破壁灵芝孢子粉提取物抑制人卵巢上皮性癌细胞生长、转移及逆转顺铂耐药的研究
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摘要
目的:卵巢癌是危害女性健康的三大妇科恶性肿瘤之一,而卵巢上皮性癌占卵巢癌的90%,由于缺乏有效的早期诊断及治疗方法,75%的患者就诊时已属晚期。目前彻底的肿瘤细胞减灭术和新型化疗是治疗卵巢癌的主要手段。约有70%的晚期患者可因化疗耐药而导致治疗失败使得卵巢癌易复发、转移和产生多药耐药性,导致卵巢癌的5年存活率尚不到30%,病死率仍居妇科恶性肿瘤的首位,成为严重威胁妇女生命的疾病。因此,寻找有效的抑制肿瘤生长、转移以及逆转耐药的有效制剂,并研究其抗肿瘤及逆转铂类化疗药耐药的机制成为当前卵巢癌治疗研究的重要课题。
近年来,天然药物以其低毒、高效的独特优势越来越受到国内外学者的广泛关注,从中草药和天然植物中提取抗肿瘤成分已成为获得有效抗癌制剂和耐药逆转剂的重要途径。灵芝孢子是一种已被广泛应用于肿瘤治疗的天然植物产品。体内外实验表明灵芝孢子具有明显抗肿瘤活性。国内外研究报道灵芝孢子对乳腺癌、肺癌、鼻咽癌、胃癌、直肠癌、前列腺癌、白血病、肝癌、黑色素瘤等肿瘤细胞的生长、侵袭、转移能力有明显的抑制作用,而且能增强顺铂的抗肿瘤效应。但至目前为止灵芝孢子在卵巢癌治疗方面的研究国内外均未见报道,为探讨灵芝孢子对人卵巢癌细胞生长、转移以及对顺铂化疗耐药的影响,本课题以多个卵巢上皮性癌细胞系为研究对象,以破壁灵芝孢子粉提取物为处理因素,应用细胞增殖抑制实验(WST-1比色法)、光学显微镜观察法、细胞计数法、Hoechst-33258染色方法、流式细胞技术、克隆形成实验、多细胞球体形成实验、细胞迁移实验、细胞侵袭实验以及western blot方法等,对其进行研究。目的在于①研究破壁灵芝孢子提取物对卵巢上皮性癌细胞的生长抑制作用、诱导凋亡作用及其机制。②研究破壁灵芝孢子提取物对卵巢上皮性癌细胞的粘附、侵袭、转移能力的抑制作用及相关机制。③探讨破壁灵芝孢子提取物对人卵巢上皮性癌顺铂耐药细胞化疗耐药的逆转作用及其机制。为灵芝孢子的进一步开发以及在卵巢上皮癌的临床应用提供理论依据。
方法:
1细胞培养:体外常规培养人卵巢上皮癌细胞株OV2008、C13*、A2780S、A2780CP、SKOV3以及人卵巢上皮细胞株IOSE398。
2破壁灵芝孢子粉工作液制备:加拿大energene天然药品有限公司提供的破壁灵芝孢子粉溶于DD-H2O配制成50mg/ml的灵芝孢子储存液。高温灭菌、离心后取上清,用0.20μm微孔滤膜过滤器过滤除菌,保存于4℃冰箱。实验前以细胞培养液稀释成相应浓度的灵芝孢子液。
3破壁灵芝孢子粉提取物对人卵巢上皮性癌细胞增殖的抑制作用:以合适的密度接种细胞,待细胞贴壁后加入不同终浓度的破壁灵芝孢子液,48h用WST-1比色法及人工计数活细胞法检测灵芝孢子粉对人卵巢上皮性癌细胞增殖的抑制作用,显微镜下观察细胞形态的改变。利用WST-1比色结果的OD值计算药物对细胞生长的抑制率,细胞抑制率=1-实验组平均OD值/对照组平均OD值×100%。IC50用Logit法计算。
4流式细胞术测定破壁灵芝孢子提取物对人卵巢上皮性癌细胞凋亡及细胞周期分布影响:接种细胞于6cm培养盘,细胞贴壁后分别加入终浓度为0.5,1,2mg/ml的灵芝孢子粉溶液,对照组为不含药物的细胞培养液。处理48h以不同的方法分别收集细胞,用流式细胞仪测定细胞凋亡及细胞周期改变。
5 Hoechst-33258染色法评估破壁灵芝孢子对人卵巢上皮性癌细胞凋亡的影响:细胞分为对照组和处理组,处理组分别加入终浓度为0.5,1,2mg/ml的灵芝孢子粉溶液,对照组加入不含药物的细胞培养液,培养细胞48h后,Hoechst-33258染色法观察细胞核变化,判断凋亡情况。
6破壁灵芝孢子提取物抑制人卵巢上皮性癌细胞生长能力测定:不同浓度破壁灵芝孢子处理人卵巢上皮性癌细胞,利用克隆形成实验、悬滴培养多细胞球体形成实验检测灵芝孢子对人卵巢上皮性癌细胞生长抑制情况,同时设不含药物的对照组进行实验。
7破壁灵芝孢子提取物抑制人卵巢上皮性癌细胞侵袭和转移能力测定:在体外用不同浓度灵芝孢子处理不同的人卵巢上皮性癌细胞,利用细胞黏附实验、细胞迁移运动(划痕)实验、细胞侵袭实验,检测人卵巢上皮性癌细胞粘附、侵袭和转移能力的变化,同时设不含药物的对照组进行实验。
8人卵巢癌顺铂耐药细胞株的顺铂耐药性及破壁灵芝孢子提取物逆转其耐药作用的测定:实验分为单独应用顺铂、单独应用破壁灵芝孢子提取物以及顺铂与灵芝孢子联合应用三组,采用WST-1方法、人工计数细胞法及显微镜观察法检测人卵巢上皮性癌细胞顺铂敏感细胞和耐药细胞增殖的抑制作用。分别计算抑制率、耐药倍数及逆转倍数,以判定顺铂的耐药性以及破壁灵芝孢子能否逆转其耐药性。耐药倍数=耐药细胞的IC50/非耐药细胞的IC50。逆转倍数=顺铂单独处理组的IC50/灵芝孢子联合顺铂处理组的IC50。
9 Western blot技术检测相关蛋白表达:细胞接种于6孔板,分成不同的实验组进行处理。
第一组:不同浓度破壁灵芝孢子液(0、1、2mg/ml)处理细胞48h后提取总蛋白蛋白样品经12%SDS -聚丙烯酰胺凝胶(检测cleaved casep- ase-3用15% SDS -聚丙烯酰胺凝胶)90V电压进行电泳分离,之后电转移至硝酸纤维素滤膜上,测定bcl-2和Bax,cleaved- caspase3 , P27凋亡相关蛋白表达情况以探讨破壁灵芝孢子抑制人卵巢上皮性癌细胞凋亡机理。
第二组:不同浓度破壁灵芝孢子液(0、0.5、1、2mg/ml)处理人卵巢上皮癌细胞48h后提取总蛋白测定粘附及转移相关蛋白E-cadherin, N-cadherin、vimentin、MMP9以及TIMP-1的表达情况了解灵芝孢子抑制人卵巢上皮癌细胞侵袭、转移的机理。
第三组:bcl-2、Akt、P-Akt、P53蛋白在人卵巢上皮癌顺铂耐药细胞株A2780CP及敏感细胞株A2780S的表达测定。细胞接种于6孔板,培养48h提取总蛋白,比较两种细胞bcl-2、Akt、P-Akt、P53蛋白的表达情况,明确顺铂耐药细胞株和敏感细胞株耐药相关蛋白的不同表达。
第四组:不同浓度破壁灵芝孢子液(0、0.5、1、2mg/ml)处理A2780CP和A2780S细胞48h后提取总蛋白测定bcl-2、Akt、P-Akt、P53蛋白的表达情况,了解单独应用破壁灵芝孢子对耐药相关蛋白的表达影响。
第五组:接种A2780CP细胞后分为破壁灵芝孢子单独处理组(破壁灵芝孢子浓度为0.5mg/ml)及破壁灵芝孢子(0.5mg/ml)与顺铂(浓度为5umol/L)联合处理组进行处理。提取处理人卵巢上皮性癌细胞48h的总蛋白测定bcl-2、Akt、P-Akt、P53蛋白的表达,以判定灵芝孢子逆转耐药的机理。均以β-actin为内参照。
?结果:
1破壁灵芝孢子粉提取物抑制人卵巢上皮性癌细胞增殖:不同浓度的破壁灵芝孢子处理人卵巢上皮性癌细胞后倒置显微镜下观察到活性细胞数目减少,并呈浓度依赖性,高浓度(大于2mg/ml)破壁灵芝孢子组可见细胞脱离培养皿壁成悬浮状态,致贴壁存活细胞数目极少。采用WST-1比色法及人工细胞计数法检测的结果与显微镜下观察到的结果相似。经WST-1结果确定0.5mg/ml的灵芝孢子可以作为低毒剂量(细胞生长率大于80%)用于逆转耐药实验。
2破壁灵芝孢子提取物诱导人卵巢上皮性癌细胞凋亡并造成细胞周期G1期阻滞:Hoechst-33258染色后可以观察到人卵巢上皮性癌细胞凋亡现象,表现为细胞核缩小,染色质浓缩,药物浓度越大凋亡细胞越多。并且流式细胞技术可以检测到破壁灵芝孢子处理人卵巢上皮性癌细胞72h后可以出现凋亡峰,凋亡率随浓度增加而升高。流式细胞技术检测结果表明破壁灵芝孢子作用人卵巢上皮性癌细胞后多数细胞聚集于G1期,并且G1期细胞数目随作用时间延长而增加。而G2/M期细胞数目较少,且G2/ M细胞随作用时间延长而减少,S期细胞数目随作用时间无明显改变。
3破壁灵芝孢子提取物抑制人卵巢上皮性癌细胞克隆形成能力:经破壁灵芝孢子处理的人卵巢上皮性癌细胞克隆形成数目明显少于对照组,并呈浓度依赖性,即随着灵芝孢子浓度逐渐增加克隆数目逐渐减少,计算克隆形成率逐渐降低,而且单个细胞克隆的大小及组成克隆的细胞数也随灵芝孢子浓度的增加而递减。
