|Year : 2019 | Volume
| Issue : 7 | Page : 1642-1646
Inhibition of the hypoxia-induced factor-1α and vascular endothelial growth factor expression through ginsenoside Rg3 in human gastric cancer cells
Bingqiang Li1, Guofeng Qu2
1 Department of General Surgery, Xuzhou Central Hospital, Affiliated to Medical College of Southeast University, Xuzhou, China
2 Department of Breast Surgery, Xuzhou Central Hospital, Affiliated to Medical College of Southeast University, Xuzhou, China
|Date of Submission||24-Jan-2017|
|Date of Decision||30-May-2017|
|Date of Acceptance||28-Nov-2019|
|Date of Web Publication||13-Jan-2020|
Dr. Guofeng Qu
Department of Breast Surgery, Xuzhou Central Hospital Affiliated to Medical College of Southeast University, Jiefang Road, Xuzhou 221009, Jiangsu Province
Source of Support: None, Conflict of Interest: None
Objective: The aim of this study is to probe in the inhibitory effects of ginsenoside Rg3 on the expression of hypoxia-induced factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in human gastric cancer cells.
Materials and Methods: Human gastric cancer BGC823 cells were divided into the control group and experiment group, and expression levels of HIF-1α and VEGF were detected by immunocytochemistry and Western blot after cells were cultured under hypoxia for different durations.
Results: Under hypoxia, expression of HIF-1α and VEGF in human gastric cancer BGC823 cells showed an increasing trend, and that was remarkably lower in experiment group than in the control group after applying Rg3, which was obvious at 12 and 24 h (P < 0.05).
Conclusion: Rg3 can inhibit expression of HIF-1α and VEGF in human gastric cancer cells and may influence abdominal implantation metastasis of gastric cancer through inhibiting its expression.
Keywords: Ginsenoside Rg3, human gastric cancer cell, hypoxia-induced factor-1α, vascular endothelial growth factor
|How to cite this article:|
Li B, Qu G. Inhibition of the hypoxia-induced factor-1α and vascular endothelial growth factor expression through ginsenoside Rg3 in human gastric cancer cells. J Can Res Ther 2019;15:1642-6
|How to cite this URL:|
Li B, Qu G. Inhibition of the hypoxia-induced factor-1α and vascular endothelial growth factor expression through ginsenoside Rg3 in human gastric cancer cells. J Can Res Ther [serial online] 2019 [cited 2020 Jan 27];15:1642-6. Available from: http://www.cancerjournal.net/text.asp?2019/15/7/1642/275567
| > Introduction|| |
Gastric cancer is one of the most common causes of cancer-related deaths in the world  with high rates of metastasis and recurrence. Tumor metastasis is a multi-step complicated process involving multiple factors, and changes in tumor microenvironment (including extracellular matrix, growth factor, chemokine, and matrix metalloprotein, etc.) play an important role in tumor cell metastasis. It is indicated in research that hypoxia can promote persistently high expression of hypoxia-induced factor-1α (HIF-1α) in tumor cells, while HIF-1α can promote the overexpression of vascular endothelial growth factor (VEGF), thus enhancing proliferation, migration, and vascular engineering of vascular endothelial cells, as well as influencing blood flow volume.
Ginsenoside Rg3 is one of the most active ingredients extracted from Ginseng, a traditional Chinese medicine. In recent years, it has been used extensively in the research field and clinical use of cancer treatment.,, Accumulating findings suggest that Rg3 can inhibit cancer cell growth, invasion, and metastasis, it also exhibits an anti-cancer effect in various tumors, such as lung cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, ovarian cancer, and bladder cancer. Preclinical studies have indicated that Rg3 inhibits tumor growth and angiogenesis through downregulating VEGF expression , and targeting hypoxia-induced multiple signaling pathways  and is also able to induce apoptosis in cancer cells.
This article focused on probing into the problem that whether Rg3 could inhibit HIF-1α and VEGF in human gastric cancer cells under hypoxia, to provide the foundation for the point that Rg3 could inhibit intra-abdominal implantation metastasis of gastric cancer.
| > Materials and Methods|| |
Ginsenoside Rg3 was purchased from Sigma and dissolved in distilled water at a concentration of 100 mmol/L. Rabbit polyclonal antibody to human HIF-1α and VEGF were bought from Proteintech; mouse anti-rabbit β-actin antibody was purchased from Elabscience; goat anti-rabbit IgG secondary antibody kit and DAB developing kit were obtained from Beyotime Biotech, China; RPMI1640 medium was purchased from HyClone; trypsin-EDTA and penicillin-streptomycin were provided by Solarbio; fetal bovine serum was provided by GIBICO; and total protein extraction kit was purchased from Omega.
