Received: 9 September 2020
DOI: 10.1002/cam4.3587|

Revised: 13 October 2020|

Accepted: 15 October 2020

ORIGINAL RESEARCH

Investigation of drugs affecting hypertension in bevacizumabtreated patients and examination of the impact on the therapeutic
effectKenta Yagi1| Marin Mitstui2 | Yoshito Zamami2,3 | Takahiro Niimura2 |Yuki Izawa-Ishizawa4 | Mitsuhiro Goda3 | Masayuki Chuma1 | Kimiko Fukunaga2
Takahiro Shibata3 | Shunsuke Ishida3 | Takumi Sakurada3 | Naoto Okada3|
3
4
4
Hirofumi Hamano | Yuya Horinouchi | Yasumasa Ikeda | Hiroaki Yanagawa1 |Keisuke Ishizawa2,3
1

Clinical Research Center for
Developmental Therapeutics, Tokushima
University Hospital, Tokushima, Japan2

Department of Clinical Pharmacology
and Therapeutics, University ofTokushima Graduate School of
Biomedical Sciences, Tokushima, Japan3

Department of Pharmacy, Tokushima
University Hospital, Tokushima, Japan
4

Department of Pharmacology, Universityof Tokushima Graduate School of
Biomedical Sciences, Tokushima, Japan
Correspondence
Yoshito Zamami, Department of ClinicalPharmacology and Therapeutics,
Tokushima University Graduate Schoolof Biomedical Sciences, Tokushima,
Japan, 2-50-1 Kuramoto-cho, Tokushima
770-8503, Japan.
Email: zamami@tokushima-u.ac.jp
Funding information
This research was supported by theJapan Research Foundation for ClinicalPharmacology Grant (Grant number
2018A10) and the Japan Society for the
Promotion of Science (JSPS) KAKENHI(Grant number 18K06785)|

Abstract
Background: In patients treated with bevacizumab, hypertension may be a biomarkerof therapeutic efficacy. However, it is not clear whether drugs that control blood pressure influence bevacizumab's efficacy. In this study, we investigated drugs that may
affect hypertension in bevacizumab-treated patients and examined the impact on thetherapeutic effect.
Patients and methods: We analyzed 3,724,555 reports from the third quarter of2010 to the second quarter of 2015. All data were obtained from the Food and Drug
Administration (FDA) Adverse Event Reporting System (FAERS) analysis. In thisretrospective cohort study, we investigated a total of 58 patients diagnosed with colorectal cancer and treated for the first time with bevacizumab containing XELOX or
mFOLFOX6 at The University of Tokushima Hospital between January 2010 and
December 2015. The effect of the treatment was evaluated according to Response
Evaluation Criteria in Solid Tumors version 1.0. Thereafter, the effect was confirmed
using Gene Expression Omnibus (GEO) and cultured cells.
Results: There are few reports in FAERS of hypertension in patients treated withomeprazole on bevacizumab. Based on the chart review, patients who used proton
pump inhibitors (PPI) had a lower response to treatment than those who did not (response rate: 25% vs 50%). Furthermore, experiments on GEO and cell lines suggested
that induction of vascular endothelial growth factor (VEGF) gene expression by PPIs
is the cause of the reduced therapeutic effect.
Conclusion: PPIs prevent hypertension in bevacizumab-treated patients but may reduce bevacizumab's anti-tumoral effects by inducing VEGF expression.
KEYWORDS
bevacizumab, colorectal cancer, FAERS, gene expression omnibus, hypertension, proton pump inhibitor

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original
work is properly cited.
© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
164

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Cancer Medicine. 2021;10:164–172.

