| Objective: Laryngeal carcinoma is the very common malignant tumor of head and neck region and accounted for 2.4 % of all tumors globally. The main pathological type of laryngeal carcinoma is laryngeal squamous cell carcinoma, LSCC. It was reported that, in the world, more than 150,000 new cases diagnosed as laryngeal carcinoma each year. In China, it was widely prevalent in the northeast area, north China area and east China area. The definite cause of laryngeal carcinoma was not yet determined. Some risk factors were believed to be linked with the development of the disease, such as smoking, drinking, air pollution, human papillomavirus(HPV) infected. Nowadays, the main treatments including surgery, radiotherapy, chemotherapy and biotherapy can have good effect on early stage laryngeal carcinoma. however, the curative effect and prognosis of advanced laryngeal carcinoma was even unsatisfactory after treatment. Considering patients’ low survival rate, many complications and poor quality of life, it is significant to find a new effective diagnosis and treatment indicator. With the rapid development of molecular biology and technology, the study on the origin, development and biological behavior of laryngeal carcinoma is becoming more and more deep, and research on the related singal pathways of origin and development of laryngeal cancer is becoming more and more attention. Genistein(Gen), also known as 4’, 5’, 7’-trihydroxy isoflavones, is one of the highest content and nutritional value of soybean isoflavones, and it is aslo a monomer with anti-cancer effect in soybean isoflavones complex. Our previous study demonstrated low dose Gen can reduce the relative expression of bcl-2 m RNA, when the concentration of Gen increased to 24μg/ml, the expression of Survivin m RNA can also be inhibited. Moreover, Genistein can also induce cycle arrest of Hep2 cells, especially in G2-phase, with the concentration of Gen increasing, the ratio of S-phase block increased. Recent studies found that Gen can induce kinds of tumor cells apoptosis, inhibit proliferation and invasive ability, and regulate tumor cell growth through the mediated PI3K/Akt signaling pathways. Vascular endothelial growth factor receptor-3(VEGFR-3) is one of the receptor tyrosine kinase, which can induce the proliferation and migration of endothelial cells, control the development of vascular and lymphatic endothelial cells, meanwhile regulate embryonic development and tumor growth and metastasis via binding with VEGF-C or VEGF-D specifically. VEGFR-3 is highly expressed in most malignant tumor cells. Recent research showed that inhibition of VEGFR-3 could induce the apoptosis of tumor cells and suppress the proliferation and invasion of tumor cells by regulating Akt, ERK and other understeam signal pathways. Previous studies confirmed that Gen had obvious effect on prostate cancer cell, breast cancer cell, gastric cancer cells, humun colon cancer cells and humun liver cancer cells, but the effect on laryngeal carcinoma cell was rarely reported. In addition, the mechanism of the effect was also unclear. Therefore, We focused on studying the effect of Gen on the function of laryngeal cancer cell, discussing the mechanism of anti-cancer and analyzing the influence of Gen on VEGFR-3/PI3K/Akt/GSK-3β pathway in laryngeal carcinoma cells, which may provide theoretical foundation for application of Gen in laryngeal carcinoma treatment and find a new way to improve the treatment effect of laryngeal cancer.Methods:Control group: ①Control group, cells were cultured in RPMI 1640 medium(complete medium) with 10% FBS for 48 h by replacing the medium every 24 h.Experimental group: we cultured cells in 3 steps and the culture time was for 18 h, 6h, 24 h, respectively. In the first 3 experimental groups, the medium we chosed in the initial and last step was complete medium. In the last 2 groups, the medium we chosed in the initial and last step was complete medium, medium contained 12μmol/L genistein, respectively. All the medium in the second step were different and as follows: ②MAZ51 group(MAZ51 is the VEGFR-3 specific inhibitor) the medium contained 5μmol/L MAZ51. ③LY294002 group(LY29402 is the PI3 K specific inhibitor) the medium contained 25μmol/L LY294002. ④Gen group, the medium contained 12μmol/L genistein. ⑤MAZ51+Gen group, the medium contained 5μmol/L MAZ51. ⑥ LY294002+Gen group, the medium contained 25μmol/L LY294002.1 To detect the apoptosis rate of Hep-2 cells in each group by flowcytomery.2 To detect the influence of genistein on the prolifertation of Hep-2 cells under different concentration(0μmol/L, 2.5μmol/L, 5μmol/L, 10μmol/L, 20μmol/L, 40μmol/L) by CCK-8 kit.3 To detect the migratory and invasive ability of Hep-2 cells in each group by Tanswell chamber.4 To detect the expression the of VEGFs m RNA under different concentration of genistein(0μmol/L, 12μmol/L, 24μmol/L) by reverse transcription-polymerase chain reaction.5 To detect the expression of VEGFR-3 m RNA and PI3 K m RNA of Hep-2 cells effected by 12μmol/L genistein by real-time polymerase chain reaction.6 To detect the changes of the protein expression and the phosphorylation of Akt and GSK-3β in each group by western blot.Results:1 Genisten, MAZ51 and LY294002 can induce the apoptosis of Hep-2 cell seperately. MAZ51+genistein group or LY294002+genistein group presented a significant stronger effect compared with genistein group separately. But MAZ51+genistein group or LY294002+genistein group had no diffrerence when it compared with MAZ51 group or LY294002 group. As known as that MAZ51 is specific blocker of VEGFR-3, while LY294002 is specific blocker of PI3 K. Gen had no effect on Hep-2 cells when VEGFR-3 or PI3 K was inhibited. So we inferred that Gen can promote apoptosis of Hep-2 cells by VEGFR-3 and PI3 K.2 Genistein inhibited the proliferation of Hep-2 cells, and the inhibitory effect was dose dependent.3 Genisten, MAZ51 and LY294002 can inhibite the migratory and invasive ability of Hep-2 cell seperately. MAZ51+genistein group or LY294002+genistein group presented a significant stronger effect compared with genistein group separately. But MAZ51+genistein group or LY294002+genistein group had no diffrerence when it compared with MAZ51 group or LY294002 group. We inferred that Gen can inhibit migration and invasion of Hep-2 cells by VEGFR-3 and PI3 K.4 The expression of VEGFR-3 was constant in Hep-2 cells, and genistein can inhibite the expression of VEGFR-3 m RNA.5 Genistein can inhibite the expression of VEGFR-3 and PI3 K m RNA, and the inhibition ratio was 32% and 63%, respectively.6 Genisten, MAZ51 or LY294002 can inhibite the phosphorylation of Akt-(Ser473), Akt-(Thr308), and GSK-3β-(Ser9) proteins. Therefore, decreased the expression of p-Akt(Thr308), p-Akt(Ser473) and p-GSK-3β(Ser9) proteins. MAZ51+genistein or LY294002+genistein enhanced the inhibition rate significantly.Conclusions:1 Genistein can induce apoptosis of Hep-2 cells, and inhibite proliferation, migration and invasion property of Hep-2 cells.2 Genistein can inhibite the expression of VEGFR-3 and PI3 K m RNA.3 Genistein can inhibite the phosphorylation of Akt and GSK-3β proteins.4 We inferred that genistein plays anti-cancer effect via VEGFR-3/PI3K/Akt/GSK-3β signaling pathway. |
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