Human gastric cancer cell line BGC823 was purchased from the tumor cell bank of Beijing Jinzijing Company. The cells were cultured in complete medium in 5% CO2 incubator at 37°C, and the passage was conducted every 3 days. Cells at the exponential phase were collected, counted, and transferred to a 60 mm culture dish to continue the culture under normoxia for 24 h. Cells were divided into control group and experiment group after stable cell adherence, PBS and Rg3 were added (with the final concentration of 100 μmol/ml), and cells were cultured in a hypoxic incubator (at 37°C, 1% CO2 and 5% CO2) for 2, 6, 12, and 24 h. Total proteins at different time points were reserved for subsequent use. Cells were grown on a 24-well plate, and the time and drug concentration were the same as above.
Cells plated on coverslips were fixed in 4% paraformaldehyde, incubated with Triton-10 for 20 min and with hydrogen peroxide for 15 min, blocked with serum working solution for 20 min, and incubated with primary antibody at 4°C overnight. The primary antibody was replaced by PBS as the negative control, and cells were incubated with secondary antibody for 30 min, followed by DAB developing, hematoxylin counterstaining, and mounting. HIF-1α expression located in the nucleus and cytoplasm, while VEGF expression located in cytoplasm and cell membrane, and distribution characteristic of positive cells was observed. Image-Pro Plus image automatic analysis system was employed; blank space calibration, together with five representative fields of view was selected under magnification of ×40; optical density (OD) values were determined for calculation, and the mean OD value of the selected fields of view was thus obtained.
Western blot analysis
The total protein extraction kit was adopted, and lysis buffer was prepared 30 min before extraction and was placed at 4°C for 5 min, followed by 30s of fierce oscillation for five loops. Cell debris and impurities were removed through centrifugation, the protein was subpackaged, and the OD value was determined by protein content detection kit; the protein was boiled at 10°C for 10 min, followed by loading for sodium dodecyl sulfate–polyacrylamide gel electrophoresis electrophoresis. ECL chemiluminescence developer was adopted and exposed. Band gray value was measured using Image J (National Institutes of Health, Bethesda, USA), with β-actin being the internal reference, and gray value ratio of the two at different time points was treated as the relative protein content.
SPSS version 17.0 software (SPSS Inc., Chicago, USA) was adopted for statistical analysis, means of two samples were compared with t-test, and means of multiple samples were compared using Chi-square test analysis. Statistical significance was defined as P < 0.05.
| > Results|| |
Expression of hypoxia-induced factor-1αand vascular endothelial growth factor in gastric cancer cells under hypoxia
The expression of HIF-1α and VEGF at different time points in the experiment group and control group showed that positive HIF-1α expression located in the nucleus and cytoplasm, which was claybank (data not shown). Protein expression quantity in the experiment group was remarkably reduced compared with that in control group after 2, 6, 12, and 24 h of culture under hypoxia, and the difference was of statistical significance [P < 0.05, [Table 1]. Positive VEGF expression located in cytoplasm and cell membrane, which was claybank and dominated by cytosolic expression (data not shown). Protein expression quantity in experiment group was notably reduced compared with that in control group after 2, 6, 12, and 24 h of culture under hypoxia, and the difference was of statistical significance [P < 0.05, [Table 2].
|Table 1: Expression levels of hypoxia induced factor-1α determined by immunocytochemistry in experiment and control groups (x̄±s)|
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|Table 2: Expression levels of vascular endothelial growth factor determined by immunocytochemistry in experiment and control groups (x̄±s)|
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Ginsenoside Rg3 inhibits expression of hypoxia-induced factor-1α and vascular endothelial growth factor by western blot analysis
Protein expression of HIF-1α and VEGF at different time points in the experiment group and control group was analyzed using ImageJ analysis software for gray value analysis and statistics. It could be discovered that protein expression quantity of HIF-1α in experiment group after 2, 6, 12, and 24 h of culture under hypoxia was markedly lowered than that in control group, with the difference being of statistical significance [P < 0.05, [Figure 1]a; while that of VEGF in experiment group after 2, 6, 12, and 24 h of culture under hypoxia was distinctly lowered than that in control group, with the difference being of statistical significance [P < 0.05, [Figure 1]b.