  

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1|

IN T RO D U C T IO N

Bevacizumab is a humanized monoclonal antibody against
the vascular endothelial growth factor (VEGF) that inhibits
tumor growth by inhibiting angiogenesis. It has shown antitumor effects in several cancer types, including colorectal
cancer, breast cancer, non-small cell lung cancer, renal cell
carcinoma, ovarian cancer, glioblastoma, and metastatic melanoma.1–3 Bevacizumab has been used in combination withchemotherapy, and has been reported to prolong both overall
survival and progression-free survival.4–6 However, bevacizumab causes a variety of adverse events, one of which is
hypertension. One clinical study reported that after bevacizumab treatment, 49.3% of patients had induced-grade 2, or
higher hypertension.3
Hypertension induced by bevacizumab administration isthought to occur due to decreased endothelial cell regenerative capacity and decreased production of vasodilators, suchas nitric oxide and prostacyclin.7 VEGF inhibition leads to
the reduction of nitric oxide synthesis, thus reducing the levels of nitric oxide in the body,7 which in turn leads to constriction of the blood vessels, and a reduction in sodium ion
renal excretion, ultimately resulting in hypertension.8 The
development of bevacizumab-induced hypertension is one of
the most important problems in continuing the bevacizumab
treatment, and even deaths have been reported due to thedevelopment of hypertension. If bevacizumab-induced hypertension develops, bevacizumab should be stopped immediately until blood pressure control is achieved.9–11
However, several retrospective studies have suggested thatamong patients treated with bevacizumab, those who developed hypertension had a better prognosis than those who did
not.8,12–15 Indeed, in patients with metastatic colorectal cancer
who received combination chemotherapy with bevacizumab,
overall response, progression-free survival, and overall survival were associated with the emergence of hypertension.13
These studies suggest that the appearance of hypertension
may be a biomarker of treatment efficacy in patients treatedwith bevacizumab. In addition, it is possible that drugs reducing the occurrence of hypertension in patients receiving
bevacizumab might have a negative effect on the efficacy ofbevacizumab treatment, however, their associations are currently not clear. Most patients treated with bevacizumab use
several concomitant drugs. Among these concomitant drugs,
there might be several drugs that could alter the blood pressure, but to the best of our knowledge, there are no studies on
the interactions of these drug combinations. This could be
probably explained partly by the fact that concomitant medications are so varied that it would be difficult to adequately
study them in studies involving a small number of centers.
The Food and Drug Administration (FDA) Adverse
Event Reporting System (FAERS) is the world's largest database created by the U.S. FDA to monitor the safety risks| 165

of approved drugs. Adverse events related to drug use arevoluntarily reported to FAERS by patients, physicians, pharmacists, and other health care providers. Currently, millionsof drug-related adverse events are registered in FAERS,
which in recent years has supported several academic studies on drug safety and development that are separate fromthe FDA’s objective of monitoring drug adverse events.
For example, FAERS analysis has shown that concomitantdrug B reduces the risk of adverse events caused by drug
A.16,17 Additionally, the Gene Expression Omnibus (GEO)
is a global data repository of next-generation sequencing,
microarray, and high-throughput functional genomics datasubmitted by research communities.18 It is also an integrated
database for systematic analyses of gene function and linking
gene information with higher-order functional information.19
We have previously analyzed large information databases,
such as FAERS and GEO to identify novel drug effects ofexisting drugs.20–22
These databases have enabled us to analyze the effectsof concomitant drugs, and to estimate the mechanisms of
their effects, which otherwise cannot be analyzed in a few
centers due to the inadequate number of cases. Therefore, inthis study, we aimed to investigate drugs that could alter theincidence of hypertension in bevacizumab-treated patientsand examine whether these drugs could affect the treatment
efficacy.

2|

RESULTS

2.1 | FAERS analysis for determining
the relationship between bevacizumab andhypertension
The FAERS database was used to investigate the occurrenceof hypertension in patients treated with bevacizumab. Of
36,95,466 patients, hypertension was reported in 85,079 patients and in 1,520 using bevacizumab. The reporting odds
ratio (ROR) for bevacizumab use and hypertension was 2.45,
95% confidence interval (CI): 2.33–2.59, p < 0.001, which indicated a strong relationship. A similar relationship was foundwhen the patients were stratified by sex and age (Table 1).
Bevacizumab was found to be associated with an increased
incidence of hypertension in FAERS, regardless of age or sex.

