Statins’ Regulation of the Virulence Factors of Helicobacter pylori and the Production of ROS May Inhibit the Development of Gastric Cancer

Conventionally, statins are used to treat high cholesterol levels. They exhibit pleiotropic effects, such as the prevention of cardiovascular disease and decreased cancer mortality. Gastric cancer (GC) is one of the most common cancers, ranking as the third leading global cause of cancer-related deaths, and is mainly attributed to chronic Helicobacter pylori infection. During their co-evolution with hosts, H. pylori has developed the ability to use the cellular components of the host to evade the immune system and multiply in intracellular niches. Certain H. pylori virulence factors, including cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA), and cholesterol-α-glucosyltransferase (CGT), have been shown to exploit host cholesterol during pathogenesis. Therefore, using statins to antagonize cholesterol synthesis might prove to be an ideal strategy for reducing the occurrence of H. pylori-related GC. This review discusses the current understanding of the interplay of H. pylori virulence factors with cholesterol and reactive oxygen species (ROS) production, which may prove to be novel therapeutic targets for the development of effective treatment strategies against H. pylori-associated GC. We also summarize the findings of several clinical studies on the association between statin therapy and the development of GC, especially in terms of cancer risk and mortality.


Introduction
Gastric cancer (GC) is a global health burden with more than a million new cases diagnosed in 2020, and the mortality rate is only surpassed by that of lung and liver cancers [1]. GC progresses in multiple stages including superficial gastritis, atrophic gastritis, intestinal metaplasia, dysplasia, and finally GC [2]. Lifestyle and behavior are exogenous risk factors closely associated with the development of GC [3]. Helicobacter

H. pylori Virulence Factors Usurp Cholesterol and Lead to GC Development
H. pylori infection induces sustained inflammation, an event that has long been linked to cancer development [29,30]. In addition to the host immune response, H. pylori virulence factors promote sustained inflammation, thus maintaining a microenvironment rich in cytokines/chemokines, reactive nitrogen species (RNS), and reactive oxygen species (ROS) that can destabilize normal cellular homeostasis [29]. Chronic inflammation exhausts resident gastric stem cells [31] and leads to the recruitment of bone marrow-derived cells that are predisposed to improper differentiation, resulting in metaplasia and dysplasia [30].
H. pylori virulence factors play an important role in inducing inflammatory responses and promoting pro-tumorigenic activities.
Vacuolating cytotoxin A (VacA) is a virulence factor well known for its ability to cause vacuoles that possess the hallmarks of late endosomes and early lysosomes in host cells [32]. Although the role of these vacuoles in GC remains unclear, it is posited that VacA-induced vacuoles disrupt normal membrane trafficking at or near late endosomes [33]. Subsequently, VacA induces autophagy and impairs transient receptor potential membrane channel mucolipin 1 (TRPML1), a key regulator of the endolysosomal pathway [34]. Consequently, the inhibition of lysosomal function or preventing the fusion of autophagosomes with lysosomes promotes bacterial resistance and multiplication of autophagosomes, which is crucial for persistent bacterial infection and induction of gastric carcinogenesis [35][36][37]. VacA triggers the production of proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin (IL)-6, which promote the expression of cyclooxygenase-2 (COX-2) in T cells, macrophages, and neutrophils [38]. In addition to participating in proinflammatory events, COX-2 catalyzes a key step in the production of prostaglandin, a group of lipids known to play a role in tumorigenesis [29].
Another virulence factor is cytotoxin-associated gene A (CagA), which is encoded by the cag-pathogenicity island (cag-PAI) and is the most extensively studied virulence factor of H. pylori for its cancer-causing actions. H. pylori strains carrying cagA (referred to as cagA-positive strains) were significantly more correlated with the exacerbation of gastric conditions and gastric adenocarcinoma than cagA-negative strains [39]. The cag-PAI gene encodes a type IV secretion system (T4SS), a protein complex that is critical for the translocation of CagA across the membrane. Following its translocation into gastric epithelial cells, CagA is localized to the inner membrane and immediately phosphorylated by members of the Src family kinase [40]. Subsequently, CagA binds to the SH2 domain-containing tyrosine phosphatase SHP2, which potentiates a downstream cascade of Erk/MAPK activity and affects the regulation of cellular proliferation, growth, and morphology, leading to deregulation of phase progression from G1 to S [41,42] and to an increase in the expression of proto-oncogenes c-fos and c-jun [43]. In addition, CagA phosphorylation induces activation of the transcriptional factor nuclear factor-kappa B (NF-κB) and production of the cytokine IL-8 in gastric epithelial cells [44,45]. NF-κB regulates many genes whose products are involved in angiogenesis, anti-apoptotic pathways, metastasis, enhanced cell cycle progression, and cytokine production. IL-8 has been reported to have an angiogenic role in several types of cancer [29].
Cholesterol-α-glucosyltransferase (CGT), encoded by the type I capsular polysaccharide biosynthesis protein J gene (capJ) catalyzes the glucosylation of cellular cholesterol into cholesteryl glucosides [46]. Cholesterol glucosylation dampens H. pylori phagocytosis and T-cell activation, leading to bacterial immune evasion [47]. Furthermore, CGT increases autophagosome formation, which enhances H. pylori survival in host macrophages by providing an intracellular niche [48,49]. This process also reduces autophagosome-lysosome fusion, which is a key step in eliminating intracellular pathogens. Furthermore, cholesterol glucosylation improves H. pylori-host cell binding and reorganizes lipid raft membranes, thereby promoting T4SS functions such as CagA translocation/phosphorylation. This activates NF-κB to promote IL-8 production, thereby aggravating inflammation [50].

