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前沿科普——灵芝三萜类化合物对前列腺癌细胞的抗癌作用
时间:2018-09-18 14:27来源:未知 作者:admin 点击:
        暨南大学医学院解剖学教研团队于2017年《Oncology Letters》上发表《灵芝三萜类化合物对前列腺癌细胞的抗癌作用》一文,团队选取由南京中科药业有限公司所研发的中科灵芝孢子油为实验材料。(每100g中科灵芝孢子油含灵芝三萜21g)
        研究结果显示了灵芝孢子油中所富含的灵芝三萜类化合物的抗肿瘤作用,灵芝三萜类化合物能有效的抑制前列腺癌细胞增殖,并诱导癌细胞凋亡。
 

Anticancer effect of triterpenes from Ganoderma lucidum in human prostate cancer cells
灵芝三萜类化合物对前列腺癌细胞的抗癌作用
暨南大学第一附属医院泌尿外科,暨南大学医学院解剖学教研室,广州,广东510630,中国
LIJUN QU  , SUMEI LI  , YUMIN ZHUO  , JIANFAN CHEN  , XIAOPING QIN  and GUOQING GUO
 
Materials and methods材料与方法

Cell culture and reagents. DU-145 human prostate cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA). DU-145 cells were maintained in F-12 medium containing penicillin (50 U/ml), streptomycin (50 U/ml) and 10% fetal bovine serum (FBS; all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37˚C in a humidified incubator containing 5% CO 2 .
        细胞培养和试剂。DU-145前列腺癌细胞来自美国典型培养物保藏中心(马纳萨斯,弗吉尼亚,美国)。DU-145细胞培养在F-12介质含中,含有青霉素(50单位/毫升),链霉素(50单位/毫升)和10%胎牛血清(胎牛血清全部来自Gibco公司和Thermo Fisher Scientific,Inc., 沃尔瑟姆,马萨诸塞州,美国)在37˚C,5%的CO 2的细胞培养箱中培养。
 
GLT was purchased from Nanjing Zhongke Pharmaceutical Co.Ltd. (Nanjing, China). The principal component of GLT is ganoderic acid H, with a purity of ~99%. GLT was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a concentration of 40 mg/ml and stored at 4˚C (the final concentration of DMSO in the controls and GLT was <0.1% to exclude its toxicity). DMSO was used as the control.
        灵芝三萜购于南京中科药业有限公司(南京,中国)。灵芝三萜类化合物的主成分为灵芝酸H,纯度约为99% 。灵芝三萜类化合物溶解在二甲基亚砜DMSO(DMSO;西格玛奥德里奇;默克集团,达姆施塔特,德国)在浓度为40毫克/毫升,保存在4˚C(对照组和灵芝三萜类化合物中DMSO最终浓度<0.1%,从而可排除其毒性)。DMSO为对照组。
 
Results结果

GLT extract inhibits the viability of prostate cancer cells in a dose‑ and time‑dependent manner. To examine the effect of GLT extract on cell viability, cultured DU-145 cells were treated with increasing concentrations of GLT (0.1, 0.5, 1 and 5 mg/ml), and the viability of cells was determined using an MTT assay. As presented in Fig. 1A, a low concentration of GLT (0.1 mg/ml) had less effect on the viability of DU-145 cells compared with the control (P>0.05). Once the concentration reached ≥0.5 mg/ml, the viability of DU‑145 cells was significantly inhibited compared with the control (P<0.05). Furthermore, as presented in Fig. 1B, 2 mg/ml GLT markedly inhibited the proliferation of DU-145 cells at 24 h after the treatment. Subsequently, 2 mg/ml GLT was selected as the treatment concentration and 0.1 mg/ml GLT as an additional control for further experiments.
        灵芝三萜类化合物(GLT)对前列腺癌细胞存活的抑制作用具有剂量和时间依存性。为了检查GLT对细胞存活率的影响,我们通过增加GLT的浓度(0.1毫克,0.5毫克,1毫克和5毫克/毫升)来培养DU-145细胞,细胞存活率则用MTT法测定。如图1A中,与对照组相比(P>0.05),低浓度的GLT(0.1毫克/毫升)对DU-145细胞存活率影响较小。一旦浓度达到≥0.5毫克/毫升,与对照组相比(P<0.05),DU‑145细胞生长明显受到抑制。此外,如图1B,在治疗后的24小时,2毫克/毫升的GLT明显抑制了DU-145细胞的增殖。随后,2毫克/毫升的GLT作为治疗浓度,0.1毫克/毫升的GLT作为进一步实验的额外对照组。

