This is the peer reviewed version of the following article: Ando-Matsuoka, R., Yagi, K., Takaoka, M., Sakajiri, Y., Shibata, T., Sawada, R., Maruo, A., Miyata, K., Aizawa, F.,
Hamano, H., Niimura, T., Izawa-Ishizawa, Y., Goda, M., Sakaguchi, S., Zamami, Y., Yamanishi, Y., & Ishizawa, K. (2023). Differential effects of proton pump inhibitors andvonoprazan on vascular endothelial growth factor expression in cancer cells. Drug Development Research, 84, 75– 83., which has been published in final form at https://doi.org/10.1002/ddr.22013. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article
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Differential effects of proton pump inhibitors and vonoprazan on vascularendothelial growth factor expression in cancer cells
Short title: Effects of PPIs and vonoprazan on VEGF expression in cancer cells
Rie Ando-Matsuoka, BS 1,2, Kenta Yagi, Ph.D 3, Mayu Takaoka, BS 1, Yuko Sakajiri, Ph.D
4

, Tomokazu Shibata, Ph.D 4, Ryusuke Sawada, Ph.D 4, Akinori Maruo, BS 5, Koji Miyata,

BS 1, Fuka Aizawa, Ph.D 2, Hirofumi Hamano, Ph.D 5, Takahiro Niimura, Ph.D 3, Yuki
Izawa-Ishizawa, MD-Ph.D 6, Mitsuhiro Goda, Ph.D 1, Satoshi Sakaguchi, MD-Ph.D 3,
Yoshito Zamami, Ph.D 1,5, Yoshihiro Yamanishi, Ph.D 4, Keisuke Ishizawa, Ph.D 1,2,3
1

University of Tokushima Graduate School of Biomedical Sciences, Department ofClinical Pharmacology and Therapeutics, Tokushima, Japan
2
3

Tokushima University Hospital, Department of Pharmacy, Tokushima, Japan
Tokushima University Hospital, Clinical Research Center for Developmental

Therapeutics, Tokushima, Japan
4

Department of Bioscience and Bioinformatics, Faculty of Computer Science andSystems Engineering, Kyushu Institute of Technology, Fukuoka, Japan
5

Okayama University Hospital, Department of Pharmacy, Okayama, Japan, Okayama,

Japan
6

University of Tokushima Graduate School of Biomedical Sciences, Department ofPharmacology, Tokushima, Japan*Corresponding author: Kenta Yagi, Tokushima University Hospital, Clinical ResearchCenter for Developmental Therapeutics, Tokushima, Japan, 2-50-1 Kuramoto-cho,
Tokushima 770-8503, JAPAN; Email: yagi.kenta@tokushima-u.ac.jp; Phone: +81-88615-8512; FAX: +81-88-633-9294.
Funding
This study was supported by grants from the Japan Society for the Promotion of Science

(JSPS) KAKENHI (grant numbers 20K16044 and 20H05799).
Conflict of interest
The authors declare no potential conflicts of interest.
Author contributions
Rie Ando-Matsuoka: Conceptualization, formal analysis, investigation, methodology,
validation, visualization, writing – original draft. Kenta Yagi: Conceptualization, funding
acquisition, project administration, writing – review editing. Mayu Takaoka: formal
analysis, investigation, validation. Yuko Sakajiri: data curation, formal analysis,
investigation, methodology. Tomokazu Shibata: data curation, formal analysis,
investigation, methodology. Ryusuke Sawada: data curation, formal analysis,
investigation, methodology. Akinori Maruo: data curation, formal analysis, investigation,
methodology. Koji Miyata: writing – review editing. Fuka Aizawa: resource, writing –review, and editing. Hirofumi Hamano: methodology, resource, writing – review andediting. Takahiro Niimura: writing – review and editing. Yuki Izawa-Ishizawa: resource,
writing – review and editing. Mitsuhiro Goda: Conceptualization, funding acquisition,
methodology, project administration, resource, supervision, writing – review and editing.
Satoshi Sakaguchi: writing – review and editing. Yoshito Zamami: writing – review andediting. Yoshihiro Yamanishi: writing – review and editing. Keisuke Ishizawa:
Conceptualization, project administration, supervision, writing – review and editing.
Acknowledgments
We would like to thank Editage (www.editage.com) for English language editing.
Lay summary
We previously reported that proton pump inhibitors (PPIs), the first-line agents in treating
peptic ulcers, increased VEGF expression; elevated VEGF expression promotes cancer
progression and diminishes the efficacy of anti-VEGF drugs. In this study, we evaluatedthe effect of vonoprazan, a novel drug for the treatment of peptic ulcers, on VEGF

