Dieckol, isolated from Ecklonia stolonifera, induces apoptosis in human hepatocellular carcinoma Hep3B cells

Phlorotannins have been reported to demonstrate several biological properties, including antioxidant activity, and activities useful in the treatment of diabetic complications and in chemoprevention of several vascular diseases. In this study, we focused on the apoptosis induced by dieckol, a marine algal phlorotannin isolated from Ecklonia stolonifera, on human hepatocellular carcinoma (HCC) Hep3B cells. Dieckol reduced the numbers of viable cells and increased the numbers of apoptotic cells in a dose-dependent manner. Immunoblotting analysis revealed that dieckol increased the expression levels of cleaved caspases-3, 7, 8, and 9, and cleaved poly(ADP-ribose) polymerase. Dieckol increased the permeability of mitochondrial membranes and the release of cytochrome c from mitochondria into the cytosol with apoptosis-inducing factor. In addition, dieckol induced increased expression of truncated Bid and Bim. The results indicate that dieckol induces apoptosis via the activation of both death receptor and mitochondrial-dependent pathways in HCC Hep3B cells.     

J7, a methyl jasmonate derivative, enhances TRAIL-mediated apoptosis through up-regulation of reactive oxygen species generation in human hepatoma HepG2 cells

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L), a member of the TNF gene superfamily, induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, some human hepatoma cells are particularly resistant to the effects of TRAIL. In this study, we show that J7, a novel methyl jasmonate analogue, sensitizes TRAIL-resistant HepG2 human hepatocarcinoma cells to TRAIL-mediated apoptosis. Our results indicate that J7 substantially enhances TRAIL-induced apoptosis, compared with treatment with either agent alone. Combined treatment with J7 and TRAIL effectively induced Bid cleavage, down-regulation of XIAP, cIAP-1 and Bcl-xL, activation of caspases, and cleavage of poly(ADP-ribose) polymerase and phopholipase γ-1. In addition, generation of reactive oxygen species (ROS) showed a significant increase in cells following exposure to J7 in a time-dependent manner. However, the cytotoxic effects induced by co-treatment with J7 and TRAIL were markedly attenuated by caspase inhibitors, indicating an important role for caspases. Administration of N-acetyl cysteine, a scavenger of ROS, also resulted in significant inhibition of apoptosis induced by combinatory treatment with J7 and TRAIL. These results support a mechanism whereby J7 plus TRAIL induces apoptosis of HepG2 human hepatoma cells through a signaling cascade involving a ROS-mediated caspase pathway.     

对甲氧基肉桂醛左氧氟沙星酰腙诱导人肝癌SMMC-7721细胞凋亡的作用

目的探讨喹诺酮酰腙类化合物诱导人肝癌SMMC-7721细胞凋亡的作用。方法用不同浓度的对甲氧基肉桂醛左氧氟酰腙(FQ-10)与SMMC-7721细胞体外培养。MTT法检测FQ-10对SMMC-7721细胞的增殖抑制作用;Hoechst33258/PI双染荧光染色法、TUNEL法及琼脂糖凝胶电泳检测细胞凋亡变化;高内涵活细胞成像系统测定细胞线粒体膜电位(△ψm)变化;Western blot方法测定caspase-9、caspase-8、caspase-3、p53、Bcl-2、Bax蛋白表达。结果 FQ-10在0.625～10.00μmol.L-1的浓度范围能抑制细胞增殖,呈时间、浓度依赖性;作用于细胞24 h的IC50值是4.40μmol.L-1;各组FQ-10作用24 h后,细胞凋亡率高于对照组(P<0.05);琼脂糖凝胶电泳可见凋亡细胞典型的梯状DNA条带,并伴有线粒体膜电位降低。荧光染色观察细胞形态学变化,显示凋亡细胞染色质凝集,细胞核碎裂成碎片等典型细胞凋亡特征性变化,晚期凋亡细胞可见特异性PI染色。与对照组比较,FQ-10作用后p53、Bax、caspase-9、caspase-3蛋白表达量增加,其中caspase-9、caspase-3活性裂解片段明显增加,caspase-8无变化,而Bcl-2蛋白表达水平下降。结论对甲氧基肉桂醛左氧氟酰腙能够激活线粒体凋亡通路,诱导人肝癌细胞凋亡。     

