5-杂氮脱氧胞苷对人膀胱癌细胞株T24增殖及其作用机制的研究
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摘要
研究背景：
据美国癌症协会统计,2006年在美国,膀胱癌在男性是继前列腺癌、肺癌和直肠癌以后排名第4位的恶性肿瘤,在女性排名第9位。大约有6万余人被临床医生诊断为膀胱癌,每年1万余人死于膀胱癌,绝大多数膀胱癌为移行细胞癌以浸润的方式生长,复发率为50%～70%。虽然膀胱癌可用局部治疗控制,但仍有10%～15%的表浅性膀胱移行细胞癌最终发展成为肌肉浸润性膀胱癌或发生其他部位转移。尽管浅表性膀胱癌生存率较高,预后较好,但膀胱部分切除后局部复发率为38%～80%,其中50%在手术后1年发生,约2／3在手术后第二年发生。膀胱肿瘤复发的主要原因：(1)肿瘤细胞在手术中残余、脱落种植是重要原因之一(2)肿瘤细胞沿膀胱肌肉壁内淋巴管扩散,实际侵犯膀胱壁肉眼所见宽广,肿瘤不能充分切除。(3)有学者认为膀胱肿瘤复发的唯一预后因素是肿瘤分期。(4)肉眼难以发现的原位癌和癌前病变的发展。(5)膀胱上皮继续受到尿内致癌物质的刺激。因此,研究膀胱癌的发病机制、早期诊断及早期治疗就显得尤为重要。随着近年来肿瘤分子生物学的发展,膀胱癌发生的分子生物学机制也有相当进展。基因机制和表观遗传学机制是肿瘤形成过程中的两大类机制,基因机制包括原癌基因激活、抑癌基因失活(点突变、缺失、插入、重排、扩增)及染色体异常,表观遗传学机制包括DNA甲基化异常,组蛋白修饰,编码RNAs(包括microRNA),染色质重塑等,甲基化异常主要指甲基基团与基因组中CpG二核苷酸的胞嘧啶环第5碳原子以共价键的形式结合,形成5-甲基胞嘧啶,引起基因表达异常而DNA序列及基因产物不变。在肿瘤细胞中,DNA的局部高甲基化与普遍的低甲基化是共存的,除点突变、基因缺失外,启动子区高甲基化导致转录失活是肿瘤中抑癌基因失活的一种表现。肿瘤抑制基因的高甲基化作为基因失活的机制首先在人类散发性视网膜母细胞Rb基因中证实,VonHipple Lindau(VHL)基因5′CpG岛的新甲基化在一种散发的肾母细胞瘤病中也被报道,p16基因在许多种肿瘤细胞株和原发肿瘤中经历了5′CpG岛的高甲基化和失活,5-叠氮胞嘧啶处理后这个基因恢复转录,p15基因也发现在神经母细胞瘤中和淋巴瘤中保持高甲基化。除了抑癌基因外,肿瘤转移抑制基因E-cadherin在乳腺癌和前列腺癌中表达也是启动子区高甲基化的结果。DNA甲基化在肿瘤形成中的作用是多方面的,一方面,5mC以很高的频率自发突变为T,甲基转移酶酶促5mC脱氨基和抑制DNA错配修复更加强了C-T的突变；另一方面,DNA甲基化基因外改变与癌基因激活、抑癌基因失活有关。由此可见,DNA甲基化改变通过基因机制和基因外机制,引起与细胞增殖和分化有关的基因表达异常,造成细胞失去对正常生长过程的控制而发生癌变,最后形成肿瘤。膀胱癌的发生、发展是一个多步骤、多阶段的过程。近年来,对抑癌基因的研究越来越受重视。其中,死亡相关蛋白激酶(DAPK)是一种通过促进凋亡或抑制生长而发挥作用的肿瘤抑制基因。DAPK定位于染色体9q34.1,有报道这一位点在头颈部肿瘤中可发生点突变,在消化道癌中发生纯合性缺失。据报道,多种肿瘤发生、发展与DAPK表达缺失有关,而DAPK基因CPG岛(CPG二核甘酸聚集区,多集中分布于基因5’端启动子区域)超甲基化是DAPK表达缺失的重要环节之一。DAPK参与肿瘤发生机制可能是：当DAPK基因发生改变后(遗传或表遗传),细胞不能正常死亡,不断分裂增殖形成肿瘤。基因启动子区的CpG岛在正常状态下一般是非甲基化的,当其发生甲基化时,常导致基因转录沉寂使一些重要基因如抑癌基因DNA修复基因等丧失功能,从而导致正常细胞的生长分化调控失常以及DNA损伤不能被及时修复,导致与多种肿瘤形成密切相关。我们可以把过甲基化的CpG当作肿瘤的标记,肿瘤发生时常出现不同程度的DNA甲基化模式改变,通过对启动子区高甲基化CpG岛的检测可以发现因DNA甲基化沉默的抑癌基因,这已成为一种寻找肿瘤相关基因的新方法。目前的实验技术可以做到能检测肿瘤抑制基因启动子的甲基化状态,并且可以DNA甲基化的抑制范围进行定量,从而为评估肿瘤风险、制定预防策略、获得早期诊断和跟踪肿瘤预后提供指导。由甲基化引起基因沉默而出现的肿瘤,可通过DNA甲基转移酶非竞争性或竞争性抑制剂抑制甲基化的发生,活化沉默的抑癌基因,从而达到治疗肿瘤的目的。去甲基化药物：5-氮胞嘧啶和5-氮-2’-脱氧胞嘧啶可以抑制甲基转移酶活性,在一些难治性肿瘤,特别是在白血病治疗方面已取得了较好的疗效。5-杂氮-2’-脱氧胞苷(5-aza-cdr)是一种DNA甲基转移酶1 (DNMT1)的抑制剂,可抑制DNMT1的功能,从而逆转基因启动子的高甲基化,使基因重新表达。很多体外研究证实,5-aza-cdr通过去甲基化作用使多种CPG岛超甲基化的抑癌基因重新表达,从而恢复抑癌功能,例如在淋巴瘤,胃癌，乳腺癌中。但5-aza-cdr对人膀胱癌T24细胞株DAPK的作用国内外未见报道。本实验将使用不同浓度的5-aza-dc在不同时间处理T24细胞株,检测其对细胞株增殖,凋亡的生物学影响及其相关机制,为膀胱癌的诊疗提供新途径。
研究目的
探讨DNA甲基转移酶抑制剂5-杂氮脱氧胞苷(5-aza-dc)对人膀胱癌细胞株T24增殖、凋亡的影响及可能的作用机制,寻找膀胱癌基因治疗的新方法。
研究方法
细胞培养肿瘤细胞培养基为RPMI1640+10%胎牛血清。在37℃、5%C02条件下的恒温培养箱中进行培养,每12-24h换液1次,视细胞生长情况,在细胞接近铺满培养瓶或培养板底时(4-5天后)进行传代。消化细胞用0.25%胰酶的PBS液(0.01μmol/L)。T24细胞在胰酶处理30秒左右后需终止消化。应用不同浓度(0.1,0.5,2.5,12.5μmol/L)的特异性DNA甲基转移酶抑制剂5-aza-dc,于不同作用时间(6,12,24,48h)处理人膀胱癌细胞株T24。采用四甲基偶氮唑蓝(MTT)法测定不同浓度的5-aza-dc在不同时间作用下T24细胞株的生长抑制率,将MTT加入经不同浓度5-aza-Cdr处理前后的膀胱癌T24细胞,DMSO充分溶解后,置全自动酶标仪于570nm波长处读取吸光度(A)值,细胞增殖能力以平均吸光度(A)值分析,以A值为纵坐标,时间(d)为横坐标,绘制生长曲线。采用流式细胞仪检测不同浓度(0.1,0.5,2.5,12.5μmol/L)的5-aza-dc在处理T24细胞24h后的凋亡率变化情况。用甲基化特异性PCR(MSP)检测药物处理前后死亡相关蛋白激酶(DAPK)启动子甲基化状态。
研究结果
1、实验组各浓度5-aza-dc对T24细胞均有生长抑制作用,各浓度组对T24细胞的抑制作用与对照组相比均具有显著性差异(P<0.01)。
2、MTT检测发现,同一作用时间下不同药物浓度的5-aza-dc (0.1,0.5,2.5, 12.5μmol/L)对T24细胞的生长抑制作用为剂量依赖性；同一浓度组对T24细胞的抑制作用在24内随作用时间的延长而增加(P<0.01),而当浓度为12.5μmol/L时,超过24h后再延长作用时间其抑制率变化不明显。检测发现浓度为12.5μmol/L的5-aza-dc作用24h时对T24细胞的生长抑制作用最明显(抑制率为24.01±0.25)。
3、流式细胞仪检测(Annexin V/PI双染法)膀胱癌T24细胞在不同浓度5-aza-dc (0.1、0.5、2.5、12.5μmol/L)作用24小时后,会出现明显凋亡。用药组与对照组比较,细胞凋亡率随药物浓度增加而增高,当浓度为12.5 u mol/L时,细胞凋亡率为(24.12±1.4)%。各浓度5-aza-dc作用的细胞凋亡率有显著差异(P<0.01),T24细胞的凋亡率存在浓度依赖性。
4、MSP法检测正常培养条件下T24细胞的DAPK基因启动子甲基化状态的结果显示：甲基化引物扩增条带为阳性,未甲基化引物扩增条带为阴性。5-aza-dc (12.5 u mol/L)作用24小时后,T24细胞甲基化引物扩增条带为阴性,未甲基化引物扩增条带为阳性。提示膀胱癌细胞株T24中DAPK基因启动子存在高甲基化,去甲基化药物5-aza-dc可成功逆转DAPK启动子高甲基化状态,使其成为未甲基化状态。
研究结论：
1、本研究证实5-aza-dc对人膀胱癌细胞系T24有显著的增殖抑制和诱导凋亡的作用,并且有一定的浓度及时间依赖性,凋亡率随着浓度的增加及培养时间的延长而明显增加。
