二甲双胍对肺腺癌生长的抑制作用及其机制研究
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摘要
肺腺癌是原发性肺癌的一种常见的病理类型,约占肺癌总发病人数的30%。由于肺腺癌多发于肺周边,且早期即可侵犯血管和淋巴管而发生远处转移,因此,多数肺腺癌在初诊时已是晚期,失去了手术治疗的时机。目前对肺腺癌患者的治疗以化疗及放疗为主。尽管随着新型辅助化疗药物及肺癌靶向药物的临床应用,肺腺癌患者的生存质量得到了显著改善,但由于肺癌病情进展快,且部分患者对化、放疗存在耐受性差、耐药性强等问题,总体治疗效果并不满意。据统计,中晚期肺癌患者的5年生存率低于15%。因此,寻找安全而有效的辅助治疗方法,以提高对肺腺癌的治疗效果是临床亟待解决的问题。
二甲双胍是一种胰岛素增敏剂,多年来被广泛应用于对2型糖尿病患者的降血糖治疗。近年来的临床研究观察到,二甲双胍对肿瘤患者似乎有一定程度的保护作用,能够降低肿瘤的发生率和肿瘤相关死亡率。这一现象近期已得到了部分基础研究的证实。通过对乳腺癌、结直肠癌及前列腺癌等的研究发现,二甲双胍具有一定程度的抗肿瘤活性。然而,二甲双胍对肺腺癌的生长增殖是否具有相似的抑制作用尚未见报道;其发挥抗肿瘤活性的机制至今仍不清楚。本课题从细胞实验出发,检测了二甲双胍对肺腺癌细胞增殖的作用及其与顺铂联合作用对肺腺癌生长的影响,并通过分子生物学的方法对其作用机制进行了研究;最后通过动物实验对前期结果加以证实,并初步评价了二甲双胍作为一种抗癌药物应用于临床的可行性。全文共包括四部分内容:
第一部分二甲双胍对肺腺癌细胞体外增殖的作用研究
目的:研究二甲双胍对体外培养的肺腺癌细胞株的增殖、细胞周期、凋亡等方面的作用,观察二甲双胍与顺铂联合使用对细胞生长的影响。方法:以0、0.5、2、8mmol/L四种浓度的二甲双胍分别对肺腺癌细胞株A549和NCI-H1299进行体外干预,在第0、24、48和72h,以MTT法检测各组细胞的增殖情况。将上述各浓度的二甲双胍与顺铂(10ug/mL)联用后,再次检测药物干预不同时间后细胞的活性变化。选取A549细胞株为主要研究对象,以二甲双胍干预48h后,采用流式细胞术检测各组细胞在细胞周期及早期凋亡率等方面的差异。结果:(1)与对照组相比,二甲双胍2mmol/L组和8mmol/L组细胞的增殖活性在药物作用24h后即明显下降,以后者的下降更为明显;0.5mmol/L组细胞在药物作用48h后也呈显著的生长抑制。随着干预时间延长,二甲双胍对各组细胞增殖的抑制更加显著。(2)二甲双胍与顺铂联合使用时,对肺腺癌细胞增殖的抑制作用较单用顺铂更强;二甲双胍的浓度越高、对细胞干预的时间越长,该抑制作用越明显。(3)二甲双胍干预A549细胞48h后,与对照组相比,0.5mmol/L组细胞的周期分布及早期凋亡率均无明显变化;2mmol/L组G1期细胞所占比重显著增高(P<0.05),而S期细胞比重显著降低(P<0.01),细胞的早期凋亡率显著增加(P<0.05);8mmol/L组G1期细胞所占比重显著升高,S期和G2/M期细胞则相应减少,细胞的早期凋亡率也明显增高(均P<0.01)。结论:二甲双胍可以使细胞周期循环阻滞于G1期,并诱导细胞的早期凋亡增加,从而有效抑制肺腺癌细胞株的体外增殖,其作用呈浓度及时间依赖性增强;二甲双胍与顺铂共同作用可以增强对肺腺癌细胞生长的抑制作用。
第二部分JNK/p38 MAPK--Caspase信号传导通路在二甲双胍诱导肺腺癌细胞凋亡中的作用研究
目的:研究二甲双胍对A549细胞内AMPK、JNK、p38 MAPK的磷酸化蛋白及Caspase家族蛋白表达水平的影响,探讨JNK/p38 MAPK--Caspase信号传导通路在二甲双胍诱导的肺腺癌细胞凋亡中的作用。方法:以5mmol/L的二甲双胍对A549细胞进行体外干预,在干预的第0、15、30和60min时,分别抽提细胞总蛋白,以Western blot技术检测细胞内AMPK、JNK、p38 MAPK的总蛋白和磷酸化蛋白的表达;在干预后第0、12、24和48h,检测剪切后的Caspase-3、8、9活性蛋白的表达水平。选择特异性的JNK通路阻断剂SP600125和p38 MAPK通路阻断剂SB202190分别对细胞进行预处理1~2h,再进行二甲双胍(5mmol/L)干预,48h后,采用流式细胞术检测细胞早期凋亡的情况。结果:(1)二甲双胍干预前后,A549细胞内AMPK、JNK和p38 MAPK的总蛋白表达强度无显著变化。二甲双胍干预前,细胞内上述蛋白的磷酸化蛋白及剪切后的Caspase-3、8、9的表达量极低;p-AMPK、p-p38和p-JNK的表达水平在药物干预15min后即明显增强,至60min后明显衰减,而相应的总蛋白则变化不明显;Caspase-8、9活性蛋白的表达水平在药物干预12h后明显增强,24h后,Caspase-3的表达也出现增强。(2)流式细胞术检测结果提示,SP600125和SB202190本身对A549细胞的凋亡无显著影响;但使用这两种抑制剂分别对细胞进行预处理后,再行二甲双胍干预,细胞的早期凋亡率较单纯二甲双胍干预组显著降低(P<0.05)。结论:JNK/p38 MAPK--Caspase信号传导通路参与了对二甲双胍诱导的肺腺癌细胞凋亡的调控。第三部分GADD153在二甲双胍诱导肺腺癌细胞凋亡中的作用研究目的:研究二甲双胍对肺腺癌A549细胞内GADD153基因表达的影响,探讨GADD153基因在二甲双胍诱导的肺腺癌细胞凋亡中的作用。方法:以5mmol/L的二甲双胍对A549细胞进行体外干预,在第0、12、24、48和72h,抽提细胞总RNA,采用Realtime-PCR技术检测各时间点A549细胞内GADD153 mRNA的相对表达量。设计并合成GADD153靶向siRNA,将其转染入A549细胞,通过RNA干扰特异性的抑制细胞内GADD153基因及蛋白的表达。