Berberine Can Target the VEGFR2/ERK Pathway to Inhibit

Angiogenesis in Glioblastoma Xenografts

Huaze Liu

Northeast Yucai School, Shenyang, Liaoning, 110179, China

Keywords:

Glioblastoma, VEGFR2/ERK Pathway, Berberine.

Abstract: The purpose of this article is to investigate the concrete effect of berberine on angiogenesis of glioblastoma

cells. The glioblastoma is the most malignant cancer with high mortality and it is also difficult to treat with

modern medical methods. The fractionated radiotherapy, interstitial radiotherapy or stereotactic radiosurgery

all have unstable effects on tumor. Previous study has reported that berberine can inhibit the angiogenesis in

glioblastoma xenografts through targeting the VEGFR2/ERK pathway. And this study investigates the

concrete effect of berberine on angiogenesis of glioblastoma cells. In the hypothetical experiment, we will

treat glioblastoma xenograft mice with increasing amounts of injected Berberine (0, 0.1, 0.3, 1 mg/ml, 10

mg/ml) for 0 min, 1 day, 7 days, 2 weeks and then measure tumor weight and size, and perform confocal

microscopy/ immunohistochemistry (IHC) for VEGFR2, or phospho-ERK and unphosphorylate ERK. The

experiment will also measure VEGFR2, phosphorylate ERK, unphosphorylate ERK by western blot. Positive

control is Temozolomide treatment.There are three most possible results: (1) Berberine inhibit the

angiogenesis of glioblastoma cells in both in vitro and in vivo cell lines; (2) berberine can only inhibit the

angiogenesis of glioblastoma cells in in vitro cell cultures; (3) berberine only inhibit the angiogenesis of

glioblastoma cells in determined human and murine glioblastoma. In conclusion, the results of this study will

provide important information for future clinical trials of berberine treatment. Future studies should focus on

improving in vivo delivery methods, finding more inhibitors of the angiogenic pathway in glioblastoma, and

exploring in detail the specific mechanisms of berberine.

1 INTRODUCTION

Malignant tumor is a potential killer of human life and

seriously endangers people's healthy life. According

to the report of World Health Organization, the

incidence and mortality rate of malignant tumors are

gradually increasing all over the world. 10 million

new malignant tumors occur each year, and 6-7

million people die from this disease, accounting for

12% of the total, which becomes the second cause of

human death. The main types of malignant tumors are

lung cancer, stomach cancer, liver cancer, colorectal

cancer and breast cancer, and their causative factors

include environmental and life factors such as air

pollution, smoking, poor living habits, food additives

and drug abuse. In recent years, with the development

of science and technology, chemotherapy for tumors

has made some progress, and the life expectancy of

tumor patients has been significantly extended, such

as in leukemia, malignant lymphoma, etc., but there

is still no effective approach for most solid tumors.

Scientific researchers have gradually realized that in

order to make a breakthrough, we must start from the

mechanism of tumor development in order to solve

the problem at root. Anti-tumor drugs are gradually

moving from traditional cytotoxic drugs to multi-

targeted drugs and exploring various new drugs. The

development of new natural drugs.

Glioblastoma is one of the most malignant

astrocytomas. The tumor is located in the subcortical

region and most grows over the cerebral hemispheres.

It usually invades several lobes and deep structures of

the brain. It can also spread through the corpus

callosum to the contralateral cerebral hemisphere.

Meanwhile, glioblastoma is the most common

primary brain tumor in adults, with an annual

incidence of 52.6 per 1 million people and

approximately 17,000 new cases diagnosed each

year. The etiology of most cases of glioblastoma and

its prevention have not been clarified. Rare risk

factors include genetic disorders (e.g.,

Liu, H.

Berberine Can Target the VEGFR2/ERK Pathway to Inhibit Angiogenesis in Glioblastoma Xenografts.

DOI: 10.5220/0011313500003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 951-956

ISBN: 978-989-758-595-1

951

neurofibromatosis and Li-Fermini syndrome) and

chemotherapy.

Angiogenesis is a process in which

physiologically new microvessels develop into a

blood supply system. Whereas Vasculogenesis

usually refers to spontaneous blood vessel formation,

Intussusception refers to the more general process of

rapid formation of new blood vessels. This process is

common in human growth and development, and in

wound healing. In addition, angiogenesis is an

important step in tumor progression, and in tumor

growth, it can be the key to the transformation of a

tumor from dormant to malignant, rapidly growing,

and potentially invasive to other tissues.