2.2 | FAERS-based search for drugs
reducing hypertension in patients treated with
bevacizumab
The FAERS was used to identify concomitant drugs thatwere associated with a reduced incidence of hypertension inpatients treated with bevacizumab. Epirubicin, Erlotinib, and

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omeprazole were found to be associated with a lower rate of
hypertension in patients treated with bevacizumab (Table 2)in the FAERS analysis. However, among them, patients using
omeprazole reported the highest incidence of hypertension.

2.3 | Demographic and clinicalcharacteristics of patients by examining their
medical records
Between January 2010 and December 2015, 62 patientsfrom The Tokushima University Hospital were treated with
bevacizumab in combination with XEROX or mFOLFOX6,
among whom 58 were included in the analysis. Table 3 shows
the patients’ characteristics in the PPI (n = 16) and non-PPI(n = 42) groups. There were no significant differences between the two groups regarding median age at diagnosis, sex,
performance status (PS), histopathological staging, concomitant regimens, or concomitant antihypertensive medications(Table 3).

In both groups, the blood pressure increased after bevacizumab treatment. The change in mean systolic blood pressure
before and after bevacizumab treatment was 13.55 mmHg in
the non-PPI group and 13.94 mmHg in the PPI group, withno significant difference (Table 4).

2.5 | Evaluating changes in signaling
pathways and gene expression associatedwith the pathway of hypertension using the
GEO and Kyoto Encyclopedia of Genes andGenomes (KEGG) databases
To investigate the mechanism of effect of bevacizumab inpatients using omeprazole, we analyzed a GEO-deposited
toxicogenomics microarray dataset (GEO Association No.
GSE59927). Using these data sets, we analyzed the differences in gene expression in rats treated orally with omeprazole (30 mg/kg, for 5 days) (Table 5). GEO analysis showedthat VEGF gene expression was upregulated in the rat livers
after oral omeprazole treatment.

2.4 | Influence of PPI-use on theeffectiveness of chemotherapy, including
bevacizumab

2.6 | Effect of PPI on VEGFA expression
using human cell lines

Response rates were measured. No patient had complete
response (CR), 25 had partial response (PR), 24 had stabledisease (SD), and 9 had progressive disease (PD). The response rate was 50% in the non-PPI group and 25% in the PPI
group; objective response rate (ORR) was 25% higher in the
non-PPI group than that in the PPI group, with no significant
difference. The disease control rate was 50% in the non-PPI
group and 25% in the PPI group. The disease control rate of
the non-PPI group was 21% higher than the PPI group but notsignificant (Table 3).

The influence of PPI on VEGF gene expression was evaluated using human vascular endothelial cells (HUVEC).
Exposure of PPIs (omeprazole, rabeprazole, lansoprazole,
pantoprazole, and esomeprazole; 100 μM) increased cellproliferation significantly in HUVEC, as did the exposureof VEGF. However, there were no significant increases with
exposure of famotidine. Thus, the effect of PPI on VEGF-A
mRNA expression was then evaluated using LS174T cells.
VEGF-A mRNA expression was significantly increased
by PPI exposure in LS174T cells. However, there was no

TABLE 1

Association between bevacizumab and the occurrence of hypertension in FDA Adverse Event Reporting System.

