顺铂诱导的急性肾损伤中肾脏组织m^(6)A甲基化水平的变化

目的·探讨N6-甲基腺嘌呤(N6-methyladenosine,m^(6)A)甲基化修饰在顺铂诱导的小鼠急性肾损伤进程中的作用。方法·选择4只C57bL/6小鼠,尾静脉注射顺铂(20 mg/kg)诱导急性肾损伤(损伤组);另取4只C57bL/6小鼠,尾静脉注射等量生理盐水(对照组)。检测2组小鼠血清肌酐及血尿素氮水平变化,观察小鼠肾脏组织切片中病理损伤情况,评估模型是否成功。进一步运用甲基化RNA免疫共沉淀技术(methylatedRNAimmunoprecipitation,MeRIP)与RNA测序技术分别检测2组小鼠肾脏组织中m^(6)A甲基化水平与RNA表达变化。运用基因本体论及京都基因和基因组数据库进行结果可视化和综合研究,并将RNA测序技术所得转录组数据与MeRIP技术检测所得表观遗传数据联合分析,寻找参与顺铂诱导急性肾损伤病理变化过程的候选基因。结果·顺铂可诱导小鼠血清肌酐与血尿素氮水平显著升高。光学显微镜观察肾组织发现广泛的肾小管空泡变性,上皮细胞剥脱,肾小管坏死,提示造模成功。MeRIP检测发现损伤组与对照组小鼠肾脏中共有2227个基因含有2981个差异化表达的m^(6)A甲基化位点(表达变化倍数≥2且P<0.05),这些基因主要富集于代谢及细胞死亡通路。表达差异化m6A甲基化位点的基因与RNA差异化表达基因的联合分析发现1002个表达趋势相同的基因,如纤维蛋白原α链、溶质载体12家族成员1和甲肝病毒细胞受体1等。结论·顺铂可诱导肾脏组织中基因mRNA上m^(6)A甲基化位点的甲基化水平变化,促进急性肾损伤进程。

磷酸化修饰对P53直接线粒体转移的影响以及在顺铂诱导的卵巢癌细胞凋亡中的作用

磷酸化修饰,用于P53直接线粒体评价,对其转移的影响和对卵巢癌凋亡(顺铂诱导)的作用。方法 以卵巢癌细胞为例,顺铂(10μmol/L)处理,时间:24h,染色:Hoechst染色法,观察顺铂敏感细胞(OV2008、A2780s)、顺铂耐药细胞(C13'、A2780cp)凋亡率差异。结果 卵巢癌细胞凋亡率:OV2008>C13',A2780s>A2780cp(P<0.05);顺铂处理24h后,在OV2008、A2780s的线粒体,见P53、Smac的蛋白水平分别呈现增高、降低现象,而Smac蛋白水平(存在胞浆)呈现上升情况。但是在C13'、A2780cp内无明显变化(P<0.05);顺铂处理24h后,出现线粒体转磷酸化的是Ov2008、A2780s,而C13'、A2780cp无变化(P<0.05)。结论 ?顺铂会诱导p53卵巢癌细胞凋亡,磷酸化修饰会影响p53活性,提高敏感细胞凋亡率。