4破壁灵芝孢子粉提取物抑制人卵巢上皮性癌细胞粘附能力:不同浓度灵芝孢子处理人卵巢上皮性癌细胞3h及6h后的粘附细胞数量较对照组均有减少。
5破壁灵芝孢子粉提取物抑制人卵巢上皮性癌细胞多细胞球体形成:在悬滴培养条件下,对照组人卵巢上皮性癌细胞呈聚集生长,于悬滴形成后24h细胞即可聚集成团,之后细胞呈立体生长形成多细胞球体,至第四天球体体积最大,呈圆形,外形规则,细胞间粘附紧密。实验组细胞间粘附性降低,形成的多细胞球体较松散,形状不规则,随灵芝孢子浓度增加形成球体的能力减低。
6破壁灵芝孢子粉提取物抑制人卵巢上皮性癌细胞迁移能力:不同浓度的破壁灵芝孢子处理人卵巢上皮性癌细胞后可观察到对照组SKOV3细胞迁移活跃,划痕后24h几近完全愈合;而灵芝孢子处理组细胞迁移能力明显受到抑制,随着药物浓度的增加抑制作用增强,即划痕的愈合面积逐渐降低,并呈一定的剂量依赖关系,2mg/ml的高浓度组显示不仅划痕的宽度无明显变化,且细胞呈现明显受抑制状态。实验组24h迁移面积较对照组明显减低,差异有统计学意义,P<0.05。
7破壁灵芝孢子提取物抑制人卵巢上皮性癌细胞侵袭能力:侵袭实验表明破壁灵芝孢子处理人卵巢上皮性癌细胞skov3后实验组与对照组比较侵袭至微孔滤膜上的细胞数明显不同,浓度为0、0.5、1和2mg/ml的破壁灵芝孢子处理后其侵袭的细胞数分别为162.8±8.1, 132.8±6.5, 65.2±3.9 and39.2±1.3,可见实验组侵袭细胞数明显低于对照组,且实验组中随灵芝孢子浓度的增加侵袭细胞数逐渐减少,即侵袭率逐渐减少,差异具有统计学意义,(P<0.05)。
8顺铂对敏感细胞株A2780S和耐药细胞株A2780CP的抑制作用:为了解顺铂对卵巢癌敏感细胞株和耐药细胞株的抑制作用,用浓度比例为4:1的顺铂分别处理A2780CP和A2780S细胞48h,经WST-1测定和倒置显微镜下所观察到两种细胞的抑制率相似,提示A2780CP对顺铂的敏感性较A2780S明显降低。采用同一浓度(5umol/L)的顺铂处理两种细胞48h后A2780S数目明显少于A2780CP,经计算顺铂对A2780S细胞的抑制率显著高于对A2780CP的抑制率,其IC50分别为10.32和29.94mg/ml,提示A2780CP对顺铂具有耐药性,耐药倍数约为2.9倍。
9破壁灵芝孢子粉提取物增强顺铂的抗肿瘤作用并逆转A2780CP细胞对顺铂的耐药性:WST-1结果显示灵芝孢子和顺铂联合应用组较单独应用顺铂组对A2780CP细胞的抑制率明显提高,其IC50由单用顺铂的29.94降低至12.16umol/L,表明灵芝孢子可以逆转A2780CP对顺铂的耐药性,逆转倍数为2.46。该结果与细胞计数及显微镜观察的结果相似。
10破壁灵芝孢子对人卵巢上皮性癌细胞凋亡、侵袭及耐药相关蛋白表达影响:
第一组:Western blot方法检测到凋亡抑制蛋白bcl-2、bcl-xl随破壁灵芝孢子浓度增加而下降,而促凋亡蛋白bax及P27、casepase-3逐渐升?高,并且35KD的Casbase-3被激活成活化的19,20KD的cleaved-casep- ase3,并随破壁灵芝孢子浓度增加表达呈升高趋势。
第二组:破壁灵芝孢子作用细胞后实验组E-cadherin、TIMP-1的表达水平均高于对照组;而N-cadherin、vimentin及MMP-9的表达水平均低于对照组,并且随灵芝孢子浓度的增加而呈浓度依赖性改变。
第三组:Western blot结果表明耐药细胞A2780CP bcl-2、AKT、P-AKT的表达明显高于A2780S,而P53的表达则低于A2780S细胞。不同浓度破壁灵芝孢子处理A2780CP后P53蛋白水平随灵芝孢子浓度增加而增加;而bcl-2、Akt、P-Akt水平则逐渐降低。破壁灵芝孢子联合顺铂处理耐药细胞较单独应用顺铂bcl-2、Akt、P-Akt的表达水平明显降低;而P53的表达水平明显升高。
结论:本文在国内外首次研究了破壁灵芝孢子提取物对人卵巢上皮性癌细胞的多种抗癌作用以及逆转人卵巢上皮癌顺铂耐药细胞株对顺铂的耐药性并初步探讨其机理。
1破壁灵芝孢子粉提取物具有抑制人卵巢上皮性癌细胞生长及诱导凋亡的作用,其机制是作用于细胞周期的G1期,造成G1期阻滞,并可以通过上调bax、P27,下调bcl-2并活化casepase-3而发挥其作用。
2破壁灵芝孢子粉提取物在体外可抑制人卵巢上皮性癌细胞侵袭和转移,其可能的机制是抑制细胞粘附、上调E-cadherin及TIMP-1表达、降调N-cadherin、vimentin及MMP-9的表达。
3破壁灵芝孢子提取物能增强人卵巢上皮癌细胞对顺铂的敏感性,有效逆转卵巢癌顺铂耐药细胞株A2780CP的耐药性,其可能的机制是抑制Akt、P-Akt及bcl-2,增强P53蛋白的表达。
综上所述,本研究证明了破壁灵芝孢子提取物对人卵巢上皮性癌细胞具有多种抗癌作用,可以抑制卵巢癌细胞生长、侵袭和转移,诱导细胞凋亡,增强卵巢癌细胞对顺铂化疗的敏感性并能逆转人卵巢癌顺铂耐药细胞株对顺铂的耐药性。因此,具有抗癌效应的灵芝孢子可以作为卵巢癌常规化疗手段的有益补充,联合新辅助化疗可以提高疗效利于更彻底的肿瘤细胞减灭术。用于术后化疗可以协同化疗药物提高化疗效果,预防及逆转对卵巢癌化疗药物顺铂的耐药,可以作为有效的辅助治疗卵巢上皮癌的抗癌制剂。本研究为灵芝孢子在卵巢上皮癌的临床应用提供了理论依据,填补了灵芝孢子在卵巢癌应用方面的空白。
Objective: Ovarian cancer is one of the three gynecological malignancy. Epithelial ovarian cancer (EOC) is the most common form of ovarian cancer accounting for 90% of it. Due to the lack of effective screening strategies and the absence of symptoms in early-stage of disease, 75% of cases have progressed to an advanced stage at the time of primary diagnosis. The most effective treatment for EOC is the surgery ( cytoreduction) plus the platinum- based chemotherapy, such as cisplatin. However, the majority(70%)of patients with advanced ovarian cancer ultimately experience chemoresistance, and recurrence of their disease and the 5-year survival rate of patients is less than 30%. ovarian cancer is the most common life-threatening malignant tumors among women and the first leading cause of death from gynecological disorder. Thus,The effective anti-tumor and reversing drug resistance of platinum product need to be developed,the effective therapeutic strategies and the investigation of the molecular mechanism responsible for the effects of anti-tumor and reversing anti-resistance are exigently demanded.