Group

Hypertension without
bevacizumab (%)

Hypertension with
bevacizumab (%)

ROR

95%CI

p-value

Total

83559/3695466 (2.26)

1520/28288 (5.37)

2.45

2.33-2.59<0.001

Male

28076/1274753 (2.2)

414/11466 (3.61)

1.66

1.51-1.84<0.001

Female51603/2090097 (2.47)

776/13658 (5.68)

2.38

2.21-2.56< 0.001<40

8342/527869 (1.58)

60/1232 (4.87)

3.19

2.46-4.14<0.00140~47758/321558 (2.41)

104/1998 (5.21)

2.22

1.82-2.71<0.00150~5913054/470547 (2.77)

214/4062 (5.27)

1.95

1.7-2.24<0.00160~6914250/472096 (3.02)

299/5476 (5.46)

1.86

1.65-2.09<0.00170~10015478/529240 (2.92)

248/4663 (5.32)

1.86

1.64-2.12<0.001Stratified by gender

Stratified by age (y)  YAGI et al.

TABLE 2

Concomitait drugs that decrease occurrence of bevacizumab-induced hypertension in FDA Adverse Event Reporting System

Drug B

Hypertension without drug
B (%)

Hypertension with drug
B (%)

ROR

95% CI

p-value

EPIRUBICIN

1512/27859 (5.43)

8/429 (1.86)

0.33

0.16-0.67<0.001ERLOTINIB

1486/27390 (5.43)

34/898 (3.79)

0.69

0.49-0.97

0.035

OMEPRAZOLE

1502/27692 (5.42)

18/596 (3.02)

0.54

0.34-0.87

0.008

significant increase in exposure of famotidine (Figure 1).
Cobalt Chloride (CoCl2) was used as positive control.

T A B L E 3 Baseline patient characteristics and Responses
Categories in the RECIST.

Median age, y(range)Non- PPI group(n = 42)

PPI group(n = 16)

p- value68 (40-86)

62 (42-82)

0.188aGender, n (%)

0.5765bMale

21 (50)10 (63)

Female21 (50)6 (38)

Ⅰ

1 (2)

0

Ⅱ

1 (2)

0

Ⅲa

5 (12)1 (6)Ⅳ

35 (83)15 (94)0

21 (50)8 (50)

1

23 (48)7 (44)

2

0 (2)

1 (6)

XELOX

21 (42)12 (75)mFOLFOX6

21 (42)4 (25)Use

7 (17)

3 (19)

Non-use35 (83)13 (81)

CR

0

0

PR

21

4

SD

17

7

PD

4

5

Objective
control rate

50%

25%

0.138cDisease control
rate

90%

69%

0.097cStage

ECOG
Performance
Status, n (%)

Combined
regimesAnti
hypertensive

0.8241c

0.7633b0.1551b

1c

Response

Abbreviations: CR, complete response; PD, progression dessease; PR, partial
response; SD, stable disease.
aMann-Whitney U test.

b
c

| 167

Chi-square test.

Fisher's exact test.

3|

DISCUSSION

In this study, we identified drugs that reduced the occurrence of hypertension in patients treated with bevacizumab
and then investigated whether these drugs could reduce
the efficacy of bevacizumab. We used FAERS to identify
that among patients treated with bevacizumab, there werefewer reports of hypertension in patients taking omeprazole. However, the other PPIs did not show an association
with the reduction in the occurrence of hypertension in patients treated with bevacizumab. This may be due to thelong period of time since omeprazole was approved andthe high number of reports compared with the number ofadverse event reports for other PPIs in the FAERS database. For large databases such as FAERS, it is difficult
to achieve unsupervised collection results and some data
may be inaccurate.20,21 Furthermore, there are no data on
individual patient characteristics. Therefore, we retrospectively investigated the medical records of patients in our
hospital.
In the clinical records study, we examined the association
between the use of PPIs and response rates. There was a 25%(though not statistically significant) reduction in the ORR
and 21% reduction in the disease control rate in patients using
PPIs. Thus, there was a reduction in the response to bevacizumab combination chemotherapy. There were no significant
differences between the two groups for factors such as stage,PS, or combination regimen.
Although FAERS analysis showed a reduced rate of bevacizumab-induced hypertension in patients using PPIs, butthere was no significant reduction in the chart reviews. Thismay be because many patients in our hospital had developed
hypertension before bevacizumab was administered and werealready satisfactorily treated with antihypertensive drugs.
Furthermore, the PPI group might have had lower numbersof hypertensive patients, which may have prevented themfrom achieving significant results. This study suggests that
PPI not only reduces the incidence of hypertension in patientstreated with bevacizumab but also reduces the efficacy of thisdrug. However, this study did not examine the duration of PPI

