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。     

Arsenic induces telomerase expression and maintains telomere length in human cord blood cells

Inorganic arsenic (iAs) is a human carcinogen, well known as a clastogenic compound. To evaluate the molecular mechanism of arsenite (iAs^III) toxicity, we investigated the effects on cell growth and apoptosis, telomere length, telomerase expression, as well as the formation of reactive oxygen species (ROS) in male and female human cord blood cells in vitro. Incubation with iAs^III at the concentration of 0.0001 μM increased telomerase mRNA and protein expression maintained both telomere length and cellular growth, and induced mRNA over-expression of the two oncogenes ras and myc. Our results suggest that female cord blood cells are more sensitive than male ones to iAs^III induced telomerase stimulation at low concentrations, possibly related to the increased expression of ras and myc oncogenes. On the contrary, at the concentration of 1 μM, iAs^III decreased telomerase expression and telomere length, and induced apoptosis, necrosis and production of reactive oxygen species. Buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, markedly increased the percentage of apoptotic cells, suggesting that GSH is fundamental for detoxification of iAs^III in cord blood cells. The reactive oxygen species (ROS) scavenger, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), protected cord blood cells from iAs^III toxicity, and prevented telomere shortening and telomerase down-modulation. It can be concluded that telomerase expression and telomere length are associated with iAs^III induced cell death, via production of reactive oxygen species, as well as with iAs^III induced effects on cell differentiation processes and rate of cell growth.     

A suramin derivative induces enterocyte-like differentiation of human colon cancer cells without lysosomal storage disorder

Suramin is a polysulfonated naphthylurea currently investigated for the treatment of advanced malignancy. We have previously reported that suramin was a potent inducer of the differentiation of the human colic adenocarcinoma cell clone HT29-D4 (Fantini et al., J Biol Chem, 1989; 264: 10282-10286). In this report, we show that chronic suramin treatment of these cells is associated with a lysosomal storage disease. We have tested five suramin-related compounds for their ability to induce HT29-D4 differentiation and we looked at their action on the lysosomal apparatus. We conclude that one of the derivatives used is a more potent differentiation inducer than suramin, while it does not elicit any perturbation of the lysosomal system.     

A synthetic curcumin derivative hydrazinobenzoylcurcumin induces autophagy in A549 lung cancer cells

Context: Curcumin exhibits growth-suppressive activity against a variety of cancer cells, but low bioavailability restricts its application in chemotherapeutic trials. Nowadays, a growing number of curcumin derivatives or analogs are known, hoping to replace curcumin and circumvent this problem. Hydrazinobenzoylcurcumin (HBC) has been synthesized and identified as a potent inhibitor of cell proliferation in previous reports.

Objective: This study presents a novel mechanism of cell autophagy induced by HBC in the human non-small lung epithelial carcinoma (A549) cells.

Materials and methods: Cells were cultured and treated with HBC at different concentrations (10–80?μM) and at different time periods (1–24?h). Microscopic analysis was used to detect the morphology changes and autophagolysosomes of A549 cells. An acridine orange staining assay was conducted to evaluate the autophagolysosomes and autophagic vacuoles was analyzed by monodansylcadaverine (MDC) and GFP-LC3 transfection analysis. Western blotting was used to assess the conversion of microtubule-associated protein light chain 3 (LC3).

Results: HBC could induce A549 cells autophagolysosomes formation in a dose and time-dependent manner and the inhibitory rate of HBC (80?μM) on the viability of A549 cells reached 76.68?±?5.81% after 24?h of treatment. Autophagic vacuoles increased in a concentration-dependent manner in HBC-treated cell. Furthermore, conversion of LC3-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles and increased fusion of autophagosomes with lysosomes suggested the occurrence of autophagy.

Conclusion: Our data indicate that HBC induced A549 cell autophagy, which is a novel cell death mechanism induced by curcumin derivatives.     