|Figure 1: Expression levels of hypoxia-induced factor-1α and vascular endothelial growth factor protein determined by Western blot in experiment and control groups. (a) Relative expression of hypoxia-induced factor-1α; (b) Relative expression of vascular endothelial growth factor. β-actin was used as internal control. Three independent experiments performed in duplicate; *P < 0.05|
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| > Discussion|| |
Metastasis and recurrence account for the major reasons for the high mortality of gastric cancer. The roles of key factors in metastasis have been increasingly recognized with the gradual deepening of research on tumor cell metastasis, which has provided a new direction for targeted and individualized drug therapy. It is found in research that novel tumor molecular markers are of crucial importance to the diagnosis and prognosis of gastric cancer patients. Patients with metastasis and recurrence are still associated with poor prognosis  despite the increasingly improved surgical and drug chemotherapy levels for gastric cancer, as well as the continuously updated systemic chemotherapeutic regimens.,, Therefore, whether first-line and second-line adjuvant chemotherapy drugs can better improve survival rate has become a key to treat gastric cancer metastasis.
In this study, it is found that the expression of HIF-1α and VEGF showed an increasing trend accompanied by the extension in culture time under hypoxia, indicating that VEGF expression under hypoxia is closely associated with HIF-1α. Protein expression quantities of HIF-1α and VEGF in the experiment group show decreasing trends as time prolongs, which is more obvious at 12 and 24 h particularly. Furthermore, the low expression of the two shows time dependency, demonstrating that Rg3 can reduce the expression of HIF-1α and VEGF, and it is speculated that Rg3 can influence VEGF expression through inhibiting HIF-1α expression. Meanwhile, Rg3 may also influence VEGF expression through other pathways. Results of this experiment are similar to those in previous research, which indicates that Rg3 contributes to inhibiting the expression of HIF-1α and VEGF, suggesting that Rg3 probably downregulates VEGF expression through suppressing HIF-1α, and decreases angiogenesis, thus influencing abdominal implantation metastasis of gastric cancer.
Hypoxia is an important influencing factor of high HIF-1α expression. Hypoxia exists in numerous tumors. The continuous increase in tumor volume will lead to changes in the tumor microenvironment, and insufficient blood supply in tumor region results in the growth state of hypoxia within tissues while oxygen enrichment within surrounding tissues. HIF-1α is a kind of cytoplasmic protein that promotes the activation of multiple genes and is involved in processes such as angiogenesis, cell proliferation, and energy metabolism. In eukaryotic cells, HIF-1α mainly regulates oxygen balance in the body and is affected and regulated by oxygen. HIF-1α is shown to express at a high level persistently as hypoxia within malignant tumor becomes more and more severe, which continuously promotes tumor growth and migration. In contrast, HIF-1α loss will give rise to reduced tumor growth and migration. It is currently believed that HIF-1α is closely associated with peritoneal metastasis of gastric cancer.,, HIF-1α expression levels at different time points under hypoxia are detected by immunocytochemistry and Western blot; in this research, the results of which indicate that the application of Rg3 renders gradually reduced HIF-1α expression in a time-dependent manner.
VEGF is a kind of glycosylated mitogen that acts on endothelial cells, the high expression of which during metastasis may induce endothelial barrier destruction, thus promoting migration of endothelial cells., It has been discovered that the removal of HIF-1α during angiogenesis could significantly reduce diffusion and chemotaxis of endothelial cells, extracellular matrix permeability, and wound healing. However, the blood vessel size and number in the gastric tumor after transfection with HIF-1α apparently increased, suggesting that HIF-1α could regulate VEGF expression, which was closely related to angiogenesis. It is reported in the literature that high HIF-1α expression in the tumor can promote angiogenesis through upregulating VEGF. The application of Rg3 leads to the gradual decrease of VEGF expression, which is closely associated with HIF-1α expression, as is found in this experiment.
Recent advances have indicated that ginsenoside Rg3 acts to reduce growth, invasion, and metastasis of cancer cells, and the mechanisms involve with VEGF and hypoxia associated multiple signaling pathways., Recent studies reported that Rg3 suppresses the phosphorylation cascade of the VEGF-dependent p38/ERK signaling., In patient with acute leukemia, ginsenoside Rg3 inhibits HIF-1α and VEGF expression through blocking the activation of PI3K/Akt and ERK1/2 pathways. Furthermore, Rg3 increases the susceptibility of patients to chemotherapy;, therefore, Rg3 has synergistic effects in clinical trials in combination with chemotherapy regimens. For instance, the survival rate of patients with advanced gastric cancer could be improved when Rg3 was applied in combination with adjuvant chemotherapy. The previous study revealed that Rg3 is also able to induce apoptosis in gastric cancer cells. Therefore, Rg3 could be promising in cancer management.
| > Conclusion|| |
Rg3 can obviously inhibit expression of HIF-1α and VEGF in human gastric cancer cells under hypoxia, the mechanism of action of which may be to downregulate VEGF expression though reducing HIF-1α, thus lowering angiogenesis in tumor and affecting tumor growth. Results in this experiment have provided theoretical support for the mechanism of action of Rg3 inhibiting implantation metastasis of gastric cancer; in addition, providing novel evidence for the target drugs for inhibiting tumor angiogenesis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016;66:7-30.
Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and mortality rates and trends-an update. Cancer Epidemiol Biomarkers Prev 2016;25:16-27.
Alizadeh AM, Shiri S, Farsinejad S. Metastasis review: From bench to bedside. Tumour Biol 2014;35:8483-523.
Dachs GU, Tozer GM. Hypoxia modulated gene expression: Angiogenesis, metastasis and therapeutic exploitation. Eur J Cancer 2000;36:1649-60.
Qiu X, Jia J. Research advances on TCM anti-tumor effects and the molecular mechanisms. J Cancer Res Ther 2014;10 Suppl 1:8-13.
Kim SM, Lee SY, Cho JS, Son SM, Choi SS, Yun YP, et al
. Combination of ginsenoside Rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of NF-kappaB. Eur J Pharmacol 2010;631:1-9.
Lee CK, Park KK, Chung AS, Chung WY. Ginsenoside Rg3 enhances the chemosensitivity of tumors to cisplatin by reducing the basal level of nuclear factor erythroid 2-related factor 2-mediated heme oxygenase-1/NAD(P)H quinone oxidoreductase-1 and prevents normal tissue damage by scavenging cisplatin-induced intracellular reactive oxygen species. Food Chem Toxicol 2012;50:2565-74.
Chen ZJ, Cheng J, Huang YP, Han SL, Liu NX, Zhu GB, et al
. Effect of adjuvant chemotherapy of ginsenoside Rg3 combined with mitomycin C and tegafur in advanced gastric cancer. Zhonghua Wei Chang Wai Ke Za Zhi 2007;10:64-6.
Xu T, Jin Z, Yuan Y, Wei H, Xu X, He S, et al
. Ginsenoside Rg3 Serves as an adjuvant chemotherapeutic agent and VEGF Inhibitor in the treatment of non-small cell lung cancer: A meta-analysis and systematic review. Evid Based Complement Alternat Med 2016;2016:7826753.
Jiang JW, Chen XM, Chen XH, Zheng SS. Ginsenoside Rg3 inhibit hepatocellular carcinoma growth via intrinsic apoptotic pathway. World J Gastroenterol 2011;17:3605-13.
Kim BM, Kim DH, Park JH, Na HK, Surh YJ. Ginsenoside Rg3 induces apoptosis of human breast cancer (MDA-MB-231) cells. J Cancer Prev 2013;18:177-85.
Wang CZ, Yuan CS. Potential role of ginseng in the treatment of colorectal cancer. Am J Chin Med 2008;36:1019-28.
Xu TM, Cui MH, Xin Y, Gu LP, Jiang X, Su MM, et al
. Inhibitory effect of ginsenoside Rg3 on ovarian cancer metastasis. Chin Med J (Engl) 2008;121:1394-7.
Lee YJ, Lee S, Ho JN, Byun SS, Hong SK, Lee SE, et al
. Synergistic antitumor effect of ginsenoside Rg3 and cisplatin in cisplatin-resistant bladder tumor cell line. Oncol Rep 2014;32:1803-8.
Kim JW, Jung SY, Kwon YH, Lee JH, Lee YM, Lee BY, et al
. Ginsenoside Rg3 attenuates tumor angiogenesis via inhibiting bioactivities of endothelial progenitor cells. Cancer Biol Ther 2012;13:504-15.
Chen MW, Yang L, Ni L, Huang C. The effects of 20(R)-Rg3 on lung carcinoma A549 cell line and endogenous VEGF secreted by tumor cells. Sichuan Da Xue Xue Bao Yi Xue Ban 2006;37:60-2.
Chen QJ, Zhang MZ, Wang LX. Gensenoside Rg3 inhibits hypoxia-induced VEGF expression in human cancer cells. Cell Physiol Biochem 2010;26:849-58.
Shen L, Shan YS, Hu HM, Price TJ, Sirohi B, Yeh KH, et al
. Management of gastric cancer in Asia: Resource-stratified guidelines. Lancet Oncol 2013;14:e535-47.
Kanda M, Kodera Y. Molecular mechanisms of peritoneal dissemination in gastric cancer. World J Gastroenterol 2016;22:6829-40.
Riquelme I, Saavedra K, Espinoza JA, Weber H, García P, Nervi B, et al
. Molecular classification of gastric cancer: Towards a pathway-driven targeted therapy. Oncotarget 2015;6:24750-79.
Kanda M, Kobayashi D, Tanaka C, Iwata N, Yamada S, Fujii T, et al
. Adverse prognostic impact of perioperative allogeneic transfusion on patients with stage II/III gastric cancer. Gastric Cancer 2016;19:255-63.
Songun I, Putter H, Kranenbarg EM, Sasako M, van de Velde CJ. Surgical treatment of gastric cancer: 15-year follow-up results of the randomised nationwide Dutch D1D2 trial. Lancet Oncol 2010;11:439-49.
Kanda M, Kodera Y, Sakamoto J. Updated evidence on adjuvant treatments for gastric cancer. Expert Rev Gastroenterol Hepatol 2015;9:1549-60.
Wang Y, Yan P, Liu Z, Yang X, Wang Y, Shen Z, et al
. MEK inhibitor can reverse the resistance to bevacizumab in A549 cells harboring Kirsten rat sarcoma oncogene homolog mutation. Thorac Cancer 2016;7:279-87.
GASTRIC (Global Advanced/Adjuvant Stomach Tumor Research International Collaboration) Group, Paoletti X, Oba K, Burzykowski T, Michiels S, Ohashi Y, et al
. Benefit of adjuvant chemotherapy for resectable gastric cancer: A meta-analysis. JAMA 2010;303:1729-37.
Vaupel P, Harrison L. Tumor hypoxia: Causative factors, compensatory mechanisms, and cellular response. Oncologist 2004;9 Suppl 5:4-9.
Muz B, de la Puente P, Azab F, Azab AK. The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia (Auckl) 2015;3:83-92.
Zhao Q, Tan BB, Li Y, Fan LQ, Yang PG, Tian Y. Enhancement of Drug Sensitivity by Knockdown of HIF-1α in Gastric Carcinoma Cells. Oncol Res 2016;23:129-36.
Ko YS, Cho SJ, Park J, Choi Y, Lee JS, Youn HD, et al
. Hypoxic inactivation of glycogen synthase kinase-3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia-inducible factor-1 signaling. APMIS 2016;124:748-56.
Miyake S, Kitajima Y, Nakamura J, Kai K, Yanagihara K, Tanaka T, et al
. HIF-1α is a crucial factor in the development of peritoneal dissemination via natural metastatic routes in scirrhous gastric cancer. Int J Oncol 2013;43:1431-40.
Zervantonakis IK, Hughes-Alford SK, Charest JL, Condeelis JS, Gertler FB, Kamm RD. Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function. Proc Natl Acad Sci U S A 2012;109:13515-20.
Hu D, Zhang M, Wang S, Wang Z. High expression of cyclooxygenase 2 is an indicator of prognosis for patients with esophageal squamous cell carcinoma after Ivor Lewis esophagectomy. Thorac Cancer 2016;7:310-5.
Tang N, Wang L, Esko J, Giordano FJ, Huang Y, Gerber HP, et al
. Loss of HIF-1alpha in endothelial cells disrupts a hypoxia-driven VEGF autocrine loop necessary for tumorigenesis. Cancer Cell 2004;6:485-95.
Zimna A, Kurpisz M. Hypoxia-inducible FACTOR-1 in physiological and pathophysiological angiogenesis: Applications and therapies. Biomed Res Int 2015;2015:549412.
Aziz F, Wang X, Liu J, Yan Q. Ginsenoside Rg3 induces FUT4-mediated apoptosis in H. pylori
CagA-treated gastric cancer cells by regulating SP1 and HSF1 expressions. Toxicol In Vitro
Keung MH, Chan LS, Kwok HH, Wong RN, Yue PY. Role of microRNA-520h in 20(R)-ginsenoside-Rg3-mediated angiosuppression. J Ginseng Res 2016;40:151-9.
Tian L, Shen D, Li X, Shan X, Wang X, Yan Q, et al
. Ginsenoside Rg3 inhibits epithelial-mesenchymal transition (EMT) and invasion of lung cancer by down-regulating FUT4. Oncotarget 2016;7:1619-32.
Zhao F, Shang Y, Zeng C, Gao D, Li K. Association of single nucleotide polymorphisms of DNA repair genes in NER pathway and susceptibility to pancreatic cancer. Int J Clin Exp Pathol 2015;8:11579-86.
Kim BJ, Nah SY, Jeon JH, So I, Kim SJ. Transient receptor potential melastatin 7 channels are involved in ginsenoside Rg3-induced apoptosis in gastric cancer cells. Basic Clin Pharmacol Toxicol 2011;109:233-9.
[Table 1], [Table 2]