2、其机制可能是使因甲基化而失活的抑癌基因重新激活从而诱导肿瘤细胞的凋亡。5-aza-dc诱导肿瘤细胞凋亡应用于肿瘤治疗,为进一步研究膀胱肿瘤的基因治疗提供了新的思路,值得进一步深入研究。
Background
According to the American Cancer Society Statistics,2006 in the United States, bladder cancer in men is secondary to prostate cancer, lung cancer and colorectal cancer after 4th of malignant tumors in female ranked No.9-bit. Approximately 6 million people are clinical doctors diagnosed with bladder cancer, every year, more than 1 million people died of bladder cancer, the vast majority of transitional cell carcinoma of the bladder is to infiltrate ways, recurrence rate is 50% to 70%. Although the available local treatment of bladder control, but there is still a 10%-15% of superficial bladder cancer eventually developed into muscle invasive bladder cancer or other parts of the transfer occurs. Despite the superficial bladder cancer survival rates are high, the prognosis is good, but the bladder resection local recurrence rate of 38% to 80%,50% in 1 year, approximately 2/3 in the second year after the operation. Bladder tumor recurrence of the main reasons:(1) tumor cells in surgery residual, falls off one of the reasons for the planting is important (2) the tumor cells along the bladder muscle intramural lymphatics diffusion, actual infringement bladder wall naked eye can see broad, resection of the tumor does not sufficiently. (3) some scholars think that the only bladder tumor recurrence is the TNM prognostic factors. (4) the naked elusive carcinoma in situ and the development of precancerous lesions. (5) bladder continue to be carcinogens in urine. Therefore, the study of bladder cancer pathogenesis, early diagnosis and early treatment has become particularly important. As in recent years the development of molecular biology of cancer, bladder cancer occur the considerable progress. Gene mechanism and epigenetic mechanism of tumor formation mechanism of the two major categories, gene mechanisms include proto-oncogene activation and deactivation of the tumor suppressor gene (mutations, deletions, insertions, rearrange, proliferation) and chromosomal abnormalities, epigenetic mechanism of epigenetic mechanisms include DNA methylation abnormalities, histone modifications, coding RNAs (including microRNA), Chromatin Remodeling, abnormal methylation mainly refers to the methyl group and genome CpG dinucleotide cytosine ring 5 carbon atoms to form covalent bond, forming 5-methylcytosine, causing abnormal gene expression and DNA sequences and gene products. In cancer cells, DNA hypermethylation of local and universal low methylation co-exist, except point mutations, gene deletion, promoter hypermethylation cause transcription inactivating is tumor suppressor gene inactivation of one. Tumor suppressor genes of hypermethylation as gene inactivation of the mechanism first human sporadic retinoblastoma RB confirmed, VonHipple Lindau (VHL) gene 5'CPG islands of new methylation in a sporadic Nephroblastoma disease has also been reported, p16 genes in many kinds of tumor cell lines and primary tumor has experienced the 5'CPG Island hypermethylation and deactivation,5-azido cytosine treated this gene recovery transcription, p15 gene also found in neuroblastoma and Lymphoma remain hypermethylation in addition to the tumor suppressor gene, E-cadherin metastasis suppressor gene in breast cancer and prostate cancer expression is promoter hypermethylation of the results. DNA methylation in tumor formation is multi-faceted, on the one hand,5mC to a high frequency of spontaneous mutations to