Kosin Med J.  2019 Dec;34(2):106-116. 10.7180/kmj.2019.34.2.106.

Genipin Inhibits Hypoxia-Induced Accumulation of HIF-1α and VEGF Expressions in Human Cervical Carcinoma Cells

• Jin CE
• Lee JH
• Kim GJ
• Lee TH

• KMID: 2467971
• DOI: http://doi.org/10.7180/kmj.2019.34.2.106

Abstract

OBJECTIVES
Hypoxia"”a characteristic of almost all types of solid tumors"”has been associated with poor outcomes in several human malignancies. Genipin"”an active constituent of Gardenia fruit"” has been reported to exert an anti-tumor effect in several cancers. In this study, we investigated inhibition of angiogenesis using Genipin-mediated hypoxia-induced hypoxia inducible factor (HIF-1) and VEGF expression in human cervical cancer cells.
METHODS
Under normoxic and hypoxic conditions, the expression of HIF-1α and VEGF in cervical cancer HeLa cells was detected by quantitative reverse transcription polymerase chain reaction and western blotting. Luciferase reporter assays were used to investigate the molecular mechanisms underlying the hypoxia-induced survivin activation.
RESULTS
Surprisingly, we found that Genipin suppressed the HIF-1α accumulation during hypoxia in human liver cancer cell line (HepG2), human prostate cancer cell line (LNCaP), colon cancer cell line (HCT116), and breast cancer cell line (MDA231). Genipin treatment also significantly reduced hypoxia-induced secretion of VEGF.
CONCLUSIONS
Suppression of HIF-1α accumulation following treatment with Genipin under hypoxia was associated with PI3K and MAPK pathways. Taken together, these results suggested that Genipin inhibits HIF-1α expression through inhibition of PI3K and MAPK signaling pathways. These results provide new insights into a potential mechanism of the anticancer properties of Genipin.

Keyword

Angiogenesis; Genipin; Hypoxia; Hypoxia-inducible factor-1; VEGF

MeSH Terms

Anoxia
Blotting, Western
Breast Neoplasms
Cell Line
Colonic Neoplasms
Gardenia
HeLa Cells
Humans*
Liver Neoplasms
Luciferases
Polymerase Chain Reaction
Prostatic Neoplasms
Reverse Transcription
Uterine Cervical Neoplasms
Vascular Endothelial Growth Factor A*
Luciferases
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Effect of hypoxia and Genipin on intracellular level of HIF-1α in HeLa cells. (A) Cells were treated with various concentrations (10–500 µM) of Genipin for 24 h. The cytotoxic effect of Genipin on HeLa cells was determined using the trypan blue dye exclusion assay as described in Materials and Methods section. Error bars represent standard error of the mean (S.E.M.) from three separate experiments. (B) Kinetics of HIF-1α accumulation under hypoxic conditions. (C) Kinetics of HIF-1α accumulation during treatment with Genipin for 8 h under hypoxic conditions. (D) Cells were exposed to hypoxia, cobalt chloride, and DFX along with Genipin for 8 h, and then, harvested. Cells lysates containing equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-HIF-1α antibody. Actin was used as a loading control. Symbols denote a response that was significantly different from the control (*P < 0.05, **P < 0.01).

• 

  Fig. 2 Effect of Genipin on HIF-1α accumulation during hypoxia in various cancer cell lines. HepG2, HCT116, LNCaP, and MDA231 cells were exposed to 1% O2alone or were treated with 100 µM Genipin in combination with 1% O2 for 8 h. Cell lysates containing equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted with anti-HIF-1α antibody. Actin was used as a loading control.

• 

  Fig. 3 Role of ERK and Akt in the accumulation of HIF-1α in HeLa cells. Cells were treated with 100 µM Genipin in combination with 1% O2 in the presence or absence of 10 µM LY294002 for 8 h. Cells were pretreated with PD98059 (10 µM) and LY294002 (10 µM) for 30 min followed by treatment with Genipin and/or hypoxia for 8 h in the presence of PD98059 and LY294002. Cell lysates containing equal amounts of protein (20 µg) were separated by SDS-PAGE and immunoblotted using anti-HIF-1α, anti-phospho-Akt (S473), or anti-Akt antibody. Actin was used as a loading control.