expression. We found that PPIs increased VEGF expression via ER-α in cancer cells,
while vonoprazan did not affect VEGF expression, suggesting that vonoprazan might bemore effective than PPIs in patients with cancer, especially those receiving anti-VEGF
agents, for improving the therapeutic effects of cancer chemotherapy.
Data Availability Statement
The data that support the findings of this study are available from the corresponding
author upon reasonable request
Precis
PPIs increase VEGF expression via ER-α in cancer; elevated VEGF expression leads tocancer progression and diminished therapeutic effect of anti-VEGF drugs. Unlike PPIs,
vonoprazan, a novel gastric acid secretion inhibitor, does not upregulate VEGF
expression in cancer cell lines, suggesting that vonoprazan may be more effective than
PPIs in patients with cancer, especially those receiving anti-VEGF agents, for improving
the therapeutic effects of cancer chemotherapy.
Abstract
Background. Proton pump inhibitors (PPIs) are potent inhibitors of gastric acid secretion,used as first-line agents in treating peptic ulcers. However, we have previously reported
that PPIs may diminish the therapeutic effect of anti-vascular endothelial growth factor(VEGF) drugs in patients with cancer. In this study, we explored the effects of vonoprazan,
a novel gastric acid secretion inhibitor used for the treatment of peptic ulcers, on thesecretion of VEGF in cancer cells and attempted to propose it as an alternative PPI forcancer chemotherapy. Methods. The effects of PPI and vonoprazan on VEGF expression
in cancer cells were compared by real-time rt-PCR and ELISA. The interaction ofvonoprazan and PPIs with transcriptional regulators by docking simulation analysis.
Results. In various cancer cell lines, including the human colorectal cancer cell line
(LS174T), PPI increased VEGF mRNA expression and VEGF protein secretion, while
this effect was not observed with vonoprazan. Molecular docking simulation analysis

showed that vonoprazan had a lower binding affinity for estrogen receptor alpha (ER-α),
one of the transcriptional regulators of VEGF, compared to PPI. Although the PPIinduced increase in VEGF expression was counteracted by pharmacological ER-αinhibition, the effect of vonoprazan on VEGF expression was unchanged. Conclusions.
Vonoprazan does not affect VEGF expression in cancer cells, which suggests thatvonoprazan might be an alternative to PPIs, with no interference with the therapeutic
effects of anti-VEGF cancer chemotherapy.
Keywords: Proton pump inhibitors, vonoprazan, lansoprazole, fulvestrant, vascularendothelial growth factor.
Total number of text pages: 31
Total number of tables: 1
Total number of figures: 5
Total number of supporting files: 3
Introduction
Cancer patients are highly prone to stomach ulcer complications because of thefrequent use of non-steroidal anti-inflammatory drugs to relieve cancer pain.1, 2 In
addition, cancer patients often experience pancytopenia and thrombocytopenia as side
effects of chemotherapy, wherein the gastrointestinal bleeding associated with peptic
ulcers can be fatal.3, 4 Bleeding is a common side effect associated with the usage ofvascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab, which are
key drugs in cancer chemotherapy.5 Therefore, preventing peptic ulcers andgastrointestinal bleeding is especially important for patients receiving anti-VEGF agentsto ensure continuous treatment.
Proton pump inhibitors (PPIs) are potent inhibitors of gastric acid secretion, usedas first-line agents in treating peptic ulcers. Therefore, PPIs are frequently used in patientswith cancer. However, we previously found that PPIs upregulate VEGF mRNA
expression; VEGF is a potent angiogenesis-promoting biomolecule. Elevated VEGF