Recently,the natural active substances attracted the attention of resear- cher at home and abroad with the characteristics of high activity and small adverse reactions. It has become a main Approaches to obtain effective anti-cancer agents and drug-resistant reversal agents by antitumor composition extraction from Chinese herbal medicine and natural plant. Ganoderma lucidum wall-broken spores,a natural plant product , have been widely used to treat various human diseases, including cancers). significant amount of research had shown that Ganoderma lucidum exerts multiple anti-tumor activity on cancer cells. There were some reporters at home and abroad in recent years that Ganoderma wall-broken spores suppressed growth and invasive behavior of cells of breast cancer,lung cancer, nasopharyngeal darcinoma,carcinoma of stomach,colorectal cancer, prostate cancer, leucoc- ythemia, hepatoma,melanoma lymphomas and enhanced cisplatin anti-tumor effect .however,the effect of Ganoderma Lucidum on epithelial ovarian cancer (EOC) has not been reported at home and at abroad. In this study, we determined if Ganoderma Lucidum regulates EOC cell activity . Using several cell lines derived from EOC, We observed the effects of ganoderma lucidum on proliferation and apoptosis in the human ovarian cell line (ov2008 cells) , and study its possible mechanism ; investigated the inhibitory effects of Ganoderma lucidum on invasion and metastasis of human ovarian cancer cells; investigated the reversal effect of Ganoderma lucidum on multidrug resistance (MDR) in the Cisplantin-resistant ovarian cancer cell line by WST-1 assay, microscope, cell counting, Hoechst-33258 staining, flow cytometry, colony formation assay, hanging drop culture, migration assay, invasion assay and western blot . It will provide the theoretical basis for further development and clinical application of ganoderma lucidum spore in the epithelial ovarian cancer.
Objective 1: To observe the effects of ganoderma lucidum on prolifer- ation and apoptosis in the human ovarian cell line, and study its possible mechanism .
Objective 2: To investigate the inhibitory effects and molecular mech- anism of Ganoderma lucidum on invasion and metastasis of human ovarian cancer cells in vitro .
Objective: 3 To investigate the reversal effect of Ganoderma lucidum on multidrug resistance (MDR) in the Cisplantin-resistant ovarian cancer cell line A2780CP.
Method:1 Cell culture:epithelial ovarian cancer cell lines OV2008, C13*, A2780s, A2780-cp and SKOV3 were obtained and cultured. Cells were grown at 37°C in an atmosphere with 5% CO2。
2 Preparation of Ganoderma lucidum:The Ganoderma lucidum used in this study was purchased from Energene Naturals Inc (Toronto, On) which contains wall-broken spores and polysaccharides. A stock solution of 50mg/ml was prepared by adding water to the power and then boiling for 5 min. After a brief centrifugation, the supernatant was collected and passed through a 0.2 m filter. The stock solution was stored at 4C.
3 Cell proliferation and viability assays: Cell proliferation and viability were determined using manual counting and WST-1 assays. For manual counting, cells were seeded in 24 well plates. After treatment with Ganoderma lucidum, the cell morphology was observed under microscope, and then viability cells number were determined by manual counting . For WST-1 assay, cells were seeded on 96-well culture plates at a density of 10,000 cells per well. After treatment with different concentrations of Ganoderma for 48h, WST-1 reagent was added to the cells and absorbance was measured at 450 nm using an ELISA plate reader. The inhibition rate of the cells was calculated as follows: inhibition rate (%)=1-the absorbance of drug-treated group / the absorbance of control group×100%.
4 Cell cycle analysis: To determine the cell cycle phase distribution, flow cytometry was performed. 48h after treatment with different concentration Ganoderma lucidum 0,0.5,1,2mg/ml , 2X106 cells were collected and washed twice with PBS, and fixed with 3 ml ice cold 70% ethanol. The cells were then washed twice with cold PBS, and resuspended with 1ml staining buffer containing 50ug/ml RNAse A and 50ug/ml propipdium iodide. Stain cells for 1 hour at room temperature in the dark. Finally, samples were analyzed by flow cytometry machine .
5 Assessment of apoptosis by Hoechst-33258 : The changes of apoptotic morphological changes in ovarian cancer cells after treated with ganoderma lucidum were detected by Hoechst 33258 DNA staining and and examined by fluorescence microscopy.Control and treated cells were fixed in 4% paraf- ormaldehyde in 1xPBS for 20 min, washed with 1XPBS, stained with Hoechst 33258 at 1μg/ml in PBS for 15 min. Stained cells were washed 2 times with 1xPBS. The changes in nuclei were observed with a fluorescent microscope through UV-filter.
6 Ganoderma Lucidum inhibited Epithelial ovarian cancer cell growth: Epithelial ovarian cancer cell lines were treated with different concentration of Ganoderma Lucidum . The cell growth were determined by Colony formation assay and Hanging drop culture for spheroid formation. Formation of cell Colony and Multicell Spheroids were examined.
7 Ganoderma Lucidum inhibited Epithelial ovarian cancer cell adhesion, migration and invasion: Epithelial ovarian cancer cell lines were treated with different dose of Ganoderma Lucidum .The ability of cell adhesion,migration and invasion were detected by adhesion assay,Wound healing assay (migr- ation assay), and invasion assay.For Wound healing assay, Ten points were marked randomly down the wound and pictures were taken at the same points at regular time intervals(0, 24,48h). Area migrated by the cells was measured by computer software Simple PCI.
8 Reversal effect of Ganoderma lucidum on cisplantin resistance in ovarian cancer cell line:Cells cultured in 24 well plate at a density of 6X104 cells per well. Group 1: Cells were treated with different concentrations of cisplatin. Group 2: Cells were treated with different concentrations of Gano- derma lucidum. Group 3: Cells were treated with both different concentrations of cisplatin and 0.5mg/ml Ganoderma lucidum. Live cell numbers were determined using manual counting and WST-1 assays. The cell morphology was observed under microscope. The cell inhibition rate, drug resistance factor and reverse factor were calculated using result of WST-1 assay. The reversing factor (RF) was calculated as follows: RF=IC50(control group)/IC50 (drug- treated group). resistance factor=IC50 (cisplantin treated group)/IC50 (cispla- tin and Ganoderma lucidum treated group)
9 Western blot analysis: Epithelial ovarian cancer cells were seeded into 6-well plates. Five experimental groups were divided. Group 1: To investigate the molecular mechanism of ganoderma lucidum on proliferation and apoptosis in the human ovarian cell line. Epithelial ovarian cancer cells were seeded into 6-well plates. The cells were treated with different dose (0、1、2mg/ml)of ganoderma lucidum. 48h after treatment, the total protein were extracted. The protein lysates were separated in 12% SDS polyacrylamide gels, transferred to Immobilon PVDF Membranes and probed with apoptosis-associated proteins such as anti-bcl-2(1:1000),bax(1:4000),cleaved- caspase-3(1:500),P27(1:1000), ECL kit was used for chemiluminescent detection of immobilized proteins.
Group 2: To study the molecular mechanism of ganoderma lucidum on invision and metastasis in the human ovarian cell line. Epithelial ovarian cancer cells were treated with different dose(0、0.5、1、2mg/ml)of ganoderma lucidum. The E-cadherin(1:500), N-cadherin(1:500),vimentin(1:1000)MMP-9 and TIMP-1 were detected by western blot after treated with Ganoderma lucidum as described above.
Group 3: To determine the expression of bcl-2, Akt, P-Akt , P53 in chemosensitive cell line A2780s and chemoresistant cell line A2780cp. The cells both A2780s and A2780cp were seeded into 6-well plate and cultured for 48h. The total proteins were extracted. The bcl-2、Akt、P-Akt、P53 was detected by western blot .
Group 4: Epithelial ovarian cancer cells were treated 48h with different dose(0、0.5、1、2mg/ml)of ganoderma lucidum. The expression of protein bcl-2, Akt , P-Akt and P53 were detected by western blot respectively.
Group 5: To Study the molecular mechanism of the reversal effect of Ganoderma lucidum on multidrug resistance (MDR) in the Cisplantin-resistant ovarian cancer cell line A2780CP. Cells cultured in 24-well plate at a density of 6X104 cells per well and treated with both 0.5 mg/ml Ganoderma lucidum and different concentrations of cisplatin for 48 hours. At the end of the experiment, the total proteins were extracted. The expression of protein bcl-2, Akt, P-Akt and P53 were detected by western blot. Protein loading was evaluated using mouse anti-β-actin.
Result:1 Ganoderma Lucidum inhibited cell growth and viability in Epithelial ovarian cancer cells: To determine the effect of Ganoderma Lucidum on ovarian cancer cells, we first treated cells with 0.1, 0.5 and 1 mg/ml of Ganoderm Lucidum. Ovarian cancer cell lines had a decrease number of cells after treatment with higher doses of Ganoderma. A significant effect on cell growth was observed at 1mg/ml Ganoderma. To confirm these results, WST-1 assays were performed in dose-dependent and time-course experiments. Again, we observed that OV2008 cells treated with 2mg/ml of Ganoderma exhibited very significant decrease in cell viability at 48h after treatment. The results of WST-1 assays showed that Ganoderma lucidum could inhibit the A2780S and A2780CP cells growth as well. WST-1 assays showed that 0.5 mg/ml of ganoderma lucidum spore can be used to do reverse resistance experiment as lower toxicity dose (cell growth rate more than 80%).
2 Ganoderma induced apoptosis and blocked cell cycle progression at G1: We determined if Ganoderma regulates apoptosis. Hoechst 33258 staining showed that ganoderma lucidum was capable of inducing apoptosis in OV2008 cells, which was characterized by features of apoptotic nuclei such as nuclear shrinkage, chromatin condensation. To examine how Ganoderma inhibited cell growth and viability, we used flow cytometry to determine the effect of Ganoderma on cell cycle progression. Analysis of cell cycle profile in control and Ganoderma-treated cells revealed that Ganoderma increased the population of cells in G1 phase induced G1 phase arrest of OV2008 cells and decreased the number of cells in G1 phase. It did not change the percentage of cells in S phase.