T, methyltransferase enzyme inhibitory amino and 5mC of DNA mismatch repair is enhanced by C-T of mutation; on the other hand, DNA methylation gene and gene activation, deactivation for tumor suppressor genes. Therefore, DNA methylation changes by gene and gene mechanism, and cell proliferation and differentiation exception for gene expression, causing cells lose control of the normal growth of malignant transformation, which formed the tumor. The incidence of bladder cancer, development is a multi-step process, multiple phase, there are a variety of oncogenes and tumor suppressor genes. In recent years, research on tumor suppressor genes is becoming more and more attention. Where death-associated protein kinase (DAPK) is a means of promoting apoptosis or inhibit growth and functioning of tumor suppressor genes. Located on chromosome DAPK 9q34.1, has been reported that a point in the head and neck tumors can occur point mutations, in digestive cancer occur in the absence of. It is reported that many kinds of cancer, development and expression for DAPK, and DAPK gene CPG Island (CPG second nucleotide accumulation, distribution to the 5'-end of gene promoter hypermethylation regional) is DAPK expression one of the important links. DAPK cancer pathogenesis may be:when DAPK gene genetic altered (or table cell genetic), does not die, constantly forming tumors mitosis. Promoter CPG Islands in a normal state general non-methylation of methylation occurs when its often result in gene transcription silence makes some important genes such as tumor suppressor genes in DNA repair genes, such as loss of functionality, resulting in a normal cell regulation and control arrhythmias as well as DNA damage cannot be repaired in a timely manner, resulting in the formation with a variety of tumors. We can put the CpG methylation as tumor markers, tumor occurs often with varying degrees of DNA methylation patterns change, by promoter CpG Island hypermethylation of the test can detect DNA methylation silent tumor suppressor gene, which has become a search for cancer-related genes. The present experimental techniques can be done to detect tumor suppressor gene promoter hypermethylation, and DNA methylation inhibition range of quantitative, so as to assess the risk of cancer, making prevention policy, access to early diagnosis and to trace the prognosis provide guidance. Caused by the methylation of gene silencing the tumor, available through DNA methyltransferase non-competitive or competitive inhibitors methylation occurs, activating the silence of the tumor suppressor gene, so as to achieve the aim of the treatment of tumors. Demethylation drugs:5-nitrogen cytosine and 5-AZA-2'-Deoxy cytosine can inhibit methyltransferase activity, in some of the intractable cancer, especially in leukemia treatment have better outcomes.5-AZA-2'-deoxycytidine (5-aza-cdr) is a DNA methyltransferase 1 (DNMT1) inhibitors can suppress the DNMT1 functionality to reverse the promoter hypermethylation, gene expression. Many of the in vitro research confirmed that the 5-aza-cdr pass to a variety of CPG Island hypermethylation of the tumor suppressor gene expression, thus restoring tumor suppressor function, such as lymphoma, gastric cancer, breast cancer. But 5-aza-cdr on bladder T24 cell lines DAPK role not seen at home and abroad. This experiment will use different concentrations of the 5-aza-dc at different times with T24 cell lines, testing its proliferation, apoptosis of biological effects and related mechanism for bladder cancer chemotherapy with new ideas.