Interplay between H. pylori and ROS Production to Induce Gastric Carcinogenesis
H. pylori CagA translocated in the cells is crucial for inducing the production of a significant amount of ROS, which is involved in the enforcement of cell cycle progression and acceleration of cell proliferation [51]. In addition, the accumulation of ROS increases oxidative stress, which damages mitochondrial DNA (mtDNA) and nuclear DNA, leading to gastric carcinogenesis [52]. Apart from ROS, nitrosative stress is another key mediator of H. pylori infection. Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) is responsible for bacteria-induced inflammatory responses [53]. H. pylori elicits NO production in macrophages and gastric epithelial cells, which convert L-arginine to L-citrulline using iNOS [54,55]. In GC patients with H. pylori infection, iNOS expression is higher than that in H. pylori-negative individuals [56]. iNOS deficiency lowered NO production by iNOS and markedly reduced H. pylori-associated GC in mice [57], indicating that overexpression of iNOS and sustainable NO levels contribute to H. pylori-induced stomach carcinogenesis.
VacA also plays a role in ROS generation. In a gastric epithelial model, H. pylori with CagA + /VacA + -induced ROS production and mtDNA mutations were significantly higher than those in a VacA-mutant strain [58]. Furthermore, infection with the VacA + strain elevates SQSTM1/p62 aggregation and disrupts autophagy to increase ROS expression in gastric epithelial cells, which may accelerate carcinogenesis [35]. Biopsies from patients with Crohn's disease showed that ATG16L1 with the T300A mutation increases susceptibility to H. pylori infection, indicating that the ATG16L1 genotype modulates autophagy responses to VacA [59]. Notably, autophagy and CagA can be degraded by VacA by reducing intracellular glutathione levels, leading to enhanced ROS accumulation and Akt phosphorylation, resulting in GC development [60]. The mechanism by which VacA and CagA decrease autophagy may provide a unique strategy for persistent H. pylori colonization in the stomach. These findings support the view that elevated ROS production due to H. pylori infection enhances DNA damage and prevents DNA repair mechanisms from functioning properly, thereby contributing to gastric carcinogenesis [6].
H. pylori has evolved to elicit detrimental effects in cells while dampening the host's defenses using strategic mechanisms [61]. H. pylori arginase competes with cellular iNOS for the substrate L-arginine, which reduces NO production [62]. In addition, arginase II produced by macrophages suppresses H. pylori-induced NO production by inhibiting iNOS expression [63]. Reducing L-arginine availability decreases H. pylori-stimulated iNOS expression and NO levels [64]. In parallel, VacA inhibits the expression of integrinlinked kinase and endothelial nitric oxide synthase, thereby decreasing ROS production in macrophage/monocyte-lineages [65]. These findings indicate that H. pylori exploits host factors to orchestrate ROS generation, resulting in simultaneous damage to cells and immune evasion.

