Mechanism of cell cycle arrest caused by histone deacetylase inhibitors in human carcinoma cells

Inhibitors of histone deacetylase (HDAC) block cell cycle progression at G1 in many cell types. We investigated the mechanism by which trichostatin A (TSA), a specific inhibitor of HDAC, induces G1 arrest in human cervix carcinoma HeLa cells. TSA treatment induced histone hyperacetylation followed by growth arrest in G as well as hypophosphorylation of pRb. The Cdk4 kinase activity was essentially unchanged during the TSA-induced G1 arrest. On the other hand, the arrest was accompanied by down-regulation of kinase activity of Cdk2, although the total protein levels of Cdk2 and its activator Cdc25A were unaffected. Upon TSA treatment, amounts of cyclin E and the CDK inhibitor p21WAF1/Cip1 were markedly increased, while that of cyclin A was reduced. The induction of p21 and down-regulation of cyclin A correlated well with the decreased Cdk2 activity and cell cycle arrest. Furthermore, gel filtration chromatography showed the association of p21 with the cyclin E-Cdk2 complex, suggesting that the activation of Cdk2 by the enhanced expression of cyclin E is blocked by the increased p21. The elevated expression of p2 is also observed in cells treated with trapoxin and FR901228, structurally unrelated histone deacetylase inhibitors. A human colorectal carcinoma cell line lacking both alleles of the p21 gene (p21-/-) was resistant to TSA several times more than the parental line (p21+/+). These results suggest that the suppression of Cdk2 kinase activity due to p21 overexpression play a critical role in HDAC inhibitor-induced growth inhibition.     

Src kinase inhibitors induce apoptosis and mediate cell cycle arrest in lymphoma cells

Src kinases are involved in multiple cellular contexts such as proliferation, adhesion, tumor invasiveness, angiogenesis, cell cycle control and apoptosis. We here demonstrate that three newly developed dual selective Src/Abl kinase inhibitors (SrcK-I) (AZM559756, AZD0530 and AZD0424) are able to induce apoptosis and cell cycle arrest in BCR-ABL, c-KIT and platelet-derived growth factor-negative lymphoma cell lines. Treatment of DOHH-2, WSU-NHL, Raji, Karpas-299, HUT78 and Jurkat cells with SrcK-I revealed that the tested substances were effective on these parameters in the cell lines DOHH-2 and WSU-NHL, whereas the other tested cell lines remained unaffected. Phosphorylation of Lyn and in particular Lck were affected most heavily by treatment with the SrcK-I. Extrinsic as well as intrinsic apoptosis pathways were activated and elicited unique expressional patterns of apoptosis-relevant proteins such as downregulation of survivin, Bcl-XL and c-FLIP. Protein levels of c-abl were downregulated and Akt phosphorylation was decreased by treatment with SrcK-I. Basal expression levels of c-Myc were notably lower in sensitive cell lines as compared with nonsensitive cell lines, possibly providing an explanation for sensitivity versus resistance against these novel substances. This study provides the first basis for establishing novel SrcK-I as weapons in the arsenal against lymphoma cells.     

含过氧桥组蛋白去乙酰化酶抑制剂的合成及抗肿瘤细胞增殖活性研究

目的设计合成含过氧桥的组蛋白去乙酰化酶(HDAC)抑制剂,并考察化合物的抗肿瘤细胞增殖活性。方法以含过氧桥的胺类化合物或醇类化合物为起始原料,经缩合、水解、缩合、脱保护等4步反应合成目标化合物,同时合成了对应的非过氧桥类似物。采用MTT法测试化合物对肿瘤细胞及正常细胞的IC50值,探讨过氧桥、不同连接键方式、碳链的长度对化合物活性的影响。结果与结论合成了15个未见文献报道的全新化合物,化合物结构经氢谱、碳谱以及高分辨质谱确证;体外抗肿瘤细胞增殖活性评价表明,羟肟酸部分是活性必需基团,碳链缩短会导致活性降低,过氧桥结构对化合物的抗肿瘤细胞增殖活性影响较大,过氧桥取代基的位阻增大有利于提高化合物的活性,化合物对肿瘤细胞的选择性优于正常细胞。     

Bortezomib treatment of ovarian cancer cells mediates endoplasmic reticulum stress,cell cycle arrest,and apoptosis

