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Cell Biology International (2009) 33, 10081019 (Printed in Great Britain)
Effects and mechanisms of proton pump inhibitors as a novel chemosensitizer on human gastric adenocarcinoma (SGC7901) cells
Min Chena1, Xiaoping Zoub*1, Hesheng Luoa**, Jun Caob, Xiaoqi Zhangb, Bin Zhangb and Wenjia Liub
aDepartment of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China
bDepartment of Gastroenterology, The Affiliated Drum Tower Hospital of Nanjing University, Medical School, Nanjing 210008, PR China
Upregulation of proton extrusion is critical for tumor cell survival in an ischemic microenvironment with a lower extracellular pH (pHe). Lower pHe and higher intracellular pH (pHi) benefit cancer cells for invasion and growth. Vacuolar H+-ATPases (V-H+-ATPases) play a critical role in regulating the transmembrane pH gradient. Proton Pump Inhibitors (PPI), mainly treating acid-related diseases, could inhibit the expression of V-H+-ATPases. We have investigated whether PPI decreases the pHi of the human gastric adenocarcinoma cell line, SGC7901, by inhibiting V-H+-ATPases so as to enhance the cytotoxicity of anti-tumor drugs. We have assessed the optimal treatment time, pretreatment dosage of PPI and the possible mechanism of action. PPI exceeding 10
Keywords: Vacuolar H+-ATPases, Tumor acidity, Proton pump inhibitors, Transmembrane pH gradient.
1Min Chen and Xiaoping Zou contributed equally to this work.
*Corresponding author. Tel.: +86 25 88304616 20601; fax: +86 25 83105206.
**Corresponding author. Tel.: +86 13618644197; fax: +86 27 88042292.
A major barrier for the effective treatment of gastric cancer is the phenomenon of Multidrug Resistance (MDR) exhibited by tumor cells (Juranka et al., 1989; Roninson, 1987). A common feature of MDR cells is a net decrease in the intracellular accumulation of drugs (Gottesman and Pastan, 1993). In fact, tumor microenvironment is characterized by reversed pH gradient, with an acidic extracellular pH (pHe) and an alkaline intracellular pH (pHi) (pHe
Vacuolar H+-ATPases (V-H+-ATPases), specific proton pumps of the cell, have an important role in maintaining a relatively neutral pHi, an acidic luminal pH, and an acidic pHe, through pumping protons into extracellular environment or lumen of some membrane-bound organelles (Nishi and Forgac, 2002). V-H+-ATPases are overexpressed in many types of metastatic cancers and positively correlated to their invasion and metastasis (Sennoune et al., 2004). Some human tumor cells, particularly those selected for MDR, also exhibit enhanced V-H+-ATPases activity (Marquardt and Center, 1991; Martinez-Zaguilan et al., 1999).
Some molecules that inhibit V-H+-ATPases and may reverse the MDR to cytotoxic drugs have been identified (Martinez-Zaguilan et al., 1993). Although their toxicity and poor results in preclinical tests have limited their development as therapeutic agents, recent insight into the mechanism of tumor acidification may provide new strategies that mainly target V-H+-ATPases (Fais et al., 2007). And Proton Pump Inhibitors (PPI) could represent a class of drugs suitable to this purpose (Barrison et al., 2001).
Since the introduction of Omeprazole in 1989, PPI have demonstrated gastric acid suppression superior to that of histamine H
We therefore investigated whether PPI could inhibit the expression of V-H+-ATPases, reverse the transmembrane pH gradient and chemosensitize SGC7901 cells to anti-tumor agents. We sought the optimal administration time, the optimal dose of PPI, and look at possible mechanisms.
2 Materials and methods
2.1 Reagents and drugs
The human gastric adenocarcinoma cell line, SGC7901, was kindly given by the Department of Oncology, Drum Tower Hospital of Nanjing University, Medical School. Nigericin was obtained from Alexia Biochem, USA. Vacuolar H+-ATPase 6V
Pantoprazole sodium salts (Altana Pharma AG D-78467 Konstanz, Germany) were resuspended in normal saline (0.85%) at 1
2.2 Cell line and cell culture
SGC7901 cells were cultured in RPMI-1640 (Hyclone, USA) supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials, China) and antibiotics (100
2.3 Western blot analysis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of V-H+-ATPase 6V
2.4 Immunofluorescence staining analysis
Dispersed single cells (2
2.5 Cytotoxic assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, MTT assay)
The cytotoxicity of PPI and/or anti-tumor drugs was determined by MTT assay (Wang et al., 2008). Cells (1
2.6 Annexin V-FITC apoptosis detection
Apoptosis detection in untreated and pretreated cells was performed by Annexin V-FITC and Propidium Iodide (PI) double staining Apoptosis Detection Kit by flow cytometry (BD Biosciences, USA), as previously described by Pfaffel-Schubart et al. (2008). Briefly, the cells were trypsinised, washed with PBS, centrifuged and resuspended with Annexin-binding buffer (500
2.7 Measurements of intracellular and extracellular pH values
pH standard buffer solutions A and B were prepared as described in Table 1, and the pH value of solution A was regulated to 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4 and 7.6. The pH value of solution B was regulated to 7.4. The 2 solutions were stored at 4
The pH standard buffer solution prescription.