氧氟沙星-绕丹宁衍生物诱导人肝癌细胞凋亡作用

目的研究氧氟沙星-绕丹宁衍生物对人肝癌SMMC-7721细胞凋亡的诱导作用。方法用不同浓度的6-(3-苄基-4-氧代-2-硫代-噻唑烷-5-叉甲基)-9-氟-3-甲基-10-(4-甲基哌嗪-1-基)-2,3-二氢-7-氧代-7-氢-吡啶并[1,2,3-de][1,4]苯并噁嗪(R3)与人肝癌SMMC-7721细胞、食管鳞癌EC-9706细胞、结肠癌CaCO-2细胞和L-02肝转化细胞体外培养。MTT法检测R3对各种细胞的增殖抑制作用;DAPI荧光染色法和TUNEL法检测细胞凋亡变化;PI染色流式细胞术检测细胞周期变化;Western blot法测定p53和caspase-3蛋白表达量的变化。结果 R3在2~20μmol·L~(-1)的浓度范围内能明显抑制SMMC-7721细胞、EC-9706细胞和CaCO-2细胞的增殖,作用于细胞24 h的IC_(50)值分别为3.893、4.181和3.408μmol·L~(-1);R3作用于L-02细胞24 h的IC_(50)值为33.959μmol·L~(-1);氧氟沙星盐酸盐作用于SMMC-7721细胞24 h的IC_(50)值为240.137μmol·L~(-1);舒尼替尼作用于SMMC-7721细胞24 h的IC_(50)值为8.075μmol·L~(-1);R3处理SMMC-7721细胞后,细胞周期被阻滞在G1-S期检测点,且细胞凋亡率明显高于对照组(P<0.05)。R3明显增加SMMC-7721细胞p53和caspase-3蛋白表达量,其中caspase-3活性裂解片段增加明显。结论氧氟沙星-绕丹宁衍生物对肿瘤细胞具有较好的选择性抑制作用,能够明显诱导人肝癌SMMC-7721细胞凋亡。     

苯甲醛左氧氟沙星席夫碱诱导人肝癌细胞凋亡作用

目的研究苯甲醛左氧氟沙星席夫碱化合物对人肝癌SMMC-7721细胞凋亡的诱导作用。方法用不同浓度的(S)-1,8-(2-甲基亚乙氧基)-6-氟-7-(4-甲基-哌嗪-1-基)-3-[S-苄基硫基-4-(对硝基苯甲叉基氨基)-1,2,4-均三唑-3-基]-喹啉(1-H)-4-酮(M18)与SMMC-7721细胞、人乳腺癌细胞MB-231、人结肠癌细胞HCT-116、人肝癌细胞HEPG-2和小鼠骨髓间充质干细胞体外培养。MTT法检测M18对各种细胞的生长抑制作用;Hoechst 33258荧光染色法、TUNEL法检测细胞凋亡变化;高内涵活细胞成像系统测定细胞线粒体膜电位(△ψm)变化;Western blot方法测定caspase-3、p53蛋白表达量的改变,以及细胞色素C在线粒体内外的分布。结果 M18在4~32μmol·L-1的浓度范围内能明显抑制SMMC-7721细胞、MB-231细胞、HCT-116细胞、HEPG-2细胞增殖,呈浓度、时间依赖关系,作用于细胞24 h的IC50值分别为8.65、9.37、12.74和9.40μmol·L-1;左氧氟沙星盐酸盐作用于SMMC-7721细胞24 h的IC50值为735.10μmol·L-1,M18作用于骨髓间充质干细胞24 h的IC50值为38.96μmol·L-1;不同浓度M18作用人肝癌SMMC-7721细胞24h,细胞凋亡率高于对照组(P<0.05)。M18作用于SMMC-7721细胞后,细胞线粒体膜电位降低,与对照组相比差异有统计学意义;M18明显增加SMMC-7721细胞的p53和caspase-3蛋白表达量,其中caspase-3活性裂解片段增加明显;M18作用使细胞线粒体内细胞色素C明显减少,胞质内细胞色素C明显增加。结论苯甲醛左氧氟沙星席夫碱能够诱导人肝癌细胞凋亡,作用与线粒体凋亡通路有关。     