Neovascularization is an important way for tumors to

obtain nutrients to ensure their own development, and

tumor angiogenesis is the result of the combination of

multiple factors induced by tumors. Tumor

angiogenesis is the result of the combined action of

multiple factors in the tumor-induced body, mainly

promoters and inhibitors, which are in a balanced

state under normal conditions. Among the various

promoting factors, vascular endothelial VEGF is an

important regulator of angiogenesis, and its receptors

(VEGFR) are mainly VEGFR-1, VEGFR-2 and

VEGFR-33. It has been suggested that inhibition of

angiogenesis is a powerful strategy for cancer

treatment, and when one of these factors is affected,

angiogenesis will not occur properly. induced

proliferation, migration, invasion and duct formation

at sub-toxic doses. In addition, HDT significantly

inhibited the in vivo production of villi allantoic

membranes without showing cytotoxicity.

Furthermore, HDT reduced not only VEGFR2

signaling in HUVECs but also hypoxia-inducible

factor (HIF)-1 expression in hepatocellular

carcinoma. The currently investigated antitumor

peptide drugs in inhibiting mechanism of

angiogenesis is a hot topic of research for most

scholars. NT4 was shown to have important effects

on endothelial cell proliferation, migration, and tube

formation, especially when induced by FGF2 and

coagulation. when induced by FGF2 and thrombin. In

addition, NT4 has an important role in the migration

and invasion of aggressive tumor cells. Therefore, the

anti-angiogenic mechanism of anti-tumor peptides

provides clues for their development as tumor-

targeting drugs. The anti-angiogenic mechanism of

antitumor peptides thus provides clues for their

development as tumor-targeting agents.

EGFR is a transmembrane receptor that, when

bound to a ligand, phosphorylates and binds some

intracellular adapter molecules or forms homo- or

heterodimers with other receptors, thereby activating

a series of downstream signaling pathways that lead

to cell proliferation, apoptosis, invasion, and

metastasis. Several solid tumors are known to occur

in association with aberrant activation of EGFR in

tumor tissue. Gefitinib competitively binds to the Mg-

ATP site in the EGFR-TK catalytic region on the cell

surface, blocking intracellular signaling, thereby

inhibiting cell proliferation and metastasis and

producing an anti-tumor effect. This inhibits cell

proliferation and metastasis, resulting in anti-tumor

effects. The drug was launched in February 2005 for

the treatment of locally advanced or metastatic non-

small cell lung cancer in patients who have received

prior chemotherapy or are unsuitable for

chemotherapy. It has also been shown to inhibit

microangiogenesis, modulate the cell cycle and

increase chemotherapy sensitivity, and in some areas

to potentiate the antitumor effects of cisplatin,

carboplatin, platinum oxalate, adriamycin, topotecan,

ralitrexed, paclitaxel, paclitaxel ester, glucosamine,

and interferon.

Alkaloid is a naturally occurring basic compound

that contains a nitrogen atom. Some compounds that

are chemically synthesized but structurally similar to

alkaloids are sometimes referred to as alkaloids. In

addition to C, H, and N, alkaloids can also contain O,

S, or other elements such as chlorine, bromine, and

phosphorus. Alkaloids are mostly derivatives of

amino acids and taste bitter and astringent. They are

often found as secondary metabolites in plants,

animals, and mushrooms. Most of the alkaloids can

be obtained from their plant extracts by acid-base

extraction. Among the plant derivatives with

biological properties, berberine, an isoquinoline

quaternary alkaloid isolated mainly from Huanglian,

has a wide range of therapeutic effects on a variety of

diseases. In recent years, berberine has been reported

to inhibit cell proliferation and be cytotoxic to cancer

cells. Therefore, many derivatives have been

synthesized to improve the efficiency and selectivity

of berberine.(Ortiz, Lombardi, Tillhon, Scovassi

2014). In this study, we tested the inhibitory activity

of berberine on angiogenesis in cell-based

experiments and in a mouse xenograft model of

human glioblastoma, and clarified the involvement of

the VEGFR2/ERK pathway (Jin, Xie, Huang & Zhao

2018).

