| Background and aims:Gastric cancer remains one of common diseases worldwide. Surgical resection is the standard treatment for patients with early-stage gastric cancer. However, most of all patients are either diagnosed at an advanced stage when the tumor is inoperable. Chemotherapy is the therapeutical approach for most cancer patients at an advanced stage. Although several randomized control trials have provided evidence that chemotherapy improves survival in these patients, the standard chemotherapeutic regimens often seriously impair the quality of life and cause serious side effects, which pose serious constraints on the use of chemotherapy. However, multidrug resistance (MDR) is a common clinical problem in patients with gastric cancer, which defines that resistance to cytotoxic compounds is associated with cross-resistance to different drugs with or without structural similarity to the primary agent. The mechanisms leading to MDR includes 1) Increased drug efflux or decreased drug uptake; 2) Decreased drug activation or increased drug inactivation; 3) Increased levels or modifications of drug targets; 4) Increased DNA repair; 5) Increased survival and apoptosis inhibition. Effluxing of chemotherapeutic drugs is frequently caused by transmembrane xenobiotic transport molecules belonging to the superfamily of ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and multidrug resistant protein 1 (MRP1). In addition, Vacuolar H+-ATPases (V-H+-ATPases) are ubiquitously expressed not only in vacuolar membranes but also in the plasma membranes of eukaryotic cells. And it plays a critical role in regulating the transmembrane pH gradient, therefore affecting the intracellular and extracellular pH values. The intracellular pH (pHi) is critical for the cytotoxicity of many anticancer agents, and thus Vacuolar H+-ATPases have been implicated in the acquisition of the MDR phenotype. As reported, Vacuolar H+-ATPases is increased in chemoresistant cancer cells and can be induced by chemotherapeuticals. Meanwhile, many cancer cells secrete lysosomal enzymes participating in degradation of the extracellular matrix, facilitating metastatic invasion. Moreover, a low pH extracellular environment also can enhance the activity of these enzymes. Vacuolar H+-ATPases are the primary responsible for the control of tumor microenvironment by proton extrusion to the extracellular medium, which play a crucial role in tumor invasion, metastasis and chemoresistance. Therefore, Vacuolar H+-ATPases may represent a potential target for novel anticancer strategy. However, the Proton pump inhibitors (PPIs) are inevitably mentioned in terms of inhibition of acidification, and have enabled improvement of various acid-related disorders, including gastroesophageal reflux disease, peptic ulcer disease, and nonsteroidal anti-inflammatory drug-induced gastropathy. In addition, PPIs might be one of potential chemosensitizer agents in chemotherapy by inhibiting Vacuolar H+-ATPases so as to reverse MDR of cancer cells. Moreover, tumor microenvironment is characterized by reversed pH gradient with anacidic pHe and an alkaline pHi compared to normal tissues. Tumor acidity may induce a selective accumulation of PPIs in the tumor tissues. By analogy with the gastric compartment, PPIs may be protonated and transformed in the active form in the acidic tumor microenvironment. Therefore, our study aimed to 1) investigate whether Pantoprazole (PPZ) might inhibit the protein expression of Vacuolar H+-ATPases so as to reverse the transmembrane pH gradient, enhancing the chemosensitivity of SGC7901; 2) investigate the optimal time, dosage and the possible mechanism of PPZ; 3) discuss whether PPZ could inhibit the protein expressions of P-gp and MRP1 so as to reverse the MDR of SGC7901/ADR; 4) discuss that PPZ might enhance the cytotoxic effects of ADR on the xenografted tumor of athymic nude mice by in vivo study.Methods:1) Human gastric adenocarcinoma cell line SGC7901 was cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum and antibiotics