expression in tumors promotes cancer progression and hematogenous metastasis byenhancing angiogenesis.6-8 It also promotes the formation of fragile blood vessels, making
it difficult for anticancer drugs to penetrate the entire tumor tissue and induce drug
resistance.8 Furthermore, it may also reduce the therapeutic effect of anti-VEGF drugs.
Thus, elevated VEGF expression is known to exert various negative effects in patientswith cancer. Our previous retrospective study revealed that the response rate in patients
receiving bevacizumab, an antiangiogenic drug, decreased due to PPIs' usage. Thesefindings indicate that PPIs may reduce the therapeutic effect of bevacizumab byincreasing VEGF expression.9 Therefore, when treating patients with bevacizumab andother anti-VEGF drugs, it is necessary to select a peptic ulcer treatment that does notaffect VEGF expression. However, alternative peptic ulcer medications, such as H2
blockers and mucosal protectants, are less effective than PPIs in treating peptic ulcers,10,
11

often making it difficult to switch to alternative drug agents.
Vonoprazan is a novel gastric acid secretion inhibitor, authorized in Japan in

2015.12 Vonoprazan is a more potent and sustained inhibitor of gastric acid secretioncompared to PPIs.13-16 In Japan, it is used as a first-line drug in peptic ulcer treatment,
similar to PPIs.15 The mechanism of action of vonoprazan differs from that of PPIs;
vonoprazan is secreted into the gastric secretory lumen from gastric cell walls. It
competitively inhibits the influx of K+ ions required for the proton pump to operate.16 In
contrast, PPIs are activated in the secretory lumen and covalently bind to the proton pump,inhibiting it non-competitively.
All PPIs share a common structure consisting of benzimidazole, sulfoxide, and pyridine.17
Because all PPIs exhibit VEGF-inducing activity, this effect may be derived from thecommon structure of PPIs. However, the chemical structure of vonoprazan differs
significantly from that of PPIs, and it is unclear how vonoprazan affects VEGF expression.
In this study, we investigated the differences in the effect of vonoprazan and PPI on VEGF
expression in cancer cells. We also examined the differences between the two kinds of
drugs in more detail, focusing on the differences in their chemical structures.

Materials and Methods
1. Materials
Lansoprazole, pantoprazole sulfide, rabeprazole sodium salt, and omeprazole werepurchased from Tokyo Chemical Industry (Tokyo, Japan). Esomeprazole magnesium
hydrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Vonoprazan Fumarate
was purchased from MedChem Express LLC (Monmouth Junction, NJ, USA) andFulvestrant from Selleck Chemicals LLC (Houston, USA).
2. Cells
The A549 human lung adenocarcinoma epithelial cell line and LS174T humancolon cancer cell line were purchased from ATCC (Manassas, VA). The MCF7 humanbreast cancer (RCB1904) and OVK18 human ovarian cancer (RCB1903) cell lines werepurchased from RIKEN BioResource Center (Ibaraki, Japan). Cells were cultured in
RPMI-1640 medium (NACALAI TESQUE, Inc., Kyoto, Japan) supplemented with 10%
heat-inactivated fetal bovine serum (Corning, Inc., NY, USA), 100 units/mL penicillin,and 100 μg/mL streptomycin (ICN Biomedicals, Inc., CA, USA) in a 5% CO2 air
incubator at 37 °C.
3. Real-time PCR
The human cancer cell lines LS174T, A549, MCF7, and OVK18, were used toinvestigate gene expression. Vonoprazan and lansoprazole were dissolved in DMSO.
Cells were seeded into 6-well plates (3.0 × 105 cells/well), incubated for 24 h, and exposedto lansoprazole (10 μM) or vonoprazan (10 μM) for 24 h. Total RNA was extracted from
cells using RNeasy Plus Mini Kit (Qiagen, Venlo, Netherlands), according to the
manufacturer’s protocol. Total RNA was reverse transcribed to cDNA using Prime
ScriptTM RT reagent Kit with gDNA Eraser (Takara Bio, Inc., Shiga, Japan). The cDNA
product was used as a template for PCR amplification in a total volume of 10 μL, and the
PCR conditions were as follows: 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s,
60 °C for 30 s, and 95 °C for 5 s. Reactions were conducted on a Step One Plus Real-time
PCR Detection System (Thermo fisher science, Waltham, MA, USA ). All data wereanalyzed with the Step One Software V2.3 (Thermo fisher science, Waltham, MA, USA).
The data were analyzed using the delta cycle threshold method and calculated based on
the cycle quantification (Cq) values; the expression of each gene was normalized toglyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gene-specific primer pairs wereused:

h-VEGFA:(F)

5′-AGGAGGAGGGCAGAATCATC-3′,

ATGTCCACCAGGGTCTCGAT-3′;

GAPDH:

(R)

(F)

5′5′-

TCGGAGTCAACGGATTTGGTC-3′, (R) 5′-AAACCATGTAGTTGAGGTCAATG-3′.
4. VEGF ELISALS174T cells were seeded into the wells of 24-well plates (2.0 × 105 cells/well)and incubated at 37 °C overnight. Cells were then incubated with lansoprazole (10 μM)
or vonoprazan (10 μM). After 24 h of incubation, the culture medium was collected andcentrifuged at 500 × g for 5 min to remove floating cells. The cell lysate was preparedusing CelLyticTM MT Cell Lysis Reagent (Sigma-Aldrich, St. Louis, MO, USA). The
protein concentration of each lysate was measured using a BCA Protein Assay Kit(Thermo Fisher Scientific, Waltham, MA, USA). The VEGF-A secretion in the culture
medium was measured using Human VEGF DuoSet ELISA Kit (R&D Systems,
Minnesota, US), according to the manufacturer’s instructions, and corrected with theprotein concentrations.
5. Molecular docking simulation with three-dimensional (3D) protein structures
The three-dimensional (3D) structure of the target estrogen receptor alpha (ERα) (PDB Code: 5DXE) was obtained from the Protein Data Bank (PDB).18 Six compounds(vonoprazan, omeprazole, esomeprazole, lansoprazole, rabeprazole, and pantoprazole)
were used as ligands for docking (Fig. 1). The chemical structure of vonoprazan was
obtained from the KEGG DRUG database,19 and those of the other five compounds wereobtained from Ma’ayanlab. Cloud.20 The target ER-α and these six compounds were
protonated at pH 7.4.21 Since lansoprazole, rabeprazole, and pantoprazole are racemic,
docking simulations were performed for R and S isomers. Docking simulations of theligands with the estrogen binding site of ER-α were performed under the following

settings. The docking configuration search was evaluated using Triangle Matcher andLondon dG as the score function. The MMFF94 force field was used to refine the docking
pose, and then GBVI/WSA dG was used for scoring.22 All processes were performed
using the Molecular Operating Environment (MOE) from the Chemical Computing
Group.
6. Statistical analysis
Statistical differences between the two groups were evaluated using Student's ttest and between more than three groups using ANOVA with the Tukey posthoc test, using
the JSTAT software package (Version 17.0; SPSS, Chicago, IL, U.S.A.). All values arereported as the mean ± S.D. The levels of significance were set at *p < 0.05.
Result
1. Effect of vonoprazan on VEGF expression
Compared to the untreated group, LS174T cells treated with 10 μM lansoprazole
exhibited a significant 1.6-fold increase in VEGF mRNA expression. In contrast, VEGF
mRNA expression did not increase when treated with 10 μM of vonoprazan (Fig. 2A).
Furthermore, the evaluation of VEGF protein secretion using ELISA revealed that VEGF
secretion was significantly increased after lansoprazole treatment but not aftervonoprazan treatment (Fig. 2B). The concentrations used in this experiment had nocytotoxic effect on cancer cells (Fig. S1).
2. Effect of vonoprazan on VEGF expression in various cancer types
In this study, OVK-18 (ovarian cancer cell line), MCF-7 (breast cancer cell line),and A549 (lung cancer cell line) were used as cancer types, for which bevacizumab isindicated. Compared with control OVK18 cells, those treated with lansoprazole exhibited
significantly upregulated VEGF mRNA expression by 2.7-fold. However, VEGF mRNA
expression in vonoprazan-treated cells was similar to that in control cells. Similarly,
compared to control MCF7 cells, lansoprazole-treated cells showed upregulated VEGF
mRNA expression by 2.0-fold, whereas vonoprazan-treated cells did not show