S-allylcysteine,a garlic derivative,suppresses proliferation and induces apoptosis in human ovarian cancer cells in vitro

Aim： To investigate the effects of S-allylcysteine （SAC）, a water-soluble garlic derivative, on human ovarian cancer cells in vitro. Methods： Human epithelial ovarian cancer cell line A2780 was tested. Cell proliferation was examined with CCK-8 and colony formation assays. Cell cycle was analyzed with flow cytometry. Cell apoptosis was studied using Hoeohst 33258 staining and Annexin V/PI staining with flow cytometry. The migration and invasion of A2780 cells were examined with transwell and wound healing assays. The expression of relevant proteins was detected with Western blot assays. Results： SAC （1-100 mmol/L） inhibited the proliferation of A2780 cells in dose- and time-dependent manners （the ICsovalue was approximately 25 mmoVL at 48 h, and less than 6.25 mmol/L at 96 h）. Furthermore, SAC dose-dependently inhibited the colony formation of A2780 cells. Treatment of A2780 cells with SAC resulted in G/S phase arrest and induced apoptosis, accompanied by decreased expression of pro-caspase-3, Parp-1 and Bcl-2, and increased expression of active caspase-3 and Bax. SAC treatment significantly reduced the migration of A2780 cells, and markedly decreased the protein expression of Wnt5a, p-AKT and c-Jun, which were the key proteins involved in proliferation and metastasis. Conclusion： SAC suppresses proliferation and induces apoptosis in A2780 ovarian cancer cells in vitro.     

Corosolic acid analogue,a natural triterpenoid saponin,induces apoptosis on human hepatocarcinoma cells through mitochondrial pathway in vitro

Context 2a,-3a,-24-Trihydroxyurs-12-en-28-oic acid (TEO, a corosolic acid analogue) is a triterpenoid saponin isolated from Actinidia valvata Dunn (Actinidiaceae), a well-known traditional Chinese medicine.

Objective This study investigated the anti-proliferation and inducing apoptosis effects of TEO in three human hepatocellular carcinoma (HCC) cell lines.

Materials and methods Cytotoxic activity of TEO was determined by the MTT assay at various concentrations from 2.5 to 40?μg/mL in BEL-7402, BEL-7404 and SMMC-7721 cell lines. Cell morphology was assessed by acridine orange/ethidium bromide and 4′-6-diamidino-2-phenylindole dihydrochloride staining and fluorescence microscopy. Cell-cycle distribution and DNA damage were determined by flow cytometry and comet assay. Mitochondrial dysfunction was assessed by JC-1 staining and transmission electron microscopy. Apoptosis changes were explored by Western blot, TNF-α and caspase-3, -8, -9 assays.

Results TEO exhibited inhibition effects on BEL-7402, BEL-7404 and SMMC-7721 cells treated for 24?h, the IC₅₀ values were 34.6, 30.8 and 30.5?μg/mL, respectively. TEO (40?μg/mL)-treated three cell lines increased by more than 21% in the G1 phase and presented the morphological change and DNA damage. TEO also declined the mitochondrial membrane potential and altered mitochondrial ultra-structure. Furthermore, caspase-3, caspase-8, caspase-9 and TNF-α were also activated. Mechanism investigation showed that TEO could decrease anti-apoptotic Bcl-2 protein expression, increase proapoptotic Bax and Bid proteins expressions and increase Bax/Bcl-2 ratio.

Conclusion Our results demonstrate for the first time that TEO inhibited growth of HCC cell lines and induced G1 phase arrest. Moreover, proapoptotic effects of TEO were mediated through the activation of TNF-α, caspases and mitochondrial pathway.     