采用Realtime-PCR和Western blot技术验证对目的基因的干扰效率。以5mmol/L的二甲双胍干预GADD153基因干扰后的A549细胞,采用流式细胞术检测药物干预48h后细胞的早期凋亡率。结果:(1)二甲双胍作用12h后,A549细胞内的GADD153 mRNA表达水平即显著增高(P<0.01);随药物干预时间的延长,该基因的表达增高更加明显,至72h升至最高,约为干预前的14.12倍。(2)GADD153 siRNA转染可以有效地抑制A549细胞内GADD153基因的表达,干扰效率达86%左右。(3)流式细胞术检测结果显示,GADD153基因干扰对于A549细胞的早期凋亡无显著影响;经二甲双胍干预48h后,GADD153干扰的A549细胞的早期凋亡率与未干扰的细胞相比明显降低(P<0.05)。结论:GADD153基因及蛋白的表达增高可促进二甲双胍诱导的肺腺癌细胞的凋亡。
第四部分二甲双胍抑制肺腺癌裸鼠皮下移植瘤生长的实验研究
目的:通过构建肺腺癌的裸鼠皮下移植瘤模型,研究二甲双胍对于肺腺癌体内生长的影响及安全性,并探讨二甲双胍在临床抗肿瘤治疗方面的价值。方法:将人肺腺癌细胞A549接种至BALB/c-nu小鼠腋背部皮下,建立移植瘤动物模型。待移植瘤长至约100mm3左右时,选取瘤块大小、形状较均一的裸鼠,随机分为3组,每组5只,以腹腔注射的方式给药治疗,每日1次,持续1个月。对照组给予PBS 0.2ml/d;met40组给予二甲双胍40mg/kg/d;met200组给予二甲双胍200mg/kg/d。观察药物干预过程中裸鼠的一般情况,每4天称量动物的体重,测量移植瘤的长短径,以计算肿块大小。治疗结束后,颈椎脱臼法处死荷瘤鼠,剥离移植瘤及肺、心、脑、肝、肾等脏器,以HE染色观察肿瘤及各器官的形态,判定肿瘤有无远处转移;以免疫组化染色检测各组移植瘤内Ki-67的表达情况;以TUNEL染色原位检测肿瘤细胞的凋亡情况。试验重复2次。结果:(1)以A549细胞接种于裸鼠右侧腋背部皮下,成功构建了肺腺癌的裸鼠皮下移植瘤模型。在为期1个月的药物干预过程中,各组裸鼠的一般状态良好,无明显异常表现,未见明显药物毒副反应。(2)在药物干预的不同时间测量移植瘤的大小,均以对照组的肿瘤体积最大,met40组其次,met200组的肿瘤体积最小。药物干预1个月后,两个二甲双胍治疗组的移植瘤平均体积均显著小于对照组(P<0.01);以对照组为参照,40mg/kg/d和200mg/kg/d剂量的二甲双胍腹腔注射给药对裸鼠移植瘤生长的抑制率分别为19.81%±6.25%和40.70%±8.98%。(3)对移植瘤标本行免疫组化染色结果显示,met40和met200组肿瘤内Ki-67抗原的阳性表达率分别为38.80%±10.41%和32.85%±10.14%,均显著低于对照组(58.18%±12.83%)(P<0.01)。(4)TUNEL染色结果显示,对照组肿瘤内仅可见散在的凋亡细胞(3.1±1.8个/HPF),而met40组和met200组肿瘤内的凋亡细胞数量显著增加(分别为13.6±3.41个/HPF和16.35±3.07个/HPF)(P<0.01),以后者增加更为明显。结论:40mg/kg/d和200mg/kg/d两种浓度的二甲双胍均可有效的抑制肺腺癌裸鼠皮下移植瘤的生长速度,使肿瘤细胞的凋亡增多、增殖活性减弱,且不会引起明显的毒副反应,提示二甲双胍对于肺腺癌患者的治疗具有较好的临床应用前景。
通过上述研究,得到结论如下:
1、二甲双胍可以通过阻滞细胞周期于G1期,并诱导细胞凋亡,从而抑制肺腺癌细胞的体外增殖;二甲双胍与顺铂联用可增强对肺腺癌细胞的生长抑制作用。
2、JNK/p38 MAPK--Caspase信号传导通路参与了对二甲双胍诱导的肺腺癌细胞凋亡的调控,阻断JNK/p38 MAPK通路可以部分地降低二甲双胍作用下肺腺癌细胞的早期凋亡率。
3、二甲双胍可以诱导肺腺癌细胞内GADD153基因及蛋白表达增高,从而促进细胞凋亡。
4、二甲双胍腹腔注射给药可以抑制肺腺癌裸鼠皮下移植瘤的生长,降低肿瘤细胞的增殖活性、促进细胞凋亡,且无明显毒副反应。
Lung cancer is the most common cancer in the world both in terms of incidence and mortality of the total. Lung adenocarcinoma is a common histopathologic type and the percentage of patient with lung adenocarcinma is 30% of all the patients with lung cancer. In most adenocarcinoma patients the tumors grows close to pleura. Though the proliferation rate is not quite high, adenocarcinoma is very angio- and lymph-invasive. Most of the lung adenocarcinoma patients are in the late stage of the disease when first diagnosed and surgical resection is not applicable. Although combined-modality therapy with radiotherapy and chemotherapy has been applied, more than 85% of the patients die within 5 years. New therapeutic modalities are therefore necessary to improve the response to treatment.