• 

  Fig. 4 Genipin inhibits production of VEGF and invasion during hypoxia. (A) HeLa cells were exposed to 1% O2 for various time periods (2–24 hours). (B) HeLa cells were treated with various concentrations of Genipin (50, 100, and 250 µM) for 16 h during hypoxia. The concentration of VEGF protein in the culture media was determined by ELISA. The assays were performed in triplicates. The results represent the mean values of VEGF concentrations and error bars represent standard error of the mean from triplicate samples. (C) Invasion indicators are expressed as the mean ± S. D. of three independent experiments. The data was evaluated for statistical significance by Student's t-test. The means noted with an asterisk were statistically different from the matched normoxic control. (D) Western blot analysis shows that Genipin inhibits invasion related proteins in HeLa cells during hypoxic condition. Symbols denote a response that was significantly different from the control (*P < 0.05, **P < 0.01).

Reference

1. Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol. 2002; 29:15–18.

2. Ferrara N, Bunting S. Vascular endothelial growth factor, a specific regulator of angiogenesis. Curr Opin Nephrol Hypertens. 1996; 5:35–44.

3. Krishnamachary B, Berg-Dixon S, Kelly B, Agani F, Feldser D, Ferreira G, et al. Regulation of colon carcinoma cell invasion by hypoxia-inducible factor 1. Cancer Res. 2003; 63:1138–1143.

4. Fang J, Zhou Q, Liu LZ, Xia C, Hu X, Shi X, et al. Apigenin inhibits tumor angiogenesis through decreasing HIF-1alpha and VEGF expression. Carcinogenesis. 2007; 28:858–864.

5. Garcia-Maceira P, Mateo J. Silibinin inhibits hypoxia-inducible factor-1alpha and mTOR/p 70S6K/4E-BP1 signalling pathway in human cervical and hepatoma cancer cells: implications for anticancer therapy. Oncogene. 2009; 28:313–324.

6. Tang X, Zhang Q, Shi S, Yen Y, Li X, Zhang Y, et al. Bisphosphonates suppress insulin-like growth factor 1-induced angiogenesis via the HIF-1alpha/VEGF signaling pathways in human breast cancer cells. Int J Cancer. 2010; 126:90–103.

7. Kallio PJ, Wilson WJ, O'Brien S, Makino Y, Poellinger L. Regulation of the hypoxia inducible transcription factor 1alpha by the ubiquitin-proteasome pathway. J Biol Chem. 1999; 274:6519–6525.

8. Stiehl DP, Jelkmann W, Wenger RH, Hellwig-Burgel T. Normoxic induction of the hypoxia-inducible factor 1alpha by insulin and interleukin-1beta involves the phosphatidylinositol 3-kinase pathway. FEBS Lett. 2002; 512:157–162.

9. Fukuda R, Hirota K, Fan F, Jung YD, Ellis LM, Semenza GL. Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem. 2002; 277:38205–38211.

10. Jiang BH, Liu LZ, Schafer R, Flynn DC, Barnett JB. A novel role for 3, 4-dichloropropionanilide (DCPA) in the inhibition of prostate cancer cell migration, proliferation, and hypoxia-inducible factor 1alpha expression. BMC Cancer. 2006; 6:204.

11. Martin KA, Blenis J. Coordinate regulation of translation by the PI 3-kinase and mTOR-pathways. Adv Cancer Res. 2002; 86:1–39.

12. Zhang Q, Tang X, Lu QY, Zhang ZF, Brown J, Le AD. Resveratrol inhibits hypoxia-induced accumulation of hypoxia-inducible factor-1alpha and VEGF expression in human tongue squamous cell carcinoma and hepatoma cells. Mol Cancer Ther. 2005; 4:1465–1474.