significantly upregulated VEGF mRNA expression. Furthermore, compared to control
A549 cells, lansoprazole-treated cells showed upregulated VEGF mRNA expression
by1.4-fold. However, vonoprazan-treated cells did not show upregulated VEGF mRNA
expression (Fig. 3).
3. Molecular docking simulations to evaluate the interaction between ER-α andvonoprazan or PPIs
Using Toxicity predictor v1.5, we evaluated the differences between theactivities of five PPIs and vonoprazan toward various transcriptional regulators via thequantitative structure-activity relationship (QSAR) method. The QSAR analysis revealed
that ER-α was one of the transcriptional regulators active in all five PPIs but not invonoprazan (Table S1).
The binding affinity of the five PPIs and vonoprazan for ER-α, one of the transcriptional
regulators of VEGF, was calculated using molecular docking simulation. The docking
poses of the ligands are displayed in Fig. 4, and the docking score (kcal/mol) for eachligand and ER-α are shown in Table 1. Most of the PPIs showed docking scorescomparable to that of estradiol, the in vivo ligand for ER-α, whereas the docking score ofvonoprazan was slightly lower than that of PPIs.
4. ER-α is involved in the upregulation of VEGF expression by PPIs
We determined the involvement of ER-α in PPI-induced VEGF up-regulation
using the ER inhibitor, fulvestrant. We evaluated VEGF mRNA expression and VEGF
protein secretion in LS174T1 treated with fulvestrant and lansoprazole or vonoprazan.
When treated with 100 μM lansoprazole, VEGF mRNA expression was significantly
increased to 5.4-fold compared to that in the control; however, this increase wassignificantly suppressed by fulvestrant (Fig. 5A). When treated with 100 μM vonoprazan,
VEGF mRNA expression did not increase compared to that in the control, with or without
fulvestrant. VEGF protein secretion was significantly elevated after lansoprazole
treatment by 2.6-fold higher than that in control. This increase was significantly
suppressed by fulvestrant to the same extent as in the control (Fig. 5B). In contrast,

compared to the control, vonoprazan treatment did not yield a significant increase, withor without fulvestrant.
Discussion
In this study, we found that PPIs increase VEGF expression, whereas vonoprazan,
a novel gastric acid secretion inhibitor, does not affect VEGF expression, indicating thatit might not curtail the efficacy of anti-VEGF drugs. Furthermore, this effect was also
observed in ovarian, breast, lung, and colorectal cancer cells, suggesting that the effect isobserved regardless of the type of cancer. Molecular docking simulation analysis revealed
that vonoprazan and PPI have different activities against ER-α, which may underly thedifferential effects on VEGF expression. These results suggest that vonoprazan does notaffect VEGF expression and may be used in patients with cancer, especially those on antiVEGF drugs, with less effect on cancer therapy than PPIs.
The effect of vonoprazan on VEGF expression was evaluated using the samecolorectal cancer cell line as in a previous study.9 The results showed that vonoprazan
does not upregulate VEGF expression at the gene and protein levels, whereas PPI does.
Bevacizumab, an anti-VEGF drug, is widely used to treat various cancers, includingcolorectal, lung, breast, and ovarian cancers. Therefore, we evaluated the effects ofvonoprazan and PPI on VEGF expression in such cancer cell lines. The results showedthat PPI increased VEGF expression in all cell lines used in this study, while vonoprazan
did not increase VEGF expression in any cell lines. This indicated that PPI might
upregulate VEGF expression in various cancer types. In contrast, vonoprazan may notaffect VEGF expression in any cancer type.
There are no detailed data on the intra-tumor concentration of PPI or vonoprazan.
In the current study, lansoprazole was used at 10 µM. Considering that the highest bloodconcentration of lansoprazole after oral administration of 30 mg of lansoprazole isapproximately 3 µM,23 and that PPIs, which are weak-basic drugs, might be easily
distributed in tumors because of the weakly acidic conditions in tumor tissue, theconcentration of lansoprazole used in this study (10 μM) could be feasible in patients withcancer. Furthermore, the expression of VEGF clearly increased after treatment with

