Ethanolic Neem (Azadirachta indica) Leaf Extract Induces Apoptosis in the Hamster Buccal Pouch Carcinogenesis Model by Modulation of Bcl-2, Bim,Caspase 8 and Caspase 3

Induction of apoptosis is one of the most active strategies in cancer chemoprevention and the ability of medicinal ‍plants in this regard has attracted major research interest. The present study was designed to investigate the apoptosis ‍inducing capacity of an ethanolic neem leaf extract (ENLE) during 7,12-dimethylbenz[a]anthracene (DMBA)-induced ‍hamster buccal pouch carcinogenesis using the apoptosis-associated proteins Bcl-2, Bim, caspase 8 and caspase 3 as ‍markers. Topical application of DMBA to the hamster cheek pouch for 14 weeks resulted in well developed squamous ‍cell carcinomas associated with increased expression of Bcl-2 and decreased expression of Bim, caspase 8 and caspase ‍3. Administration of ENLE inhibited DMBA-induced hamster buccal pouch (HBP) carcinogenesis, as revealed by ‍the absence of neoplasms, with induction of Bim and caspases 8 and 3 and inhibition of Bcl-2 expression. Our results ‍suggest that the chemopreventive effects of ENLE may be mediated by induction of apoptosis.     

Induction of apoptosis by phenethyl isothiocyanate in cells overexpressing Bcl-XL

Isothiocyanates are a class of phytochemicals able to induce apoptosis in numerous cells including Jurkat T-lymphoma cells overexpressing the oncoprotein Bcl-2. To test if isothiocyanates are also effective against other anti-apoptotic members of the Bcl-2 family we generated Jurkat cells stably overexpressing Bcl-X(L). Phenethyl isothiocyanate (PEITC) was cytotoxic to these cells, with an LD(50) ranging from 9 to 18 microM depending on the level of Bcl-X(L) expression. Apoptosis induction in response to PEITC was confirmed by caspase activation and phosphatidylserine exposure. Isothiocyanates specifically target cysteine residues, therefore we tested the hypothesis that PEITC directly impairs Bcl-2 and Bcl-X(L) activity by interacting with their conserved cysteine residues. Jurkat cells overexpressing double cysteine mutants of Bcl-2 were generated, but they remained sensitive to PEITC. We conclude that PEITC antagonizes the action of anti-apoptotic Bcl-2 family members via an indirect mechanism.     

寻常性银屑病患者皮损中Caspase-3和bcl-xL的表达

目的 探讨Caspase-3和bcl-xL在银屑病皮损中的表达及意义。方法 采用免疫组化法对26例寻常性银屑病患者的皮损及其周边外观正常皮肤和10例正常人对照皮肤中Caspase-3和bcl-xL的表达进行了检测。结果 正常人对照皮肤、银屑病皮损及其周边外观正常皮肤的表皮均表达Caspase-3,其中皮损处的表达明显增强(P<0.0001);除皮损表皮中散在表达bcl-xL外,正常人对照皮肤、银屑病皮损周边外观正常皮肤的表皮中不表达bcl-xL。结论 Caspase-3和bcl-xL可能通过诱导银屑病皮损角质形成细胞的凋亡而参与了银屑病的发病。     

咖啡酸对神经毒素MPP+诱发大鼠小脑颗粒细胞凋亡的保护作用

目的:探讨咖啡酸(CA) 是否对1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium ion, MPP ) 诱导的大鼠小脑颗粒细胞凋亡具有保护作用.方法: 用咖啡酸孵育大鼠小脑颗粒细胞, 经MPP 处理后, 用MTT法测定细胞存活率, 荧光法检测caspase-3的活性.结果:咖啡酸对MPP 致小脑颗粒细胞存活率降低和caspase-3活性增强具有很好的抑制作用,并呈现稳定的量效和时效关系.结论:咖啡酸对MPP 诱导小脑颗粒细胞凋亡具有明显的保护作用.     

