Effect And The Related Pathway Of Apigenin On VEGF Expression In Human Breast Cancer Cells

Effect and the related pathway of apigenin on VEGF expression in human breast cancer cellsObjectiveBreast cancer is one of the main carcinomas of women, and has the characters of easy relapse and multi-pathway metastasis. Although, the mechanism participating in the progression of breast cancer is not clear now, more and more research about the effects of some autocrine or paracrine factors on the genesis and development of breast cancer shows that, in the progression of breast cancer, VEGF play a very important role. Inhibiting the role of VEGF in the angiogenesis and growth is becoming an important target in breast cancer therapy. HIF-1 is a transcription activating factor generated by cells under stress and hypoxia. HIF-1 is overexpressed in many human cancers. HIF-1 is composed of HIF-1αand HIF-1βsubunits, and theαsubunit is regulate by the oxygen concentration. Recent study showed that the regulation of HIF- 1αcan also be independent of the oxygen environment. Oncogenic mutations such as the loss of function of VHL, p53, and PTEN, all can induce HIF-1αexpression. Growth factors, cytokines, and other signaling molecules can stimulate HIF-1αprotein synthesis via activation of the PI3K/AKT or MAPK pathways. PI3K is a heterodimeric enzyme composed of a 110 kDa catalytic subunit and an 85 kDa regulatory subunit. The best-known downstream target of PI3K is the serinethreonine kinase AKT, which transmits survival signals from growth factors. Recent research demonstrated that PI3K/AKT signaling is required for VEGF expression through HIF-1 in response to growth factor stimulation and oncogene activation. Apigenin is used as a healthy food supplement and has recently been shown to possess anti- tumor properties. Nevertheless, its mechanism is unclear. In breast cancer, the catalytic subunit p110αof PI3K is increased in copy numbers, and PI3K catalytic subunit expression positively correlated with the expression of VEGF in breast cancers. VEGF and HIF-1αare both expressed in breast cancer; VEGF expression correlates with HIF-1αexpression, suggesting that HIF-1αcontributes to the overexpression of VEGF in breast cancer. In this study, we used MDA-MB-231 cell to investigate the possible anti-tumor mechanism of apigenin. We found that apigenin significantly inhibited the expression of HIF-1αand of VEGF in the breast cancer cells. Therefore, we investigated the possible mechanism by which apigenin inhibited VEGF production.Methods1. ELISA was used to determine the protein level of VEGF in MDAMB-231 cellCollect the supematants without serum of the cells treated by apigenin of different concentration for 15h. VEGF protein in the supernatant was measured by the method of ELISA according to manufacturer's instructions.2. MTT assay was used to detect the effect of apigenin on the cell viabilityThe MDA-MB-231 cells, seeded in 96-well plates, were harvested with fresh medium for 24 hours, then treated with different concentrations of apigenin for 15 hours. Detect the cell proliferation state according to the procedures of MTT Method.3. RT-PCR was used to detect mRNA levels of VEGF in MDAMB-231 cellThe total cellular RNA of the cells treated by apigenin of different concentrations for 6h was extracted using Trizol reagent. Aliquots of total RNA were separated in agarose gel (1%) to detect its integrity. The RT-PCR amplification was performed using the AMV-one step RT-PCR amplifying kit.4. The double luciferase system was used to measure the transcript- tion activity of VEGF in MDA-MB-231 cellMDA-MB-231 cells, seeded in 6-well plates, were co-transfected with pRL-TK (intro-control) and pWAP11wt, or with pRL-TK (intro-control) and pWAP11wt and pCEP4-HIF-1αwith LipofectamineTM 2000 for 12 h. The cells were harvested with fresh medium for 24 hours, then treated with different concentrations of apigenin for 15 hours. The total protein of the cells were extracted by passive lysate. Use the double double luciferase report analysis kit to illuminate, and use the fluorescence detect instrument to quantitate the illumination, pCEP4-HIF-1αwas transferred into to explore the reverse effect of HIF-1αon the inhibitory effect of apigenin on the transcription activity of VEGF.5. Western Blotting was used to detect the time-dependent and dosedependent effect of apigenin on the protein levels of HIF-1α, p-AKT, p-ERK1/2, and p53The differently treated cells were lysed in lysate. Equal amounts of protein were resolved in sample buffer, separated on SDS-PAGE and transferred to a nitro-cellulose membrane, blocked under room temperature over night. The membranes were then incubated with specific antibodies for 2 hours, washed with 0.2mol/LNaOH to remove the remained antibodies, incubated with the second antibodies marked by alkaline phosphatase for 2 hours, colored with alkaline phosphatase coloration liquid, then imaged the results by the gel imagination apparatus.Results1. Apigenin reduced the secretion of VEGF in MDA-MB-231 cellThe ELISA data showed that after the cells were treated by apigenin of different concentrations for 15 hours, the levels of the VEGF protein in the medium decreased significantly, which worked in a dose-dependent manner.2. Apigenin inhibited mRNA expression of VEGF in MDA-MB-231 cell The RT-PCR results showed that apigenin inhibited VEGF mRNA expression in MDA-MB-231 cell. 51μM apigenin decreased the mRNA levels of VEGF, and following the increasing of the concentration of apigenin, the reduction of the expression of VEGF mRNA is more prominent.3. The MTT data showedThat apigenin treatment for 15h had little effect on proliferation of MDA-MB-231 cells. These results suggest that the inhibition of VEGF production by apigenin is not through the inhibition of cell proliferation.4. Through analyzing the effect of apigenin on the promoter reporter gene of VEGF, named pMAP11wt, results showedThat apigenin inhibited the transcription activity of VEGF, and following the increasing of the concentration of apigenin, the inhibition effect is more prominent.5. Apigenin inhibited the expression of HIF-1αprotein in MDA-MB-231 cellThe expression of HIF-1αin MDA-MB-231 cells was inhibited by apigenin in a dose- and time-dependent manner, by not only inhibiting the transcription activity of HIF- 1α, but also decreasing HIF-1αprotein stability.6. Apigenin induced the expression of p53 protein in MDA-MB-231 cell, inhibited the expression of p-AKT, but has no various effect on the expression of p-ERK 1/2.ConclusionApigenin probably inhibit the production of VEGF in human breast cancer cell by decreasing the transcription activity of VEGF promoter, inhibiting VEGF mRNA level and decreasing the VEGF protein level excreted by the cell to outside, and this effect is not related to the proliferation state of the cell; Apigenin probably inhibit the expression of HIF-1αby the effect of PI3K/AKT pathway and induction of p53 expression on the HIF-1αmRNA level and the stability of HIF- 1αprotein.