多西他赛与氟尿嘧啶诱导化疗联合同期放化疗治疗局部晚期鼻咽癌的临床分析

目的研究局部晚期鼻咽癌患者实施顺铂+氟尿嘧啶诱导化疗与同期放化疗联合治疗的临床效果。方法选择70例于该院接受局部晚期鼻咽癌治疗的患者,纳入时间为2017年11月—2018年12月,采用随机数字表法将其分为实验组与参照组,每组均35例。其中参照组采用多西他赛+顺铂诱导化疗与同期放化疗联合治疗,实验组行顺铂+氟尿嘧啶诱导化疗与同期放化疗联合治疗,对比2组患者临床疗效、放化疗不良反应、复发及转移情况。结果实验组局部晚期鼻咽癌临床总有效率(85.71%)与参照组(60.00%)相比较高,组间差异有统计学意义(χ^2=5.851 3,P<0.05)。实验组白细胞异常、血小板异常、肝肾功能损伤、恶心呕吐、腹痛腹泻、口腔黏膜炎、放射脑炎、黏膜坏死发生率分别为:85.71%、77.14%、71.43%、85.71%、82.86%、45.71%、22.86%、42.86%,参照组分别为48.57%、45.71%、40.00%、82.86%、42.86%、48.57%、20.00%、17.14%(χ^2=10.943 5、7.295 4、7.005 7、0.107 8、11.993 0、0.057 3、0.084 8、0.084 8,P=0.000 9、0.006 9、0.008 1、0.742 5、0.000 5、0.810 7、0.770 8、0.770 8)。2组恶心呕吐、口腔黏膜炎、放射脑炎、黏膜坏死等放化疗不良反应比例差异无统计学意义(P>0.05),与参照组相比,实验组白细胞异常、血小板异常、肝肾功能损伤、腹痛腹泻等放化疗不良反应率较高,差异有统计学意义(P<0.05)。2组局部晚期鼻咽癌复发或转移比例(65.71%、60.00%)差异无统计学意义(χ^2=0.244 7,P>0.05)。结论多西他赛+顺铂诱导化疗方式在治疗局部晚期鼻咽癌方面具有显著疗效,但其不良反应发生率相对较高,临床中应予以谨慎选择。

N-乙酰半胱氨酸对顺铂导致肾毒性的保护作用及作用机制分析

目的:通过动物实验分析探讨N-乙酰半胱氨酸对于顺铂诱导的肾毒性和肾损伤的保护功能及其机制,为临床药理研究提供参考。方法:选取60只BALB/c小鼠,雌雄各半,喂养7 d后腹腔注射20 mg/kg的顺铂,持续注射3 d诱导并建立急性肾损伤小鼠模型(AKI),后随机分为6组,A组给予5 mg/kg顺铂,B组给予250 μg/(100 g·d)的N-乙酰半胱氨酸,C组给予5 mg/kg顺铂+250 μg/(100 g·d)的N-乙酰半胱氨酸;D组给予500 μg/(100 g·d)的N-乙酰半胱氨酸;E组给予5 mg/kg 顺铂+500 μg/(100 g·d)的N-乙酰半胱氨酸,F组注射生理盐水做对照,连续治疗7 d后,抽取小鼠眼球血,测定生化指标、炎症因子水平;做肾脏病理切片图评价肾损伤水平,并加以比较。结果:在给药前,6组小鼠的血肌酐(Scr)、尿素氮(BUN)、肾损伤评分(RIS)、肿瘤坏死因子α(TNF-α)、谷胱甘肽(GSH)水平比较差异无统计学意义(P>0.05);给药后,6组上述指标差异有统计学意义(P<0.05):①血清Scr、BUN、TNF-α和肾损伤评分RIS:A组>F组>C组>E组>B组>D组,与治疗前相比,A组显著增加(P<0.05);B组、D组、E组显著降低(P<0.05),C组、F组差异无统计学意义(P>0.05);②血清GSH:A组<F组<C组<E组<B组<D组,与治疗前相比,A组显著降低(P<0.05);B组、D组、E组显著增加(P<0.05),C组、F组差异无统计学意义(P>0.05)。结论:N-乙酰半胱氨酸可减轻因顺铂导致的小鼠肾毒性和肾损伤程度,对肾脏具有保护作用,且动物实验的疗效与N-乙酰半胱氨酸的剂量有高度相关性,其机制则与N-乙酰半胱氨酸具有抑制炎症反应和肾组织细胞凋亡、抗氧化应激等药理作用有关。

Cisplatin pretreatment enhances anti-tumor activity of cytokine-induced killer cells

AIM： To investigate whether cisplatin （DDP） enhances the anti-tumor activity of cytokine- induced killer （CIK） cells in a murine colon adenocarcinoma model. METHODS： Tumor size and weight served as indicators of therapeutic response. Immunohistochemistry was performed to observe intratumoral lymphocyte infiltration and tumor microvessel density. Changes in the percentage of regulatory T （Treg） cells within the spleens of tumor-bearing mice preconditioned with DDP were monitored using flow cytometry. RESULTS： A marked T cell-dependent, synergistic anti- tumor effect of the combined therapy was observed （1968 ± 491 mm3 ys 3872 ＋ 216 mm3; P = 0,003）, Preconditioning chemotherapy with DDP augmented the infiltration of CD3＋ T lymphocytes into the tumor mass and reduced the percentage of both intratumoral and splenic Treg cells. CONCLUSION： Preconditioning with DDP markedly enhances the efficacy of adoptively transferred CIK cells, providing a potential clinical modality for the treatment of patients with colorectal cancer.

