Reversal Effects of Pantoprazole on Multidrug Resistance in Human Gastric Adenocarcinoma Cells by Down-Regulating the V-ATPases/mTOR/HIF-1 alpha/P-gp and MRP1 Signaling Pathway In Vitro and In Vivo

• Type of Material: Article
• Authors: Min Chen, Nanjing University; Shu-Ling Huang, Nanjing University; Xiao-Qi Zhang, Nanjing University; Bin Zhang, Nanjing University; Hao Zhu, Nanjing University; Vincent W. Yang, Emory University; Xiao-Ping Zou, Nanjing University
• Language: English
• Date: 2012-07-01
• Publisher: Wiley: 12 months
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2012 Wiley Periodicals, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1002/jcb.24122
• Title of Journal or Parent Work: Journal of Cellular Biochemistry
• ISSN: 0730-2312
• Volume: 113
• Issue: 7
• Start Page: 2474
• End Page: 2487
• Grant/Funding Information: This work was granted by National Science Foundation Grant no. 81071816 and no. 81101814 and simultaneously supported by the fundamental research funds for the central universities no. 021414340018.
• Keywords: Biochemistry & Molecular Biology; APOPTOSIS; CANCER; PROTON PUMP INHIBITORS; CYTOTOXICITY; TRANSMEMBRANE pH GRADIENT; PANTOPRAZOLE SODIUM; TUMOR-CELLS; P-GLYCOPROTEIN; MULTIDRUG RESISTANCE; Cell Biology; Life Sciences & Biomedicine; VACUOLAR H+-ATPases; VACUOLAR (H+)-ATPASES; HL-60 CELLS; Science & Technology; MECHANISMS; PROTEIN MRP1; ATP-BINDING CASSETTE TRANSPORTER SUPERFAMILY
• Research Categories: Chemistry, Biochemistry; Health Sciences, Oncology

Abstract

To investigate reversal effects of pantoprazole (PPZ) on multidrug resistance (MDR) in human gastric adenocarcinoma cells in vivo and in vitro. Human gastric adenocarcinoma cell SGC7901 was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5% CO2 atmosphere at 37°C. Adriamycin (ADR)-resistant cells were cultured with addition of 0.8μg/ml of ADR maintaining MDR phenotype. ADR was used to calculate ADR releasing index; CCK-8 Assay was performed to evaluate the cytotoxicity of anti-tumor drugs; BCECF-AM pH-sensitive fluorescent probe was used to measure intracellular pH (pHi) value of cells, whereas pH value of medium was considered as extracellular pH (pHe) value; Western blotting and immunofluorescent staining analyses were employed to determine protein expressions and intracellular distributions of vacuolar H+-ATPases (V-ATPases), mTOR, HIF-1α, P-glycoprotein (P-gp), and multidrug resistant protein 1 (MRP1); SGC7901 and SGC7901/ADR cells were inoculated in athymic nude mice. Thereafter, effects of ADR with or without PPZ pretreatment were compared by determining the tumor size and weight, apoptotic cells in tumor tissues were detected by TUNEL assay. At concentrations greater than 20μg/ml, PPZ pretreatment reduced ADR releasing index and significantly enhanced intracellular ADR concentration of SGC7901 (P<0.01). Similarly, PPZ pretreatment significantly decreased ADR releasing index of SGC7901/ADR dose-dependently (P<0.01). PPZ pretreatment also decreased cell viabilities of SGG7901 and SGC7901/ADR dose-dependently. After 24-h PPZ pretreatment, administration of chemotherapeutic agents demonstrated maximal cytotoxic effects on SGC7901 and SGC7901/ADR cells (P<0.05). The resistance index in PPZ pretreatment group was significantly lower than that in non-PPZ pretreatment group (3.71 vs. 14.80). PPZ at concentration >10μg/ml significantly decreased pHi in SGC7901 and SGC7901/ADR cells and diminished or reversed transmembrane pH gradient (P<0.05). PPZ pretreatment also significantly inhibited protein expressions of V-ATPases, mTOR, HIF-1α, P-gp, and MRP1, and alter intracellular expressions in parent and ADR-resistant cells (P<0.05). In vivo experiments further confirmed that PPZ pretreatment could enhance anti-tumor effects of ADR on xenografted tumor of nude mice and also improve the apoptotic index in xenografted tumor tissues. PPZ pretreatment enhances the cytotoxic effects of anti-tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR by downregulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway.