Statins Attenuate Helicobacter pylori CagA Translocation and Reduce Incidence of Gastric Cancer: In Vitro and Population-Based Case-Control Studies

Gastric cancer is the second leading cause of cancer-related death worldwide. The correlation of Helicobacter pylori and the etiology of gastric cancer was substantially certain. Cholesterol-rich microdomains (also called lipid rafts), which provide platforms for signaling, are associated with H. pylori-induced pathogenesis leading to gastric cancer. Patients who have been prescribed statins, inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase, have exhibited a reduced risk of several types of cancer. However, no studies have addressed the effect of statins on H. pylori-associated gastric cancer from the antineoplastic perspective. In this study, we showed that treatment of gastric epithelial cells with simvastatin reduced the level of cellular cholesterol and led to attenuation of translocation and phosphorylation of H. pylori cytotoxin-associated gene A (CagA), which is recognized as a major determinant of gastric cancer development. Additionally, a nationwide case-control study based on data from the Taiwanese National Health Insurance Research Database (NHIRD) was conducted. A population-based case-control study revealed that patients who used simvastatin exhibited a significantly reduced risk of gastric cancer (adjusted odds ratio (OR) = 0.76, 95% confidence interval (CI) = 0.70–0.83). In patients exhibiting H. pylori infection who were prescribed simvastatin, the adjusted OR for gastric cancer was 0.25 (95% CI = 0.12–0.50). Our results combined an in vitro study with a nationwide population analysis reveal that statin use might be a feasible approach to prevent H. pylori-associated gastric cancer.

Introduction purchased from Invitrogen (Carlsbad, CA, USA). Luciferase substrate and β-galactosidase expression vector were purchased from Promega (Madison, MA, USA). Simvastatin, lovastatin, RhoA inhibitor (Y27632) and all other chemicals were of the highest grade commercially available and purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cell and bacterial culture. Human gastric epithelial cells (AGS cells, ATCC CRL 1739) were cultured in F12 (GibcoBRL, NY, USA). MKN45 cells (JCRB0254, RIKEN Cell Bank, Japan) were cultured in Dulbecco's minimum essential medium (HyClone, Logan, UT, USA). TSGH9201 cells were cultured in RPMI1640 medium (Gibco Laboratories, Grand Island, NY, USA). Ten percent de-complemented fetal bovine serum (Hyclone) was added to all cultures. Penicillin and streptomycin (GibcoBRL) were used if necessary. Antibiotics were not added to the cell culture medium in the H. pylori-infected assay. H. pylori 26695 (ATCC 700392) were routinely cultured on Brucella blood agar plates (Becton Dickinson, Franklin Lakes, NJ, USA) containing 10% sheep blood under 5% CO 2 and 10% O 2 conditions at 37°C for 2-3 days.
Analysis of cell viability and cellular cholesterol. Gastric epithelial cells were treated with or without various concentrations of simvastatin (0, 10, 20, and 50 μM) for 1 h. Trypan blue staining was used to measure the effects of statin on cell viability [20]. After treatment with simvastatin, the cells were washed with phosphate-buffered saline (PBS) and disrupted through ultrasonication (three 10-s bursts at room temperature). The cellular cholesterol was measured using an Amplex Red cholesterol assay kit (Molecular Probes) according to the manufacturer's instructions [21].
Analysis of translocated CagA and phophorylated CagA. Immunoprecipitates for analysis of H. pylori CagA translocation and phophorylation were prepared using the relevant techniques [20]. The immunoprecipitates were subjected to 6.5% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Pall, East Hills, NY, USA) for immunoblot analysis. CagA was probed using mouse anti-CagA antibodies (Santa Cruz Biotechnology) and tyrosine-phosphorylated CagA was probed using mouse antiphosphotyrosine antibodies (4G10) (Upstate Biotechnology, Billerica, MA, USA). To ensure equal loading of each prepared sample, β-actin from whole-cell lysates was stained using goat antiactin antibodies (Santa Cruz Biotechnology). The relevant proteins were visualized using enhanced chemiluminescence reagents (GE Healthcare, Buckinghamshire, UK) and were detected by exposure to X-ray film (Kodak, Boca Raton, FL, USA).