Bcl-2 cleavages at two adjacent sites by different caspases promote cisplatin-induced apoptosis

The protein encoded by bcl-2 proto-oncogene plays an important role in the mitochondria-mediated apoptotic pathway. Although the general role of Bcl-2 is anti-apoptotic, previous work showed that Bcl-2 fragments cleaved by caspases could promote apoptotic process. We report herein that Bcl-2 protein was cleaved to produce two fragments of around 23 kDa in human hepatocarcinoma BEL-7404 cells or in Bcl-2 overexpressing CHO cells induced by cisplatin. Treating cells with the general caspase inhibitor z-VAD-fmk blocked the induced cleavage of Bcl-2. Mutagenesis analyses showed that Bcl-2 was cleaved by caspases at two adjacent recognition sites in the loop domain （YEWD31↓AGD34↓V）, which could be inhibited by caspase-8 and -3 inhibitors, respectively. Overexpression of the carboxyl terminal 23 kDa fragments increased the sensitivity of CHO cells to cisplatin-induced apoptosis. These results indicate that Bcl-2 can be cleaved into two close fragments by different caspases during cisplatin-induced apoptosis, both of which contribute to the acceleration ofapoptotic process.

ROLE OF ERK1/2 KINASE IN CISPLATIN-INDUCED APOPTOSIS IN HUMAN OVARIAN CARCINOMA CELLS

Objective To investigate the role of extracellular regulated kinase (ERK1/2) pathway in cisplatin-induced apoptosis in human ovarian carcinoma cells. Methods Cisplatin-induced apoptosis were stained with DAPI and was assessed microscopically in human epithelial adenocarcinoma ovarian cell line SKOV3 cells. ERK activation was determined by Western blotting using an anti-phospho-ERK antibody to detect ERK activity. The effect of PD98059 on ERK activity induced by cisplatin was detected by MTT assay. Results Marked apoptosis of SKOV3 cells resulted from 48 hours treatment with 20 μg/mL cisplatin. Strong activation of ERK was led to by 15 μg/mL cisplatin. Dose response and time course of cisplatin induced apoptosis in SKOV3 cells. Cisplatin-induced ERK activation occurred at 12 hours and increased to highest induction at 24 hours by Western blotting. The effect of PD 98059 on ERK activity induced by cisplatin at the concentration of 100 μmol/L PD 98059. Statistically significant decreased in cell survival were observed with 100 μmol/L PD 98059 at 15 and 20 μg/mL cisplatin (P< 0.05). Conclusions Cisplatin activates the ERK signaling pathway in ovarian cancer cell line SKOV3. Inhibition of ERK acti-vity enhances sensitivity to cisplatin cytotoxity in ovarian cancer cell line SKOV3. Evaluation of ERK activity could be useful in predicting which ovarian cancer will response most favorably to cisplatin therapy.

Casticin combination with Cisplatin in sub-toxic concentration induced apoptosis of human ovarian cancer HO-8910 cells in vitro

Objective: The aim of the study was to investigate the effect of Casticin (CAS) combination with Cisplatin (DDP) in sub-toxic concentration on apoptosis of human ovarian cancer HO-8910 cells in vitro and unravel the associated mechanisms. Methods: Human ovarian cancer HO-8910 cells were cultured in vitro. The inhibitory effect of CAS combination with DDP in sub-toxic concentration on viability of human ovarian cancer HO-8910 cells was evaluated by the MTT assay. Morphological changes of cell apoptosis were detected by Hoechst 33258 staining assay. Cell apoptosis rate was analyzed by flow cytometry. The protein expression level was analyzed by Western blot. Results: CAS in sub-toxic concentration and DDP in sub-toxic concentration could slightly inhibit Human ovarian cancer HO-8910 cells, but CAS combination with DDP in sub-toxic concentration significantly inhibited the growth of HO-8910 cells, and growth inhibition rate was increased drastically compared with the control group (P﹤0.01), and the inhibiting effect showed synergistic action. Human ovarian cancer HO-8910 cells showed the typical morphological changes of apoptosis and apoptosis rate markedly increased when they were exposed to CAS combination with DDP in sub-toxic concentration for 48 h. Western blot showed that the expression of bcl-2 protein was down-regulated and protein level of caspase-3 was activated by CAS combination with DDP in sub-toxic concentration. Conclusion: CAS combination with DDP in sub-toxic concentration could inhibit the cells growth and lead to cell apoptosis in human ovarian cancer HO-8910 cells. And the down-regulation of bcl-2 protein expression and activation of caspase-3 protein might contribute to CAS combination with DDP in sub-toxic concentration in human cancer HO-8910 cells.