Bortezomib, an approved drug for the treatment of certain haematological neoplasms, is currently being tested in clinical trials as a potential therapeutic agent against several types of solid cancer, including ovarian cancer. We have analyzed the effect of bortezomib on ovarian cancer cells and tissue explants either as a single agent or in combination with carboplatin, taxol, or TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). Bortezomib alone efficiently induced apoptosis in ovarian cancer cells. Apoptosis was preceded by an upregulation of the endoplasmic reticulum stress sensor ATF3, and increased the expression of cytoplasmic heat shock proteins. Bortezomib enhanced the sensitivity of ovarian cancer cells and tissue explants to an apoptosis-inducing TRAIL receptor antibody by upregulating the TRAIL receptor DR5. In contrast to the synergistic effect observed for TRAIL, the efficacy of the taxol treatment was reduced by bortezomib, and bortezomib inhibited the G2/M phase accumulation of ovarian cancer cells treated with taxol. Bortezomib alone or in combination with taxol induced a cell cycle arrest within the S phase, and downregulation of cdk1, a cyclin-dependent kinase that is necessary for the entry into the M phase. Thus, bortezomib can be regarded as a promising agent for the treatment of ovarian cancer and could either be administered as a single agent or in combination with TRAIL. However, a combination treatment with taxanes may not be beneficial and may even be less effective.     

Zerumbone inhibits interleukin-6 and induces apoptosis and cell cycle arrest in ovarian and cervical cancer cells

Interleukin-6 is one of the factors affecting sensitivity to cytotoxic agents. Therefore, the current study was designed to investigate the role of IL-6 and IL6 receptors in the cytotoxic effects of zerumbone in ovarian and cervical cancer cell lines (Caov-3 and HeLa, respectively). Exposure of both cancer cells to zerumbone or cisplatin demonstrated growth inhibition at a dose-dependent manner as determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,Sdiphenyltetrazolium bromide) reduction assay. Both laser scanning confocal microscopy and TUNEL assay showed typical apoptotic features in treated cells. The studies conducted seems to suggest that zerumbone induces cell death by stimulating apoptosis better than cisplatin, based on the significantly higher percentage of apoptotic cells in zerumbone's treated cancer cells as compared to cisplatin. In addition, zerumbone and cisplatin arrest cancer cells at G2/M phase as analyzed by flow cytometry. Our results indicated that zerumbone significantly decreased the levels of IL-6 secreted by both cancer cells. In contrast, HeLa and Caov-3 cells were still sensitive to cisplatin and zerumbone, even in the presence of exogenous IL-6. However, membrane-bound IL-6 receptor is still intact after zerumbone treatment as demonstrated using an immune-fluorescence technique. This study concludes that the compound, zerumbone inhibits both cancer cell growth through the induction of apoptosis, arrests cell cycle at G2/M phase and inhibits the secretion levels of IL-6 in both cancer cells. Therefore, zerumbone is a potential candidate as a useful chemotherapeutic agent in treating both cervical and ovarian cancers in future.     

Synthesis of apicidin-derived quinolone derivatives: parasite-selective histone deacetylase inhibitors and antiproliferative agents

Apicidin's indole was efficiently converted into a series of N-substituted quinolone derivatives by indole N-alkylation followed by a two-step, one-pot, ozonolysis/aldol condensation protocol. The new quinolones exhibited good parasite selectivity and potency both at the level of their molecular target, histone deacetylase, and in their whole cell antiproliferative activity in vitro.     

新型组蛋白去乙酰化酶抑制剂致U251细胞周期阻滞、凋亡及其机制的研究

目的探讨新型组蛋白去乙酰化酶抑制剂(HDACi)2,2,3,3-四甲基环丙酰硫脲(TCCT)诱导人脑胶质瘤U251细胞周期阻滞、凋亡及其作用机制。方法以不同药物浓度与U251细胞共同培养48 h后,采用MTT法检测药物作用48 h后肿瘤细胞的增殖。药物作用24 h后,采用RT-PCR检测肿瘤细胞的p21WAF1/CIP1与Cyclin D1 mRNA的表达,Western blot检测HDAC3、HDAC4、Cyclin D1与p21WAF1/CIP1蛋白的表达,PI单染法分析细胞周期,Annexin V-PI双染法检测肿瘤细胞凋亡。结果 TCCT对U251细胞增殖具有明显抑制作用,药物干预48 h时的IC50为(0.461±0.108)mmol·L-1,并呈现剂量依赖性。TCCT药物干预24 h后U251细胞p21WAF1/CIP1mRNA表达上调,Cyclin D1 mRNA下调;组蛋白去乙酰化酶3(HDAC3)和组蛋白去乙酰化酶4(HDAC4)表达下调,Cyc-lin D1蛋白表达弱下调,p21WAF1/CIP1蛋白表达上调;S期细胞比例明显提高(P<0.05);细胞凋亡率明显增高(P<0.01)。结论 TCCT对U251细胞增殖有明显的抑制作用,引起肿瘤细胞S期阻滞和凋亡。其作用机制可能与其下调HDAC3、HDAC4表达,促进组蛋白乙酰化,而影响p21WAF1/CIP1和Cyclin D1的基因、蛋白的表达有关。     