Transient transfection of NF-κB reporter gene and luciferase activity assay. AGS cells were grown in 12-well plates for 20 h and transfected with a NF-κB-luc reporter plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) [21]. After a 24-h incubation for transfection, cells were cocultured in the presence of 25 μM simvastatin and thereafter infected with H. pylori at an multiplicities of infection (MOI) of 100 for 6 h. The cells were scraped from dishes and an equal volume of luciferase substrate (Promega) was added to the samples. The luminescence was measured using a microplate luminometer (Biotek, Winooski, VT, USA). Luciferase activity was normalized to transfection efficiency, which was determined using the β-galactosidase activity of a cotransfected β-galactosidase expression vector (Promega).
Measurement of IL-8. AGS cells were treated with 25 μM simvastatin and thereafter infected with H. pylori at an MOI of 100 and incubated for 16 h. The supernatants from cell cultures were collected and the levels of IL-8 were determined using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions [27].
Analysis of H. pylori-induced hummingbird phenotype of human gastric epithelial cells. AGS cells (1 × 10 6 cells) were cultured in 12-well plates at 37°C for 20 h. After one wash with PBS, cells were treated with simvastatin (25 μM), then infected with H. pylori 26695 at an MOI of 100 for 6 h. Elongated cells were defined as cells that were typically elongated and had thin needle-like protrusions > 20 μm long. All samples were cultured in triplicate in 3 independent experiments. The proportion of elongated cells was determined and cells with the hummingbird phenotype were counted [20].

B. Population-Based Case-Control Study
Data source. The National Health Insurance (NHI) program was implemented in 1995 and covers approximately 99% of the Taiwanese population, and has contracts with 97% of all medical providers [28]. The details of NHI program have been comprehensively described in previous reports [29,30]. The database provides diagnostic codes in the format of the International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM). The database encrypts the patients' personal information for privacy protection and provides researchers with anonymous identification numbers associated with the relevant claim information, which includes the patient's sex, date of birth, registry of medical services, and medication prescriptions. Patient consent is not required for accessing the database. This study was approved by the Institutional Review Board of China Medical University (CMU-REC-101-012). Our IRB specifically waived the requirement for consent.
Sampled participants. In this population-based case-control study, patients aged at least 20 years, with recently diagnosed gastric cancer (ICD-9-CM code 151), were selected from the Catastrophic Illnesses Patient Database (CIPD) as the case group during the period 2005-2010. The NHI offers a catastrophic illness program that exempts patients from co-paying for the corresponding medical services, and the CIPD includes cancer patients. The index date for each case was the date of diagnosis of gastric cancer. Control subjects were identified from the Longitudinal Health Insurance Database 2000 (LHID 2000), a database containing the claims data from 1996 to 2011 for 1 million people randomly sampled from 2000 Registry of Beneficiaries of the Taiwan NHI program. For each case, one control patient, frequency-matched for sex, age (in 5-y bands), and year of gastric cancer diagnosis, was selected as the control group. Patients with any other cancer (ICD-9-CM codes 140-208) diagnosed before the index date were excluded. To explore the association between individual statins and gastric-cancer risk, the medication history of two statins commercially available before the index date, simvastatin and lovastatin, was analyzed.
Statistical analysis. The differences in sex, age (20-39 y, 40-64 y, 65-74 y, and ! 75 y), medication history of simvastatin and lovastatin, and co-morbidities were compared between the gastric-cancer cases and the controls by using a chi-square test. We used a t test for continuous variables. Univariate and multivariate unconditional logistic regression model was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs). The ORs and 95% CIs were measured to explore the risk of gastric-cancer associated with medication use and comorbidities. The multivariate analysis was performed to adjust for possible confounders (including co-morbidities of H. pylori infection, gastric diseases, gastroesophageal reflux disease, gastric polyp, cirrhosis, and gastritis). The defined daily dose (DDD), recommended by the World Health Organization (WHO), was assumed to be the average maintenance dose per day of a drug. The annual mean DDD was calculated by diving the total cumulative DDD by the number of days in a year. Patients were classified into 3 groups according to the first quartile and second quartile of dose distribution. All analyses were conducted using SAS statistical software (Version 9.3 for Windows; SAS Institute, Inc., Cary, NC, USA). All statistical tests were performed at the 2-tailed significance level of 0.05.