The biguanide metformin is one of the mostly used drugs for treatment of type 2 diabetes. By reducing hepatic gluconeogebesis and increasing glucose uptake in skeletal muscles, metformin can lower blood glucose levels while inducing no risk of hypoglycemia and very rarely causes lactic acidosis. Recent clinical studies have revealed that metformin treatment is associated with reduced cancer risk and improved prognosis. Evans et al did a pilot observational study reporting that among patients with type 2 diabetes, those treated with metformin have a lower incidence of cancer compared with those untreated. In an independent study, using administrative databases of a population-based cohort, Bowker et al reported that type 2 diabetes patients who use metformin have a lower cancer-related mortality compared with those that use sulfonylureas or insulin. There is also growing experimental evidence that metformin may impede the growth of human tumors, including prostate cancer, breast carcinoma, colon carcinoma, ovarian cancer, et al. However, until now, the relation between lung adenocarcinoma and metformin has not been studied and the precise anti-tumor mechanisms of the anti-neoplastic effects of metformin are not completely understood.
In the present study, we evaluated the effects of metformin on the growth of lung adenocarcinoma in vitro and in vivo, and its relationship to AMPK activation, JNK/p38 MAPK--Caspase signaling pathways activation and growth arrest and DNA damage -inducible transcription factor (GADD) 153. Our results open new therapeutic possibilities for the use of metformin as an anti-neoplasm agent. Four parts are included in this study:
Part one Effects of metformin on proliferation in lung adenocarcinoma cell lines A549 and NCI-H1299 in vitro
Objective To study the effects of metformin on cell viability, cell cycle, apoptosis and the synergism with cispatin in lung adenocarcinoma cell lines A549 and NCI-H1299 in vitro. Methods and materials Cells were treated with metformin (0, 0.5, 2 or 8 mM) in vitro together with cisplatin (10ug/mL) or not. Cell growth was determined using Methylthiazolyldiphenyl-tetrazolium bromide (MTT) following the manufacturer’s protocol at the time of 0, 24, 48 and 72 hours after treatment. After treatment with metformin for 48 hours, apoptosis and cell cycle of A549 cells were exemined by Flow cytometry. Apoptotic cells were analyzed by double staining with
fluoresceinisothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) . Cell cycle was analyzed by measuring the amount of PI-labeled DNA in ethanol-fixed cells. Results The cell viability started to decrease as early as 24 hours later after metformin treatment. Metformin induced significant proliferation inhibition on both A549 and NCI-H1299 cell lines in a dose- and time-dependent manner. When applied in combination with metformin, the cytotoxicity of cisplatin on both cells was stronger. After treatment for 48 hours, metformin at 2 and 8mM caused significant increases both in numbers of apoptotic (annexin+PI–) A549 cells and G1 phases fraction (P<0.05). While the apoptotic rate and cell-cycle progression of A549 cells treated with 0.5mM metformin did not change much compared to those of the control cells. Conclusions Metformin can inhibit the growth of lung adenocarcinoma cell lines significantly and potentiate cisplatin in a dose- and time-dependent manner in vitro. Cell-cycle progression is arrested in G1 stage and apoptosis is indued after metformin treatment.
Part two Role of JNK/p38 MAPK--Caspase signaling pathways in metformin-induced apoptosis of lung adenocarcinoma cell line A549
Objective To detect the effects of metformin on the phosphorylation of AMPK, JNK and p38 MAPK and the expression of cleaved Caspase-3, 8, 9 proteins in A549 cells, and investigate the correlation between JNK/p38 MAPK--Caspase signaling pathways and cell apoptosis. Methods and materials A549 cells were collected and lysed for total protein after treated with metformin (5 mM) for 0, 15, 30 and 60 minutes. Western blot analysis was performed to detect the expressions of pan- and pho- AMPK, JNK and p38 proteins. Cleaved Caspase-3, 8, 9 proteins were also detected. SP600125 and SB202190, the specific inhibitor for JNK and p38 MAPK signaling pathways respectively, were chosen to treat cells prior to metformin treatment and apoptotic rates of A549 cells were then analyzed by Flow cytometry.