Statins Lower GC Risk by Reducing H. pylori Survival and Inhibition of Virulence Factor Actions
Statins are competitive inhibitors that block the conversion site of HMG-CoA reductase to prevent substrate access and effectively inhibit the conversion of HMG-CoA into mevalonic acid [66]. With a reduced level of mevalonic acid, the cholesterol synthesis pathway is interrupted in the liver [67]. This triggers the production of microsomal 3-hydroxy-3-methylglutaryl-CoA reductase and cell-surface low-density lipoprotein (LDL) receptors, which assist in lowering the level of circulating LDL in the bloodstream to 20-55% [68]. By inhibiting mevalonate synthesis, statins inhibit the production of mevalonate-derived intermediates, which are involved in the posttranslational modification of proteins crucial for intracellular signaling, cell growth, and cellular differentiation [69,70].
Simvastatin is a class of statins that has been reported to reduce the level of cellular cholesterol in macrophages and gastric epithelial cells [13,26]. As both CagA translocation and phosphorylation depend on adequate cholesterol levels, treatment with simvastatin significantly reduces CagA translocation into gastric epithelial cells [13], which may in turn attenuate CagA-induced oncogenesis ( Figure 1). In addition, treatment of cells with cholesterol-depleting agents reduces VacA internalization and cholesterol likely plays an important role in VacA entry into cells [71]. It is possible that statins may inhibit VacA internalization by gastric epithelial cells, subsequently inhibiting its pro-tumorigenic effects. Our recent study further demonstrated that the cholesterolreducing effect of statins interrupts the cholesterol-dependent cellular evasion strategies of H. pylori mediated by CGT and reduces the bacterial load in macrophages [26]. Most importantly, several studies have reported a wide range of actions performed by statins that could reduce cancer incidence besides the inhibition of the internalization of H. pylori virulence factors. These include reduction in the plasma concentration of inflammatory cytokines, attenuation of proliferative response, and modulation of immune responses [69]. CGT and reduces the bacterial load in macrophages [26]. Most importantly, several studies have reported a wide range of actions performed by statins that could reduce cancer incidence besides the inhibition of the internalization of H. pylori virulence factors. These include reduction in the plasma concentration of inflammatory cytokines, attenuation of proliferative response, and modulation of immune responses [69].

Statins Modulate MicroRNAs and Exosome Levels
MicroRNAs (miRNAs) are short, noncoding segments of RNA involved in the regulation of gene expression at the post-translational level [72]. Zambrano et al. investigated the effects of low dose short-term statin treatment and found that statin affects the expression of certain miRNAs [73]. Although simvastatin did not significantly impact the expression of the 86 miRNAs studied, it appeared to up-regulate several miRNAs that are involved in tumor progression in subjects who exhibited a high reduction in the level of LDLC. In addition, atorvastatin was associated with poor expression of some miRNAs studied, and it possibly downregulated the level of miRNA-33, a miRNA that reduces fatty acid metabolism and cholesterol transport [73,74].
Other than miRNAs, exosomes have been reported to lower cholesterol levels through the use of statins. Exosomes are extracellular vesicles that function in cell-to-cell signaling, and simvastatin was found to repress exosomal formation and secretion [75].

Statins Modulate MicroRNAs and Exosome Levels
MicroRNAs (miRNAs) are short, noncoding segments of RNA involved in the regulation of gene expression at the post-translational level [72]. Zambrano et al. investigated the effects of low dose short-term statin treatment and found that statin affects the expression of certain miRNAs [73]. Although simvastatin did not significantly impact the expression of the 86 miRNAs studied, it appeared to up-regulate several miRNAs that are involved in tumor progression in subjects who exhibited a high reduction in the level of LDLC. In addition, atorvastatin was associated with poor expression of some miRNAs studied, and it possibly downregulated the level of miRNA-33, a miRNA that reduces fatty acid metabolism and cholesterol transport [73,74].
Other than miRNAs, exosomes have been reported to lower cholesterol levels through the use of statins. Exosomes are extracellular vesicles that function in cell-to-cell signaling, and simvastatin was found to repress exosomal formation and secretion [75]. As cholesterol is an integral component of exosomal membrane, it is natural to attribute this outcome to its cholesterol-lowering effect; however, the results of the study revealed that alternative pathways may be at play instead. Because exosomes are thought to have proinflammatory activities, inhibition of exosome biogenesis and secretion may reduce inflammation, which suggests a possible chemopreventive mechanism of simvastatin.
In addition to regulating cholesterol synthesis, the virulence factors of H. pylori, and ROS responses, it is possible that statins reduce the risk of H. pylori-associated GC via the regulation of miRNAs and exosomes. For example, miRNA-146 and miRNA-155 are induced after H. pylori infection to regulate inflammation [76,77]. Another miRNA, let-7b is reduced in GC cells in a CagA-dependent manner, which may lead to the downregulation of TLR4 [78]. Additionally, miRNA-451, which inhibits the macrophage migration inhibitory factor (MIF) and functions as a tumor suppressor, is also downregulated after H. pylori infection in GC cells [79]. miRNA-29a is downregulated in GC cells to promote cell cycle progression and proliferation [80]. However, statins are known to reverse the functions of these miRNAs, suggesting that these drugs may be useful for treating GC caused by H. pylori. Meanwhile, simvastatin is known to suppress exosome formation [75], which allows cells to deliver various miRNAs and CagA for the pathogenesis of H. pylori.