2 METHOD AND MATERIALS

I predict that Berberine inhibits angiogenesis in

glioblastoma xenografts by targeting the

VEGFR2/ERK pathway. I will treat glioblastoma

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

952

xenograft mice will increasing amounts of injected

Berberine (0, 0.1, 0.3, 1 mg/ml, 10 mg/ml) for 0 min,

1 day, 7 days, 2 weeks and then measure tumor weight

and size, and perform confocal microscopy/

immunohistochemistry (IHC) for VEGFR2, or

phospho-ERK and unphosphorylate ERK. Also

measure VEGFR2, phosphorylate ERK,

unphosphorylate ERK by western blot. Positive

control is Temozolomide treatment.

① Reagents

Berberine: Berberine powder was dissolved in

phosphate buffered saline (PBS), then sterilized using

a 0.22 μm pore filter and stored at 4 °C until use.

Temozolomide: Compounds were resuspended in

DMSO and stored at room temperature (O6BG) or -

20℃ (all others).

② Immunocytochemistry

Immunocytochemistry of VEGFR2, or phospho-

ERK and unphospho-ERK were performed as

described previously.

3 POSSIBLE RESULT

Possible Result 1: Berberine was applied to inhibit

the VGEFR2/ERK pathway in defined human and

murine glioblastoma cell lines, cell lines from

clinical samples and cell lines from in vivo animal

models. (Luan, 2020)

Berberine inhibits glioblastoma in all of the in

vitro and in vivo cell samples, decreasing VEGF-C or

VEGF-D binding to VEGFR2, and decreasing

activity of ERK outside the cells, as shown in table 1.

The proliferations of cell samples are inhibited

significantly. The animal experiments display that

berberine has therapeutic effect on angiogenesis of

glioblastoma cells, as shown in table 2. In the

simulation experiment, to investigate the possible

molecular mechanism of berberine-induced

inhibition of angiogenesis, this experiment will

analyze the protein expression of VEGFR2 and

MAPK pathways by Western blots. The total

expression of VEGFR2 will not change after

berberine treatment, while the phosphorylation of

VEGFR2 will be significantly reduced (p ˂ 0.001,

Figure 1). Likewise, phosphorylation of ERK and p38

will also reduce after berberine treatment (p ˂ 0.001

and p ˂ 0.01, respectively, Figure 1). (Jin, Xie, Huang

& Zhao 2018)

Figure 1: Molecular mechanisms involved in

antiangiogenic effect of berberine. Tumor tissue from

ectopic xenograft model was isolated and homogenized for

Western blot analysis. ***p< 0.001 vs. vehicle group, **p<

0.01 vs. vehicle group. N = 6 for each group (Jin, Xie,

Huang & Zhao 2018) .

Possible Result 2: Berberine was applied to

inhibit the VGEFR2/ERK pathway in defined

human and murine glioblastoma cell lines, cell

lines from clinical samples, but not in cell lines

from in vivo animal models. (Wang, Zhou, Xu,

Song, Qian, Lv, Luan 2019)

Berberine inhibits glioblastoma in all of the in

vitro cell samples, decreasing VEGF-C or VEGF-D

binding to VEGFR2, and decreasing activity of ERK

outside the cells, as shown in table 1. The

proliferations of in vitro cell samples are inhibited

significantly. However, the berberine does not

successfully decrease in vivo VEGF-C or VEGF-D

binding to VEGFR2 or decreasing activity of ERK

outside the cells, or the animal experiments do not

display a significant therapeutic effect of berberine

inhibits VEGFR2/ERK pathway of angiogenesis, as

shown in table 2.

Possible Result 3: Berberine was applied to

inhibit the VGEFR2/ERK pathway in identified

human and murine glioblastoma cell lines, but not

cell lines derived from clinical samples.

Berberine inhibits glioblastoma in CUTLL1,

HPBALL, NS2, and W44, as shown in table 1. The

proliferations of these in vitro cell samples are

inhibited significantly. However, the berberine does

not successfully decrease VEGF-C or VEGF-D

binding to VEGFR2, or decrease activity of ERK

outside the cells in the clinical samples. The animal

experiment involving the NOD-CID model will fail

as described in Possible Result 2 since this model has

glioblastoma cells from clinical samples, as shown in

table 2.

Possible Result 4: Application of berberine

inhibited the VGEFR2/ERK pathway in defined

human cell lines and clinical samples, but had no

effect on murine cell lines.

Berberine inhibits glioblastoma in CUTLL1,

HPBALL, and the majority of the clinical samples

Berberine Can Target the VEGFR2/ERK Pathway to Inhibit Angiogenesis in Glioblastoma Xenografts

953

and reduces VEGF-C or VEGF-D binding to

VEGFR2 and decrease activity of ERK outside the

cells, as shown in table 1. The proliferations of these

in vitro cell samples are inhibited significantly.

