate on esophageal cancer was explored in our study. The cell growth inhibitory effect of ADM on Eca109/ABCG2 cells was significantly increased by combining Art and ADM. Art could significantly reduce the ABCG2 expression of Eca109/ABCG2 cells which could inhibit the drugs excretion and increase the ADM content in Eca109/ABCG2 cells, so artesunate could reverse the ADM drug resistance of Eca109/ABCG2 cells.5 The model of esophageal cancer xenograft in nude mice was successfully established, which could provide an ideal model for the research of esophageal cancer drug-resistance. The growth inhibitory effect of ADM on esophageal cancer xenograft in vivo was enhanced by combining artesunate without causing obvious side effects in treated mice, which coule provide some experimental reference for artesunate as resistance-reversal agent. Artesunate could become high-performance and harmfulless resistance-reversal agent in clinic.