苦瓜素对小鼠柯萨奇B3病毒性心肌炎caspase 3作用的研究

目的观察苦瓜素对病毒性心肌炎心肌组织caspase 3活性、基因转录及相应蛋白质表达的影响,探索苦瓜素治疗病毒性心肌炎的机制。方法实验小鼠随机分为苦瓜素治疗组、生理盐水对照组及正常对照组和正常小鼠苦瓜素处理组。苦瓜素剂量为25 mg.kg-1.d-1,1次/d,疗程7 d。第15天后取小鼠心肌进行caspase 3活性测定、HE染色心肌病理检查、心肌细胞凋亡检查、RT-PCR检测、caspase 3,mRNA转录水平,免疫组化与免疫印迹测定caspase 3蛋白表达。结果生理盐水对照组小鼠心肌组织caspase 3活性明显增高、mRNA转录水平与相应蛋白质表达水平亦显著增加;苦瓜素治疗组caspase 3活性明显抑制,caspase 3 mR-NA转录水平明显低于生理盐水对照组(0.012±0.008 vs 0.043±0.015,t=4.37,P<0.01),凋亡细胞明显减少。结论苦瓜素通过抑制caspase 3基因转录与蛋白质表达、抑制其活性,对Balb/c小鼠CVB3病毒性心肌炎具有明显的治疗作用。     

Antiproliferative activity and standardization of Tecomella undulata bark extract on K562 cells

Ethnopharmacological relevance

The bark of Tecomella undulata is primarily used in the treatment of syphilis, painful swellings and cancer by traditional healers. Also, it is claimed to be useful in treating urinary discharges, enlargement of spleen, leucorrhoea, leukoderma, tumors, liver disorders, gonorrhea, gout and promotes wound healing in Indian traditional system of medicine.

Aim

To establish a scientific validation for the antitumor effects of Tecomella undulata bark and explore the mechanistic pathway in chronic myeloid leukemia cell line, K562. The study was further extended to standardize the extract using quercetin as biomarker.

Methods

Induction of apoptosis by chloroform extract of Tecomella undulata bark (CTUB) was determined by MTT, Annexin V and caspase activation assays. The cell cycle analysis was done by flow cytometer and nuclear staining by DAPI. The standardization of the extract was performed through reverse phase-HPLC method under PDA detection.

Result

Results clearly showed the induction of apoptosis by CTUB in K562 cells. The effect was found to be dose dependent, having IC₅₀ of 30 μg/ml with activation of FAS, FADD, caspase 8, caspase 3/7 and fragmentation of DNA. The bioactive CTUB was determined to possess 0.03% (w/w) of quercetin.

Conclusion

The investigation clearly demonstrated the potential antitumor effect of CTUB, thereby validating the traditional claim. Quercetin, known to have anticancer activity is being reported and quantified for the first time from the bark of Tecomella undulata.     

大鼠缺血再灌流脑区半胱氨酸蛋白酶-3活化与神经元凋亡的关系

目的　探讨缺血再灌流脑区半胱氨酸蛋白酶 3(caspase 3)活性变化与神经元凋亡的关系。方法　采用WesternBlotting、原位细胞凋亡检测和免疫组化染色等方法,观察大鼠大脑中动脉栓塞 2h后再灌流 1、6、12、24h时,颞顶叶皮层缺血脑区caspase 3表达和活化、多 (ADP 核糖 )聚合酶(PARP)表达和切割灭活及神经元凋亡程度的变化。结果　再灌流 1、6、12及 24h组,缺血脑区的caspase 3前体及其 12 000切割片段含量的相对灰度值分别为 16 72±2 96、28 36±3 51、51 15±3 10、76 14±3 45及 8 17±2 31、15 36±1 39、31 23±5 43、58 95±6 28;PARP及其 24 000切割片段含量的相对灰度值分别为 12 63±3 02、22 65±4 38、30 81±3 16、67 49±8 59及 6 02±0 73、12 86±2 30、20 76±3 16、27 36±2 63;PARP阳性神经元及凋亡神经元的密度 (细胞数 /0 1mm2 )分别为 68 00±6 67、91 40±9 56、112 20±11 78、127 00±11 51及 83 31±7 53、100 70±6 16、121 53±7 21、197 36±11 78。以上 6种指标的变化彼此间均呈正相关 (r= 0 805 ~0 942, P<0 01 );除 6h与 12h组间PARP含量的差异及 6h与 12h组间和 12h与 24h组间PARP阳性神经元密度的差异无统计学意义外,上述 6种指标的其余组间差异均有统计学意义 (P<0 0     