Statin Reduces Cellular Cholesterol
To analyze whether statins affect cellular cholesterol, AGS cells were pretreated with simvastatin (0-50 μM) and infected with wild-type H. pylori 26695; the levels of cellular cholesterol were then determined. As indicated in Fig 1A, simvastatin-treated cells exhibited a significantly reduced level of cellular cholesterol in a concentration-dependent manner. The viability of H. pylori and the cells was barely affected by treatment with simvastatin ( Fig 1B).

Statin Mitigates H. pylori-Induced Pathogenesis
The translocated/phosphorylated CagA in gastric epithelial cells is associated with activation of NF-κB and production of IL-8, followed by induction of hummingbird phenotype formation [10]. To investigate the mechanism responsible for statin-mediated inhibition of H. pylori CagA functions, NF-κB luciferase activity and IL-8 production were analyzed. The results revealed that treatment of cells with 25 μM simvastatin and subsequent infection with H. pylori reduced the levels of NF-κB promoter activity and secretion of IL-8 (Fig 3). We then examined whether simvastatin, in addition to inhibiting translocation and phosphorylation of CagA, also specifically attenuates CagA-induced responses by evaluating the hummingbird phenotype of cells. AGS cells were pretreated with or without simvastatin (25 μM) and subsequently infected with H. pylori. Approximately 60% of cells represented the hummingbird phenotype when compared with the uninfected cells (Fig 4). In cells pretreated with 25 μM simvastatin, the proportion of H. pylori-induced cell elongation was substantially reduced. Because H. pylori CagA can stimulate RhoA-dependent activation of NF-κB [31,32], we further investigated the role of the RhoA inhibitor in our findings. As shown in S1 Fig, H. pylori-induced NF-κB promoter activity was markedly reduced in cells treated with either simvastatin or RhoA inhibitor compared to that in the untreated group. In parallel, treatment with simvastatin and RhoA   inhibitor also significantly attenuated H. pylori CagA-induced hummingbird phenotype (S2 Fig). These results are in agreement with those of previous reports where RhoA was shown to mediate the inhibitory effect of statin [33,34], indicating that statin not only reduced cellular cholesterol but also inhibited cholesterol pathway intermediates metabolites. Taken together, our findings indicated that simvastatin reduced cellular cholesterol and inhibited the cholesterol pathway intermediates that mitigated the geranylgeranylated RhoA-dependent activation of NF-κB, leading to attenuation of CagA translocation/phosphorylation levels and reduction in the hummingbird phenotype of H. pylori-infected cells.

Demographic Analyses
A total of 19728 patients with recently diagnosed gastric cancer were identified from 2005 to 2010 and 19727 patients comprised the control group. Among the enrolled patients, 63.0% were men and 61.0% were aged 65 years or older ( Table 1). The mean age (± SD) was 67.7 ± 14.2 years for patients diagnosed with gastric cancer and 67.3 ± 14.4 years for the control patients. The proportion of patients with medication history of simvastatin and lovastatin was lower in the gastric-cancer group than in the control group. Patients diagnosed with gastric cancer exhibited a significantly higher prevalence of co-morbidities than did the controls including H. pylori infection (3.41% vs 0.38%), gastric diseases (75.3% vs 40.4%),

Statin Use Reduces the Risk of Gastric Cancer
Patients who used simvastatin exhibited a significantly reduced risk of gastric cancer (adjusted OR = 0.76, 95% CI = 0.70-0.83) (  Table 4). The results indicated that in patients diagnosed with H. pylori infection, the adjusted OR for gastric cancer in patients prescribed simvastatin was 0.25 (95% CI = 0.12-0.50), compared with those not prescribed simvastatin. The same trend was also observed in patients treated with lovastatin, but the reduction in risk was nonsignificant. We further analyzed whether the use of statin was associated with the occurrence of different types of gastric cancers. As shown in Table 5, patients who used simvastatin exhibited a significantly reduced risk of the proximal type, distal type, and other types of gastric cancers. In addition, patients who were prescribed lovastatin showed a lower risk of other types of gastric cancer compared to those who did not use lovastatin.