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YAGI et al.

mean systolic blood pressure (SD)before treatment withbevasizumab [mmHg]

after treatment withbevasizumab [mmHg]

p- value

Non-PPI group

130 (15.33)

143 (19.55)

0.979a

PPI group

120 (5.39)

134 (4.56)

T A B L E 4 Systolic blood pressure
before and after bevacizumab treatment in
non-proton pump inhibitors (PPI) and PPIgroups.

aMann-Whitney U test.

T A B L E 5 Gene Expression Omnibus data showing the effects ofomeprazole on the expression levels of vascular endothelial growth
factor signaling-related genes.
Gene title

Omeprazole

Corn Oilp-valuevascular endothelial growth
factor

11.57

11.07

0.048

SPHK1 (sphingosine kinasetype 1) interacting protein(Hs.) (DBSS)

5.05

5.69

0.003

neuroblastoma RAS viral(v-ras) oncogene homolog

7.31

7.53

0.006

v-raf-1 murine leukemia viraloncogene homolog 1

10.52

11.01

0.012

nuclear factor of activatedT-cells, cytoplasmic,
calcineurin-dependent 4

4.44

6.54

0.000

ras-related C3 botulinum
toxin substrate 1

10.79

10.43

0.005

nitric oxide synthase (DBSS)

5.23

5.78

0.001

n = 32 for vehicle; n = 3 for omeprazole-treated group.

dosing, and further investigation is needed to determine theextent to which PPI-use influences the effects and adverse
events of bevacizumab.
We investigated the molecular mechanisms by which the
combination of PPI and bevacizumab was found to be associated with poor antitumor effect. We analyzed the GEO databases and found that PPI induces VEGF gene expression.
It has also been previously suggested that drugs that inhibit
VEGF expression reduce VEGF-induced angiogenesis.23
This suggests that PPI might reduce the therapeutic effect ofbevacizumab by inducing the expression of VEGF.
Therefore, we then investigated the effects of PPIs on
tumor growth and angiogenesis using the human cell lines.
We found that PPIs promoted cell proliferation in the vascular endothelial cell line HUVEC. The inhibition of angiogenesis was related to the mechanism of tumor growth
inhibition by bevacizumab,24,25 suggesting that the promotion of angiogenesis by PPIs in tumor tissues might have led
to a lower response rate in patients using PPI. Next, using
the colon cancer cell line LS174T, we revealed that PPIs
promoted the expression of the VEGF mRNA expression
in the tumor cells. These results indicated that PPI induced

