摘要
背景：喉癌是头颈部最常见的恶性肿瘤之一,男性发病居多,每年新发病人为3.5～5.5/10万,女性为0.6/10万,越发达国家的发病率越高,死亡率约为2.4/10万,女性0.3/10万~([1])。喉癌虽可通过手术、化疗、放疗等多种手段进行综合治疗,但治愈率仍有待提高。目前国内外已研究的抗肿瘤药众多,但尚未发现理想的、高效、低毒的抗喉癌药物,因此有必要研究一些高效、低毒、乃至天然的抗肿瘤药物丰富喉癌的防治手段。
流行病学研究提示,多吃西兰花、花椰菜等芸苔属十字花科蔬菜,可明显降低膀胱癌、胰腺癌、结肠癌、肺癌、胃癌、等恶性肿瘤的发病[2-5]。作为十字花科蔬菜中的主要抗瘤成分,吲哚-3-甲醇(indole-3-carbinol,I3C)日益成为研究的热点~([6])。多项研究提示,I3C可通过诱导肿瘤细胞G1期阻滞~([7,8])、诱发肿瘤细胞凋亡~([10]),~([11])、抗肿瘤细胞侵润转移~([12])如、调节激素受体~([9])等多个方面发挥抗肿瘤作用。有研究发现,I3C通过下调cyclin D1, cyclin E等蛋白激酶而诱导细胞周期阻滞,调节抗凋亡的相关的基因与蛋白的表达,如下调Bcl-2, Bcl-xL, survivin,上调Bax,激活caspase-3和caspase-9,从而诱发细胞凋亡~([9,14])
磷醇酰肌醇3-激酶/丝氨酸苏氨酸激酶(PI3K/Akt)通路是体内重要的生长因子通路,可激活抗细胞凋亡机制,葡萄糖代谢及蛋白合成等过程,从而促进细胞的生长和增殖~([18])。在许多恶性肿瘤中该信号转导通路发生异常激活。比较清楚的机制包括(一)酪氨酸激酶介导的PI3K的激活,如磷酸化的酪氨酸激酶与亚单位p85结合,或者突变的Ras直接与PI3K结合,导致PI3K的激活；(二)体细胞基因突变,比如,PTEN抑癌基因的突变,使PI3K通路无法关闭,最近发现PIK3CA基因突变出现在30%的上皮肿瘤；上述的酪氨酸激酶介导的PI3K的异常激活和体细胞基因突变可导致细胞异常增殖引起肿瘤,如卵巢癌、乳腺癌、胰腺癌、肺癌及结肠癌等恶性肿瘤的发生发展~([18,22,19])。
目前I3C应用于喉癌治疗和作用机制的研究相关报道较少,在喉癌中,I3C、PI3K/Akt两者的相关研究尚未见报道。
目的：本研究试图通过体外实验及体内试验,研究I3C对喉癌Hep-2细胞增殖凋亡及移植瘤生长的影响,并探讨I3C与PI3K/Akt通路关键蛋白及下游蛋白的关系,探讨I3C对喉癌的作用机制,为喉癌防治寻找新方向,为将来可能的临床用药研究提供理论基础。
方法：
1.体外实验研究I3C对喉癌Hep-2细胞系的影响
培养喉癌Hep-2细胞系,用不同浓度的I3C处理喉癌Hep-2细胞,CCK8法检测喉癌Hep-2细胞增殖的影响,Hochest33258染色观察喉癌Hep-2细胞形态变化,AnnexinV-PI双染检测喉癌Hep-2细胞凋亡情况。Western blot检测Hep-2细胞株中PIK/Akt信号通路关键蛋白及下游蛋白PI3K p110α、PI3K p110β、PI3K classⅢ PDK1、Akt、p-Akt及p-c-Raf、GSK3-β的表达。
2.体内实验研究I3C对喉癌Hep-2细胞系的影响
培养喉癌Hep-2细胞系,构建BALB/C荷瘤裸鼠。将BALB/C荷瘤裸鼠随机分成I3C预处理组、I3C处理组、对照组,分别按计划于接种前给予、接种后给予或不给予含I3C饲料。8周后比较各组移植瘤的大小。处死裸鼠,取移植瘤检测PIK/AKT信号通路关键蛋白及下游蛋白PI3K p110α、PI3Kp110β、PI3K classⅢ、 PDK1、Akt、p-Akt及p-c-Raf、GSK3-β的表达。取裸鼠心、肝、肾等重要组织器官切片、HE染色,观察有无损害。
结果：
1.体外实验研究I3C对喉癌Hep-2细胞系的影响
50μM、100μM、150μM各种浓度的I3C均能抑制喉癌Hep-2细胞增殖(P<0.01),100μM,150μM I3C能促进细胞凋亡(P<0.01)。I3C处理48小时Hochest33258染色,发现喉癌Hep-2细胞核染色增强、核固缩碎裂,呈凋亡改变。I3C抑制Hep-2细胞增殖、诱导细胞凋亡,呈现明显的剂量依赖性。I3C诱导细胞凋亡需要一定的浓度。I3C处理48小时,喉癌Hep-2细胞PI3K/AKT信号通路蛋白及下游蛋白PI3Kp110α,PI3K p110β、PI3K classⅢ、PDK/Akt、p-Akt、p-c-Raf GSK3-β的表达均明显下调,证实I3C对喉癌Hep-2细胞的抗瘤作用,可能与下调PI3K/AKT信号通路的关键蛋白及下游蛋白的表达有关。
2.体内实验研究I3C对喉癌Hep-2细胞系的影响
喂食含0.5%I3C的饲料的预处理组和处理组裸鼠移植瘤体积明显小于对照组(P<0.05),移植瘤组织中PI3K p110α、PI3K p110β、PI3K classⅢ、Akt、p-Akt、 p-c-Raf、GSK3-β表达均明显下调。裸鼠心、肝、肾等重要组织脏器无损害。
结论：I3C在体外实验及体内实验中,均可有效发挥抗喉癌Hep-2细胞作用。I3C发挥抑瘤作用,可能是通过下调PI3K/Akt信号通路关键蛋白及下游蛋白的表达来实现的。I3C对正常组织无损伤。因其高效无毒,I3C可作为喉癌的靶向治疗和新药筛选的对象。
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 malignancies
To 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 VITRO
In 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 VIVO
In 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 VITRO
I3C 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 VIVO
The 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.
引文
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