Discussion
The inhibitors of HMG-CoA reductase, commonly known as statins, are widely prescribed for lowering serum cholesterol. Statins are associated with multiple protective functions including reducing the risk of hepatocellular carcinoma [35], enhancing chemosensitivity in colorectal cancer [36], and reducing the risk of death in patients with prostate cancer [37]. The results of this study consistently indicated that the use of statins may reduce gastric cancer risk. The results of previous studies implying an inverse association between statin use and the risk of gastric cancer have been nonsignificant [38,39]. In addition, these reports were based on small sample sizes and did not adjust for potential confounders. In contrast, the totality of epidemiologic evidence synthesized in recent meta-analyses on this topic support a statistically significant inverse association between statin use and gastric cancer risk [40,41]. We used a nationwide database and extended the period from 2005 to 2010 for further analysis. A total of 19728 patients with gastric cancer and 19727 patients without gastric cancer were enrolled in this study. Additionally, the multivariate analysis was adjusted for several possible confounders, including co-morbidities of H. pylori infection, gastric diseases, gastroesophageal reflux disease, and gastritis. Therefore, the results of the present study were highly reliable and consistent with the findings of Chiu et al, who reported the association of statin use with reduced gastric cancer risk by conducting a population-based case-control study [26]. The mechanisms underlying the immunoregulatory effects of statins have been studied by other researchers. Exposure of murine macrophages to statins attenuated lipopolysaccharide (LPS)-induced proinflammatory cytokine production [42]. Moreover, LPS-challenge serum levels of IL-1β and TNF-α, and monocyte-activating Toll-like receptor 2 (TLR2) and TLR4 were decreased in humans treated with statins [43]. Treatment of Staphylococcus aureus with high concentrations of statins resulted in considerable antimicrobial activity [44]. However, in this study, bacterial viability did not influence the treatment of H. pylori with 0-50 μM statins (Fig 1A). CagA is the major determining factor for NF-κB activation followed by IL-8 secretion, and is critical for H. pylori-induced gastric inflammation [10]. Previous studies have indicated that depletion of cholesterol by lipid raft-disrupting/usurping agents significantly attenuates CagA-mediated pathogenesis consistently [20,21]. Therefore, it is possible that the anti-H. pylori-induced-inflammation action of statins was mediated by a depletion of cholesterol which might inhibit CagA-functions in host cells rather than H. pylori growth.