3 Ganoderma lucidum inhibited epithelial ovarian cancer cell colony formation: colony formation assays were used to test the effect of Ganoderma lucidum on cell colony growth. Skov3 cells were treated without or with Ganoderma and plated. At 7 days after plating, colonies were stained and photographed. The result showed Ganoderma lucidum strongly inhibited colony formation in a dose-dependent manner. Cloning formation rate reduced gradually. Moreover, the size and the number of single cell colony decreased in a dose-dependent manner under inverted microscope.
4 Ganoderma lucidum inhibited epithelial ovarian cancer cell adhesion ability: Cell adhesion is an important step in the initiation of tumor formation and matastasis. We tested the effects of Ganoderma lucidum spores on ?epithelial ovarian cancer cell adhesion. Ovarian cancer skov3 cells were incubated with Ganoderma lucidum spores for 3h and 6h, unattached cells were removed and the plates were washed. The attached cells were pictured and counted. Our experiments indicated that Ganoderma lucidum spores inhibited epithelial ovarian cancer cell adhesion ability. The number of adhesion cells decreased in a dose-dependent manner under inverted microscope and cell counting.
5 Ganoderma lucidum inhibited epithelial ovarian cancer cell multicell spheroid formation ability: The effect of Ganoderma lucidum on spheroid formation was assessed using a hanging drop culture. When droplets of skov3 cells were plated on the inner cover of culture dish, cells proliferate, aggregate at 24h after hanging drop, and form tight spheroids at day 4 after hanging drop. When cells were treated with 1 and 2 mg/ml Ganoderma, they only formed lose spheroids and irregular spheroids. The ability of spheroid formation reduced gradually with the increasing of dose of Ganoderma lucidum spore. The result indicated that Ganoderma lucidum inhibited epithelial ovarian cancer cell multicell spheroid formation in a dose dependent manner.
6 Ganoderma lucidum inhibited epithelial ovarian cancer cell migration ability: Since Ganoderma lucidum inhibits cell growth and viability in ovarian cancer cells, to determine if Ganoderma lucidum inhibits cell migration, the migration assays were performed. The skov3 cells were treated without or with different doses of Ganoderma lucidum and a wound healing assay was carried out. Briefly, cells were cultured in 6-well plates until cells reached confluence and a wound was made using a 200μl pipette tip. After scratching, the culture medium was refreshed and different doses of Ganoderma lucidum (0, 0.5, 1, 2 mg/ml) were added. Ten points were marked randomly down the wound and pictures were taken at the same points before and 24h after incubation with Ganoderma lucidum. The area migrated by the cells was measured by computer software Simple PCI. We found that wound were almost closure at 24h after hanging drop in untreated group and the migration ?ability were inhibited in treated group in a dose-dependent manner. After treated with 2mg/ml of Ganoderma lucidum, the wounds almost no difference between 24hour and at 0hour after scratching. The experiment suggested Ganoderma lucidum strongly inhibited wound closure in skov3 cell lines.
7 Ganoderma lucidum inhibited epithelial ovarian cancer cell invasion ability: In cell invasion assay, matrigel poured on the surface of porous filter membrane formed a structure similar to the natural basal membrane. The ability of the cells to penetrate matrigel reflected its invasive capability. The results showed that the average number of skov3 cells that penetrated the membrane was 162.8±8.1, 132.8±6.5, 65.2±3.9 and 39.2±1.3respectively. Statistical analysis showed that there was significant difference between the cell invasion ability of control and that of Ganoderma lucidum treated cells (P>0.05), indicating that Ganoderma lucidum inhibited epithelial ovarian cancer cell invasion ability.
8 Cisplantin inhibited epithelial ovarian cancer chemosensitive cell line A2780S and chemoresistant cells line A2780-cp proliferation: To determine inhibition effect of Cisplatin on epithelial ovarian cancer chemosensitive cell line A2780S and chemoresistant cells line A2780-cp, A2780S cells were treated with 0,1,2.5,5umol/L of cisplatin and A2780-cp cells were treated with 0,5,10,20umol/L of cisplatin. At 48h after treatment, Cell proliferation and viability were determined using WST-1 assays; and morphology and number of cell were determined by microscope and manual counting. The result showed that the number of A2780-cp was similar to A2780S after treated with different dose ciaplatin. A2780-cp and A2780S were treated with the same dose of cisplatin. We found that the number of A2780S less than that of A2780-cp obviously at 48h after treatment and the cisplatin inhibition rate to A2780S was significant higher than that of A2780-cp. The IC50 of A2780S and A2780-cp was 10.32 and 29.94mg/ml respectively. The result indicated that A2780CP cell line result in resistance to cisplatin, the resistance factor was 2.9.
9 Ganoderma lucidum enhanced the anti-tumor effect of cisplatin and Reversal the drug resistance of cisplatin on ovarian cancer chemoresistant cells A2780-cp: Since we observed that Ganodema lucidum was effective in inhibiting growth/viability in both chemosensitive and chemoresistant cells, we examined if it will enhance the sensitivity of ovarian cancer cells to a chemo-therapeutic agent, cisplatin and Reversal their chemoresistance to cisplatin. A2780-cp cells were treated with different concentration of cisplatin in the presence or absence of a low dose of Ganoderma lucidum. The Ganoderma lucidum treatment alone slightly decreased cell number. However, when added with cisplatin, it significantly enhanced the effect of cisplatin on inhibiting cell growth. The median inhibitory concentration(IC50) reduced from 29.94 to12.16umol/L. These results indicated that Ganoderma lucidum can reverse the drug resistance of chemoresistant cells A2780-cp to Cisplantin. The RF(reversing factor) was2.46. The results were consistent with of cell counting and microscope.
10 Effects of Ganoderma lucidum on proteins expression on apoptosis, invasion, matastasis and drug-resistance in epithelial ovarian cancer cell line:
Group1: The results of Western blot suggested that the Bax,P27 protein level was increased after treatment with ganoderma lucidum and the levels of bcl-2 decreased in a dose-dependent manner. The 35 kD proenzyme caspase-3 was cleaved to its active 19 kD form after treatment .
Group2: To study the molecular mechanism of Ganoderma lucidum inhibiting effect on invasion and metastasis in epithelial ovarian cancer. The E-cadherin, N-cadherin and vimentin were detected by western blot. The expression of E-cadherin and TIMP-1 of human ovarian cancer cells were increased, that of N-cadherin, vimentin and MMP-9 was decreased in dose-dependent manner.
Group3: To understand the mechanisms underlying the chemo-enhancing effect of Ganoderma lucidum, we tested if it could regulate Akt,P-Akt ,bcl-2 and p53 expression and activation. The western blot was carried out to detect Akt,P-Akt ,bcl-2 and p53 .The results of western blot suggested that The expression of Akt,P-Akt ,bcl-2 in A2780CP cell was significantly higher than that in A2780S cell, whereas the expression of P53 showed decre- ased .The P53 protein level was increased and the level of Akt,P-Akt ,bcl-2 was decreased after treatment with Ganoderma lucidum . Moreover,when A2780CP cells were exposed to Ganoderma lucidum and Cisplantin simultaneously,the level of Akt,P-Akt,bcl-2 reduced markedly and the P53 increased than that exposed to Cisplantin alone. Conclusion:To the best of our knowledge, this is the first report of Ganoderma lucidum on epithelial ovarian cancer cells. Here, we demonstrated that Ganoderma lucidum has potent anti-tumor effects on chemosensitive and chemoresistant ovarian cancer cells and reversing drug-resistance effect on chemoresistant ovarian cancer cells.
Conclusion 1:Ganoderma lucidum inhibited cell proliferation and indu- ced apoptosis in human ovarian cancer cells through inducing cell cycle G1phase arrest and up-regulation of bax , p27, down-regulation of bcl-2 and activation of caspase-3.
Conclusion 2:Ganoderma lucidum may inhibit invasion and metastasis in human ovarian cancer cells by inhibiting adhesion, migration, invasion and increasing the expression of E-cadherin and TIMP-1 of human ovarian cancer cells, decreasing that of N-cadherin, vimentin and MMP-9.
Conclusion3:Ganoderma lucidum can enhance chemosensitivity of epi- thelial ovarian cancer cells to cisplatin and reverse the resistance of A2780- CP cells to Cisplantin by downregulating the expression of Akt,P-Akt,bcl-2 and upregulating the expression of P-53 in A2780CP cells. Ganoderma lucidum may be a promising multidrug resistance modulator.
Taken together, we have demonstrated that Ganoderma lucidum exerts multiple anti-tumor effects on ovarian cancer cells such as inhibit proliferation, adhesion, migration, invasion and induce apoptosis. We have also provided evidence that Ganoderma lucidum enhances chemosensitivity of epithelial ovarian cancer cells to cisplatin and reverses resistance to cisplatin in chemoresistance cell line A2780CP. Our findings suggest that Ganoderma lucidum may be useful in treatment of chemosensitive and chemoresistant epithelial ovarian cancer. Therefore, Ganoderma lucidum spore can be as useful supplementary means of ovarian cancer conventional chemotherapy with its anti-cancer properties and can improve the curative effect, benefit to the cytoreductive surgery (surgical debulking) if combining neoadjuvant chemotherapy. Combining chemotherapeutics, Ganoderma lucidum can enhance anti-cancer effect and prevent and reverse chemoresistance after surgery. Ganoderma lucidum may be served as a possible therapeutic agent as a dietary supplement for cancer patients.