Results The expressions of pho-AMPK, pho-JNK and pho-p38 were detected after 15 min of metformin treatment. The levels of cleaved Caspase-8, 9 proteins elevated after 12 h of metformin treatment. After treatment for 30 min, the expression level of cleaved Caspase-3 protein also elevated. After pretreated with SP600125 or SB202190, the metformin-induced apoptotic rates of A549 cells significantly decreased (P<0.05), though still higher than those of the cells treated with SP600125 or SB202190 only.
Conclusions JNK/p38 MAPK--Caspase signaling pathways are involved in metformin-induced apoptosis in lung adenocarcinoma cell line A549.
Part three GADD153 is involved in metformin-induced A549 cell apoptosis
Objective To detect the effects of metformin on the expression of GADD153 mRNA and protein in A549 cells, and to investigate the role of GADD153 gene in metformin-induced A549 cell apoptosis.
Methods and materials A549 cells were treated with metformin (5mM) for 0, 12, 24, 48 and 72 hours. The expression of GADD153 mRNA in A549 cells was detected by Realtime-PCR. GADD153 siRNA (50nM) or negative control siRNA (50nM) was transient transfected into human lung adenocarcinoma cell line A549 cells by INTERFERinTM siRNA transfection reagent. At 48h after transfection, cells were collected and the total RNA and protein were extracted. Realtime-PCR and western blot were performed to detect the level of GADD153 mRNA and protein respectively to assess the efficiency of GADD153 gene silencing. A549 cells were then divided into four groups: Negative control siRNA (siNEG) group, GADD153 siRNA (siGADD153) group, siNEG+metformin group and siGADD153+metformin group. The cells of the last two groups were treated with metformin (5mM) at 10h after siRNA transfection. PBS was used instead of metformin to the cells of siNEG group and siGADD153 group. After siRNA transfection and metformin or PBS treatment for another 48 hours, cell apoptotic rate of every group was detected by Flow cytometry.
Results Realtime-PCR analysis showed that the expression of GADD153 mRNA in A549 cells increased significantly at 12h after metformin treatment (P<0.01) and kept increasing to a level as high as 14.12-fold of the original level after treatment for 72h. Realtime-PCR and western blot analysis showed that both GADD153 mRNA and protein expression were significantly decreased after transient transfected with specific GADD153 siRNA. The cell apoptitic rate of siGADD153+metformin group was significantly lower than that of siNEG+metformin group (P<0.05), though no significant difference was detected between siGADD153 group and siNEG group (P=0.591).
Conclusions The apoptosis-inducing effect of metformin in lung adenocarcinoma cells is partly due to the up-regulation of GADD153 mRNA and protein in the cells under metformin treatment.
Part four Repressive effects of metformin on the growth of xenograft of lung adenocarcinoma in nude mice
Objective To study the suppressive effect of metformin on the growth of lung adenocarcinoma xenograft in nude mice and its security and to evaluate the potential value of metformin as a novel anti-neoclassic agent for the treatment of lung adenocarcinoma.
Methods and materials A549 cells were harvested, washed twice in PBS, and resuspended in 200ul PBS before being inoculated subcutaneouly in 4- to 6-week-old females BALB/c nude mice on the right flank (2×107 cells per site). Treatment with metformin was started when the average tumor volumes reached 100mm3. Five mice were used per group. Metformin (200 mg or 40 mg×kg body weight) was dissolved in 200ul PBS and administered daily with i.p. injections. The control group received vehicle only (200ul PBS). Animal weight and tumor volume (mm3) were measured every 4 days and estimated from caliper measurements using the formulaπ/6×A×B2 (A is larger diameter, B is smaller diameter). After treatment for a month, the mice were all sacrified and paraffin sections of tumors were used for immunohistochemical analyses with hemotoxylin and eosin stain and proliferation with Ki-67 and apoptosis with TUNEL staining.
Results No severe adverse effect was observed among the xenograft mice during metformin treatment. The growth of lung adenocarcinoma xenograft of met40 group and met200 group was significantly suppressed compared with that of the control group at every point of the treatment course. When the treatment ended after a month, the mice of met200 group had the smallest average tumor volume among the three groups, followed by those of met40 group. Compared with control group, the inhibition ratio of metformin at the concentration of 40 mg/kg/d and 200 mg/kg/d to the growth of tumor was 19.81%±6.25% and 40.70%±8.98%, respectively. The average positive rate of Ki-67 in tumors of the control, met40 and met200 group was 58.18%±12.83%, 38.80%±10.41% and 32.85%±10.14%, respectively. The average number of positive cells per HPF in tumors of the control, met40 and met200 group was 3.1±1.8, 13.6±3.41 and 16.35±3.07, respectively. Both the positive rate of Ki-67 and number of apoptotic cells in met40 and met200 group were significantly higher than those of the control group (P<0.01). Conclusions Metformin can inhibit the growth of lung adenocarcinoma xenograft in nude mice by attenuating the expression of the proliferation marker Ki-67 and inducing tumor cell apoptosis without any severe adverse effect.