Statins are known to reduce the risk of severe bacterial infections, including Chlamydia pneumonia [45], Clostridium difficile [46], Streptococcus pneumonia [47], and Staphylococcus aureus [48]. The immunomodulatory properties of statins only partly explain the potential antiinfection mechanism. This paper indicates that statins reduced NF-κB promoter activity and IL-8 production (Fig 3). These results are supported by previous studies indicating that statins might influence transcriptional factors, AP-1 and NF-κB, thereby reducing the secretion of proinflammatory cytokines [49]. Additionally, statin-treatment was observed to inhibit H. pylori-induced gastritis in mice [50]. Long-term statin therapy reduces the severity of chronic gastritis, even in the presence of H. pylori infection [51]. Statins can be used as adjuvants to improve the H. pylori eradication rates of standard antibiotics [52]. Although these studies have explained the pleiotropic functions exerted by statins, the exact molecular mechanism(s) of eradication of H. pylori and reduced H. pylori-associated diseases required further investigation. The incidence and mortality rate of gastric cancer remain high worldwide. The association of statins with a reduced risk of gastric cancer has been reported by epidemiological meta-analysis studies [26,41,53]. There are several mechanisms of antineoplastic actions of statins, including upregulation of p21 and p27 [54], and inhibition of MYC phosphorylation/activation [55], which might attenuate mitosis and lead to apoptosis. Although the correlation of H. pylori etiology and gastric cancer is known, the exact mechanism of statins' influence on H. pyloriassociated gastric cancer remains unclear. H. pylori CagA is a bacterial oncoprotein that can contribute to the development of H. pylori-associated neoplasms in mammals [11]. Furthermore, it is well documented that the delivery of CagA into cells by bacterial TFSS requires membrane cholesterol [5,13,20]. Therefore, it was suggested that inhibition of cholesterol reduces CagA delivery, which might lead to the failure of H. pylori-induced carcinogenesis. The results of this study revealed that treatment of cells with statins reduces cellular cholesterol and inhibits H. pylori CagA actions, which might exert a protective effect against gastric cancer.
Studies on the various effects of statins are contradictory. Some studies have indicated that statins do not reduce the risk of gastric cancer [38,56]. In a study on H. pylori-infected rodent models, pitavastatin was ineffective in inhibiting gastric carcinogenesis [57]. This might have been due to the use of distinct types of statin for analysis because pitavastatin was not commercially available in Taiwan [53].
In this study, we found that simvastatin reduced the risks of both distal and proximal gastric cancer ( Table 5). The data indicated that simvastatin might influence not only H. pylori-associated distal gastric cancer, but might also affect H. pylori-independent proximal gastric cancer. We further showed simvastatin use resulted in much lower odds ratios for distal gastric cancer than for proximal cancer (OR = 0.65 vs. 0.82). Our results demonstrated that the inhibition of cancer development by simvastatin was more pronounced in distal than in proximal gastric cancer, suggesting that simvastatin may affect H. pylori-induced pathogenesis. Although H. pylori infection is associated with distal gastric cancer, it is not the only risk factor for development of gastric cancer. The main risk factors for distal gastric cancer include H. pylori infection and diet, while gastroesophageal reflux disease and obesity play important roles in proximal gastric cancer [58]. Previous studies reported that statin use reduces the risk of Barrett's esophagus, which is a complication of gastroesophageal reflux disease [59] and associated with obesity [60]. Moreover, the mevalonate pathway has recently been reported as truly oncogenic [61], indicating that mevalonate pathway inhibitors, such as statins, are bona fide anticancer drugs [62]. These lines of evidence may explain why statin use could reduce the incidence rate of proximal gastric cancer independent of H. pylori infection. Long-term follow-up studies or randomized controlled trials are necessary to elucidate the role of statins in the prevention of different types of gastric cancer.
Some limitations might have been present in those studies, including potential confounders, concomitant use of other chemopreventive drugs, short enrolment periods, and small sample sizes. In particular, individuals of higher socioeconomic classes are more likely to be prescribed statins. A social gradient in statin use may reflect social inequalities in health care use or health care quality, as well as systematic differences in factors such as smoking, nutrition, physical activity, and obesity [63]. In addition, most of the tests performed to confirm H. pylori infection (such as serology, biopsy, urea breath test or a combination of both) are not covered by the insurance system. The data regarding H. pylori infection was under-estimated using the NHIRD. Therefore, large and long-term clinical studies on statin usage are required to further clarify statins' mechanism of inhibiting H. pylori-associated gastric cancer.
In conclusion, we determined that H. pylori infection is associated with a high risk of gastric cancer, which is attenuated by the prescription of statins. It was also demonstrated that statins inhibit H. pylori-induced pathogenesis by preventing CagA actions in host cells. Therefore, statins, which are cholesterol-lowering agents and proven to be extremely safe, can be used as chemopreventive drugs for protecting against H. pylori-gastric cancer.