| Background:Laryngeal cancer is one of the leading malignant tumors of the head and neck and is reported to have a higher incidence in highly developed countries. Male patients are at particular risk, with an annual rate of incidence of～3.5.5.5/100,000cases. By contrast, female patients are at a markedly lower risk with an annual rate of0.6/100,000cases. The mortality rate among men was～2.4/100,000cases and in women, the rate was～0.3/10million cases. There are various options for treatment of laryngeal cancer including the conventional approaches of surgery, chemotherapy and radiotherapy. However, the curative effect and rate remain poor and require significant improvement.Although a number of chemotherapeutic drugs are available for the treatment of cancer, a highly effective and less toxic approach for treating laryngeal cancer is lacking. One potential resource for a new generation of therapeutics for the prevention and treatment of laryngeal cancer may be natural substances. For example, epidemiological studies suggest that the intake of broccoli, cauliflower and other cruciferous vegetables can significantly reduce the incidence of cancers of the bladder, pancreas, colon, lung and stomach. Indole-3-carbinol (I3C) has recently been identified as an important tumoricidal component found in cruciferous vegetables and particularly in members of the genus Brassica. A number of studies have shown that I3C induces cell cycle arrest in the G1phase in cancer cells, promotes apoptosis and prevents tumor invasion and metastasis. It has been found that I3C promotes cell cycle arrest by downregulating cyclin-proteins such as cyclin Dl and cyclin E. Additionally,it is thought that IC3induces apoptosis by mechanisms that depend on downregulation of the anti-apoptotic genes Bcl-2, Bcl-xL and survivin and by enhancing expression of Bax and by functionally activating caspase-3and caspase-9.The phosphatidylinositol-3kinase/serine-threonine kinase (PI3K/Akt) signaling pathway is involved in the activation of anti-apoptotic mechanisms, glucose metabolism and protein synthesis, all of which influence cell growth and proliferation. Abnormal activation of the PI3K/Akt pathway is apparent that tyrosine kinase-mediated activation of PI3K may be important; for example, in the context of phosphorylated tyrosine kinase interacting with the p85subunit or in the context of mutated Ras-binding to PI3K, which leads to the activation of PI3K. In addition, somatic mutations may also play an important role. For example, mutation in the PTEN tumor-suppressor gene may disrupt the ability of PTEN to switch off the PI3K pathway. Recently a PIK3CA mutation was found to occur in～30%of epithelial tumors. Abnormal activation of PI3K and somatic mutations can collectively drive uncontrolled growth and cell proliferation during the development of tumors, including ovarian, breast, pancreatic, lung and colon cancer or indeed other malignanciesTo the best of our knowledge there are few reports describing the application of I3C in the treatment of laryngeal cancer. Similarly, there currently exists no report concerning the mechanism of action of I3C and its putative relationship with PI3K/Akt.Objective:The present study was designed, in both in vitro and in vivo experiments, to assess the effect of I3C on the proliferation and apoptosis of laryngeal carcinoma cell lines Hep-2, and the impact of xenograft tumor growth, and its relationship with key proteins of the PI3K/Akt pathway and downstream protein investigate the mechanism of action of I3C on laryngeal, so as to seek for novel directions for laryngeal cancer prevention and treatment and provide evidence for future researches on clinical treatments.Methods:1. IN VITROIn the in-vitro experiment, designed to assess the effect of I3C on proliferation and apoptosis of Hep-2cell lines, the cultured Hep-2cells were treated with I3C at various concentrations, and then the proliferation of Hep-2cells was determined with the CCK8method, the changes in morphology of Hep-2cells were detected using the Hochest33258staining method, and the apoptosis of Hep-2cells was determined using the Annexin-V/PI double staining method. Western blotting was performed to determine the expression of PI3K/AKT signaling pathway-related proteins including PI3K p110α, PI3K class Ⅲ, PDK1, Akt, p-Akt, p-c-Raf and GSK3-β.2. IN VIVOIn the in-vivo experiment, designed to evaluate the effect of I3C on growth of Hep-2cells-transplanted tumors, BALB/c mice models bearing Hep-2cells-transplanted tumors were established, and randomly assigned into the I3C pretreatment group,I3C treatment group, and control group, which were given feed containing I3C prior to cell transplantation, after cell transplantation, and feed without I3C, respectively. After8weeks of treatment, the sizes of the transplanted tumors in the three groups were compared. Nude mice were sacrificed, and the expression of PI3K/AKT signaling pathway-related proteins including PI3K p110α, PI3K p110β,PI3K class Ⅲ, PDK1, Akt, p-Akt, p-c-Raf and GSK3-β in the transplanted tumors was determined. The heart, liver and kidney tissues of nude mice were cut into sections and stained with hematoxylin and eosin (HE), and the damages of these tissues were observed under a microscope.Results:1. IN VITROI3C at concentrations of50,100and150μmol/L inhibited the proliferation of Hep-2cells(P<0.01), and I3C at concentrations of100and150μmol/L promoted apoptosis of Hep-2cells (P<0.01). Hochest33258staining method showed enhanced nuclear staining, nuclear condensation and fragmentation in Hep-2cells treated with I3C for48h, appearing apoptotic changes. I3C inhibited Hep-2cell proliferation and induced apoptosis in an obviously dose-dependent manner.13C induced apoptosis need to reach a certain concentration.After I3C treatment, significant reductions in expression of PI3K/AKT signaling pathway-related proteins including PI3K p110α, PI3K p110β,PI3K class III, PDK1, Akt, p-Akt, p-c-Raf and GSK3-β were detected in Hep-2cells, indicating that I3C had anti-tumor activity against laryngeal carcinoma Hep-2cell lines.2. IN VIVOThe sizes of transplanted tumors from the mice in the I3C pretreatment group and I3C treatment group were significantly smaller than that in the control group (P<0.05), and the expression of PI3K p110a, PI3K class III, PDK1, Akt, p-Akt, p-c-Raf and GSK3-β proteins significantly down-regulated in the transplanted tumors. There were no damages of heart, liver and kidney of the nude mice.Conclusions:I3C effectively inhibits Hep-2cell proliferation and induces apoptosis of Hep-2cells in vitro and in vivo, however, it has no toxicity to normal cells. The anti-rumor action of I3C may be associated with BC-induced down-regulation of PI3K/Akt signaling pathway-related proteins expression, which provides evidence for targeted therapy of tumors and screening of novel anti-cancer drugs. |
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