GLT suppresses the migration and invasion of prostate cancer cells. The present study investigated the effect of GLT on cell migration and invasion. First, a wound-healing assay was performed. As presented in Fig. 2, the control group cells migrated across the wound, which was healed after 24 h; however, 2 mg/ml GLT significantly inhibited this process and 0.1 mg/ml GLT exhibited no effect. Furthermore, cell migration and invasion were assessed using a Transwell system. DU-145 cells treated with or without GLT were allowed to migrate through a Transwell membrane into complete medium. Equal quantities of cells were transferred to the membrane surface and cell migration was assessed within 24 h. Compared with the control or 0.1 mg/ml GLT-treated cells, 2 mg/ml GLT led to a decreased level of cell migration (Fig. 3A and B). To evaluate cell invasion, DU-145 cells were plated on the Matrigel surface. As presented in Fig. 3C and D, 2 mg/ml GLT treatment significantly decreased DU‑145 cell invasion. In addition, migration/invasion-associated genes, MMPs, have previously been implicated in prostate cancer metastasis (17,18). Thus, the mRNA expression level of MMP-2 and MMP-9 were investigated. As presented in Fig. 4, the results of RT‑PCR indicated that 2 mg/ml GLT significantly suppressed the expression levels of MMPs. These results revealed that a high dose of GLT inhibited prostate cancer cell migration and invasion via the suppression of MMPs. GLT induces prostate cancer cell apoptosis. It was investigated whether GLT was able to induce the apoptosis of prostate cancer cells. The apoptosis rate was determined by annexin V-FITC and PI double staining. Compared with the control group, 0.1 mg/ml GLT administration revealed no effect, whereas the apoptosis rate with 2 mg/ml GLT treatment was markedly increased (Fig. 5). These results suggested that GLT inhibited viability, migration and invasion, and also induces the apoptosis of prostate cancer cells in vitro.
        灵芝三萜类化合物(GLT)抑制前列腺癌细胞的侵袭和迁移。本文研究了GTL对细胞迁移和侵袭的效果。首先,进行伤口愈合试验。如图2,对照组中的细胞穿过伤口转移了,伤口是 24小时后痊愈;然而,2毫克/毫升的GLT明显抑制这一过程,而0.1毫克/毫升得GLT 基本无效果。此外,采用Transwell共培养系统评估细胞迁移和侵袭。在有或没有GTL的情况下,DU-145细胞可以通过Transwell膜迁移进入完整的培养基。同等数量的细胞被转移到膜表面,细胞迁移在24 小时内进行了评估,与对照组或0.1毫克/毫升浓度GTL组相比,2毫克/毫升GLT组降低细胞的迁移水平(图3a,3b)。为了检测细胞侵袭程度,细胞DU-145被置于基质胶表面。如图3c和D所示,2毫克/毫升GLT治疗显着降低DU‑145细胞的侵袭。此外,与迁移和侵袭相关的基因-MMPs,先前已被证实与前列腺癌转移相关17,18。因此,对MMP-2和MMP-9的mrna表达水平进行了研究。如图4, RT‑PCR结果表明,2毫克/毫升GLT显著抑制MMPs的表达水平。这些结果表明,高剂量的GLT抑制前列腺癌细胞迁移和侵袭通过抑制基质金属蛋白酶。GLT诱导前列腺癌细胞凋亡。GLT是否能诱导前列腺癌细胞凋亡已进行了研究。细胞凋亡率测定采用膜联蛋白V-FITC和PI双染色。与对照组相比,0.1毫克/毫升GLT显示没有影响,而2毫克/毫升GLT治疗细胞凋亡率明显增加(图5)。这些结果表明,GLT抑制细胞活性、迁移和侵袭,并诱导体外培养的前列腺癌细胞凋亡。