近年来,天然药物以其低毒、高效的独特优势越来越受到国内外学者的广泛关注,从中草药和天然植物中提取抗肿瘤成分已成为获得有效抗癌制剂和耐药逆转剂的重要途径。灵芝孢子是一种已被广泛应用于肿瘤治疗的天然植物产品。体内外实验表明灵芝孢子具有明显抗肿瘤活性。国内外研究报道灵芝孢子对乳腺癌、肺癌、鼻咽癌、胃癌、直肠癌、前列腺癌、白血病、肝癌、黑色素瘤等肿瘤细胞的生长、侵袭、转移能力有明显的抑制作用,而且能增强顺铂的抗肿瘤效应。但至目前为止灵芝孢子在卵巢癌治疗方面的研究国内外均未见报道,为探讨灵芝孢子对人卵巢癌细胞生长、转移以及对顺铂化疗耐药的影响,本课题以多个卵巢上皮性癌细胞系为研究对象,以破壁灵芝孢子粉提取物为处理因素,应用细胞增殖抑制实验(WST-1比色法)、光学显微镜观察法、细胞计数法、Hoechst-33258染色方法、流式细胞技术、克隆形成实验、多细胞球体形成实验、细胞迁移实验、细胞侵袭实验以及western blot方法等,对其进行研究。目的在于①研究破壁灵芝孢子提取物对卵巢上皮性癌细胞的生长抑制作用、诱导凋亡作用及其机制。②研究破壁灵芝孢子提取物对卵巢上皮性癌细胞的粘附、侵袭、转移能力的抑制作用及相关机制。③探讨破壁灵芝孢子提取物对人卵巢上皮性癌顺铂耐药细胞化疗耐药的逆转作用及其机制。为灵芝孢子的进一步开发以及在卵巢上皮癌的临床应用提供理论依据。
方法:
1细胞培养:体外常规培养人卵巢上皮癌细胞株OV2008、C13*、A2780S、A2780CP、SKOV3以及人卵巢上皮细胞株IOSE398。
2破壁灵芝孢子粉工作液制备:加拿大energene天然药品有限公司提供的破壁灵芝孢子粉溶于DD-H2O配制成50mg/ml的灵芝孢子储存液。高温灭菌、离心后取上清,用0.20μm微孔滤膜过滤器过滤除菌,保存于4℃冰箱。实验前以细胞培养液稀释成相应浓度的灵芝孢子液。
3破壁灵芝孢子粉提取物对人卵巢上皮性癌细胞增殖的抑制作用:以合适的密度接种细胞,待细胞贴壁后加入不同终浓度的破壁灵芝孢子液,48h用WST-1比色法及人工计数活细胞法检测灵芝孢子粉对人卵巢上皮性癌细胞增殖的抑制作用,显微镜下观察细胞形态的改变。利用WST-1比色结果的OD值计算药物对细胞生长的抑制率,细胞抑制率=1-实验组平均OD值/对照组平均OD值×100%。IC50用Logit法计算。
4流式细胞术测定破壁灵芝孢子提取物对人卵巢上皮性癌细胞凋亡及细胞周期分布影响:接种细胞于6cm培养盘,细胞贴壁后分别加入终浓度为0.5,1,2mg/ml的灵芝孢子粉溶液,对照组为不含药物的细胞培养液。处理48h以不同的方法分别收集细胞,用流式细胞仪测定细胞凋亡及细胞周期改变。
5 Hoechst-33258染色法评估破壁灵芝孢子对人卵巢上皮性癌细胞凋亡的影响:细胞分为对照组和处理组,处理组分别加入终浓度为0.5,1,2mg/ml的灵芝孢子粉溶液,对照组加入不含药物的细胞培养液,培养细胞48h后,Hoechst-33258染色法观察细胞核变化,判断凋亡情况。
6破壁灵芝孢子提取物抑制人卵巢上皮性癌细胞生长能力测定:不同浓度破壁灵芝孢子处理人卵巢上皮性癌细胞,利用克隆形成实验、悬滴培养多细胞球体形成实验检测灵芝孢子对人卵巢上皮性癌细胞生长抑制情况,同时设不含药物的对照组进行实验。
7破壁灵芝孢子提取物抑制人卵巢上皮性癌细胞侵袭和转移能力测定:在体外用不同浓度灵芝孢子处理不同的人卵巢上皮性癌细胞,利用细胞黏附实验、细胞迁移运动(划痕)实验、细胞侵袭实验,检测人卵巢上皮性癌细胞粘附、侵袭和转移能力的变化,同时设不含药物的对照组进行实验。
8人卵巢癌顺铂耐药细胞株的顺铂耐药性及破壁灵芝孢子提取物逆转其耐药作用的测定:实验分为单独应用顺铂、单独应用破壁灵芝孢子提取物以及顺铂与灵芝孢子联合应用三组,采用WST-1方法、人工计数细胞法及显微镜观察法检测人卵巢上皮性癌细胞顺铂敏感细胞和耐药细胞增殖的抑制作用。分别计算抑制率、耐药倍数及逆转倍数,以判定顺铂的耐药性以及破壁灵芝孢子能否逆转其耐药性。耐药倍数=耐药细胞的IC50/非耐药细胞的IC50。逆转倍数=顺铂单独处理组的IC50/灵芝孢子联合顺铂处理组的IC50。
9 Western blot技术检测相关蛋白表达:细胞接种于6孔板,分成不同的实验组进行处理。
第一组:不同浓度破壁灵芝孢子液(0、1、2mg/ml)处理细胞48h后提取总蛋白蛋白样品经12%SDS -聚丙烯酰胺凝胶(检测cleaved casep- ase-3用15% SDS -聚丙烯酰胺凝胶)90V电压进行电泳分离,之后电转移至硝酸纤维素滤膜上,测定bcl-2和Bax,cleaved- caspase3 , P27凋亡相关蛋白表达情况以探讨破壁灵芝孢子抑制人卵巢上皮性癌细胞凋亡机理。
第二组:不同浓度破壁灵芝孢子液(0、0.5、1、2mg/ml)处理人卵巢上皮癌细胞48h后提取总蛋白测定粘附及转移相关蛋白E-cadherin, N-cadherin、vimentin、MMP9以及TIMP-1的表达情况了解灵芝孢子抑制人卵巢上皮癌细胞侵袭、转移的机理。
第三组:bcl-2、Akt、P-Akt、P53蛋白在人卵巢上皮癌顺铂耐药细胞株A2780CP及敏感细胞株A2780S的表达测定。细胞接种于6孔板,培养48h提取总蛋白,比较两种细胞bcl-2、Akt、P-Akt、P53蛋白的表达情况,明确顺铂耐药细胞株和敏感细胞株耐药相关蛋白的不同表达。
第四组:不同浓度破壁灵芝孢子液(0、0.5、1、2mg/ml)处理A2780CP和A2780S细胞48h后提取总蛋白测定bcl-2、Akt、P-Akt、P53蛋白的表达情况,了解单独应用破壁灵芝孢子对耐药相关蛋白的表达影响。
第五组:接种A2780CP细胞后分为破壁灵芝孢子单独处理组(破壁灵芝孢子浓度为0.5mg/ml)及破壁灵芝孢子(0.5mg/ml)与顺铂(浓度为5umol/L)联合处理组进行处理。提取处理人卵巢上皮性癌细胞48h的总蛋白测定bcl-2、Akt、P-Akt、P53蛋白的表达,以判定灵芝孢子逆转耐药的机理。均以β-actin为内参照。
?结果:
1破壁灵芝孢子粉提取物抑制人卵巢上皮性癌细胞增殖:不同浓度的破壁灵芝孢子处理人卵巢上皮性癌细胞后倒置显微镜下观察到活性细胞数目减少,并呈浓度依赖性,高浓度(大于2mg/ml)破壁灵芝孢子组可见细胞脱离培养皿壁成悬浮状态,致贴壁存活细胞数目极少。采用WST-1比色法及人工细胞计数法检测的结果与显微镜下观察到的结果相似。经WST-1结果确定0.5mg/ml的灵芝孢子可以作为低毒剂量(细胞生长率大于80%)用于逆转耐药实验。
2破壁灵芝孢子提取物诱导人卵巢上皮性癌细胞凋亡并造成细胞周期G1期阻滞:Hoechst-33258染色后可以观察到人卵巢上皮性癌细胞凋亡现象,表现为细胞核缩小,染色质浓缩,药物浓度越大凋亡细胞越多。并且流式细胞技术可以检测到破壁灵芝孢子处理人卵巢上皮性癌细胞72h后可以出现凋亡峰,凋亡率随浓度增加而升高。流式细胞技术检测结果表明破壁灵芝孢子作用人卵巢上皮性癌细胞后多数细胞聚集于G1期,并且G1期细胞数目随作用时间延长而增加。而G2/M期细胞数目较少,且G2/ M细胞随作用时间延长而减少,S期细胞数目随作用时间无明显改变。
3破壁灵芝孢子提取物抑制人卵巢上皮性癌细胞克隆形成能力:经破壁灵芝孢子处理的人卵巢上皮性癌细胞克隆形成数目明显少于对照组,并呈浓度依赖性,即随着灵芝孢子浓度逐渐增加克隆数目逐渐减少,计算克隆形成率逐渐降低,而且单个细胞克隆的大小及组成克隆的细胞数也随灵芝孢子浓度的增加而递减。
4破壁灵芝孢子粉提取物抑制人卵巢上皮性癌细胞粘附能力:不同浓度灵芝孢子处理人卵巢上皮性癌细胞3h及6h后的粘附细胞数量较对照组均有减少。
5破壁灵芝孢子粉提取物抑制人卵巢上皮性癌细胞多细胞球体形成:在悬滴培养条件下,对照组人卵巢上皮性癌细胞呈聚集生长,于悬滴形成后24h细胞即可聚集成团,之后细胞呈立体生长形成多细胞球体,至第四天球体体积最大,呈圆形,外形规则,细胞间粘附紧密。实验组细胞间粘附性降低,形成的多细胞球体较松散,形状不规则,随灵芝孢子浓度增加形成球体的能力减低。
6破壁灵芝孢子粉提取物抑制人卵巢上皮性癌细胞迁移能力:不同浓度的破壁灵芝孢子处理人卵巢上皮性癌细胞后可观察到对照组SKOV3细胞迁移活跃,划痕后24h几近完全愈合;而灵芝孢子处理组细胞迁移能力明显受到抑制,随着药物浓度的增加抑制作用增强,即划痕的愈合面积逐渐降低,并呈一定的剂量依赖关系,2mg/ml的高浓度组显示不仅划痕的宽度无明显变化,且细胞呈现明显受抑制状态。实验组24h迁移面积较对照组明显减低,差异有统计学意义,P<0.05。
7破壁灵芝孢子提取物抑制人卵巢上皮性癌细胞侵袭能力:侵袭实验表明破壁灵芝孢子处理人卵巢上皮性癌细胞skov3后实验组与对照组比较侵袭至微孔滤膜上的细胞数明显不同,浓度为0、0.5、1和2mg/ml的破壁灵芝孢子处理后其侵袭的细胞数分别为162.8±8.1, 132.8±6.5, 65.2±3.9 and39.2±1.3,可见实验组侵袭细胞数明显低于对照组,且实验组中随灵芝孢子浓度的增加侵袭细胞数逐渐减少,即侵袭率逐渐减少,差异具有统计学意义,(P<0.05)。
8顺铂对敏感细胞株A2780S和耐药细胞株A2780CP的抑制作用:为了解顺铂对卵巢癌敏感细胞株和耐药细胞株的抑制作用,用浓度比例为4:1的顺铂分别处理A2780CP和A2780S细胞48h,经WST-1测定和倒置显微镜下所观察到两种细胞的抑制率相似,提示A2780CP对顺铂的敏感性较A2780S明显降低。采用同一浓度(5umol/L)的顺铂处理两种细胞48h后A2780S数目明显少于A2780CP,经计算顺铂对A2780S细胞的抑制率显著高于对A2780CP的抑制率,其IC50分别为10.32和29.94mg/ml,提示A2780CP对顺铂具有耐药性,耐药倍数约为2.9倍。
9破壁灵芝孢子粉提取物增强顺铂的抗肿瘤作用并逆转A2780CP细胞对顺铂的耐药性:WST-1结果显示灵芝孢子和顺铂联合应用组较单独应用顺铂组对A2780CP细胞的抑制率明显提高,其IC50由单用顺铂的29.94降低至12.16umol/L,表明灵芝孢子可以逆转A2780CP对顺铂的耐药性,逆转倍数为2.46。该结果与细胞计数及显微镜观察的结果相似。