呫吨酮并吡啶衍生物XP-16诱导人肺癌A549细胞凋亡

目的探讨呫吨酮并吡啶衍生物5,9-二(2-吡咯烷基乙酰氨基)-7H-吡啶并[4,3-c]呫吨-7-酮(XP-16)对人肺癌A549细胞的抗肿瘤作用及其可能作用机制。方法通过MTT法、细胞形态学和克隆实验观察XP-16对A549细胞增殖的影响;应用Hoechst 33258和PI双染法观察细胞凋亡;荧光分光光度计检测细胞内钙([Ca2+]i)及线粒体膜电位;qRT-PCR检测Bad和金属硫蛋白1A(metallothionein 1A,MT-1A)mRNA表达。结果 XP-16能抑制A549细胞增殖,呈剂量和时间依赖性。XP-16作用A549细胞24 h后,A549细胞出现染色质聚集、核碎裂等典型的凋亡形态学改变;随XP-16剂量的增加,A549细胞凋亡百分率逐渐增大。XP-16作用后,A549细胞的[Ca2+]i和线粒体膜电位降低、Bad和MT-1A mRNA的表达增加。结论 XP-16具有诱导A549细胞凋亡的作用,可能与其降低[Ca2+]i和线粒体膜电位有关。MT-1A表达的上调可能是[Ca2+]i降低的结果。     

Cisplatin-induced apoptosis in Hep3B cells: mitochondria-dependent and -independent pathways

Human hepatoma cell lines undergo apoptosis after treatment with cisplatin (CP), by mechanisms that are not fully understood, although our previous study demonstrated that Fas-dependent or -independent pathways are involved. To elucidate the mechanisms of CP-induced apoptosis in Hep3B cells, which are Fas- and p53-negative, we investigated mitochondria associated pathways, the involvement of NF-kappaB, and p73 activation. Results of Western blot and flow cytometry assay revealed that the translocation of Bax, resulted in the loss of mitochondrial membrane potential (Deltaphi(m)) and the efflux of cytochrome c and of second mitochondria-derived activator of caspase/DIABLO from mitochondria into the cytosol. Caspase-3, -8 and -9 were activated by CP treatment, however, CP-induced apoptosis was not completely blocked by pretreating with the pan-caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(O-methyl)-fluoromethylketone, indicating that caspase-independent apoptotic pathways might also be involved. RNase protection assay confirmed that NF-kappaB downregulation leading to the suppression of its target genes, such as XIAP and TRAF2, and p73 accumulation were also observed in Hep3B cells treated with CP. CP-induced apoptosis was inhibited to some extent by transiently overexpressed p73 dominant negative and XIAP, but not by p73DN or XIAP alone. In conclusion, this study demonstrates that CP-induced apoptosis in Hep3B cells is associated with mitochondrial dysregulation, NF-kappaB downregulation and p73 accumulation.     

左氧氟喹诺酮查尔酮类衍生物对人肝癌SMMC-7721细胞凋亡和自噬的影响

目的 研究左氧氟喹诺酮查尔酮类衍生物6-氟-7-(4-甲基-哌嗪-1-基)-1,8-(3,1-氧丙基)-3-[2-(4-甲氧苯甲酰基)-乙烯-1-基]-喹啉-4-(1H)-酮(HGQ5)对人肝癌细胞凋亡和自噬的诱导作用.方法 采用MTT法检测SMMC-7721细胞的增殖,TUNEL法测定细胞的凋亡率,采用自噬抑制剂氯喹处理细胞来验证HGQ5对细胞增殖和凋亡的影响;用Western blot法检测细胞自噬标志性蛋白LC3B的表达.结果 HGQ5对SMMC-7721细胞的增殖具显著抑制作用,24、48 h的IC50分别为5.0、4.8 μmol· L-;各给药组细胞经HGQ5作用24 h后,细胞凋亡率显著高于对照组的;HGQ5导致细胞中LC3B-Ⅱ的表达量增加,6～24 h达到最高,之后明显下降;氯喹可增强低剂量HGQ5(0.3～ 2.5 μmol·L-)对SMMC-7721细胞增殖的抑制作用,但降低高剂量HGQ5(5～ 10 μmol·L-1)对SMMC-7721细胞增殖的抑制作用.结论 HGQ5能够显著诱导SMMC-7721细胞的凋亡和自噬,低剂量HGQ5作用于SMMC-7721细胞时,自噬对细胞具有保护作用;高浓度HGQ5作用时,自噬作用则降低了细胞增殖率,促进了细胞凋亡.     