However, applying berberine to NS2, and W44 does

not successfully decrease the reduces VEGF-C or

VEGF-D binding to VEGFR2 and decrease activity

of ERK outside the cells. Since the animal model is

constructed using human cell lines, there are still

possibility that berberine will decrease in vivo VEGF-

C or VEGF-D binding to VEGFR2 and decrease

activity of ERK outside the cells in animal

experiments, as shown in table 2.

Possible Result 5: Application of berberine did

not inhibit the VGEFR2/ERK pathway in any cell

line.

The berberine does not significantly reduce

VEGF-C or VEGF-D binding to VEGFR2 and

decrease activity of ERK outside the cells in any cell

lines, as shown in table 1. The animal experiment will

not be successfully conducted in this scenario, as

shown in table 2. Additional Possible Results on

VEGFR2/ERK Pathway Different from Previous

Researches

Possible Results 6: Berberine inhibits the

VGEFR2/ERK pathway, but does not have effects

on angiogenesis levels. (Luan 2020)

The level of angiogenesis of glioblastoma

determined by VGEFR2/ERK pathway is low. The

cell proliferation decreases. However, the level of

angiogenesis of tumors does not change significantly,

as shown in table 1 and table 2.

Table 1: Possible Results.

Cell Line

Result Ⅰ

Result

Ⅱ

Result

Ⅲ

Result

Ⅳ

Result Ⅴ Result Ⅵ

Tumor size decreases with

Increasing

erberine

+ - - -

inhibition(+)

Negative

inhibition(-)

VEGFR WB level

+ - + -

reduction(-)

Phospho

-AKT decrease + +

- - Partly agree -

Note. “+” represents a significant decrease in cell proliferations. “-” represent not significantly different from negative control.

Table 2: Possible Results on Cell Proliferation.

Cell Line

Result Ⅰ Result Ⅱ

Result

Ⅲ

Result

Ⅳ

Result Ⅴ

Res

ult

Ⅵ

VEFG/HIF-1 ex

ression + + + + + +

enesis of HUVECs + + + + - -

Tumo

growth in vivo + + - - - -

PI3

athwa

YNYNY N

Note. “+” represents a significant decrease in cell proliferations. “-” represent not significantly different from negative control.

4 CONCLUSION

The Possible Results 1 are consistent with previous

studies on the effect of berberine on glioblastoma

angiogenesis. To gain insight into the structure and

function of berberine, the specific gene regulatory

mechanisms of berberine should be further

investigated. The relationship between berberine and

RNA interference with VGEFR should also be

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

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studied to investigate more specific pathways that can

be used to treat glioblastoma angiogenesis.

Preclinical testing on more complex and

representative animal models should also be

conducted before transitioning to clinical testing of

berberine treatment. Better delivery platforms, such

as nanodrugs or mechanisms involving endocytosis,

should also be applied in order to improve this

therapeutic approach.

What makes the failure of the experiments in vivo

in Possible Result 2 is most likely due to unsuccessful

delivery of berberine: either the cells in the animal did

not take up berberine or berberine was not maintained

in vivo long enough to perform its function. The final

result would indicate a high expression level of

berberine and a low expression level of berberine. In

order to improve this experiment, an efficient and

reliable delivery method should be developed. The

experiment can be repeated again with the traditional

retroviral infection method as it has proven successful

in previous experiments. The safety level of retroviral

gene therapy should be improved prior to clinical

trials. In addition, pharmacological angiogenic

inhibitors of glioblastoma could be developed for

berberine.

Only if the berberine reduces the activity of

angiogenesis in the tumor, the berberine treatment

targeting the VGEFR2/BRK pathway will potentially

have therapeutic effects and should be carried on to

clinical trial. Possible Result 3 suggests that berberine

does not qualify as a universal treatment for

glioblastoma because a subset of clinical samples do

not have abnormal gap expression or because they

have different types of gaps and angiogenic

mechanisms. This requires future studies to reassess

the relationship between berberine, the

VGEFR2/ERK pathway, and the general type of

glioblastoma.

The unlike Possible Results 4 and 5 indicate

potential systematic errors in the experimental

designs. Possible Result 4 indicate the berberine used

in this experiment cannot be applied on mice. The

redesign of potential drug will be required for future

studies on animal cell lines. Possible Results 5 is

likely to be caused by the offtarget of the berberine

used in this experiment.