Discussion讨论

The results of the present study demonstrated that GLT inhibits the growth of prostate cancer cells, suppresses the migration and invasion and induces apoptosis via the inhibition of MMP expression. The elucidation of the anticancer mechanism underlying GLT may contribute to the clinical usage of active compounds isolated from G. lucidum. G. lucidum has been used as a preventive medicine in Asia for centuries (19). Various biologically active compounds have been isolated from G. lucidum, including polysaccharides, phenols, lipids and triterpenes. Polysaccharides possess antioxidant (20), immunomodulatory (21) and antitumor characteristics (22). Also, polysaccharides activate the immune response via the stimulation of production of inflammation mediators (5,23). Phenols have antioxidant properties (24) and lipids are able to inhibit the growth of hepatoma and sarcoma (25). Triterpenes demonstrate cytotoxicity towards hepatoma, cervical cancer and lung carcinoma cells (26-28). Besides the antitumor activity, GLT serve a role in a variety of other biological process, including anti-human immunodeficiency virus (HIV) and anti‑HIV‑1 protease activity (29,30), neurotrophic activity (31) and anti-obesity activity (32). The results of the present study identified that GLT inhibited the metastasis and induced the apoptosis of prostate cancer cells. These results are consistent with those of previous studies that identified the anti‑prostate cancer effect of G. lucidum (15,16).
        目前的研究结果表明,灵芝三萜类化合物抑制前列腺癌细胞的生长,同时抑制细胞迁移和侵袭,并通过抑制MMP的表达来诱导细胞凋亡。灵芝三萜化合物抗癌机制的阐明可能有助于灵芝活性成分的临床使用。数百年来,灵芝在亚洲地区一直被用作预防药物(19)。各种具有生物活性成分的化合物已从灵芝分离出来,包括多糖类化合物、酚类化合物、脂类化合物和三萜类化合物。多糖具有抗氧化(20)、免疫调节(21)和抗肿瘤特性(22)。同时,多糖通过刺激炎症介质来激活免疫应答(5,23)。酚类化合物具有抗氧化作用(24),脂类化合物能抑制肝癌和肉瘤的生长(25)。三萜类化合物,已表明对肝癌、宫颈癌和肺癌细胞有细胞毒作用(26-28)。除了抗肿瘤活性,灵芝三萜类化合物在多种其他生物过程中具有作用,包括抗人类免疫缺陷病毒(HIV)和抗HIV-1蛋白酶活性(29,30)、神经营养活性(31)和抗肥胖活性(32)。目前的研究结果确定灵芝三萜类化合物可抑制前列腺癌细胞转移并诱导其凋亡。这些结果与以前关于灵芝具有抗前列腺癌作用的研究一致(15,16)。
 
The identification of biologically active components of G. lucidum is important for the mechanistic characterization of their specific activity and the results of the present study further revealed that triterpenes may be the active compounds responsible for the antitumor effect of G. lucidum. In addition, there is certain evidence that specific components in the natural herbal products interact with each other to function as the whole product (33). The present study did not identify other compounds extracted from G. lucidum, which requires further investigation.
        对于灵芝特定生物活性的机理表征和目前研究将进一步显示灵芝三萜类化合物可能是灵芝抗肿瘤作用的活性化合物来说,灵芝生物活性成分的鉴定是十分重要的。此外,有一定的证据表明,天然草药产品中的特定成分相互作用,可以形成一个完整的产品(33)。本研究没有对从灵芝中提取的其他化合物进行验证,这需要进一步研究。