Baicalin induces human mucoepidermoid carcinoma Mc3 cells apoptosis in vitro and in vivo

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor in salivary glands and high-grade MEC in particular demonstrates little response to chemotherapy which has been used largely for palliative treatment of metastatic disease. Baicalin, one of the main active compounds of Scutellaria baicalensis, possesses anti-inflammatory, antioxidant and antitumor properties. In the present study, we investigated the growth inhibiting and apoptosis-inducing effects of baicalin on a highly metastatic human mucoepidermoid carcinoma cell line Mc3 for the first time. Baicalin exerted dose- and time-dependent antiproliferative potential against Mc3 cells as assessed by MTT assay. Baicalin treatment of Mc3 cells resulted in an accumulation of cells at the G₀/G₁ and G₂/M phase with a concomitant decrease in cells processing to S phase as assessed by flow cytometry. Furthermore, baicalin induced apoptosis of Mc3 cells as determined by annexin V binding and PI dual staining, DNA fragmentation, nuclear condensation and in vivo tumor inhabitation. Rhodamine 123 assay indicated that baicalin caused cytotoxicity and induced apoptosis through decreasing the mitochondrial membrane potential in Mc3 cells. Our results suggest that baicalin seems to be very attractive as a new anticancer drug and a potential chemotherapeutic agent against human high-grade mucoepidermoid carcinoma.     

Supernatant of bacterial fermented soybean induces apoptosis of human hepatocellular carcinoma Hep 3B cells via activation of caspase 8 and mitochondria.

SC-1, the aqueous phase of soybean fermentation products by bacteria (Bacillus subtilis and Bacillus brevis), significantly inhibited the growth and clonogenesity of human hepatocellular (Hep 3B), mouse hepatocellular (ML-1), and human colorectal (HCT 116 and HT-29) carcinoma cells. Cytotoxicity of SC-1 in Hep 3B cells was through the process of apoptosis characterizing by increase in cell population of sub-G(1) phase, fragmentation of DNA, and change of nuclear morphology. Treatment of Hep 3B cells with SC-1 activated caspase 8 and caspase 3. Elevation of nuclear DNA fragmentation factor 40 (DFF40) and cleavage form of poly(ADP-ribose) polymerase (PARP) were also observed. SC-1 also activated intrinsic pathway via increase of pro-apoptotic (tBid, Bak and Bax) and decrease of anti-apoptotic (Bcl-2 and Bcl-x(L)) proteins on mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c and Smac (second mitochondria-derived activator of caspase/direct IAP binding protein with low PI) from mitochondria, and activation of caspase 9. Inhibition on protein expression of Ku70 in cytosol and cyclooxygenase (COX)-2, but not COX-1, in whole cell lystes were revealed in SC-1-treated Hep 3B cells. These results suggest caspase 8, Ku70 and mitochondria are involved in the antitumor mechanism of SC-1 in Hep 3B cells.     