Objective
(1) To analyze the proliferation and the apoptosis of T24 cells before and after treatment with 5-Aza-CdR by using different concentrations.
(2) To detect the biological effect of the DNA methyltransferase of human bladder cell cancer T24 lines.To investigate the effect and the possible mechanism of 5-Aza-2'-deoxycytidine (5-aza-dc) on the growth of bladder cancer T24 cells, and to find a new target for bladder cancer genetic therapy.
Methods
Cell culture medium for tumor cells RPMI1640 + 10% fetal bovine serum. At 37℃,5%CO2 conditions in the incubator, each of the 12-24h change 1 time depending on cell growth, in cell close to the shops flasks or training Board (4-5 days) for generations. Digestion cells with 0.25% of pancreatic lipase PBS fluid (usingμm o 1/L). T24 cells in the pancreas to handle 30 seconds after termination of digestion. Apply different concentrations (0.1,0.5,2.5,12.5μm o 1/L) of specific DNA methyltransferase inhibitor 5-aza-dc, to the different roles of time (6,12,24,48h) deal with bladder cancer cell lines T24. Using four methyl blue (MTT) method for the determination of different concentrations of the 5-aza-dc at different times of T24 cell line rate, the MTT joined via different concentrations of 5 a Aza-CdR treatment before and after the bladder T24 cells, DMSO fully dissolved, reset the automatic 570nm wavelength of the enzyme to read absorbance (A) the value, cell proliferation ability to average absorbance (A) analysis of the value to A value of ordinate, time (d) as jian, draw the growth curve. Using flow cytometry different concentrations (0.1, 0.5,2.5,12.5μm o 1/L) of 5-aza-dc in 24h after T24 cell apoptosis rate changes. Use the methylation specific PCR (MSP) to detect drug treatment before and after death-associated protein kinase (DAPK) the methylation status.
Results
1、The proliferation of T24 cell was inhibited significantly by 5-aza-dc from 0.1 to 12.5μmol/L accordingly.Compared with control group,there is significant difference in experimental groups(P<0.01).
2、It was found by MTT assay that the growth inhibitory action of 5-aza-dc at different levels (0.1,0.5,2.5,12.5umol/l) on T24 cell was dose-dependent at the same action time. The inhibitory action of the same concentration group on T24 cells increased with time within 24 hours (P<0.01), however, at the level of 12.5 umol/L, the change of inhibitory rate became not so distinct beyond 24 hours. The test showed that 5-aza-dc had the most significant inhibitory action on the growth of T24 cells at 24 hours with the concentration of 12.5umol/L.
3、Distinct apoptosis of bladder cancer T24 cells 24 hours after 5-aza-dc at different concentrations (0.1,0.5,2.5 andl2.5umol/L) was given was observed by Flow cytometry assay (Annexin V/PI double stain). The apoptosis rate in administration group increased with the increase of drug concentration compared to that in control group and the apoptosis rate was (24.12±1.4)% at the concentration of 12.5umol/L. Above results indicated that the cellular apoptosis rates at different levels was of significant difference (p<0.01) and the apoptosis rate of T24 cells was dose-dependent.
4、With MSP assay, the result of the methylation status of the promoter of DARK gene in T24 cell under normal cultivation conditions showed that the amplification band of methylated primer was positive and the amplification band of non-methylated primer was negative. Twenty four hours after 5-aza-da (12.5umol/L) was given, the amplification band of methylated primer in T24 cell was negative and the amplification band of non-methylated primer was positive. That suggested that the promoter of DARK gene in bladder cancer T24 cells was highly methylated and such high-methylation state of DARK promoter could be inverted into its non-methylation state by the demethylation effect of 5-aza-da.
Conclusion
1、This study evidenced that 5-aza-da had significant effects of proliferation inhibition and apoptosis induction on human bladder cancer T24 cell line in concentration and time dependent manner; the apoptosis rate distinctly increased with the increase of concentration and the elongation of cultivation time.
2、The action mechanism of the drug might be inducing the apoptosis by re-activating tumor suppressor gene, which had been inactivated by methylation. The induction of apoptosis by 5-aza-da might be applied to the clinical treatment for tumors and offer a new idea for further studies on the gene therapy for bladder tumor, which was worthy of further exploration.