lansoprazole, although some lansoprazole was degraded in the medium owing to its
instability in an aqueous solution. While the effects of short-term stimulation were
evaluated in this study, PPIs are essentially long-term oral medications. Even at low
concentrations, long-term exposure to PPIs may increase VEGF expression. Considering
these factors, we reasonably speculate that lansoprazole is likely to increase VEGF
expression in cancer patients. The highest blood concentration of vonoprazan after oral
administration of 20 mg was approximately 0.05 μM,24 indicating that the clinicalconcentration of vonoprazan is lower than the concentration used in the current study.
Although vonoprazan is a basic drug that is stable under weakly acidic conditions intumors and thus may easily distribute, it is unlikely that vonoprazan increases VEGF
expression in patients with cancer since no effect of vonoprazan on VEGF expression wasobserved up to 100 μM in the current study.
Although both lansoprazole and vonoprazan are gastric acid secretion inhibitors,
their effects on VEGF expression differ. This suggests that lansoprazole-induced VEGF
up-regulation may be due to a different mechanism than the original pharmacologicaleffect of proton pump inhibition.
Our previous study has also shown that five PPIs currently in clinical use(lansoprazole, omeprazole, esomeprazole, rabeprazole, and pantoprazole) commonly
increase VEGF expression.9 All PPIs have a structure consisting of a series ofbenzimidazole, sulfoxide, and pyridine. PPI-induced VEGF upregulation may be
attributed to this common structure. Therefore, we focused on the differences between thechemical structures of PPI and vonoprazan.
Using Toxicity predictor v1.5, the activities of five PPIs and vonoprazan forvarious transcriptional regulators were evaluated by the QSAR method. We searched forthe transcriptional regulator wherein the five PPIs had activity in common andvonoprazan showed no activity (Table S1). The results showed that ER-α was atranscriptional regulator with different activities in PPIs and vonoprazan. ER-α has been
known to upregulate VEGF expression during the female menstrual cycle.25 Furthermore,
ER-α is reportedly involved in VEGF upregulation in various cancers,26-29, and VEGF
expression is induced by estradiol (E2), a ligand for ER-α.30-32 Therefore, in this study,

we focused on ER-α to reveal the difference in the effects of PPI and vonoprazan on
VEGF expression.
The affinity of PPI and vonoprazan for ER-α was calculated by docking
simulation, and it was found that the docking score of PPI was comparable to that of E2,
an in vivo ligand. The docking score of vonoprazan was lower than that of PPI, suggestingthat vonoprazan has a relatively low stimulatory effect compared with PPIs.
Furthermore, inhibition of ER-α by fulvestrant significantly suppressedlansoprazole-induced up-regulation of the mRNA expression and protein secretion of
VEGF. Lansoprazole also up-regulates the expression of EBAG9 and cathepsin D, thetarget genes of ER-α but vonoprazan did not (Fig S2). These findings indicated that the
PPI-induced up-regulation of VEGF expression might be mediated by ER-α. However,fulvestrant treatment did not completely suppress the lansoprazole-induced VEGF mRNA
upregulation, suggesting that transcriptional regulators other than ER-α may be involvedin this phenomenon. The QSAR analysis also revealed that glucocorticoid receptors (GR)function as transcriptional regulators with different activities in PPI and vonoprazan.
Although few reports described the direct regulation of VEGF expression, GR has beenreported to modulate angiogenesis, suggesting that GR might also be involved in thisphenomenon.36,37
Since we have not performed animal experiments or clinical research in thisstudy, it is unclear how the difference in the effects of lansoprazole and vonoprazan on
VEGF expression affects the therapeutic effect of anti-VEGF drugs and the exacerbationof cancer. A future, more detailed study integrating clinical data is warranted. In addition,although the binding affinity can be evaluated by docking simulation, theagonist/antagonist activity of the ligand after binding cannot be assessed. The
experiments using fulvestrant revealed that the activity for ER-α differs between PPI andvonoprazan. However, the mechanism of this phenomenon needs to be investigated in
more detail in the future.
Conclusion
PPIs increase VEGF expression via ER-α in cancer; elevated VEGF expression