VEGF expression in tumor cells might have promoted theangiogenesis and cancer cell growth. This could be themechanism that led to the reduced therapeutic efficacy ofbevacizumab in the medical records study. This effect may
also vary according to cancer type and should be further
investigated for other types of cancer. However, it has beenreported that PPIs have an enhanced antitumor effect in pancreatic cancer.26 Therefore, for anti-tumor agents other thanbevacizumab, PPIs may have a synergistic effect by mechanisms other than VEGF.
There are possible limitations in this study. FAERS analysis
includes reporting bias and unmeasured confounding factors.
Furthermore, not all adverse events observed in clinical practice are included in the database. Under reporting or selective
reporting might be included. Because the FAERS database
contains missing data, incorrect drug names, and duplicate
data, we removed or corrected such data before the analysis.
The FAERS analysis showed that some variables were also
limited. We also did not consider race, duration of treatment,
drug dosage, or co-administration of other medications.
Although hypertension may have occurred prior to treatment
as an underlying condition, the treatment time and the othermedications were not listed in the FAERS database. Whilethese aforementioned factors might affect the results, the useof self-reporting systems can be useful in detecting potential
drug signals. Moreover this was a single-center study and waslimited to a specific type of cancer, thus, a multicenter study
with a larger cohort is required. Additionally, the relationshipbetween the PPI and the treatment effect of bevacizumab has
not been examined because not enough cases have been collected for evaluation. Therefore, further studies are needed toconclude the possibility of PPIs decreasing the therapeutic effect of bevacizumab. Furthermore, further studies, including
multicenter and randomized trials, may be considered.
However, we should never dismiss the use of PPIs. PPIs
are very important in many diseases such as gastric ulcers
and reflux esophagitis. However, the long-term use of PPIs
may be a risk factor for stomach cancer,27,28 and shorter prognosis for lung cancer patients receiving immune checkpoint
inhibitors.29,30 Thus, although PPIs are currently effective ingastric conditions and are frequently used, they may not be
used properly.31,32 It may be necessary to avoid long-term
administration of PPI which should be administered to only
those who truly require it.

  

YAGI et al.

Cell viability(fold change)2
1.5** ** **** **B

H-VEGFA mRNA
(fold change)

A

**

0.5
0

***

2

***

**

1.5

1

| 169

1

0.5

Cont VEGF Ome
100ng/ml

Rab

Lan

Pan

Eso

Fam

0

PPI (10 μM)

Cont

Eso

Pan

Ome

Fam

CoCl2

PPI (10 μM)

F I G U R E 1 Effect of proton pump inhibitors to cell survival and promotion of angiogenesis. (A) Cell viability in HUVEC(n = 5) and(B) h-VEGFA mRNA expression in LS174T(n = 7) treated with PPIs or Famotidine or CoCl2 at 100 µM for 24 h. Ome; omeprazole, Eso;
esomeprazole, Pant; pantoprazole and Famo; famotidine. Data are expressed as mean fold change from controm±SEM (**p < 0.01, ***p < 0.001).

In conclusion, the use of PPIs might reduce the antitumor
activity of bevacizumab in patients receiving bevacizumab.

4
4.1

|

M ATE R IA L S A N D ME T HODS|

Analysis of the FAERS database

The FAERS data sets for the third quarter of 2010 to the second quarter of 2015 were obtained from the FDA website(https://www.fda.gov/Drugs/​Guida​nceCo​mplia​nceRe​gulat​
oryIn​forma​tion/Surve​illan​ce/Adver​seDru​gEffe​cts/ucm08​
2193.htm). Following the FDA’s recommended method, weused the most recent case numbers to exclude duplicate reports(out of a total of 4331802 reports) and analyzed the remaining
3724555 reports. The definition of hypertension was basedon the 10 terms from the Standardized Medical Dictionary
for Regulatory Activities (MedDRA) Queries [Hypertension](SMQ code: 20000147), published in MedDRA/J ver 19.0,
by the International Council for Harmonisation.
FAERS reports were classified into four groups: (a) received bevacizumab and reported hypertension, (b) received
bevacizumab but did not report hypertension, (c) did not receive bevacizumab but reported hypertension, and (d) did notreceive bevacizumab and did not report hypertension. The
ROR was used to signal detection and was calculated using
the following equation.16,33

Calculation for ROR: ROR = (a∕b) ∕ (c∕d) ,95%CI = exp (log (OR) ± 1.96 square 1∕a + 1∕b + 1∕c + 1∕d)
Letters a, b, c, and d indicate the number of individuals ineach group. If the lower limit of the 95% CI was >1, it was
assumed that there was a relationship between the drug useof interest and hypertension report. Conversely, if the upper

limit of the 95% CI <1, we assumed that there was an association between the drug use of interest and reduced hypertension report.