Clinical analysis of preoperative induction chemotherapy with gemcitabine combined with cisplatin for locally advanced non-small cell lung cancer

Objective:The purpose of this study was to assess the curative effect and adverse reaction of preoperative induction chemotherapy with gemcitabine combined with cisplatin for locally advanced non-small cell lung cancer(NSCLC).Methods:This prospective randomized controlled trial included 115 patients with locally advanced NSCLC were randomly divided into experimental and control groups and were treated from January 2007 to January 2010.The experimental group of 63 cases was treated with two cycles of induction chemotherapy before operation,radical surgery had been performed about three weeks after completion of chemotherapy,followed by received two cycles of chemotherapy.And the control group(52 cases) was treated at first with radical surgery,then treated with four cycles of chemotherapy.Two groups of the cases received routine thoracic radiotherapy with a total dose of 45 Gy.One cycle of gemcitabine combined with cisplatin regimen included gemcitabine 1000 mg/m2 on day 1 and day 8 and cisplatin 25 mg/m2 on day 1,day 2 and day 3 by intravenous infusion,with 21 days as one cycle.The tumor recurrence was evaluated by chest CT and abdominal B-ultrasound.Efficacy and toxicity results were compared by two groups.Results:All patients were followed up for three months to two years.The surgical stage of the experimental group reduced,two-years disease-free survival and postoperative recovery in the experimental group were better than in the control group,the difference was statistical significant.Toxicity and side effect after chemotherapy were mainly bone marrow suppression and gastrointestinal reactions,other complications included thrombocytopenia,leukopenia,anemia,liver and kidney dysfunction were no significant difference in two groups.Conclusion:Preoperative induction chemotherapy with gemcitabine combined with cisplatin for locally advanced lung cancer can reduce the surgical staging and extend the postoperative disease-free survival.

Induction of apoptosis of human ovarian cancer cells by SGI-1776 combination with DDP in sub-toxic concentration in vitro

Objective： The aim of the study was to investigate the effect of SG1-1776 combination with DDP in sub-toxic concentration on induction of apoptosis of human ovarian cancer HO-8910 ceiis in vitro and to unravei the associated mechanisms. Methods： Human ovarian cancer HO-8910 cells were cultured in vitro. The inhibitory effect of SG1-1776 combination with DDP in sub-toxic concentration on induction on viability of human ovarian cancer HO-8910 cells was evaiuated by the MTT assay. Cell apoptosis rate was analyzed by flow cytometry. The proteins expression level related to apoptosis were analyzed by Western blot. Results： SG1-1776 combination with DDP in sub-toxic concentration significantiy inhibited the proliferation of human ovarian cancer HO-8910 cells, and proliferation inhibition rate was increased drastically compared with normai saline （NS） group or DDP group in sub-toxic concentration or SG1-1776 group in sub-toxic concentration （P 〈 0.01）. Apoptosis rate markedly increased after the treatment of SG1-1776 combination with DDP in sub-toxic concentration for 48 h. Western blot showed that the expression of bcl-2 protein was down-regulated and protein level of Bax and Cyto-c were depressed by SG1-1776 combination with DDP in sub-toxic concentration. Cenclusion： SG1-1776 combination with DDP in sub-toxic concentration could inhibit the cell proliferation and lead to cell apoptosis inhuman ovarian cancer HO-8910 cells, and its mechanism may be related to through mitochondrial apoptotic pathway.