The inhibition of the viability of prostate cancer cells by GANODERMA LUCIDUM TRITERPENES may be caused by the induction of apoptosis. Apoptosis is a physiological process where cells are removed when they experience critical DNA damage (34,35). Inhibition of apoptosis, rather than enhanced cell proliferation, is particularly important for the development of cancer (36,37). The results of the present study revealed that GANODERMA LUCIDUM TRITERPENES significantly induced apoptosis, confirmed by annexin V staining and flow cytometry. Generally, apoptosis can be divided into early and late stages. During the late stage of apoptosis, DNA fragmentation occurs following reactive oxygen species generation, caspase-3 activation and mitochondrial dysfunction (38). Additionally, apoptosis can be divided into the extrinsic and intrinsic pathways. The extrinsic apoptotic pathway can be initiated by death receptors (39) and the intrinsic pathway is also called the mitochondrion-dependent pathway (40). The majority of stimuli induce apoptosis via the mitochondrial pathway, which induces mitochondrial outer membrane permeabilization and these processes are regulated by members of the B cell lymphoma-2 (Bcl-2) protein family. This family may be subdivided into pro-apoptotic (Bcl-2-associated X protein and Bcl-2 homologous antagonist killer), pro-Bcl-2 homology 3-only proteins (including BH3-interacting domain death agonist, Bcl-2-like protein 11 and p53-upregulated modulator of apoptosis) and anti-apoptotic proteins ,including Bcl-2 and B cell lymphoma extra-large (Bcl-xL) (35). Decreased expression levels of anti-apoptotic proteins and increased expression levels or activation of pro-apoptotic proteins have been demonstrated to be critical for the induction of apoptosis (35,41). DU-145 prostate cancer cells have increased expression levels of Bcl-2 and Bcl-xL, protecting cells from apoptosis (42,43). The results of the present study revealed that GANODERMA LUCIDUM TRITERPENES induced apoptosis; however, the specific underlying molecular mechanisms remain under investigation. The results of the present study suggested that GANODERMA LUCIDUM TRITERPENES may decrease the expression of anti-apoptotic proteins and increase the expression levels of, or activate, pro-apoptotic proteins, thus inducing cell death.
        灵芝三萜类化合物对前列腺癌细胞活性的抑制作用可能是通过诱导细胞凋亡而引起的。细胞凋亡是一种生理过程,在这一过程中,当DNA损伤时,细胞则会消失(34,35)。抑制细胞凋亡不是促进细胞增殖,它对肿瘤的发展尤为重要(36,37)。目前的研究表明,灵芝三萜类化合物明显诱导细胞凋亡,经由Annexin V染色和流式细胞仪检测确定。细胞凋亡一般分为早期和晚期两个阶段。在细胞凋亡后期,随着活性氧生成、caspase-3活化和线粒体功能障碍,会发生DNA断裂(38)。此外,细胞凋亡可分为外源性和内源性途径。外源性凋亡通路可由死亡受体启动(39),内源性通路又称线粒体依赖通路(40)。大部分刺激通过线粒体途径诱导细胞凋亡,诱导线粒体外膜通透性,这些过程是由B细胞淋巴瘤-2(Bcl-2)蛋白家族成员进行调节。这个家庭可以细分为促凋亡(Bcl-2相关X蛋白和Bcl-2同源拮抗剂杀手),pro-bcl-2同源3-only蛋白(含BH3结构域凋亡诱导蛋白、BCL-2-like蛋白11和p53上调凋亡调制器)和抗凋亡蛋白Bcl-2,包括bcl-2和B细胞淋巴瘤(Bcl-xL)(35)。抗凋亡蛋白表达水平的下降,以及促凋亡蛋白表达水平升高或激活已被证明是诱导细胞凋亡的关键(35,41)。前列腺癌细胞DU-145细胞的Bcl-2和Bcl-xl表达水平增高,可保护细胞免于凋亡(42,43)。目前的研究表明,灵芝三萜类化合物可诱导细胞凋亡,而具体的分子机制仍在研究中。目前的研究表明,灵芝三萜类化合物可降低抗凋亡蛋白的表达,提高促凋亡蛋白的表达水平,或激活促凋亡蛋白,从而诱导细胞死亡。
 
The results of the present study revealed the antitumor effect of GANODERMA LUCIDUM TRITERPENES. GANODERMA LUCIDUM TRITERPENES administration inhibits the proliferation of human prostate cancer cells and induced apoptosis. Additional detailed signaling mechanisms and in vivo studies are required to establish GANODERMA LUCIDUM TRITERPENES as a potential clinical agent for the prevention and/or treatment of prostate cancer.
        本研究结果显示了灵芝三萜类化合物的抗肿瘤作用。灵芝三萜类化合物抑制前列腺癌细胞增殖并诱导细胞凋亡。附加的详细信号转导机制和体内研究需要以灵芝三萜类化合物作为潜在临床药剂对前列腺癌进行预防和/或治疗。
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