4.2| Analysis of the medical chart review

In this retrospective study, we investigated patients’ medical records. This data was collected from the medical records of patients who visited our hospital. Patients aged
≥18 years who were diagnosed with colorectal cancer at The
Tokushima University Hospital between January 2010 and
December 2015 and were treated with bevacizumab combination XELOX, or mFOLFOX6 for the first time, and patientswith evaluated lesions using Response Evaluation Criteria in
Solid Tumors (RECIST) were included. Patients who weretreated with <1 course of bevacizumab, failed to follow-up,
and had missing values were excluded. This study was approved by the Ethics Committee of Tokushima University
Hospital (Approval Number: 2962-1).
The following data were extracted from patient charts: age
at diagnosis, sex, cancer type, stage at diagnosis (using the
6th edition AJCC system), Eastern Cooperative Oncology
Group PS, bevacizumab dose, treatment duration, blood pressure, and PPI-use. Patients were considered to have received
concomitant PPIs if they had been prescribed PPIs continuously during the bevacizumab treatment period. Blood pressure was measured by the nurse prior to administration of
bevacizumab for each cycle. The event of hypertension inpatients receiving bevacizumab was defined according to
the Common Terminology Criteria for Adverse Events v 4.0(CTCAE v 4.0): an increase in blood pressure of <20 mmHg,
an increase in blood pressure to ≥150/100 mmHg, or hypertension requiring initiation or increase in therapeutic medication. Those who were taking antihypertensive medication

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|   

before bevacizumab treatment were considered “taking antihypertensive medication” and those who started antihypertensive medication after bevacizumab treatment wereconsidered “not taking antihypertensive medication.” Thestage of cancer was extracted from the medical records, andrelapse cases were considered as stage IV.
Treatment response was assessed according to RECIST
version 1.0 as follows: CR, PR, SD, and PD. The primary
endpoint was assessed using the ORR, which is the numberof patients achieving CR or PR divided by the number of patients evaluated. Disease control was defined as the numberof patients achieving CR, PR, and SD divided by the numberof patients assessed.

4.3 | Analysis of the GEO and KEGG
PATHWAY databases
Microarray gene expression data were obtained from the
National Center for Biotechnology Information (NCBI) GEO
database (http://www.ncbi.nlm.nih.gov/geo/) (accession
number GSE59927).We analyzed expression data using samples analyzed in the livers of rats receiving oral omeprazole(30 mg/kg, once daily/5 days) or vehicle (carboxymethylcellulose), focusing on genes involved in the VEGF signaling
pathway extracted from the KEGG PATHWAY database(entry number: MAP04370 http://www.genome.jp/dbget​-bin/www_bget?pathw​ay:map04370). The expression levelsof each gene were shown in relation to controls by inverting
the log2-transformed microarray data to the original scale.
We chose a rat model and not a human model for thisanalysis because in reality, the patients were treated with anumber of drugs in combination. Thus, it was ethically difficult to administer PPIs alone to humans, and to obtain tissue
samples for comparison before and after PPI administration.
Therefore, to exclude the effects of concomitant drugs, invivo experiments were needed to investigate the effects ofthese drugs.

4.4 | Real-time PCR and cellproliferation assay
The LS174T (human colon cancer cells) and HUVEC were
cultured in α-MEM supplemented with 5% fetal bovine
serum and 10 mg/mL of 1% (v/v) penicillin streptomycin(Sigma Aldrich, MO, USA). All cells were incubated at 37°C
in a humidified atmosphere containing 5% (v/v) CO2.
HUVEC were cultured in 96-well plates and then exposed
to PPI: omeprazole, esomeprazole, pantoprazole, rabeprazole, lansoprazole (100 μM), famotidine (100 μM), CoCl2(100 μM), and DMSO (0.1% v/v) for 24 hours, followed byincubation at 37°C in a humidified atmosphere containing

YAGI et al.