据美国癌症协会统计,2006年在美国,膀胱癌在男性是继前列腺癌、肺癌和直肠癌以后排名第4位的恶性肿瘤,在女性排名第9位。大约有6万余人被临床医生诊断为膀胱癌,每年1万余人死于膀胱癌,绝大多数膀胱癌为移行细胞癌以浸润的方式生长,复发率为50%～70%。虽然膀胱癌可用局部治疗控制,但仍有10%～15%的表浅性膀胱移行细胞癌最终发展成为肌肉浸润性膀胱癌或发生其他部位转移。尽管浅表性膀胱癌生存率较高,预后较好,但膀胱部分切除后局部复发率为38%～80%,其中50%在手术后1年发生,约2／3在手术后第二年发生。膀胱肿瘤复发的主要原因：(1)肿瘤细胞在手术中残余、脱落种植是重要原因之一(2)肿瘤细胞沿膀胱肌肉壁内淋巴管扩散,实际侵犯膀胱壁肉眼所见宽广,肿瘤不能充分切除。(3)有学者认为膀胱肿瘤复发的唯一预后因素是肿瘤分期。(4)肉眼难以发现的原位癌和癌前病变的发展。(5)膀胱上皮继续受到尿内致癌物质的刺激。因此,研究膀胱癌的发病机制、早期诊断及早期治疗就显得尤为重要。随着近年来肿瘤分子生物学的发展,膀胱癌发生的分子生物学机制也有相当进展。基因机制和表观遗传学机制是肿瘤形成过程中的两大类机制,基因机制包括原癌基因激活、抑癌基因失活(点突变、缺失、插入、重排、扩增)及染色体异常,表观遗传学机制包括DNA甲基化异常,组蛋白修饰,编码RNAs(包括microRNA),染色质重塑等,甲基化异常主要指甲基基团与基因组中CpG二核苷酸的胞嘧啶环第5碳原子以共价键的形式结合,形成5-甲基胞嘧啶,引起基因表达异常而DNA序列及基因产物不变。在肿瘤细胞中,DNA的局部高甲基化与普遍的低甲基化是共存的,除点突变、基因缺失外,启动子区高甲基化导致转录失活是肿瘤中抑癌基因失活的一种表现。肿瘤抑制基因的高甲基化作为基因失活的机制首先在人类散发性视网膜母细胞Rb基因中证实,VonHipple Lindau(VHL)基因5′CpG岛的新甲基化在一种散发的肾母细胞瘤病中也被报道,p16基因在许多种肿瘤细胞株和原发肿瘤中经历了5′CpG岛的高甲基化和失活,5-叠氮胞嘧啶处理后这个基因恢复转录,p15基因也发现在神经母细胞瘤中和淋巴瘤中保持高甲基化。除了抑癌基因外,肿瘤转移抑制基因E-cadherin在乳腺癌和前列腺癌中表达也是启动子区高甲基化的结果。DNA甲基化在肿瘤形成中的作用是多方面的,一方面,5mC以很高的频率自发突变为T,甲基转移酶酶促5mC脱氨基和抑制DNA错配修复更加强了C-T的突变；另一方面,DNA甲基化基因外改变与癌基因激活、抑癌基因失活有关。由此可见,DNA甲基化改变通过基因机制和基因外机制,引起与细胞增殖和分化有关的基因表达异常,造成细胞失去对正常生长过程的控制而发生癌变,最后形成肿瘤。膀胱癌的发生、发展是一个多步骤、多阶段的过程。近年来,对抑癌基因的研究越来越受重视。其中,死亡相关蛋白激酶(DAPK)是一种通过促进凋亡或抑制生长而发挥作用的肿瘤抑制基因。DAPK定位于染色体9q34.1,有报道这一位点在头颈部肿瘤中可发生点突变,在消化道癌中发生纯合性缺失。据报道,多种肿瘤发生、发展与DAPK表达缺失有关,而DAPK基因CPG岛(CPG二核甘酸聚集区,多集中分布于基因5’端启动子区域)超甲基化是DAPK表达缺失的重要环节之一。DAPK参与肿瘤发生机制可能是：当DAPK基因发生改变后(遗传或表遗传),细胞不能正常死亡,不断分裂增殖形成肿瘤。基因启动子区的CpG岛在正常状态下一般是非甲基化的,当其发生甲基化时,常导致基因转录沉寂使一些重要基因如抑癌基因DNA修复基因等丧失功能,从而导致正常细胞的生长分化调控失常以及DNA损伤不能被及时修复,导致与多种肿瘤形成密切相关。我们可以把过甲基化的CpG当作肿瘤的标记,肿瘤发生时常出现不同程度的DNA甲基化模式改变,通过对启动子区高甲基化CpG岛的检测可以发现因DNA甲基化沉默的抑癌基因,这已成为一种寻找肿瘤相关基因的新方法。目前的实验技术可以做到能检测肿瘤抑制基因启动子的甲基化状态,并且可以DNA甲基化的抑制范围进行定量,从而为评估肿瘤风险、制定预防策略、获得早期诊断和跟踪肿瘤预后提供指导。由甲基化引起基因沉默而出现的肿瘤,可通过DNA甲基转移酶非竞争性或竞争性抑制剂抑制甲基化的发生,活化沉默的抑癌基因,从而达到治疗肿瘤的目的。去甲基化药物：5-氮胞嘧啶和5-氮-2’-脱氧胞嘧啶可以抑制甲基转移酶活性,在一些难治性肿瘤,特别是在白血病治疗方面已取得了较好的疗效。5-杂氮-2’-脱氧胞苷(5-aza-cdr)是一种DNA甲基转移酶1 (DNMT1)的抑制剂,可抑制DNMT1的功能,从而逆转基因启动子的高甲基化,使基因重新表达。很多体外研究证实,5-aza-cdr通过去甲基化作用使多种CPG岛超甲基化的抑癌基因重新表达,从而恢复抑癌功能,例如在淋巴瘤,胃癌，乳腺癌中。但5-aza-cdr对人膀胱癌T24细胞株DAPK的作用国内外未见报道。本实验将使用不同浓度的5-aza-dc在不同时间处理T24细胞株,检测其对细胞株增殖,凋亡的生物学影响及其相关机制,为膀胱癌的诊疗提供新途径。
研究目的
探讨DNA甲基转移酶抑制剂5-杂氮脱氧胞苷(5-aza-dc)对人膀胱癌细胞株T24增殖、凋亡的影响及可能的作用机制,寻找膀胱癌基因治疗的新方法。
研究方法
细胞培养肿瘤细胞培养基为RPMI1640+10%胎牛血清。在37℃、5%C02条件下的恒温培养箱中进行培养,每12-24h换液1次,视细胞生长情况,在细胞接近铺满培养瓶或培养板底时(4-5天后)进行传代。消化细胞用0.25%胰酶的PBS液(0.01μmol/L)。T24细胞在胰酶处理30秒左右后需终止消化。应用不同浓度(0.1,0.5,2.5,12.5μmol/L)的特异性DNA甲基转移酶抑制剂5-aza-dc,于不同作用时间(6,12,24,48h)处理人膀胱癌细胞株T24。采用四甲基偶氮唑蓝(MTT)法测定不同浓度的5-aza-dc在不同时间作用下T24细胞株的生长抑制率,将MTT加入经不同浓度5-aza-Cdr处理前后的膀胱癌T24细胞,DMSO充分溶解后,置全自动酶标仪于570nm波长处读取吸光度(A)值,细胞增殖能力以平均吸光度(A)值分析,以A值为纵坐标,时间(d)为横坐标,绘制生长曲线。采用流式细胞仪检测不同浓度(0.1,0.5,2.5,12.5μmol/L)的5-aza-dc在处理T24细胞24h后的凋亡率变化情况。用甲基化特异性PCR(MSP)检测药物处理前后死亡相关蛋白激酶(DAPK)启动子甲基化状态。
研究结果
1、实验组各浓度5-aza-dc对T24细胞均有生长抑制作用,各浓度组对T24细胞的抑制作用与对照组相比均具有显著性差异(P<0.01)。
2、MTT检测发现,同一作用时间下不同药物浓度的5-aza-dc (0.1,0.5,2.5, 12.5μmol/L)对T24细胞的生长抑制作用为剂量依赖性；同一浓度组对T24细胞的抑制作用在24内随作用时间的延长而增加(P<0.01),而当浓度为12.5μmol/L时,超过24h后再延长作用时间其抑制率变化不明显。检测发现浓度为12.5μmol/L的5-aza-dc作用24h时对T24细胞的生长抑制作用最明显(抑制率为24.01±0.25)。
3、流式细胞仪检测(Annexin V/PI双染法)膀胱癌T24细胞在不同浓度5-aza-dc (0.1、0.5、2.5、12.5μmol/L)作用24小时后,会出现明显凋亡。用药组与对照组比较,细胞凋亡率随药物浓度增加而增高,当浓度为12.5 u mol/L时,细胞凋亡率为(24.12±1.4)%。各浓度5-aza-dc作用的细胞凋亡率有显著差异(P<0.01),T24细胞的凋亡率存在浓度依赖性。
4、MSP法检测正常培养条件下T24细胞的DAPK基因启动子甲基化状态的结果显示：甲基化引物扩增条带为阳性,未甲基化引物扩增条带为阴性。5-aza-dc (12.5 u mol/L)作用24小时后,T24细胞甲基化引物扩增条带为阴性,未甲基化引物扩增条带为阳性。提示膀胱癌细胞株T24中DAPK基因启动子存在高甲基化,去甲基化药物5-aza-dc可成功逆转DAPK启动子高甲基化状态,使其成为未甲基化状态。
研究结论：
1、本研究证实5-aza-dc对人膀胱癌细胞系T24有显著的增殖抑制和诱导凋亡的作用,并且有一定的浓度及时间依赖性,凋亡率随着浓度的增加及培养时间的延长而明显增加。