The pHi value was measured in the monolayers using the pH-sensitive fluorescent probe 2′, 7′-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF) as previously described (Xia et al., 1999). The first step was to establish a standard curve. Cells were cultured for 24
The pHe values of culture medium at 0
2.8 Intracellular Adriamycin (ADR) concentration analysis
Fluorescence intensity of intracellular ADR was determined by flow cytometry. The wavelengths of excitation light and emission light were 488
2.9 Statistical analysis
The data were expressed as mean
3.1 Protein expression of V-H+-ATPases by western blot analysis
The expression of V-H+-ATPases in SGC7901 cells was examined by western blot analysis. After 24
Effects of PPI pretreatment with different concentrations on Vacuolar-H+-ATPases expression of SGC7901.*P<
Effects of PPI pretreatment on Vacuolar-H+-ATPase expression of SGC7901 at different time points. *
3.2 Intracellular distribution of V-H+-ATPases via immunofluorescence analysis
As an inhibitor of V-H+-ATPases, PPI pretreatment (10
Effects of PPI pretreatment on the intracellular distributions of Vacuolar-H+-ATPases in SGC7901. A) Intracellular Distribution of Vacuolar-H+-ATPases in SGC7901 before PPI pretreatment (×200). B) Intracellular Distribution of Vacuolar-H+-ATPases in SGC7901 before PPI pretreatment using double staining (×200) DAPI stained for nuclei (DAPI: 0.5
3.3 Effects of anti-tumor drugs with presence or absence of PPI pretreatment on the cell viability using MTT assay
The design and results of combination strategy including PPI and/or anti-tumor drugs are summarized in Fig. 4. The cell viability in the chemo group and other 3 PPI
Comparison of the cell viability of SGC7901 after the treatment of PPI and/or anti-tumor drugs. The PPI group: After 24-h incubation, the PPI solution was added into cells. The chemo group: After 48-hr incubation, 5-Fu and cisplatin were added into cells for 24 h. The PPI+chemo group-1: After 48-h incubation, PPI, 5-Fu and cisplatin were simultaneously added into cells for 24 h. The PPI+chemo group-2: PPI was added into cells after 36-hr culturing and then at 48 h 5-Fu and cisplatin were added for 24 h. The PPI+chemo group-3: PPI was added into cells after 24-h culturing and then at 48 h 5-Fu and cisplatin were added for 24 h. MTT ASSAY was performed to detect the cell viability at 72 h. Dosage: PPI (10
3.4 Apoptosis detection
A quantitative analysis of the fluorescent signals was performed by Fluorescence Activated Cell Sorting (FACS). The results are summarized in Fig. 5A and B. In PPI
A) Comparison of the apoptosis rate of SGC7901 cells after the treatment of PPI and/or anti-tumor drugs, a) The control group: No treatment, b) The chemo group: 5-Fu
3.5 Measurements of the intracellular and extracellular pH values
The standard curve of fluorescence intensity versus the pHi value of SGC7901 is shown in Fig. 6A. According to regression analysis, the mathematic model was established following the formula: Y
A) Standard curve of fluorescence intensity of BCECF versus the pHi value of SGC7901. B) Comparison of the fluorescent intensity of BCECF in SGC7901 before and after 24-h PPI pretreatment A. the control group (×200) B. the PPI group (×200). The cells with presence or absence of 24–h PPI pretreatment (10 μg/ml) were incubated with the solution B containing BCECF (5 μM) for 1 h to visualize under a fluorescent microscope. C) Changes of the pHi value of SGC7901 after PPI pretreatment with different concentrations at different time points. #
Fig. 7 shows the pHi and pHe values at 0 and 24
A) Comparison of extracellular and intracellular pH values of SGC7901 after 0-h PPI pretreatment with different concentrations. #
A) Comparisons of pH gradients in SGC7901 at 0
Scatterplot of intracellular pH value versus Vacuolar H+-ATPase expression in SGC7901.