5% (v/v) CO2. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H
tetrazolium bromide (MTT) was then added, and the cell proliferation rate was measured.
LS174T cells were used to investigate gene expression.
Cells were seeded in 6-well plates and incubated for 24 hours,
then exposed to PPI: esomeprazole, pantoprazole, omeprazole, (10 μM), famotidine (100 μM), CoCl2 (100 μM), andDMSO (0.1% v/v) for 24 hours. Total RNA was extracted
from cells using ISOGEN (NIPPON GENE) according to
the manufacturer's protocol. Total RNA was reverse transcribed to cDNA using Prime ScriptTM RT reagent Kit with
gDNA Eraser (TaKaRa). The cDNA product was used as atemplate for Polymerase Chain Reaction (PCR) amplification
in a total volume of 10 µl, and the PCR condition were as
follows: 95°C for 2 minutes, followed by 40 cycles at 95°C
for 5 seconds, 60°C for 30 seconds, and 95°C for 5 seconds.
Reactions were conducted on a CFX96 Real-time PCR
Detection System (Bio-Rad). All data were analyzed with the
CFX Manager Software (Bio-Rad). The data were analyzed
using the delta cycle threshold method and calculated basedon the Cq values; the expression of each gene was normalized
to 36B4. Gene-specific primer pairs were used: h-VEGFA:(F) 5'-AGGAGGAGGGCAGAATCATC-3’, (R) 5'-ATGTCCACCAGGGTCTCGAT-3’; 36B4: (F) 5'-GCTCCAAGCAGATGCAGCA-3’, (R) 5'-CCGGATGTGAGGCAGCAG-3'.

4.5| Statistical analysis

For FAERS analysis, Microsoft Access 2013 was used for the
FAERS database construction and R ver 3.2.1 (R Foundationfor Statistical Computing, Vienna, Austria) was used for statistical analysis using the Fisher's exact test. For the retrospective chart review, the Mann-Whitney U-test was used as
the distribution was not normal. Differences between categorical values were estimated using the chi-square or Fisher's
exact test for comparisons with cells <5. Data obtained fromthe pathway analysis of RT-PCR experiments were performed using the Dunnett's test. All recorded P values were
two-sided, and P values <0.05 were considered significant.
The continuous variable case records are shown as mean
± standard deviation. The demographic characteristics were
evaluated with descriptive statistics. Student's t test with
Welch's correction was used when the data distribution was
normal, and the Mann-Whitney U-test was used when the
distribution was not normal.
ACKNOWLEDGMENTS
The authors thank the support received from the
Department of Clinical Pharmacology and Therapeutics,
Tokushima University Graduate School of Biomedical
Sciences, Tokushima, Japan and are grateful to Saito H
and Moriguchi H for helpful discussions. Additionally, the  

YAGI et al.

authors also thank Editage (www.edita​ge.com) for English
language editing.

9.

CONFLICT OF INTEREST
The authors have no conflicts of interest directly relevant tothe content of this article.

10.

AUTHOR CONTRIBUTIONSYZ is the principal investigator and guarantor of the paper,
had full access to all the data in the study, and takes responsibility for the integrity of the data and the accuracy of thedata analysis. KY designed the research and drafted the manuscript. MM, TN, YI, MG, MC, KF, TS, SI, TS, NO, HH,
YH, YI, HY, and KI contributed to the acquisition, analysis,or interpretation of data and critically reviewed and revised
the article for important intellectual content. All authors approved the final manuscript and decided to submit the article
for publication.
ORCID
Kenta Yagi https://orcid.org/0000-0003-0869-5786
Naoto Okada https://orcid.org/0000-0003-2476-9191

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How to cite this article: Yagi K, Mitstui M, Zamami
Y, et al. Investigation of drugs affecting hypertension
in bevacizumab-treated patients and examination of
the impact on the therapeutic effect. Cancer Med.
2021;10:164–172. https://doi.org/10.1002/cam4.3587