三氮唑席夫碱衍生物对人肝癌细胞分化和凋亡的影响

目的探讨三氮唑席夫碱衍生物对人肝癌细胞BEL-7402的诱导分化和凋亡作用。方法用不同剂量的3-吡啶-3-基-4-[(4-羟基-3-甲氧基-苯亚甲基)氨基]-5-甲硫基-1,2,4-三唑(LH-37)与BEL-7402细胞共同培养,以MTT法检测细胞增殖和LH-37对细胞增殖的抑制作用;RT-PCR方法检测细胞甲胎蛋白(α-FP)和白蛋白(Alb)mRNA表达;ELISA法测定细胞α-FP含量;免疫组织化学检测细胞内激活型Caspase-3表达;Western blot检测Caspase-3和Caspase-9蛋白表达;可见光法测定超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活力;荧光显微镜观察凋亡细胞形态;Annexin-Ⅴ和PI双染流式细胞术测定凋亡细胞数。结果LH-37在10μmol·L-1~1mmol·L-1的浓度范围能抑制细胞增殖,且抑制率随浓度的增加而增高(P<0·05或P<0·01),10μmol·L-1和1·0μmol·L-1的LH-37使细胞Alb mRNA表达量增高,α-FP mRNA和蛋白质表达量明显下降,Caspase-3和Caspase-9表达量明显增加,Caspase-3阳性细胞明显增加;药物浓度达100μmol·L-1作用48h后,BEL-7402细胞皱缩,呈现凋亡各期改变,细胞凋亡率高于对照组(P<0·05)。结论三氮唑席夫碱衍生物对人肝癌BEL-7402细胞具有抗增殖、促分化和凋亡作用,作用可能与过氧化氢的氧化损伤作用有关。     

Piperonal ciprofloxacin hydrazone induces growth arrest and apoptosis of human hepatocarcinoma SMMC-7721 cells

Aim:

To investigate the cytotoxic effects of piperonal ciprofloxacin hydrazone (QNT4), a novel antibacterial fluoroquinolone derivative, against human hepatocarcinoma SMMC-7721 cells.

Methods:

Human hepatocarcinoma cells (SMMC-7721), human breast adenocarcinoma cells (MCF-7) and human colon adenocarcinoma cells (HCT-8) were tested. The effects of QNT4 on cell proliferation were examined using MTT assay. Cell apoptosis was determined using Hoechst 33258 fluorescence staining, TUNEL assay and agarose gel electrophoresis. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. Mitochondrial membrane potential (Δψm) was measured using a high content screening imaging system. Protein expression of caspase-9, caspase-8, caspase-3, p53, Bcl-2, Bax, and cytochrome c was detected with Western blot analysis.

Results:

Treatment with QNT4 (0.625–10 μmol/L) potently inhibited the proliferation of the cancer cells in time- and dose-dependent manners (the IC₅₀ value at 24 h in SMMC-7721 cells, MCF-7 cells and HCT-8 cells was 2.956±0.024, 3.710±0.027, and 3.694±0.030 μmol/L, respectively). Treatment of SMMC-7721 cells with QNT4 (0.2146, 2.964, and 4.600 μmol/L) for 24 h dose-dependently increased the percentage of apoptotic cells, elicited characteristic DNA “ladder” bands, and decreased the mitochondrial membrane potential. QNT4 dose-dependently increased topoisomerase II-mediated DNA breaks while inhibiting DNA relegation, thus keeping the DNA in fragments. Treatment of SMMC-7721 cells with QNT4 significantly increased cytochrome c in the cytosol, and decreased cytochrome c in the mitochondrial compartment. QNT4 (3–7.39 μmol/L) significantly increased the protein expression of p53, Bax, caspase-9, caspase-3, and the cleaved activated forms of caspase-9 and caspase-3 in SMMC-7721 cells. In contrast, the expression of Bcl-2 was decreased, while caspase-8 had no significant change.

Conclusion:

QNT4 induced the apoptosis of SMMC-7721 cells via inhibiting topoisomerase II activity and modulating mitochondrial-dependent pathways.     

Isoobtusilactone A induces both caspase-dependent and -independent apoptosis in Hep G2 cells.

Isoobtusilactone A, a constituent isolated from the leaves of Cinnamomum kotoense, has been demonstrated by us earlier to be an agent capable of inducing apoptotic cell death of Hep G2 cells. In order to clarify if caspases alone were the sole mediator for eliciting this apoptotic process, a broad caspases inhibitor, Z-VAD.fmk, was utilized to explore this possibility. Interestingly, although Z-VAD.fmk was demonstrated to be capable of completely inhibiting isoobtusilactone A-induced oligonucleosomal DNA fragmentation, yet it could only prevent limited amount of cells from becoming apoptosis-prone. These data implied that some other mechanism(s) might be involved. Thus, the involvement of apoptosis-inducing factor (AIF), a mediator arbitrating caspase-independent apoptosis, in isoobtusilactone A-induced apoptotic process was examined. These findings indicated that isoobtusilactone A could elicit the nuclear translocation of AIF that accompanied the occurrence of large-scale DNA fragmentation. Reduction of AIF expression by AIF-siRNA transfection suppressed large-scale DNA fragmentation. Interestingly, inhibition of AIF expression by AIF-siRNA could not prevent isoobtusilactone A-induced oligonucleosomal DNA fragmentation. In the same vein, when the cells were simultaneously combined pretreatment with AIF-siRNA and Z-VAD.fmk, both large-scale DNA and oligonucleosomal DNA fragmentations could nearly be prevented. Taken together, these findings suggested that isoobtusilactone A-induced apoptotic cell death was mediated via both caspase-dependent and -independent pathways.     