Cholesterol-Independent Beneficial Effects of Statins in Cancer Therapy
In addition to their cholesterol-lowering function, statins exert pleiotropic therapeutic effects, which have been demonstrated to reduce the risk of several types of cancer. Statins possess anti-cancer properties, mainly by virtue of their high NO production, which is essential for tumor cytotoxicity [81]. For example, fluvastatin and simvastatin have been shown to be cytotoxic to human breast cancer cells by elevating iNOS activity and inhibiting geranylgeranylation [82]. Activation of iNOS increases NO levels, which arrests the cell cycle in the G1 phase and downregulates cyclin D1, leading to synergistically enhanced statin-induced cancer cell death [83]. In addition, lovastatin and simvastatin increase mitochondrial membrane potential (∆Ψ) and modulate mitochondrial metabolism in several cancer cells independent of cholesterol content [84]. Nonsteroidal anti-inflammatory drugs (NSAID) have been found to cause gastropathy, which was attributed to redox imbalance [85]. Notably, statins exert a protective effect against NSAIDinduced lesions by increasing NO production and prostaglandin expression [86]. Overall, the NO-mediated proapoptotic, tumoricidal, and antiproliferative effects of statins confer anti-cancer properties.
Although ROS is activated as the cellular defense mechanism against bacterial invasion, H. pylori has evolved a variety of strategies, including antioxidant and DNA repair enzymes for facilitating its long-term survival within host cells [63,87]. However, persistent H. pylori infection enhances genetic instability and high mutations are generated during the repair processes, leading to gastric carcinogenesis [88]. In contrast, the excessive ROS induced by statins is irreversible, which leads to the accumulation of these ROS/RNS at toxic levels and results in profound cell death for therapeutic benefits [89,90].