2、其机制可能是使因甲基化而失活的抑癌基因重新激活从而诱导肿瘤细胞的凋亡。5-aza-dc诱导肿瘤细胞凋亡应用于肿瘤治疗,为进一步研究膀胱肿瘤的基因治疗提供了新的思路,值得进一步深入研究。
Background
According to the American Cancer Society Statistics,2006 in the United States, bladder cancer in men is secondary to prostate cancer, lung cancer and colorectal cancer after 4th of malignant tumors in female ranked No.9-bit. Approximately 6 million people are clinical doctors diagnosed with bladder cancer, every year, more than 1 million people died of bladder cancer, the vast majority of transitional cell carcinoma of the bladder is to infiltrate ways, recurrence rate is 50% to 70%. Although the available local treatment of bladder control, but there is still a 10%-15% of superficial bladder cancer eventually developed into muscle invasive bladder cancer or other parts of the transfer occurs. Despite the superficial bladder cancer survival rates are high, the prognosis is good, but the bladder resection local recurrence rate of 38% to 80%,50% in 1 year, approximately 2/3 in the second year after the operation. Bladder tumor recurrence of the main reasons:(1) tumor cells in surgery residual, falls off one of the reasons for the planting is important (2) the tumor cells along the bladder muscle intramural lymphatics diffusion, actual infringement bladder wall naked eye can see broad, resection of the tumor does not sufficiently. (3) some scholars think that the only bladder tumor recurrence is the TNM prognostic factors. (4) the naked elusive carcinoma in situ and the development of precancerous lesions. (5) bladder continue to be carcinogens in urine. Therefore, the study of bladder cancer pathogenesis, early diagnosis and early treatment has become particularly important. As in recent years the development of molecular biology of cancer, bladder cancer occur the considerable progress. Gene mechanism and epigenetic mechanism of tumor formation mechanism of the two major categories, gene mechanisms include proto-oncogene activation and deactivation of the tumor suppressor gene (mutations, deletions, insertions, rearrange, proliferation) and chromosomal abnormalities, epigenetic mechanism of epigenetic mechanisms include DNA methylation abnormalities, histone modifications, coding RNAs (including microRNA), Chromatin Remodeling, abnormal methylation mainly refers to the methyl group and genome CpG dinucleotide cytosine ring 5 carbon atoms to form covalent bond, forming 5-methylcytosine, causing abnormal gene expression and DNA sequences and gene products. In cancer cells, DNA hypermethylation of local and universal low methylation co-exist, except point mutations, gene deletion, promoter hypermethylation cause transcription inactivating is tumor suppressor gene inactivation of one. Tumor suppressor genes of hypermethylation as gene inactivation of the mechanism first human sporadic retinoblastoma RB confirmed, VonHipple Lindau (VHL) gene 5'CPG islands of new methylation in a sporadic Nephroblastoma disease has also been reported, p16 genes in many kinds of tumor cell lines and primary tumor has experienced the 5'CPG Island hypermethylation and deactivation,5-azido cytosine treated this gene recovery transcription, p15 gene also found in neuroblastoma and Lymphoma remain hypermethylation in addition to the tumor suppressor gene, E-cadherin metastasis suppressor gene in breast cancer and prostate cancer expression is promoter hypermethylation of the results. DNA methylation in tumor formation is multi-faceted, on the one hand,5mC to a high frequency of spontaneous mutations to T, methyltransferase enzyme