Synthetic polysulfane derivatives induce cell cycle arrest and apoptotic cell death in human hematopoietic cancer cells

Natural polysulfanes including diallyltrisulfide (DATS) and diallyltetrasulfide (DATTS) from garlic possess antimicrobial, chemopreventive and anticancer properties. However these compounds exhibit chemical instability and reduced solubility, which prevents their potential clinical applicability. We synthesized six DATS and DATTS derivatives, based on the polysulfane motif, expected to exhibit improved physical and chemical properties and verified their biological activity on human leukemia cells.We identified four novel cytotoxic compounds (IC₅₀ values: compound 1, 24.96 ± 12.37 μM; compound 2, 22.82 ± 4.20 μM; compound 3, 3.86 ± 1.64 μM and compound 5, 40.62 ± 10.07 μM, compared to DATTS: IC₅₀: 9.33 ± 3.86 μM). These polysulfanes possess excellent differential toxicity, as they did not affect proliferating mononuclear blood cells from healthy donors.We further demonstrated ability of active compounds to induce apoptosis in leukemia cells by analysis of nuclear fragmentation and of cleavage of effector and executioner caspases. Apoptosis was preceded by accumulation of cells in G2/M phase with a pro-metaphase-like nuclear pattern as well as microtubular alterations. Prolonged and persistent arrest of cancer cells in early mitosis by the benzyl derivative identifies this compound as the most stable and effective one for further mechanistic and in vivo studies.     

Antiproliferative activity of aucubin is through cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells

Aucubin, an iridoid glycoside isolated from the leaves of Aucuba japonica, inhibits human non-small cell lung cancer A549 cells by blocking cell cycle progression in the G(0)/G(1) phase and inducing apoptosis. An ELISA showed that the G(0)/G(1) phase arrest is due to p53-mediated induction of p21. Enhancement of Fas and its two ligands, membrane-bound and soluble Fas ligand, may be responsible for the apoptotic effect induced by aucubin. The present study shows, for the first time, that the induction of p53 and activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of aucubin in A549 cells.     

QSAR study on aminophenylbenzamides and acrylamides as histone deacetylase inhibitors: An insight into the structural basis of antiproliferative activity

Histone deacetylases have emerged as important drug target with a multitude of therapeutic potentials for their inhibitors. With the purpose of designing new chemical entities with enhanced inhibitory potencies against histone deacetylases, a two-dimensional (2D) quantitative structure-activity relationship (QSAR) study was carried out on aminophenylbenzamides and acrylamide derivatives as inhibitors of these deacetylases. The developed model was validated by standard QSAR parameters and through a detailed structural analysis of how it reproduces and explains the differences in the experimentally known activity data. The model showed a good correlative and predictive ability, with a cross-validated correlation coefficient of 0.594. The conventional and predictive correlation coefficients were found to be 0.725 and 0.577 for the antiproliferative activity of aminophenylbenzamides and acrylamide series, respectively. The study indicated that their antiproliferative activity is largely explained by the steric factors of the substituents, highlighting the role of the size and shape of the inhibitor in forming effective binding interactions with histone deacetylase. The models could be usefully employed to design inhibitors with greatly enhanced potency and selectivity.     

Luteolin induced G2 phase cell cycle arrest and apoptosis on non-small cell lung cancer cells

In this study, we investigated the underlying molecular mechanism for the potent cell cycle inhibition and pro-apoptotic effect of luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone) on human non-small-cell lung carcinoma cell line A549. MTT assay showed that luteolin had obvious cytotoxicity on A549 with IC₅₀ of 40.2 μM at 48 h. Pro-apoptotic effect of luteolin on A549 cells was demonstrated by Hoechst 33258 staining assay and annexin V-FITC/PI double staining analysis. A great quantity of apoptotic cells and increasing G2 phase cells were observed by flow cytometry. Western blotting assay revealed that luteolin activated JNK, increased Bax, promoted procaspase-9 cleavage and activated caspase-3 at last. Assay using TNFα, an active agent of NF-κB, showed that pretreatment of A549 cells with luteolin could inhibit TNFα induced trans-nuclear of NF-κB. In summary, luteolin displayed a significant cytotoxic effect through cell cycle arrest and apoptosis induction in A549 cells. Pro-apoptotic effect was implemented via activating JNK and inhibiting translocation of NF-κB (p65). These results suggested that luteolin might have therapeutic potential against NSCLC.     