Statin Use Reduces GC Risk
Statins were originally used to lower cholesterol levels to prevent cardiovascular disease. In addition to cholesterol restriction, statins are potential drugs for cancer therapy. To investigate the direct relationship between statins and anti-cancer activity, its effect on several types of cancers has been analyzed [91]. The results showed that statins induce cancer cell death by triggering the apoptotic pathway, not only in cell models but also in murine GC xenografts [92,93]. These findings suggest that statins may possess anticancer activity; however, very few clinical studies have reported that statins are anti-cancer drugs. This review further discussed the role of statin use in GC prevention and treatment (Tables 1 and 2, respectively). Chiu et al. conducted a population-based case-control study in Taiwan, which showed a lower risk of GC in statin users than in non-users (OR = 0.68, 95% CI = 0.49-0.95) [18]. Notably, a dose-dependent effect was observed between statin use and GC risk. Lee et al. conducted a clinical study to examine the association between statin use and GC by analyzing patients with diabetes [27]. Their results showed that prescription for any statins exhibited a significant inverse association with GC. Additionally, the duration of statin use was positively correlated with a reduction in the risk of GC in patients with diabetes. These anti-GC effects warrant further research on the potential clinical use of statins.  H. pylori infection is closely associated with GC incidence [4,99,100]. Membrane cholesterol-rich microdomains provide specific regions for H. pylori virulence factorinduced pathogenesis and GC development [47,101,102]. Statins may reduce cholesterol levels and attenuate bacterial virulence factor actions, which may alleviate H. pylori-associated diseases, making them a plausible therapeutic option to treat H. pylori-induced GC. Our recent study reported that statin treatment reduced CagA translocation/phosphorylation levels and mitigated H. pylori-induced pathogenesis [13]. These results suggest that statins can decrease several pathogenic effects caused by H. pylori virulence factors. We then conducted population-based case-control studies by analyzing the Taiwan National Health Insurance Research Database and demonstrated that patients who received simvastatin exhibited a remarkably low incidence of GC [13]. GC risk reduction is especially significant in patients with H. pylori infection compared to that in statin non-users (adjusted OR = 0.25, 95% CI = 0.12-0.50). A similar trend was observed with the use of other types of statins. In addition to the type of statin prescribed, the defined daily dose is a factor related to the efficacy of GC risk reduction. These results demonstrate that statin use significantly reduces the incidence of GC, particularly in patients with H. pylori infection. However, the mechanism by which statins lower the risk of H. pylori-related GC requires further investigation.
Conversely, research by Toyoda's group indicated that pitavastatin failed to suppress GC in murine models [94]. This study examined the relationship between pitavastatin and H. pylori-associated gastric carcinogenesis by adding pitavastatin to the diet of H. pylori-infected Mongolian gerbils. Compared to the control group, the incidence of H. pylori-associated gastric adenocarcinomas was not reduced in pitavastatin-treated mice. Serum total cholesterol also increased in the experimental groups treated with pitavastatin compared to that in the untreated controls. These results indicate that pitavastatin is ineffective in suppressing H. pylori-induced GC in murine models.
Although H. pylori infection is an important causative agent of GC, the possibility of developing GC still exists [103]. Statins interfere with H. pylori infection and suppress the delivery of virulence factors to the cells [13,26]. H. pylori infection could be a confounding factor that may cause bias in clinical studies. To eliminate the confounding effect of H. pylori status, Cheung et al. investigated the effect of statins in H. pylori-eradicated GC by analyzing the clinical data and reporting system in Hong Kong [96]. Competing risk regression with propensity score matching revealed that statin prescription was related to reducing the risk of GC in patients who had received H. pylori eradication therapy. The risk difference was 2.6 times lower in GC cases (95% CI = 1.56-3.12) per 10,000 personyears in statin users than in statin non-users. These results support the use of statins as chemopreventive agents for the treatment of GC in H. pylori-eradicated patients.
Spence et al. analyzed the relationship between statin use and GC mortality in England [14]. Two independent databases, the UK Clinical Practice Research Datalink in England and the Prescribing Information System in Scotland were investigated. These two databases recorded statin prescription and death information identified from the national mortality records. Combined cohorts and hazard ratio (HR) analysis showed that patients with GC who received statins exhibited a reduction in mortality (adjusted HR = 0.83, 95% CI = 0.74-0.92). Cancer-specific mortality was also reduced for patients who were prescribed statins before diagnosis with GC (adjusted HR = 0.91, 95% CI = 0.84-0.98). However, the doses of statins used in the treatment of patients with GC were not analyzed.
The association between statin use and lower GC risk remains elusive, and some discrepancies have emerged. A study by Cho et al. showed that statins were able to reduce mortality in GC but failed to decrease its incidence [98]. These findings are inconsistent with results from previous systematic reviews and meta-analyses, which may be because different study populations and settings were analyzed [17]. Although recent research has focused on using statins as an agent in GC treatment, whether statins can be used in the prevention of GC requires further supporting evidence. Further research that includes a large cohort with different populations should be conducted to clarify whether the clinical use of statins in the treatment of GC is feasible.

Conclusions and Perspectives
Most studies conducted in Western and Eastern countries to investigate the relationship between statin use and the development of GC have reported similar results, which suggest that the use of statins can reduce the risk of GC. However, most of these studies were still in the preclinical stage or were only conducted in the form of database analysis. The mechanisms by which statins inhibit H. pylori infection, especially the link between statin treatment and the manipulation of autophagy to eliminate H. pylori, remain unclear and need to be investigated further [26]. Moreover, the mechanisms by which statins increase ROS levels and regulate oxidative stress to promote GC cell death need to be elucidated. The current information from in vivo studies is insufficient, and further investigations should be conducted to determine whether statins can potentially be used for the treatment of GC.
Some studies have reported that the use of statins is associated with reducing the risk of other types of cancer. Data mining conducted using databases from the Food and Drug Administration (FDA) Adverse Event Reporting System and the Japan Medical Data Center, examined the association between the use of statins and different types of cancer, including colorectal, lung, pancreatic, gastric, esophageal, breast, and prostate cancers, as well as hematological malignancies and melanoma [104]. These results indicate that different statin categories target different types of cancer. For example, simvastatin exhibits the best efficacy in reducing the risk of GC, whereas it is positively correlated with the risk of pancreatic cancer. As conflicting results were obtained in various clinical observation studies, it is necessary to perform a large-scale prospective trial on the effects of statins in cancer therapy, which will assist physicians in determining the type of statin that is suitable for a specific type of cancer and in understanding the side effects of their use before administration to patients. In addition to further investigating the relationship between the use of statins and GC, the potential applications of statins for the treatment of other types of cancer are worth examining.