inhibitory amino and 5mC of DNA mismatch repair is enhanced by C-T of mutation; on the other hand, DNA methylation gene and gene activation, deactivation for tumor suppressor genes. Therefore, DNA methylation changes by gene and gene mechanism, and cell proliferation and differentiation exception for gene expression, causing cells lose control of the normal growth of malignant transformation, which formed the tumor. The incidence of bladder cancer, development is a multi-step process, multiple phase, there are a variety of oncogenes and tumor suppressor genes. In recent years, research on tumor suppressor genes is becoming more and more attention. Where death-associated protein kinase (DAPK) is a means of promoting apoptosis or inhibit growth and functioning of tumor suppressor genes. Located on chromosome DAPK 9q34.1, has been reported that a point in the head and neck tumors can occur point mutations, in digestive cancer occur in the absence of. It is reported that many kinds of cancer, development and expression for DAPK, and DAPK gene CPG Island (CPG second nucleotide accumulation, distribution to the 5'-end of gene promoter hypermethylation regional) is DAPK expression one of the important links. DAPK cancer pathogenesis may be:when DAPK gene genetic altered (or table cell genetic), does not die, constantly forming tumors mitosis. Promoter CPG Islands in a normal state general non-methylation of methylation occurs when its often result in gene transcription silence makes some important genes such as tumor suppressor genes in DNA repair genes, such as loss of functionality, resulting in a normal cell regulation and control arrhythmias as well as DNA damage cannot be repaired in a timely manner, resulting in the formation with a variety of tumors. We can put the CpG methylation as tumor markers, tumor occurs often with varying degrees of DNA methylation patterns change, by promoter CpG Island hypermethylation of the test can detect DNA methylation silent tumor suppressor gene, which has become a search for cancer-related genes. The present experimental techniques can be done to detect tumor suppressor gene promoter hypermethylation, and DNA methylation inhibition range of quantitative, so as to assess the risk of cancer, making prevention policy, access to early diagnosis and to trace the prognosis provide guidance. Caused by the methylation of gene silencing the tumor, available through DNA methyltransferase non-competitive or competitive inhibitors methylation occurs, activating the silence of the tumor suppressor gene, so as to achieve the aim of the treatment of tumors. Demethylation drugs:5-nitrogen cytosine and 5-AZA-2'-Deoxy cytosine can inhibit methyltransferase activity, in some of the intractable cancer, especially in leukemia treatment have better outcomes.5-AZA-2'-deoxycytidine (5-aza-cdr) is a DNA methyltransferase 1 (DNMT1) inhibitors can suppress the DNMT1 functionality to reverse the promoter hypermethylation, gene expression. Many of the in vitro research confirmed that the 5-aza-cdr pass to a variety of CPG Island hypermethylation of the tumor suppressor gene expression, thus restoring tumor suppressor function, such as lymphoma, gastric cancer, breast cancer. But 5-aza-cdr on bladder T24 cell lines DAPK role not seen at home and abroad. This experiment will use different concentrations of the 5-aza-dc at different times with T24 cell lines, testing its proliferation, apoptosis of biological effects and related mechanism for bladder cancer chemotherapy with new ideas.