紫草素促进食管癌细胞凋亡及细胞周期阻滞的作用机制研究

目的研究紫草素促进食管癌细胞凋亡及细胞周期阻滞的作用及机制。方法 2019年 1月至 2020年 6月期间开展细胞实验,培养食管癌 Eca109细胞,分为不含药物杜氏改良 Eagle培养基( DMEM)处理的对照组,含不同剂量紫草素 DMEM处理的紫草素组,含 2.0 μmol/L紫草素和 10 μg/L胰岛素样生长因子 -1(IGF-1)DMEM处理的 2.0 μmol/L紫草素 +10μg/L IGF-1组。检测细胞凋亡率、细胞周期及凋亡基因活化的胱天蛋白酶 -3(cleaved caspase-3)、细胞周期蛋白 D1(cyclin D1)、磷酸化磷酸肌醇 3激酶( PI3K)、蛋白激酶 B(AKT)的表达量。结果 0.5 μmol/L紫草素组、 1.0 μmol/L紫草素组、 2.0 μmol/L紫草素组的细胞凋亡率、细胞周期 G1期比例、 cleaved caspase-3的表达量［ 0.5 μmol/L紫草素组( 0.64±0.14)、 1.0 μmol/L紫草素组( 0.81±0.19)、2.0 μmol/L紫草素组( 1.02±0.24)］高于对照组 0.41±0.07,细胞周期 S期、 G2期比例及 cyclin D1［0.5 μmol/L紫草素组( 0.80±0.16)、 1.0 μmol/L紫草素组( 0.68±0.13)、 2.0 μmol/L紫草素组( 0.45±0.09)］、 p-PI3K、p-AKT表达量低于对照组［ cyclin D1表达量(0.91±0.18)］(P<0.05); 2.0 μmol/L紫草素 +10 μg/L IGF-1组的细胞凋亡率、细胞周期 G1期比例、 cleaved caspase-3的表达量低于 2.0 μmol/L紫草素组,细胞周期 G2期比例及 cyclin D1、p-PI3K、p-AKT表达量高于 2.0 μmol/L紫草素组( P<0.05)。结论紫草素具有促进食管癌细胞凋亡及细胞周期阻滞的作用,该作用与抑制 PI3K/AKT通路激活有关。     

鱼藤素诱导肺癌细胞凋亡和细胞周期阻滞的分子机制

目的探究鱼藤素是否能够阻滞细胞周期和细胞迁移,抑制非小细胞肺癌细胞的增殖。方法鱼藤素(1. 5625、3. 125、6. 25、12. 5、25、50μmol·L~(-1))处理H1299细胞不同时间后,CCK-8法检测细胞存活率,划痕实验测定划痕宽度及愈合率;鱼藤素(1. 5、3、6μmol·L~(-1))处理24 h,PI单染测定细胞周期,Annexin V-FITC/PI双染检测凋亡率,q PCR检测药物处理后的H1299细胞中CDK4、CDK6和CCND1的基因表达情况。结果鱼藤素能够降低H1299细胞的存活率,作用24、48、72 h的IC50值分别为(5. 47±0. 97)、(4. 01±0. 45)、(2. 86±0. 19)μmol·L~(-1),并抑制细胞的迁移与愈合能力。PI单染流式细胞术检测到其能阻滞细胞周期,使细胞阻滞在G0/G1期,流式双染实验测得其可诱导细胞的凋亡,q PCR发现鱼藤素可下调CDK4、CDK6和CCND1的基因表达。结论鱼藤素可抑制H1299细胞的增殖,抑制细胞的运动迁移能力,阻滞细胞周期,诱导细胞凋亡,且鱼藤素可通过下调细胞周期调控系统中的CDK4、CDK6、CCND1基因表达来调控细胞周期,达到抗肿瘤作用。     

Induction of G1 cell cycle arrest and apoptosis by berberine in bladder cancer cells

The dried root of Saussurea lappa Clarke (Compositae) has been used as a traditional medicine. Dehydrocostus lactone is one of the main bioactive constituents of this medicinal plant. In the present study, the protective effect of dehydrocostus lactone against antimycin A (an inhibitor of mitochondrial complex III)-induced cytotoxicity was investigated in osteoblastic MC3T3-E1 cells. Pre-treatment with dehydrocostus lactone prior to antimycin A exposure significantly prevented mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, cytochrome c release, intracellular calcium elevation and potassium loss, and reactive oxygen species production induced by antimycin A. These results suggest that dehydrocostus lactone protects osteoblastic MC3T3-E1 cells from antimycin A-induced cell damage through the improved mitochondrial function.