10破壁灵芝孢子对人卵巢上皮性癌细胞凋亡、侵袭及耐药相关蛋白表达影响:
第一组:Western blot方法检测到凋亡抑制蛋白bcl-2、bcl-xl随破壁灵芝孢子浓度增加而下降,而促凋亡蛋白bax及P27、casepase-3逐渐升?高,并且35KD的Casbase-3被激活成活化的19,20KD的cleaved-casep- ase3,并随破壁灵芝孢子浓度增加表达呈升高趋势。
第二组:破壁灵芝孢子作用细胞后实验组E-cadherin、TIMP-1的表达水平均高于对照组;而N-cadherin、vimentin及MMP-9的表达水平均低于对照组,并且随灵芝孢子浓度的增加而呈浓度依赖性改变。
第三组:Western blot结果表明耐药细胞A2780CP bcl-2、AKT、P-AKT的表达明显高于A2780S,而P53的表达则低于A2780S细胞。不同浓度破壁灵芝孢子处理A2780CP后P53蛋白水平随灵芝孢子浓度增加而增加;而bcl-2、Akt、P-Akt水平则逐渐降低。破壁灵芝孢子联合顺铂处理耐药细胞较单独应用顺铂bcl-2、Akt、P-Akt的表达水平明显降低;而P53的表达水平明显升高。
结论:本文在国内外首次研究了破壁灵芝孢子提取物对人卵巢上皮性癌细胞的多种抗癌作用以及逆转人卵巢上皮癌顺铂耐药细胞株对顺铂的耐药性并初步探讨其机理。
1破壁灵芝孢子粉提取物具有抑制人卵巢上皮性癌细胞生长及诱导凋亡的作用,其机制是作用于细胞周期的G1期,造成G1期阻滞,并可以通过上调bax、P27,下调bcl-2并活化casepase-3而发挥其作用。
2破壁灵芝孢子粉提取物在体外可抑制人卵巢上皮性癌细胞侵袭和转移,其可能的机制是抑制细胞粘附、上调E-cadherin及TIMP-1表达、降调N-cadherin、vimentin及MMP-9的表达。
3破壁灵芝孢子提取物能增强人卵巢上皮癌细胞对顺铂的敏感性,有效逆转卵巢癌顺铂耐药细胞株A2780CP的耐药性,其可能的机制是抑制Akt、P-Akt及bcl-2,增强P53蛋白的表达。
综上所述,本研究证明了破壁灵芝孢子提取物对人卵巢上皮性癌细胞具有多种抗癌作用,可以抑制卵巢癌细胞生长、侵袭和转移,诱导细胞凋亡,增强卵巢癌细胞对顺铂化疗的敏感性并能逆转人卵巢癌顺铂耐药细胞株对顺铂的耐药性。因此,具有抗癌效应的灵芝孢子可以作为卵巢癌常规化疗手段的有益补充,联合新辅助化疗可以提高疗效利于更彻底的肿瘤细胞减灭术。用于术后化疗可以协同化疗药物提高化疗效果,预防及逆转对卵巢癌化疗药物顺铂的耐药,可以作为有效的辅助治疗卵巢上皮癌的抗癌制剂。本研究为灵芝孢子在卵巢上皮癌的临床应用提供了理论依据,填补了灵芝孢子在卵巢癌应用方面的空白。
Objective: Ovarian cancer is one of the three gynecological malignancy. Epithelial ovarian cancer (EOC) is the most common form of ovarian cancer accounting for 90% of it. Due to the lack of effective screening strategies and the absence of symptoms in early-stage of disease, 75% of cases have progressed to an advanced stage at the time of primary diagnosis. The most effective treatment for EOC is the surgery ( cytoreduction) plus the platinum- based chemotherapy, such as cisplatin. However, the majority(70%)of patients with advanced ovarian cancer ultimately experience chemoresistance, and recurrence of their disease and the 5-year survival rate of patients is less than 30%. ovarian cancer is the most common life-threatening malignant tumors among women and the first leading cause of death from gynecological disorder. Thus,The effective anti-tumor and reversing drug resistance of platinum product need to be developed,the effective therapeutic strategies and the investigation of the molecular mechanism responsible for the effects of anti-tumor and reversing anti-resistance are exigently demanded.
Recently,the natural active substances attracted the attention of resear- cher at home and abroad with the characteristics of high activity and small adverse reactions. It has become a main Approaches to obtain effective anti-cancer agents and drug-resistant reversal agents by antitumor composition extraction from Chinese herbal medicine and natural plant. Ganoderma lucidum wall-broken spores,a natural plant product , have been widely used to treat various human diseases, including cancers). significant amount of research had shown that Ganoderma lucidum exerts multiple anti-tumor activity on cancer cells. There were some reporters at home and abroad in recent years that Ganoderma wall-broken spores suppressed growth and invasive behavior of cells of breast cancer,lung cancer, nasopharyngeal darcinoma,carcinoma of stomach,colorectal cancer, prostate cancer, leucoc- ythemia, hepatoma,melanoma lymphomas and enhanced cisplatin anti-tumor effect .however,the effect of Ganoderma Lucidum on epithelial ovarian cancer (EOC) has not been reported at home and at abroad. In this study, we determined if Ganoderma Lucidum regulates EOC cell activity . Using several cell lines derived from EOC, We observed the effects of ganoderma lucidum on proliferation and apoptosis in the human ovarian cell line (ov2008 cells) , and study its possible mechanism ; investigated the inhibitory effects of Ganoderma lucidum on invasion and metastasis of human ovarian cancer cells; investigated the reversal effect of Ganoderma lucidum on multidrug resistance (MDR) in the Cisplantin-resistant ovarian cancer cell line by WST-1 assay, microscope, cell counting, Hoechst-33258 staining, flow cytometry, colony formation assay, hanging drop culture, migration assay, invasion assay and western blot . It will provide the theoretical basis for further development and clinical application of ganoderma lucidum spore in the epithelial ovarian cancer.