The Possible Results 6 contradicts with the

current understanding of berberine’s effects on

VGEFR/ERK pathway. Result 6 indicates that an

alternative pathway that is crucial for the

angiogenesis of glioblastoma maintenance. Future

studies should use experiments like dual-luciferase

reporter assay to verify the relationship between

berberine and other potential glioblastoma

oncogenes.

In conclusion, white tyrosine kinases occupy a

very important position in the cell signaling pathway,

regulating a series of physiological and biochemical

processes such as cell growth, differentiation and

death. More than 50% of proto-oncogenes and

oncogenes are tyrosine kinases, and their abnormal

expression usually leads to disruption of cell

proliferation regulation, resulting in tumorigenesis.

More than 20 different families of receptor and non-

receptor tyrosine kinases have been used as targets for

antitumor drug screening, including epidermal

growth factor receptor (EGFR), vascular endothelial

growth factor receptor (VEGFR), platelet-derived

growth factor receptor (PDGFR), fibroblast growth

factor receptor (FGFR), insulin receptor (InsR), Src ,

Abl, etc. (representative drugs: Imatinib, Gefitinib,

Erlotinib, Exatinib, Sorafenib, Sunitinib, Crizotinib).

Recently, another challenging concept has been

proposed for molecularly targeted antitumor drug

therapy: the strategy of mul-tipletargeted tyrosine

kinase inhibition. Based on the complexity of tumor

development, the vast majority of tumors do not rely

on a single signaling pathway to maintain their

growth and survival; there are crossovers and

compensations between signaling pathways. Multi-

targeted drugs can achieve the dual function of

synergistic treatment and overcoming drug resistance

by inhibiting multiple signaling pathways or multiple

molecules upstream and downstream in one pathway

(representative drugs: lapatinib, afatinib, daclatinib,

axitinib, ceritinib, etc.).

Mechanism of tumor neovascularization

inhibition: Targeting VEGFR, FGFR, EGFR and

other receptor tyrosine kinase inhibitors with tumor

neovascularization-promoting effects represents

another important direction in antitumor targeted

drug research - inhibition of tumor

neovascularization. Blocking tumor

neovascularization to varying degrees can slow down

the growth of solid tumor tissue (representative drugs:

bevacizumab, sorafenib, sunitinib).

Stimulated by cancer cell growth, VEGF binds to

specific endothelial cell receptors, leading to

angiogenesis and the production of new blood vessels

to feed tumor tissue. VEGF inhibitors destroy tumor

tissue by blocking this process. Everolimus and

pazopanib are used clinically in the treatment of renal

cell carcinoma, and bevacizumab, which has the

structure of a human-derived antibody structural

region and the complementary decision region of a

murine-derived monoclonal antibody that binds

VEGF, is used in the treatment of non-small cell lung

Berberine Can Target the VEGFR2/ERK Pathway to Inhibit Angiogenesis in Glioblastoma Xenografts

955

cancer. Due to the tolerability of this class of drugs,

tumor recurrence and metastasis can occur after

discontinuation of the drug, rendering the treatment

ineffective. Adverse effects include increased blood

pressure, delayed wound healing, bleeding,

thrombosis, intestinal perforation, heart failure, and

heart disease. Some of these drugs currently interfere

with the regulation of the activity of other cellular

pathways. The mechanism of action is not well

understood.

In addition, HER-2 has a transmembrane tyrosine

kinase receptor, and its positive expression is closely

related to tumor cell development, progression and

prognosis, of which only the intracellular ligand-

binding region has tyrosine kinase activity. Her-2

oncogene amplification causes receptor

overexpression, activates the intracellular region and

phosphorylates at the tyrosine kinase site, activates

the downstream PI3K/Akt and MAPK pathways, and

regulates tumor cell proliferation, differentiation,

migration and apoptosis. Trastuzumab is a humanized

monoclonal antibody that binds to the Her-2

oncogene expression product P185 protein on tumor

cell membranes to produce anti-cancer effects.

Currently, it is mainly used for the treatment of lung

cancer, gastric cancer, breast cancer, ovarian cancer

and kidney cancer, etc. It has a wide spectrum of anti-

tumor, high efficiency and low toxicity. Due to the

emergence of tumor cell drug resistance

phenomenon, with the investigation of its anti-tumor

and tumor drug resistance reversal mechanism, the

screening of drug-resistant target markers will help to

develop combination drug regimens and provide a

new way out for Her-2-positive tumor patients.

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