In summary, we found from this study that metformin can inhibit the proliferation of lung adenocarcinoma cell lines and potentiate cisplatin in a dose- and time-dependent manner in vitro. Both JNK/p38 MAPK--Caspase signaling pathways and GADD153 gene are involved in metformin-induced apoptosis. When applied to the mice with xenograft tumor, metformin can markedly inhibit the growth of the tumor while causing no severe adverse effect. Our results indicate that metformin has anti-neoplastic activity and may be used as an adjunctive agent for the treatment of lung adenocarcinoma. The detailed mechanisms of the anti-neoplastic activity of metformin should be further studied.
二甲双胍是一种胰岛素增敏剂,多年来被广泛应用于对2型糖尿病患者的降血糖治疗。近年来的临床研究观察到,二甲双胍对肿瘤患者似乎有一定程度的保护作用,能够降低肿瘤的发生率和肿瘤相关死亡率。这一现象近期已得到了部分基础研究的证实。通过对乳腺癌、结直肠癌及前列腺癌等的研究发现,二甲双胍具有一定程度的抗肿瘤活性。然而,二甲双胍对肺腺癌的生长增殖是否具有相似的抑制作用尚未见报道;其发挥抗肿瘤活性的机制至今仍不清楚。本课题从细胞实验出发,检测了二甲双胍对肺腺癌细胞增殖的作用及其与顺铂联合作用对肺腺癌生长的影响,并通过分子生物学的方法对其作用机制进行了研究;最后通过动物实验对前期结果加以证实,并初步评价了二甲双胍作为一种抗癌药物应用于临床的可行性。全文共包括四部分内容:
第一部分二甲双胍对肺腺癌细胞体外增殖的作用研究
目的:研究二甲双胍对体外培养的肺腺癌细胞株的增殖、细胞周期、凋亡等方面的作用,观察二甲双胍与顺铂联合使用对细胞生长的影响。方法:以0、0.5、2、8mmol/L四种浓度的二甲双胍分别对肺腺癌细胞株A549和NCI-H1299进行体外干预,在第0、24、48和72h,以MTT法检测各组细胞的增殖情况。将上述各浓度的二甲双胍与顺铂(10ug/mL)联用后,再次检测药物干预不同时间后细胞的活性变化。选取A549细胞株为主要研究对象,以二甲双胍干预48h后,采用流式细胞术检测各组细胞在细胞周期及早期凋亡率等方面的差异。结果:(1)与对照组相比,二甲双胍2mmol/L组和8mmol/L组细胞的增殖活性在药物作用24h后即明显下降,以后者的下降更为明显;0.5mmol/L组细胞在药物作用48h后也呈显著的生长抑制。随着干预时间延长,二甲双胍对各组细胞增殖的抑制更加显著。(2)二甲双胍与顺铂联合使用时,对肺腺癌细胞增殖的抑制作用较单用顺铂更强;二甲双胍的浓度越高、对细胞干预的时间越长,该抑制作用越明显。(3)二甲双胍干预A549细胞48h后,与对照组相比,0.5mmol/L组细胞的周期分布及早期凋亡率均无明显变化;2mmol/L组G1期细胞所占比重显著增高(P<0.05),而S期细胞比重显著降低(P<0.01),细胞的早期凋亡率显著增加(P<0.05);8mmol/L组G1期细胞所占比重显著升高,S期和G2/M期细胞则相应减少,细胞的早期凋亡率也明显增高(均P<0.01)。结论:二甲双胍可以使细胞周期循环阻滞于G1期,并诱导细胞的早期凋亡增加,从而有效抑制肺腺癌细胞株的体外增殖,其作用呈浓度及时间依赖性增强;二甲双胍与顺铂共同作用可以增强对肺腺癌细胞生长的抑制作用。
第二部分JNK/p38 MAPK--Caspase信号传导通路在二甲双胍诱导肺腺癌细胞凋亡中的作用研究
目的:研究二甲双胍对A549细胞内AMPK、JNK、p38 MAPK的磷酸化蛋白及Caspase家族蛋白表达水平的影响,探讨JNK/p38 MAPK--Caspase信号传导通路在二甲双胍诱导的肺腺癌细胞凋亡中的作用。方法:以5mmol/L的二甲双胍对A549细胞进行体外干预,在干预的第0、15、30和60min时,分别抽提细胞总蛋白,以Western blot技术检测细胞内AMPK、JNK、p38 MAPK的总蛋白和磷酸化蛋白的表达;在干预后第0、12、24和48h,检测剪切后的Caspase-3、8、9活性蛋白的表达水平。选择特异性的JNK通路阻断剂SP600125和p38 MAPK通路阻断剂SB202190分别对细胞进行预处理1~2h,再进行二甲双胍(5mmol/L)干预,48h后,采用流式细胞术检测细胞早期凋亡的情况。结果:(1)二甲双胍干预前后,A549细胞内AMPK、JNK和p38 MAPK的总蛋白表达强度无显著变化。二甲双胍干预前,细胞内上述蛋白的磷酸化蛋白及剪切后的Caspase-3、8、9的表达量极低;p-AMPK、p-p38和p-JNK的表达水平在药物干预15min后即明显增强,至60min后明显衰减,而相应的总蛋白则变化不明显;Caspase-8、9活性蛋白的表达水平在药物干预12h后明显增强,24h后,Caspase-3的表达也出现增强。(2)流式细胞术检测结果提示,SP600125和SB202190本身对A549细胞的凋亡无显著影响;但使用这两种抑制剂分别对细胞进行预处理后,再行二甲双胍干预,细胞的早期凋亡率较单纯二甲双胍干预组显著降低(P<0.05)。结论:JNK/p38 MAPK--Caspase信号传导通路参与了对二甲双胍诱导的肺腺癌细胞凋亡的调控。第三部分GADD153在二甲双胍诱导肺腺癌细胞凋亡中的作用研究目的:研究二甲双胍对肺腺癌A549细胞内GADD153基因表达的影响,探讨GADD153基因在二甲双胍诱导的肺腺癌细胞凋亡中的作用。