Objective 1: To observe the effects of ganoderma lucidum on prolifer- ation and apoptosis in the human ovarian cell line, and study its possible mechanism .
Objective 2: To investigate the inhibitory effects and molecular mech- anism of Ganoderma lucidum on invasion and metastasis of human ovarian cancer cells in vitro .
Objective: 3 To investigate the reversal effect of Ganoderma lucidum on multidrug resistance (MDR) in the Cisplantin-resistant ovarian cancer cell line A2780CP.
Method:1 Cell culture:epithelial ovarian cancer cell lines OV2008, C13*, A2780s, A2780-cp and SKOV3 were obtained and cultured. Cells were grown at 37°C in an atmosphere with 5% CO2。
2 Preparation of Ganoderma lucidum:The Ganoderma lucidum used in this study was purchased from Energene Naturals Inc (Toronto, On) which contains wall-broken spores and polysaccharides. A stock solution of 50mg/ml was prepared by adding water to the power and then boiling for 5 min. After a brief centrifugation, the supernatant was collected and passed through a 0.2 m filter. The stock solution was stored at 4C.
3 Cell proliferation and viability assays: Cell proliferation and viability were determined using manual counting and WST-1 assays. For manual counting, cells were seeded in 24 well plates. After treatment with Ganoderma lucidum, the cell morphology was observed under microscope, and then viability cells number were determined by manual counting . For WST-1 assay, cells were seeded on 96-well culture plates at a density of 10,000 cells per well. After treatment with different concentrations of Ganoderma for 48h, WST-1 reagent was added to the cells and absorbance was measured at 450 nm using an ELISA plate reader. The inhibition rate of the cells was calculated as follows: inhibition rate (%)=1-the absorbance of drug-treated group / the absorbance of control group×100%.
4 Cell cycle analysis: To determine the cell cycle phase distribution, flow cytometry was performed. 48h after treatment with different concentration Ganoderma lucidum 0,0.5,1,2mg/ml , 2X106 cells were collected and washed twice with PBS, and fixed with 3 ml ice cold 70% ethanol. The cells were then washed twice with cold PBS, and resuspended with 1ml staining buffer containing 50ug/ml RNAse A and 50ug/ml propipdium iodide. Stain cells for 1 hour at room temperature in the dark. Finally, samples were analyzed by flow cytometry machine .
5 Assessment of apoptosis by Hoechst-33258 : The changes of apoptotic morphological changes in ovarian cancer cells after treated with ganoderma lucidum were detected by Hoechst 33258 DNA staining and and examined by fluorescence microscopy.Control and treated cells were fixed in 4% paraf- ormaldehyde in 1xPBS for 20 min, washed with 1XPBS, stained with Hoechst 33258 at 1μg/ml in PBS for 15 min. Stained cells were washed 2 times with 1xPBS. The changes in nuclei were observed with a fluorescent microscope through UV-filter.
6 Ganoderma Lucidum inhibited Epithelial ovarian cancer cell growth: Epithelial ovarian cancer cell lines were treated with different concentration of Ganoderma Lucidum . The cell growth were determined by Colony formation assay and Hanging drop culture for spheroid formation. Formation of cell Colony and Multicell Spheroids were examined.
7 Ganoderma Lucidum inhibited Epithelial ovarian cancer cell adhesion, migration and invasion: Epithelial ovarian cancer cell lines were treated with different dose of Ganoderma Lucidum .The ability of cell adhesion,migration and invasion were detected by adhesion assay,Wound healing assay (migr- ation assay), and invasion assay.For Wound healing assay, Ten points were marked randomly down the wound and pictures were taken at the same points at regular time intervals(0, 24,48h). Area migrated by the cells was measured by computer software Simple PCI.
8 Reversal effect of Ganoderma lucidum on cisplantin resistance in ovarian cancer cell line:Cells cultured in 24 well plate at a density of 6X104 cells per well. Group 1: Cells were treated with different concentrations of cisplatin. Group 2: Cells were treated with different concentrations of Gano- derma lucidum. Group 3: Cells were treated with both different concentrations of cisplatin and 0.5mg/ml Ganoderma lucidum. Live cell numbers were determined using manual counting and WST-1 assays. The cell morphology was observed under microscope. The cell inhibition rate, drug resistance factor and reverse factor were calculated using result of WST-1 assay. The reversing factor (RF) was calculated as follows: RF=IC50(control group)/IC50 (drug- treated group). resistance factor=IC50 (cisplantin treated group)/IC50 (cispla- tin and Ganoderma lucidum treated group)
9 Western blot analysis: Epithelial ovarian cancer cells were seeded into 6-well plates. Five experimental groups were divided. Group 1: To investigate the molecular mechanism of ganoderma lucidum on proliferation and apoptosis in the human ovarian cell line. Epithelial ovarian cancer cells were seeded into 6-well plates. The cells were treated with different dose (0、1、2mg/ml)of ganoderma lucidum. 48h after treatment, the total protein were extracted. The protein lysates were separated in 12% SDS polyacrylamide gels, transferred to Immobilon PVDF Membranes and probed with apoptosis-associated proteins such as anti-bcl-2(1:1000),bax(1:4000),cleaved- caspase-3(1:500),P27(1:1000), ECL kit was used for chemiluminescent detection of immobilized proteins.
Group 2: To study the molecular mechanism of ganoderma lucidum on invision and metastasis in the human ovarian cell line. Epithelial ovarian cancer cells were treated with different dose(0、0.5、1、2mg/ml)of ganoderma lucidum. The E-cadherin(1:500), N-cadherin(1:500),vimentin(1:1000)MMP-9 and TIMP-1 were detected by western blot after treated with Ganoderma lucidum as described above.
Group 3: To determine the expression of bcl-2, Akt, P-Akt , P53 in chemosensitive cell line A2780s and chemoresistant cell line A2780cp. The cells both A2780s and A2780cp were seeded into 6-well plate and cultured for 48h. The total proteins were extracted. The bcl-2、Akt、P-Akt、P53 was detected by western blot .
Group 4: Epithelial ovarian cancer cells were treated 48h with different dose(0、0.5、1、2mg/ml)of ganoderma lucidum. The expression of protein bcl-2, Akt , P-Akt and P53 were detected by western blot respectively.
Group 5: To Study the molecular mechanism of the reversal effect of Ganoderma lucidum on multidrug resistance (MDR) in the Cisplantin-resistant ovarian cancer cell line A2780CP. Cells cultured in 24-well plate at a density of 6X104 cells per well and treated with both 0.5 mg/ml Ganoderma lucidum and different concentrations of cisplatin for 48 hours. At the end of the experiment, the total proteins were extracted. The expression of protein bcl-2, Akt, P-Akt and P53 were detected by western blot. Protein loading was evaluated using mouse anti-β-actin.
Result:1 Ganoderma Lucidum inhibited cell growth and viability in Epithelial ovarian cancer cells: To determine the effect of Ganoderma Lucidum on ovarian cancer cells, we first treated cells with 0.1, 0.5 and 1 mg/ml of Ganoderm Lucidum. Ovarian cancer cell lines had a decrease number of cells after treatment with higher doses of Ganoderma. A significant effect on cell growth was observed at 1mg/ml Ganoderma. To confirm these results, WST-1 assays were performed in dose-dependent and time-course experiments. Again, we observed that OV2008 cells treated with 2mg/ml of Ganoderma exhibited very significant decrease in cell viability at 48h after treatment. The results of WST-1 assays showed that Ganoderma lucidum could inhibit the A2780S and A2780CP cells growth as well. WST-1 assays showed that 0.5 mg/ml of ganoderma lucidum spore can be used to do reverse resistance experiment as lower toxicity dose (cell growth rate more than 80%).
2 Ganoderma induced apoptosis and blocked cell cycle progression at G1: We determined if Ganoderma regulates apoptosis. Hoechst 33258 staining showed that ganoderma lucidum was capable of inducing apoptosis in OV2008 cells, which was characterized by features of apoptotic nuclei such as nuclear shrinkage, chromatin condensation. To examine how Ganoderma inhibited cell growth and viability, we used flow cytometry to determine the effect of Ganoderma on cell cycle progression. Analysis of cell cycle profile in control and Ganoderma-treated cells revealed that Ganoderma increased the population of cells in G1 phase induced G1 phase arrest of OV2008 cells and decreased the number of cells in G1 phase. It did not change the percentage of cells in S phase.