leads to cancer progression and diminished therapeutic effect of anti-VEGF drugs. Unlike
PPIs, vonoprazan, a novel gastric acid secretion inhibitor, did not upregulate VEGF
expression in cancer cell lines, suggesting that vonoprazan may be more effective than
PPIs in patients with cancer, especially those receiving anti-VEGF agents, in improving
the therapeutic effect of cancer chemotherapy.

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Figure legends
Figure 1. Six chemical structures, including their racemic structures.
(A) Vonoprazan, (B) omeprazole, (C) esomeprazole, (D) R-lansoprazole, (E) Slansoprazole, (F) R-rabeprazole, (G) S-rabeprazole, (H) R-pantoprazole, and (I) Spantoprazole.
Figure 2. Effects of lansoprazole or vonoprazan on the expression of VEGF
LS174T cells were treated with lansoprazole (Lanso) or vonoprazan (Vono) at aconcentration of 10 μM each. Twenty-four hours later, RNA was collected, and VEGF
mRNA expression was evaluated (a). LS174T cells were treated with lansoprazole orvonoprazan at 10 μM each. Twenty-four hours later, culture supernatants were collected,
and the VEGF concentration in the medium was determined. The total protein
concentration in the cell lysate was measured to correct for variations in cell number (b).
Data are expressed as mean ± SD; **p < 0.01
Figure 3. Effects of lansoprazole or vonoprazan in various cancer cell lines
A549, OVK18, and MCF7 cell lines were treated with 10 μM lansoprazole (Lanso) or
vonoprazan (Vono), and 24 h later, RNA was collected, and VEGF mRNA expression was
evaluated. Data are expressed as mean ± SD; **p < 0.01

Figure 4. Docking poses of compounds
Compounds and estrogen receptor alpha (ER-α) are shown as a green rod and blue-ribbon
models, respectively. (A) Overall structure of ER-α docked with estradiol (PDB:5DXE).
(B–J) Docking pose of vonoprazan, Omeprazole, Esomeprazole, R-Lansoprazole, SLansoprazole, R-Rabeprazole, S-Rabeprazole, R-Pantoprazole, and S-Pantoprazole.
Figure 5. Effects of an Estrogen receptor alpha (ER-α) inhibitor on the change of
VEGF expression by lansoprazole or vonoprazan
LS174T cells were treated with lansoprazole (Lanso) or vonoprazan (Vono) at aconcentration of 100 μM or fulvestrant (Ful) at 1 μM. After 24 h, RNA was collected, andVEGF mRNA expression was assessed (a). At 24 h after adding lansoprazole, Vono, or
Ful, culture supernatants were collected, and VEGF concentration was measured. The
total protein concentration in lysate was measured to correct for variations in cell numbers.
(b) Data are expressed as mean ± SD; *p < 0.05, **p < 0.01
Table 1. Docking Scores of the following chemical structures with the estrogen
receptor alphaChemical Structure

Docking Score(GBVI/WSA dG)

Vonoprazan-8.44

Omeprazole-8.81

Esomeprazole-8.86

R-Lansoprazole-8.84

S-Lansoprazole-8.75

R-Rabeprazole-9.39

S-Rabeprazole-8.98

R-Pantoprazole-9.39

S-Pantoprazole-9.01

Estradiol-9.04

Supporting information list
Figure S1. In vitro cytotoxicity of proton pump inhibitors (PPIs) and vonoprazan in cancer
cells.
Figure S2. Effect of lansoprazole or vonoprazan on the expression of ER-α target genes
Table S1. Toxicity predictor analysis.

Figure1

Figure 2

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Figure 4

Figure 5