方法:以5mmol/L的二甲双胍对A549细胞进行体外干预,在第0、12、24、48和72h,抽提细胞总RNA,采用Realtime-PCR技术检测各时间点A549细胞内GADD153 mRNA的相对表达量。设计并合成GADD153靶向siRNA,将其转染入A549细胞,通过RNA干扰特异性的抑制细胞内GADD153基因及蛋白的表达。采用Realtime-PCR和Western blot技术验证对目的基因的干扰效率。以5mmol/L的二甲双胍干预GADD153基因干扰后的A549细胞,采用流式细胞术检测药物干预48h后细胞的早期凋亡率。结果:(1)二甲双胍作用12h后,A549细胞内的GADD153 mRNA表达水平即显著增高(P<0.01);随药物干预时间的延长,该基因的表达增高更加明显,至72h升至最高,约为干预前的14.12倍。(2)GADD153 siRNA转染可以有效地抑制A549细胞内GADD153基因的表达,干扰效率达86%左右。(3)流式细胞术检测结果显示,GADD153基因干扰对于A549细胞的早期凋亡无显著影响;经二甲双胍干预48h后,GADD153干扰的A549细胞的早期凋亡率与未干扰的细胞相比明显降低(P<0.05)。结论:GADD153基因及蛋白的表达增高可促进二甲双胍诱导的肺腺癌细胞的凋亡。
第四部分二甲双胍抑制肺腺癌裸鼠皮下移植瘤生长的实验研究
目的:通过构建肺腺癌的裸鼠皮下移植瘤模型,研究二甲双胍对于肺腺癌体内生长的影响及安全性,并探讨二甲双胍在临床抗肿瘤治疗方面的价值。方法:将人肺腺癌细胞A549接种至BALB/c-nu小鼠腋背部皮下,建立移植瘤动物模型。待移植瘤长至约100mm3左右时,选取瘤块大小、形状较均一的裸鼠,随机分为3组,每组5只,以腹腔注射的方式给药治疗,每日1次,持续1个月。对照组给予PBS 0.2ml/d;met40组给予二甲双胍40mg/kg/d;met200组给予二甲双胍200mg/kg/d。观察药物干预过程中裸鼠的一般情况,每4天称量动物的体重,测量移植瘤的长短径,以计算肿块大小。治疗结束后,颈椎脱臼法处死荷瘤鼠,剥离移植瘤及肺、心、脑、肝、肾等脏器,以HE染色观察肿瘤及各器官的形态,判定肿瘤有无远处转移;以免疫组化染色检测各组移植瘤内Ki-67的表达情况;以TUNEL染色原位检测肿瘤细胞的凋亡情况。试验重复2次。结果:(1)以A549细胞接种于裸鼠右侧腋背部皮下,成功构建了肺腺癌的裸鼠皮下移植瘤模型。在为期1个月的药物干预过程中,各组裸鼠的一般状态良好,无明显异常表现,未见明显药物毒副反应。(2)在药物干预的不同时间测量移植瘤的大小,均以对照组的肿瘤体积最大,met40组其次,met200组的肿瘤体积最小。药物干预1个月后,两个二甲双胍治疗组的移植瘤平均体积均显著小于对照组(P<0.01);以对照组为参照,40mg/kg/d和200mg/kg/d剂量的二甲双胍腹腔注射给药对裸鼠移植瘤生长的抑制率分别为19.81%±6.25%和40.70%±8.98%。(3)对移植瘤标本行免疫组化染色结果显示,met40和met200组肿瘤内Ki-67抗原的阳性表达率分别为38.80%±10.41%和32.85%±10.14%,均显著低于对照组(58.18%±12.83%)(P<0.01)。(4)TUNEL染色结果显示,对照组肿瘤内仅可见散在的凋亡细胞(3.1±1.8个/HPF),而met40组和met200组肿瘤内的凋亡细胞数量显著增加(分别为13.6±3.41个/HPF和16.35±3.07个/HPF)(P<0.01),以后者增加更为明显。结论:40mg/kg/d和200mg/kg/d两种浓度的二甲双胍均可有效的抑制肺腺癌裸鼠皮下移植瘤的生长速度,使肿瘤细胞的凋亡增多、增殖活性减弱,且不会引起明显的毒副反应,提示二甲双胍对于肺腺癌患者的治疗具有较好的临床应用前景。
通过上述研究,得到结论如下:
1、二甲双胍可以通过阻滞细胞周期于G1期,并诱导细胞凋亡,从而抑制肺腺癌细胞的体外增殖;二甲双胍与顺铂联用可增强对肺腺癌细胞的生长抑制作用。
2、JNK/p38 MAPK--Caspase信号传导通路参与了对二甲双胍诱导的肺腺癌细胞凋亡的调控,阻断JNK/p38 MAPK通路可以部分地降低二甲双胍作用下肺腺癌细胞的早期凋亡率。
3、二甲双胍可以诱导肺腺癌细胞内GADD153基因及蛋白表达增高,从而促进细胞凋亡。
4、二甲双胍腹腔注射给药可以抑制肺腺癌裸鼠皮下移植瘤的生长,降低肿瘤细胞的增殖活性、促进细胞凋亡,且无明显毒副反应。
Lung cancer is the most common cancer in the world both in terms of incidence and mortality of the total. Lung adenocarcinoma is a common histopathologic type and the percentage of patient with lung adenocarcinma is 30% of all the patients with lung cancer. In most adenocarcinoma patients the tumors grows close to pleura. Though the proliferation rate is not quite high, adenocarcinoma is very angio- and lymph-invasive. Most of the lung adenocarcinoma patients are in the late stage of the disease when first diagnosed and surgical resection is not applicable. Although combined-modality therapy with radiotherapy and chemotherapy has been applied, more than 85% of the patients die within 5 years. New therapeutic modalities are therefore necessary to improve the response to treatment.