in a humidified 5%CO2 atmosphere at 37℃. The Adriamycin-resistant cells were cultured in the same medium, with the addition of 0.8μg/ml of adriamycin to maintain the MDR phenotype; 2) Adriamycin (ADR) was used as probe to evaluate drug accumulation and retention in gastric cancer cells so as to calculating ADR releasing index; 3) Annexin V-FITC-PI double staining was used to detect the apoptosis rate of cells; 4) MTT Assay was performed to evaluate the cytotoxicity of anti-tumor drugs with or without PPI pretreatment; 5) BCECF-AM pH-sensitive fluorescent probe was used to measure the pHi value of cells, whereas the pH value of medium measured by the microprocessor pH meter was considered as the extracellular pH (pHe) value; 6) Western blotting and immunofluorescent staining analyses were employed to determine the protein expressions and intracellular distributions of V-H+-ATPases, P-gp and MRP1 before and after PPZ pretreatment; 7) SGC7901 and SGC7901/ADR cells were planted on the athymic nude mice. Then the effects of Adriamycin with or without PPZ pretreatment were compared by determining the tumor size, weight and by calculating the apoptotic index in tumor tissues by TUNEL assay.Results:1) After 24 hr, the expression of Vacuolar H+-ATPases in cells pretreated with PPZ of 10μg/ml (1.19±0.03) or 100μg/ml (0.70±0.03) was significantly lower than that in the control group (1.53±0.05), but this expression was increased by pretreatment with PPZ of 1μg/ml (2.29±0.06, P<0.05); 2) The inhibitory effects of PPZ (10μg/ml) on Vacuolar H+-ATPases were observed at 6 hr (0.32±0.02) and 12 hr (0.13±0.02); 3) And PPZ pretreament for 24 hr (10μg/ml) could alter the intracellular distribution of Vacuolar H+-ATPases; 4) The intracellular pH value in cells pretreated with PPZ of 10μg/ml (7.44±0.09) or 100μg/ml (7.31±0.06) was significantly decreased in comparison with untreated cells (7.5±0.05, P<0.01); 5) After administration of anti-tumor drugs, the viability in SGC7901 cells untreated with PPZ (74.3%±1.77%, P<0.05), while the total and early apoptotic rates in former cells (80.8%±1.16%and 77.5%±1.13%, respectively) were significantly higher than those in later cells (26.4%±1.19%and 23.2%±0.92%, respectively, P<0.01); 6) And the ADR releasing index in cells treated with PPZ (20,50 and 100μg/ml) for 24 hr was obviously lower than that in the blank control (0.164±0.013,0.162±0.015,0.152±0.012 vs 0.277±0.011, respectively, P<0.01). PPZ pretreatment at the dosage of greater than 20μg/ml also could significantly decrease the ADR releasing index of Adriamycin-resistant SGC7901 so as to increase the intracellular concentration of ADR in a dose-dependent manner (P<0.01); 7) Meanwhile, PPZ also could inhibit cell viability of SGC7901 and SGC7901/ADR cells dose-dependently, especially in the 50,80 and 100μg/ml PPZ group (P<0.01); 8) Pretreatment with PPZ for 24 hr could enhance the cytotoxic effects of antitumor agents on SGC7901 and SGC7901/ADR cells; 9) After 24-hr PPZ pretreatment, the resistance index was significantly lower than without PPZ pretreatment (3.71 vs.14.80); 10) PPZ (more than 10μg/ml) also could decrease the pHi value and increase the pHe value of SGC7901 and SGC7901/ADR cells so as to reverse the transmembrane pH gradient, whereas PPZ at the concentration of 1μg/ml could not exert the similar effects; 11) PPZ pretreatment also could significantly inhibit the protein expressions of V-H+-ATPases, P-gp and MRP1, and alter their intracellular distributions in parent and adriamycin-resistant SGC7901 cells (P<0.05); 12) In vivo experiments further confirmed that PPZ pretreament could enhance the antitumor effects of Adriamycin on the xenografted tumor of nude mice and also improve the apoptotic index in the xenografted tumor tissues.Conclusions:PPZ pretreatment could enhances the cytotoxic effects of anti-tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR. Therefore, PPZ might be utilized as a novel chemosensitizer in the future tumor treatment. |
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