心肌营养素-1基因治疗大鼠脑损伤后心肌营养素-1和caspase-3的表达变化

目的 观察外源性心肌营养素-1（CT-1）基因被载体重组腺病毒（Adv）导入损伤大脑细胞后在细胞中表达的情况及其对凋亡基因半胱氨酸天冬氨酸特异性蛋白酶-3（caspase-3）表达的影响，了解CT-1对损伤大脑神经元的作用和机制。方法 建立大鼠创伤性脑损伤（TBI）模型，于损伤脑区局部注射Adv—CT-1，应用免疫组化技术检测伤后及治疗后CT-1和caspase-3基因的表达变化。结果 TBI后1，3，7d，大脑皮层和海马等损伤脑区CT-1基因表达轻度增加；伤后12h、1，3，7d，在大脑皮层和海马等损伤脑区caspase-3基因表达明显增加，1d达高峰，3—7d逐渐降低。Adv—CT-1转染损伤脑区治疗后12h-7d，大脑皮层和海马等损伤脑区CT—1表达明显增加，7d达高峰；治疗后12h～7d在大脑皮层和海马等损伤脑区caspase-3基因表达较损伤对照组明显减少。结论 TBI后CT-1基因表达轻度增加，可能是机体对脑损伤后的反馈性保护机制；伤后caspase-3基因表达增加，是创伤后神经细胞凋亡的内在原因。伤后Adv—CT—1转染治疗可使CT-1基因在受损大脑中表达增加，而CT-1可下调凋亡基因caspase-3的表达，这可能是CT—1保护损伤神经元、促进神经功能恢复的内在机制。     

热休克预处理抑制过氧化氢所致C2C12肌原细胞释放天冬氨酸特异性半胱氨酸蛋白酶激活物及细胞凋亡

为探讨热休克预处理对过氧化氢所致C2C12肌原细胞凋亡和第二种线粒体释放的天冬氨酸特异性半胱氨酸蛋白酶激活物从线粒体释放的影响，采用0.5mmol／L过氧化氢作用于C2C12肌原细胞。Hoechst33258荧光染色，观察细胞形态学改变并计算凋亡百分率，抽提DNA作琼脂糖电泳，以确定细胞凋亡情况。采用天冬氨酸特异性半胱氨酸蛋白酶活性定量检测试剂盒及蛋白质印迹观察天冬氨酸特异性半胱氨酸蛋白酶3，天冬氨酸特异性半胱氨酸蛋白酶9的活化情况。采用免疫荧光及细胞成分分离后蛋白质印迹检测第二种线粒体释放的天冬氨酸特异性半胱氨酸蛋白酶激活物从线粒体的释放。结果发现，过氧化氢处理1～2h，第二种线粒体释放的天冬氨酸特异性半胱氨酸蛋白酶激活物逐渐从线粒体释放入胞浆；处理4h天冬氨酸特异性半胱氨酸蛋白酶3，天冬氨酸特异性半胱氨酸蛋白酶9活性升高，12h达高峰；处理24h细胞明显出现凋亡，凋亡百分率明显升高。热休克预处理可诱导C2C12肌原细胞中热休克蛋白90，热休克蛋白70及αβ-晶状体蛋白的表达增高：抑制第二种线粒体释放的天冬氨酸特异性半胱氨酸蛋白酶激活物释放、天冬氨酸特异性半胱氨酸蛋白酶3，天冬氨酸特异性半胱氨酸蛋白酶9的活化及细胞凋亡的发生。结果提示，线粒体信号通路在过氧化氢所致的心肌细胞凋亡中发挥重要作用；热休克预处理通过抑制上述通路而减轻过氧化氢所致的细胞凋亡，其机制与其诱导热休克蛋白表达、阻断第二种线粒体释放的天冬氨酸特异性半胱氨酸蛋白酶激活物从线粒体向胞浆释放、从而抑制天冬氨酸特异性半胱氨酸蛋白酶3，天冬氨酸特异性半胱氨酸蛋白酶9的活化有关。