Epothilone B induces extrinsic pathway of apoptosis in human SKOV-3 ovarian cancer cells

The molecular mechanisms underlying epothilone B (EpoB) induced apoptosis were investigated in SKOV-3 human ovarian cancer cells. The aim of this research was to compare EpoB’s, which belongs to the new class of anticancer drugs, with paclitaxel’s (PTX) ability to induce apoptosis. The mode of cell death was assessed colorimetrically, fluorimetrically and by immunoblot analyses through measuring DNA fragmentation, the level of intracellular calcium, the level of cytochrome c, TRAIL, the cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of caspase-9, -8 and -3.EpoB leads to an increase of the cytosolic level of cytochrome c after 4 h of cell treatment. After 24 and 48 h of cell treatment the level of intracellular calcium also increased by about 21% and 24% respectively. Moreover, EpoB, similarly to PTX, promoted the expression of TRAIL in lymphocytes, although high TRAIL expression on tumor cells was detected only after adding EpoB to SKOV-3 cells. EpoB mediates caspases-8 and -3 activation, which is independent of the reduction in the amount of caspase-9. Epitope-specific monoclonal and polyclonal antibodies revealed characteristic apoptotic changes that included cleavage of the 116 kDa PARP polypeptide to 25 kDa fragments. The results of our study show that EpoB induces mainly the extrinsic pathway.     

BFPP, a phloroglucinol derivative, induces cell apoptosis in human chondrosarcoma cells through endoplasmic reticulum stress

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(2-fluorophenylacetyl)phloroglucinol; BFPP) in human chondrosarcoma cells. BFPP induced cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353 but not in primary chondrocytes. BFPP triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels, and increased glucose-regulated protein 78 (GRP78) expression, but failed to show the same effects on GRP94 expression. BFPP also increased calpain expression and activity. Transfection of cells with GRP78 or calpain siRNA reduced BFPP-mediated cell apoptosis in JJ012 cells. Importantly, animal studies have revealed a dramatic 50% reduction in tumor volume after 21 days of treatment. This study demonstrates novel anticancer activity of BFPP against human chondrosarcoma cells and in murine tumor models.     

A natural compound,methyl angolensate,induces mitochondrial pathway of apoptosis in Daudi cells

Natural products discovered from medicinal plants have played an important role in the treatment of cancer. In an effort to identify novel small molecules which can affect the proliferation of lymphoma cells, we tested methyl angolensate (MA), a plant derived tetranortriterpenoid, purified from the crude extract of the root callus of Soymida febrifuga commonly known as Indian red wood tree. We have tested MA for its cytotoxic properties on Burkitt’s lymphoma cell lines, using various cellular assays. We observed that MA induces cytotoxicity in Daudi cells in a dose-dependent manner using trypan blue, MTT and LDH assays. We find that the treatment with MA led to activation of DNA double-strand break repair proteins including KU70 and KU80, suggesting the activation of nonhomologous DNA end joining pathway in surviving cells. Further, we find that methyl angolensate could induce apoptosis by cell cycle analysis, annexin V-FITC staining, DNA fragmentation and PARP cleavage. Besides, MA treatment led to reactive oxygen species generation and loss of mitochondrial transmembrane potential. These results suggest the activation of mitochondrial pathway of apoptosis. Hence, we identify MA as a potential chemotherapeutic agent against Daudi cells.