3.6 Effects of PPI with various concentrations on intracellular ADR concentration
PPI pretreatment could reduce the ADR-releasing index in SGC7901 (Fig. 10). The ADR-releasing index decreased as the PPI concentration increased. PPI exceeding 20
Changes of ADR-releasing index after 24-h PPI pretreatment with different concentrations in SGC7901. *
Comparison of intracellular ADR fluorescent intensity of SGC7901 before and after 24-h PPI pretreatment. A. the control group (×200) B. the PPI group (×200). The cells with or without 24-h PPI pretreatment (20 μg/ml) were incubated with RPMI-1640 containing ADR (10 μM) for 1 h to visualize under a fluorescent microscope.
Evidence is accumulating that hypoxia and acidity are involved in cancer progression and in the sensitivity of tumors to chemotherapy (Raghunand and Gillies, 2000). Besides, hypoxia and acidity may also contribute to the progression from benign to malignant growth (De Milito and Fais, 2005a). Tumor acidity, in particular, has a role in resistance to chemotherapy (Raghunand et al., 2001), proliferation (Morita et al., 1992) and metastatic behavior (Martinez-Zaguilan et al., 1996).
As to chemoresistance, many anticancer drugs are classified as either weak basic or molecules whose binding to cellular structures is pH dependent. Accumulation of these drugs within tumor cells should be affected by the transmembrane pH gradient (Raghunand et al., 1999). Thus lower pHe and higher pHi perhaps contribute to promoting MDR when drugs which partition across the membrane would be protonated and ironically trapped in the cytosol or acid vesicles in their biologically active form (Simon et al., 1994). Thus agents that disrupt or normalize the pH gradient in tumors may reverse MDR and/or directly inhibit growth (De Milito and Fais, 2005b). In the case of proliferation, the unfavorable environment may favor the selection of tumor cells able to survive in acidic and hypoxic conditions. However, the normal cells are almost unable to survive in the same microenvironment (Morita et al., 1992; Cosse and Michiels, 2008). As to the metastatic behavior, many tumor cells secrete lysosomal enzymes that participate in degradation of the extracellular matrix, which is necessary for metastatic invasion. Because these enzymes have a lower pH optimum, their activity is greatly enhanced by an acidic extracellular environment (Otero-Rey et al., 2008). Therefore, how to inhibit tumor acidity might be the new strategy in future chemotherapy.
V-H+-ATPases is a large, complex enzyme, composed of a cytosolic V
According to our results, PPI exceeding 10
In addition, PPI could induce apoptosis in tumor cells. As indicated by Yeo et al. (2004), PPI selectively induces in vivo and in vitro apoptotic cell death in gastric cancer, suggesting that PPI could be used for selective anticancer effects which may be caused by suppressing ERK phosphorylation. Other research has indicated the potential use of PPI as antineoplastic agents towards human B-cell tumors (De Milito et al., 2007). However, in our research we investigated the role of PPI as a chemosensitizer so that relatively lower dosage (10
We also studied the optimal time of administration of PPI on SGC7901. Firstly, the chemo drugs combined with PPI pretreatment were more effective than administration of chemo drugs or PPI alone, indicating their synergistic effects. Then among the PPI
Another interesting finding was that simultaneous administration of PPI and the chemo drugs was obviously less effective than administration of anti-tumor drugs after 24
In detecting apoptosis, PPI (10
We also confirmed that the ADR-releasing index obviously decreased when the PPI concentration was more than 20
In conclusion, PPI inhibits the expression of V-H+-ATPases, and reverses the transmembrane pH gradient so as to sensitize the SGC7901 cells to the anti-tumor drugs. Overexpression of V-H+-ATPases might play a crucial role in MDR of gastric cancer so that downregulation of V-H+-ATPases expression by PPI could facilitate the chemosensitivity of SGC7901 to the anti-tumor drugs. However, the possible relevant signaling pathway of PPI involved in enhancing the chemosensitivity needs further research. Further detailed investigation should also be performed to investigate the relationship between inhibition of V-H+-ATPases and reversal of MDR.
Special thanks to Yong Liu and Junhao Chen for their technical assistances in flow cytometry. We also thank Li Wang for collecting materials and references.
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Received 28 December 2008/9 April 2009; accepted 7 May 2009doi:10.1016/j.cellbi.2009.05.004