3 Ganoderma lucidum inhibited epithelial ovarian cancer cell colony formation: colony formation assays were used to test the effect of Ganoderma lucidum on cell colony growth. Skov3 cells were treated without or with Ganoderma and plated. At 7 days after plating, colonies were stained and photographed. The result showed Ganoderma lucidum strongly inhibited colony formation in a dose-dependent manner. Cloning formation rate reduced gradually. Moreover, the size and the number of single cell colony decreased in a dose-dependent manner under inverted microscope.
4 Ganoderma lucidum inhibited epithelial ovarian cancer cell adhesion ability: Cell adhesion is an important step in the initiation of tumor formation and matastasis. We tested the effects of Ganoderma lucidum spores on ?epithelial ovarian cancer cell adhesion. Ovarian cancer skov3 cells were incubated with Ganoderma lucidum spores for 3h and 6h, unattached cells were removed and the plates were washed. The attached cells were pictured and counted. Our experiments indicated that Ganoderma lucidum spores inhibited epithelial ovarian cancer cell adhesion ability. The number of adhesion cells decreased in a dose-dependent manner under inverted microscope and cell counting.
5 Ganoderma lucidum inhibited epithelial ovarian cancer cell multicell spheroid formation ability: The effect of Ganoderma lucidum on spheroid formation was assessed using a hanging drop culture. When droplets of skov3 cells were plated on the inner cover of culture dish, cells proliferate, aggregate at 24h after hanging drop, and form tight spheroids at day 4 after hanging drop. When cells were treated with 1 and 2 mg/ml Ganoderma, they only formed lose spheroids and irregular spheroids. The ability of spheroid formation reduced gradually with the increasing of dose of Ganoderma lucidum spore. The result indicated that Ganoderma lucidum inhibited epithelial ovarian cancer cell multicell spheroid formation in a dose dependent manner.
6 Ganoderma lucidum inhibited epithelial ovarian cancer cell migration ability: Since Ganoderma lucidum inhibits cell growth and viability in ovarian cancer cells, to determine if Ganoderma lucidum inhibits cell migration, the migration assays were performed. The skov3 cells were treated without or with different doses of Ganoderma lucidum and a wound healing assay was carried out. Briefly, cells were cultured in 6-well plates until cells reached confluence and a wound was made using a 200μl pipette tip. After scratching, the culture medium was refreshed and different doses of Ganoderma lucidum (0, 0.5, 1, 2 mg/ml) were added. Ten points were marked randomly down the wound and pictures were taken at the same points before and 24h after incubation with Ganoderma lucidum. The area migrated by the cells was measured by computer software Simple PCI. We found that wound were almost closure at 24h after hanging drop in untreated group and the migration ?ability were inhibited in treated group in a dose-dependent manner. After treated with 2mg/ml of Ganoderma lucidum, the wounds almost no difference between 24hour and at 0hour after scratching. The experiment suggested Ganoderma lucidum strongly inhibited wound closure in skov3 cell lines.
7 Ganoderma lucidum inhibited epithelial ovarian cancer cell invasion ability: In cell invasion assay, matrigel poured on the surface of porous filter membrane formed a structure similar to the natural basal membrane. The ability of the cells to penetrate matrigel reflected its invasive capability. The results showed that the average number of skov3 cells that penetrated the membrane was 162.8±8.1, 132.8±6.5, 65.2±3.9 and 39.2±1.3respectively. Statistical analysis showed that there was significant difference between the cell invasion ability of control and that of Ganoderma lucidum treated cells (P>0.05), indicating that Ganoderma lucidum inhibited epithelial ovarian cancer cell invasion ability.
8 Cisplantin inhibited epithelial ovarian cancer chemosensitive cell line A2780S and chemoresistant cells line A2780-cp proliferation: To determine inhibition effect of Cisplatin on epithelial ovarian cancer chemosensitive cell line A2780S and chemoresistant cells line A2780-cp, A2780S cells were treated with 0,1,2.5,5umol/L of cisplatin and A2780-cp cells were treated with 0,5,10,20umol/L of cisplatin. At 48h after treatment, Cell proliferation and viability were determined using WST-1 assays; and morphology and number of cell were determined by microscope and manual counting. The result showed that the number of A2780-cp was similar to A2780S after treated with different dose ciaplatin. A2780-cp and A2780S were treated with the same dose of cisplatin. We found that the number of A2780S less than that of A2780-cp obviously at 48h after treatment and the cisplatin inhibition rate to A2780S was significant higher than that of A2780-cp. The IC50 of A2780S and A2780-cp was 10.32 and 29.94mg/ml respectively. The result indicated that A2780CP cell line result in resistance to cisplatin, the resistance factor was 2.9.
9 Ganoderma lucidum enhanced the anti-tumor effect of cisplatin and Reversal the drug resistance of cisplatin on ovarian cancer chemoresistant cells A2780-cp: Since we observed that Ganodema lucidum was effective in inhibiting growth/viability in both chemosensitive and chemoresistant cells, we examined if it will enhance the sensitivity of ovarian cancer cells to a chemo-therapeutic agent, cisplatin and Reversal their chemoresistance to cisplatin. A2780-cp cells were treated with different concentration of cisplatin in the presence or absence of a low dose of Ganoderma lucidum. The Ganoderma lucidum treatment alone slightly decreased cell number. However, when added with cisplatin, it significantly enhanced the effect of cisplatin on inhibiting cell growth. The median inhibitory concentration(IC50) reduced from 29.94 to12.16umol/L. These results indicated that Ganoderma lucidum can reverse the drug resistance of chemoresistant cells A2780-cp to Cisplantin. The RF(reversing factor) was2.46. The results were consistent with of cell counting and microscope.
10 Effects of Ganoderma lucidum on proteins expression on apoptosis, invasion, matastasis and drug-resistance in epithelial ovarian cancer cell line:
Group1: The results of Western blot suggested that the Bax,P27 protein level was increased after treatment with ganoderma lucidum and the levels of bcl-2 decreased in a dose-dependent manner. The 35 kD proenzyme caspase-3 was cleaved to its active 19 kD form after treatment .
Group2: To study the molecular mechanism of Ganoderma lucidum inhibiting effect on invasion and metastasis in epithelial ovarian cancer. The E-cadherin, N-cadherin and vimentin were detected by western blot. The expression of E-cadherin and TIMP-1 of human ovarian cancer cells were increased, that of N-cadherin, vimentin and MMP-9 was decreased in dose-dependent manner.
Group3: To understand the mechanisms underlying the chemo-enhancing effect of Ganoderma lucidum, we tested if it could regulate Akt,P-Akt ,bcl-2 and p53 expression and activation. The western blot was carried out to detect Akt,P-Akt ,bcl-2 and p53 .The results of western blot suggested that The expression of Akt,P-Akt ,bcl-2 in A2780CP cell was significantly higher than that in A2780S cell, whereas the expression of P53 showed decre- ased .The P53 protein level was increased and the level of Akt,P-Akt ,bcl-2 was decreased after treatment with Ganoderma lucidum . Moreover,when A2780CP cells were exposed to Ganoderma lucidum and Cisplantin simultaneously,the level of Akt,P-Akt,bcl-2 reduced markedly and the P53 increased than that exposed to Cisplantin alone. Conclusion:To the best of our knowledge, this is the first report of Ganoderma lucidum on epithelial ovarian cancer cells. Here, we demonstrated that Ganoderma lucidum has potent anti-tumor effects on chemosensitive and chemoresistant ovarian cancer cells and reversing drug-resistance effect on chemoresistant ovarian cancer cells.
Conclusion 1:Ganoderma lucidum inhibited cell proliferation and indu- ced apoptosis in human ovarian cancer cells through inducing cell cycle G1phase arrest and up-regulation of bax , p27, down-regulation of bcl-2 and activation of caspase-3.
Conclusion 2:Ganoderma lucidum may inhibit invasion and metastasis in human ovarian cancer cells by inhibiting adhesion, migration, invasion and increasing the expression of E-cadherin and TIMP-1 of human ovarian cancer cells, decreasing that of N-cadherin, vimentin and MMP-9.
Conclusion3:Ganoderma lucidum can enhance chemosensitivity of epi- thelial ovarian cancer cells to cisplatin and reverse the resistance of A2780- CP cells to Cisplantin by downregulating the expression of Akt,P-Akt,bcl-2 and upregulating the expression of P-53 in A2780CP cells. Ganoderma lucidum may be a promising multidrug resistance modulator.
Taken together, we have demonstrated that Ganoderma lucidum exerts multiple anti-tumor effects on ovarian cancer cells such as inhibit proliferation, adhesion, migration, invasion and induce apoptosis. We have also provided evidence that Ganoderma lucidum enhances chemosensitivity of epithelial ovarian cancer cells to cisplatin and reverses resistance to cisplatin in chemoresistance cell line A2780CP. Our findings suggest that Ganoderma lucidum may be useful in treatment of chemosensitive and chemoresistant epithelial ovarian cancer. Therefore, Ganoderma lucidum spore can be as useful supplementary means of ovarian cancer conventional chemotherapy with its anti-cancer properties and can improve the curative effect, benefit to the cytoreductive surgery (surgical debulking) if combining neoadjuvant chemotherapy. Combining chemotherapeutics, Ganoderma lucidum can enhance anti-cancer effect and prevent and reverse chemoresistance after surgery. Ganoderma lucidum may be served as a possible therapeutic agent as a dietary supplement for cancer patients.
引文
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