The biguanide metformin is one of the mostly used drugs for treatment of type 2 diabetes. By reducing hepatic gluconeogebesis and increasing glucose uptake in skeletal muscles, metformin can lower blood glucose levels while inducing no risk of hypoglycemia and very rarely causes lactic acidosis. Recent clinical studies have revealed that metformin treatment is associated with reduced cancer risk and improved prognosis. Evans et al did a pilot observational study reporting that among patients with type 2 diabetes, those treated with metformin have a lower incidence of cancer compared with those untreated. In an independent study, using administrative databases of a population-based cohort, Bowker et al reported that type 2 diabetes patients who use metformin have a lower cancer-related mortality compared with those that use sulfonylureas or insulin. There is also growing experimental evidence that metformin may impede the growth of human tumors, including prostate cancer, breast carcinoma, colon carcinoma, ovarian cancer, et al. However, until now, the relation between lung adenocarcinoma and metformin has not been studied and the precise anti-tumor mechanisms of the anti-neoplastic effects of metformin are not completely understood.
In the present study, we evaluated the effects of metformin on the growth of lung adenocarcinoma in vitro and in vivo, and its relationship to AMPK activation, JNK/p38 MAPK--Caspase signaling pathways activation and growth arrest and DNA damage -inducible transcription factor (GADD) 153. Our results open new therapeutic possibilities for the use of metformin as an anti-neoplasm agent. Four parts are included in this study:
Part one Effects of metformin on proliferation in lung adenocarcinoma cell lines A549 and NCI-H1299 in vitro
Objective To study the effects of metformin on cell viability, cell cycle, apoptosis and the synergism with cispatin in lung adenocarcinoma cell lines A549 and NCI-H1299 in vitro. Methods and materials Cells were treated with metformin (0, 0.5, 2 or 8 mM) in vitro together with cisplatin (10ug/mL) or not. Cell growth was determined using Methylthiazolyldiphenyl-tetrazolium bromide (MTT) following the manufacturer’s protocol at the time of 0, 24, 48 and 72 hours after treatment. After treatment with metformin for 48 hours, apoptosis and cell cycle of A549 cells were exemined by Flow cytometry. Apoptotic cells were analyzed by double staining with
fluoresceinisothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) . Cell cycle was analyzed by measuring the amount of PI-labeled DNA in ethanol-fixed cells. Results The cell viability started to decrease as early as 24 hours later after metformin treatment. Metformin induced significant proliferation inhibition on both A549 and NCI-H1299 cell lines in a dose- and time-dependent manner. When applied in combination with metformin, the cytotoxicity of cisplatin on both cells was stronger. After treatment for 48 hours, metformin at 2 and 8mM caused significant increases both in numbers of apoptotic (annexin+PI–) A549 cells and G1 phases fraction (P<0.05). While the apoptotic rate and cell-cycle progression of A549 cells treated with 0.5mM metformin did not change much compared to those of the control cells. Conclusions Metformin can inhibit the growth of lung adenocarcinoma cell lines significantly and potentiate cisplatin in a dose- and time-dependent manner in vitro. Cell-cycle progression is arrested in G1 stage and apoptosis is indued after metformin treatment.
Part two Role of JNK/p38 MAPK--Caspase signaling pathways in metformin-induced apoptosis of lung adenocarcinoma cell line A549
Objective To detect the effects of metformin on the phosphorylation of AMPK, JNK and p38 MAPK and the expression of cleaved Caspase-3, 8, 9 proteins in A549 cells, and investigate the correlation between JNK/p38 MAPK--Caspase signaling pathways and cell apoptosis. Methods and materials A549 cells were collected and lysed for total protein after treated with metformin (5 mM) for 0, 15, 30 and 60 minutes. Western blot analysis was performed to detect the expressions of pan- and pho- AMPK, JNK and p38 proteins. Cleaved Caspase-3, 8, 9 proteins were also detected. SP600125 and SB202190, the specific inhibitor for JNK and p38 MAPK signaling pathways respectively, were chosen to treat cells prior to metformin treatment and apoptotic rates of A549 cells were then analyzed by Flow cytometry.
Results The expressions of pho-AMPK, pho-JNK and pho-p38 were detected after 15 min of metformin treatment. The levels of cleaved Caspase-8, 9 proteins elevated after 12 h of metformin treatment. After treatment for 30 min, the expression level of cleaved Caspase-3 protein also elevated. After pretreated with SP600125 or SB202190, the metformin-induced apoptotic rates of A549 cells significantly decreased (P<0.05), though still higher than those of the cells treated with SP600125 or SB202190 only.