Objective
(1) To analyze the proliferation and the apoptosis of T24 cells before and after treatment with 5-Aza-CdR by using different concentrations.
(2) To detect the biological effect of the DNA methyltransferase of human bladder cell cancer T24 lines.To investigate the effect and the possible mechanism of 5-Aza-2'-deoxycytidine (5-aza-dc) on the growth of bladder cancer T24 cells, and to find a new target for bladder cancer genetic therapy.
Methods
Cell culture medium for tumor cells RPMI1640 + 10% fetal bovine serum. At 37℃,5%CO2 conditions in the incubator, each of the 12-24h change 1 time depending on cell growth, in cell close to the shops flasks or training Board (4-5 days) for generations. Digestion cells with 0.25% of pancreatic lipase PBS fluid (usingμm o 1/L). T24 cells in the pancreas to handle 30 seconds after termination of digestion. Apply different concentrations (0.1,0.5,2.5,12.5μm o 1/L) of specific DNA methyltransferase inhibitor 5-aza-dc, to the different roles of time (6,12,24,48h) deal with bladder cancer cell lines T24. Using four methyl blue (MTT) method for the determination of different concentrations of the 5-aza-dc at different times of T24 cell line rate, the MTT joined via different concentrations of 5 a Aza-CdR treatment before and after the bladder T24 cells, DMSO fully dissolved, reset the automatic 570nm wavelength of the enzyme to read absorbance (A) the value, cell proliferation ability to average absorbance (A) analysis of the value to A value of ordinate, time (d) as jian, draw the growth curve. Using flow cytometry different concentrations (0.1, 0.5,2.5,12.5μm o 1/L) of 5-aza-dc in 24h after T24 cell apoptosis rate changes. Use the methylation specific PCR (MSP) to detect drug treatment before and after death-associated protein kinase (DAPK) the methylation status.
Results
1、The proliferation of T24 cell was inhibited significantly by 5-aza-dc from 0.1 to 12.5μmol/L accordingly.Compared with control group,there is significant difference in experimental groups(P<0.01).
2、It was found by MTT assay that the growth inhibitory action of 5-aza-dc at different levels (0.1,0.5,2.5,12.5umol/l) on T24 cell was dose-dependent at the same action time. The inhibitory action of the same concentration group on T24 cells increased with time within 24 hours (P<0.01), however, at the level of 12.5 umol/L, the change of inhibitory rate became not so distinct beyond 24 hours. The test showed that 5-aza-dc had the most significant inhibitory action on the growth of T24 cells at 24 hours with the concentration of 12.5umol/L.
3、Distinct apoptosis of bladder cancer T24 cells 24 hours after 5-aza-dc at different concentrations (0.1,0.5,2.5 andl2.5umol/L) was given was observed by Flow cytometry assay (Annexin V/PI double stain). The apoptosis rate in administration group increased with the increase of drug concentration compared to that in control group and the apoptosis rate was (24.12±1.4)% at the concentration of 12.5umol/L. Above results indicated that the cellular apoptosis rates at different levels was of significant difference (p<0.01) and the apoptosis rate of T24 cells was dose-dependent.
4、With MSP assay, the result of the methylation status of the promoter of DARK gene in T24 cell under normal cultivation conditions showed that the amplification band of methylated primer was positive and the amplification band of non-methylated primer was negative. Twenty four hours after 5-aza-da (12.5umol/L) was given, the amplification band of methylated primer in T24 cell was negative and the amplification band of non-methylated primer was positive. That suggested that the promoter of DARK gene in bladder cancer T24 cells was highly methylated and such high-methylation state of DARK promoter could be inverted into its non-methylation state by the demethylation effect of 5-aza-da.
Conclusion
1、This study evidenced that 5-aza-da had significant effects of proliferation inhibition and apoptosis induction on human bladder cancer T24 cell line in concentration and time dependent manner; the apoptosis rate distinctly increased with the increase of concentration and the elongation of cultivation time.
2、The action mechanism of the drug might be inducing the apoptosis by re-activating tumor suppressor gene, which had been inactivated by methylation. The induction of apoptosis by 5-aza-da might be applied to the clinical treatment for tumors and offer a new idea for further studies on the gene therapy for bladder tumor, which was worthy of further exploration.
引文
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