Conclusions JNK/p38 MAPK--Caspase signaling pathways are involved in metformin-induced apoptosis in lung adenocarcinoma cell line A549.
Part three GADD153 is involved in metformin-induced A549 cell apoptosis
Objective To detect the effects of metformin on the expression of GADD153 mRNA and protein in A549 cells, and to investigate the role of GADD153 gene in metformin-induced A549 cell apoptosis.
Methods and materials A549 cells were treated with metformin (5mM) for 0, 12, 24, 48 and 72 hours. The expression of GADD153 mRNA in A549 cells was detected by Realtime-PCR. GADD153 siRNA (50nM) or negative control siRNA (50nM) was transient transfected into human lung adenocarcinoma cell line A549 cells by INTERFERinTM siRNA transfection reagent. At 48h after transfection, cells were collected and the total RNA and protein were extracted. Realtime-PCR and western blot were performed to detect the level of GADD153 mRNA and protein respectively to assess the efficiency of GADD153 gene silencing. A549 cells were then divided into four groups: Negative control siRNA (siNEG) group, GADD153 siRNA (siGADD153) group, siNEG+metformin group and siGADD153+metformin group. The cells of the last two groups were treated with metformin (5mM) at 10h after siRNA transfection. PBS was used instead of metformin to the cells of siNEG group and siGADD153 group. After siRNA transfection and metformin or PBS treatment for another 48 hours, cell apoptotic rate of every group was detected by Flow cytometry.
Results Realtime-PCR analysis showed that the expression of GADD153 mRNA in A549 cells increased significantly at 12h after metformin treatment (P<0.01) and kept increasing to a level as high as 14.12-fold of the original level after treatment for 72h. Realtime-PCR and western blot analysis showed that both GADD153 mRNA and protein expression were significantly decreased after transient transfected with specific GADD153 siRNA. The cell apoptitic rate of siGADD153+metformin group was significantly lower than that of siNEG+metformin group (P<0.05), though no significant difference was detected between siGADD153 group and siNEG group (P=0.591).
Conclusions The apoptosis-inducing effect of metformin in lung adenocarcinoma cells is partly due to the up-regulation of GADD153 mRNA and protein in the cells under metformin treatment.
Part four Repressive effects of metformin on the growth of xenograft of lung adenocarcinoma in nude mice
Objective To study the suppressive effect of metformin on the growth of lung adenocarcinoma xenograft in nude mice and its security and to evaluate the potential value of metformin as a novel anti-neoclassic agent for the treatment of lung adenocarcinoma.
Methods and materials A549 cells were harvested, washed twice in PBS, and resuspended in 200ul PBS before being inoculated subcutaneouly in 4- to 6-week-old females BALB/c nude mice on the right flank (2×107 cells per site). Treatment with metformin was started when the average tumor volumes reached 100mm3. Five mice were used per group. Metformin (200 mg or 40 mg×kg body weight) was dissolved in 200ul PBS and administered daily with i.p. injections. The control group received vehicle only (200ul PBS). Animal weight and tumor volume (mm3) were measured every 4 days and estimated from caliper measurements using the formulaπ/6×A×B2 (A is larger diameter, B is smaller diameter). After treatment for a month, the mice were all sacrified and paraffin sections of tumors were used for immunohistochemical analyses with hemotoxylin and eosin stain and proliferation with Ki-67 and apoptosis with TUNEL staining.
Results No severe adverse effect was observed among the xenograft mice during metformin treatment. The growth of lung adenocarcinoma xenograft of met40 group and met200 group was significantly suppressed compared with that of the control group at every point of the treatment course. When the treatment ended after a month, the mice of met200 group had the smallest average tumor volume among the three groups, followed by those of met40 group. Compared with control group, the inhibition ratio of metformin at the concentration of 40 mg/kg/d and 200 mg/kg/d to the growth of tumor was 19.81%±6.25% and 40.70%±8.98%, respectively. The average positive rate of Ki-67 in tumors of the control, met40 and met200 group was 58.18%±12.83%, 38.80%±10.41% and 32.85%±10.14%, respectively. The average number of positive cells per HPF in tumors of the control, met40 and met200 group was 3.1±1.8, 13.6±3.41 and 16.35±3.07, respectively. Both the positive rate of Ki-67 and number of apoptotic cells in met40 and met200 group were significantly higher than those of the control group (P<0.01). Conclusions Metformin can inhibit the growth of lung adenocarcinoma xenograft in nude mice by attenuating the expression of the proliferation marker Ki-67 and inducing tumor cell apoptosis without any severe adverse effect.
In summary, we found from this study that metformin can inhibit the proliferation of lung adenocarcinoma cell lines and potentiate cisplatin in a dose- and time-dependent manner in vitro. Both JNK/p38 MAPK--Caspase signaling pathways and GADD153 gene are involved in metformin-induced apoptosis. When applied to the mice with xenograft tumor, metformin can markedly inhibit the growth of the tumor while causing no severe adverse effect. Our results indicate that metformin has anti-neoplastic activity and may be used as an adjunctive agent for the treatment of lung adenocarcinoma. The detailed mechanisms of the anti-neoplastic activity of metformin should be further studied.
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