A novel fusicoccin derivative preferentially targets hypoxic tumor cells and inhibits tumor growth in xenografts

Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Tumor hypoxia is also associated with a more malignant phenotype and poor survival in cancer patients. Recent progress in our understanding of the biology of tumor cells under hypoxia has led to increased attention on targeting hypoxia for cancer therapy. We report here that a novel fusicoccin derivative (ISIR-042), but not its parent or related compounds such as fusicoccin A and cotylenin A, is more cytotoxic to hypoxic cells than to normoxic cells. The hypoxia-induced accumulation of hypoxia-inducible factor (HIF)-1α and the phosphorylation of Akt were effectively inhibited by treatment with ISIR-042, suggesting that the preferential cytotoxicity toward hypoxic cells is associated with a reduction of HIF-1α and Akt activation. ISIR-042 inhibited the growth of human pancreatic cancer MIAPaCa-2 cells while sparing normal endothelial cells, and significantly inhibited the growth of MIAPaCa-2 cells as xenografts without apparent adverse effects. Pancreatic cancer cells expressing CD24 and CD44 exhibited characteristics of stem cells. Treatment with gemcitabine increased this stem cell-enriched population, and this effect was significantly inhibited by ISIR-042, suggesting that ISIR- 042 preferentially inhibits stem/progenitors in pancreatic cancer cell lines compared with chemotherapeutic agents. These results suggest that ISIR-042 may be a potential therapeutic agent for hypoxic tumors such as pancreatic cancer.     

A methyl jasmonate derivative,J-7, induces apoptosis in human hepatocarcinoma Hep3B cells in vitro

The pro-apoptotic activity of J-7, a synthetic methyl jasmonate derivative, on the Hep3B human hepatocarcinoma cell line was investigated. Treatment of Hep3B cells with J-7 resulted in growth inhibition and the induction of apoptosis as measured by trypan blue-excluding cells, MTT assay, nuclear staining, DNA fragmentation, and flow cytometry analysis. The increased apoptotic events in Hep3B cells caused by J-7 were associated with the alteration in the ratio of Bax/Bcl-2 protein expression. J-7 treatment induced the expression of death receptor-related proteins such as death receptor 5, which triggered the activation of caspase-8 and the down-regulation of the whole Bid expression. In addition, the apoptosis induction by J-7 was correlated with the activation of caspase-9 and caspase-3, down-regulation IAP family proteins such as XIAP and cIAP-1, and concomitant degradation of poly (ADP-ribose) polymerase. However, the cytotoxic and apoptotic effects induced by J-7 were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. Furthermore, blocking the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase pathways showed increased apoptosis and the activation of caspases in J-7-induced apoptosis. The results indicated that J-7 induces the apoptosis of Hep3B cells through a signaling cascade of death-receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways, which are associated with the activation of the mitogen-activated protein kinases signal pathway.     

A thermally responsive biopolymer conjugated to an acid-sensitive derivative of paclitaxel stabilizes microtubules, arrests cell cycle, and induces apoptosis

Poor aqueous solubility limits the therapeutic index of paclitaxel as an anti-cancer drug. Synthesis of soluble prodrugs of paclitaxel, or conjugation of the drug to macromolecular carriers have been reported to increase its water-solubility. Macromolecular drug carriers have an added advantage of targeting the drug to the tumor site due to the abnormal tumor blood and lymphatic vasculature. This study describes a thermally responsive macromolecular carrier, elastin-like polypeptide (ELP) for the delivery of paclitaxel. Paclitaxel was bound to ELP by conjugation with the 6-maleimidocaproyl hydrazone derivative of paclitaxel, an acid-sensitive paclitaxel prodrug, for the potential treatment of breast cancer. Focused hyperthermia above a specific transition temperature at the site of a tumor causes ELP to aggregate and accumulate, thereby increasing the local concentration of the drug cargo. The paclitaxel prodrug described here bears an acid-sensitive linker that is cleavable at the lysosomal/endosomal pH, which allows a controlled intracellular release of the drug. The ELP-delivered paclitaxel in the presence of hyperthermia inhibits MCF-7 cell proliferation by stabilizing the microtubule structures, arresting the cells at the G2/M stage, and inducing apoptosis in a manner similar to conventional paclitaxel. It also inhibits proliferation of a paclitaxel resistant MCF-7 cell line. These data provide an in vitro proof of concept for the use of ELP as a delivery vehicle of paclitaxel.     

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.     

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.     

Novel combretastatin A-4 derivative XN0502 induces cell cycle arrest and apoptosis in A549 cells

Combretastatin A-4 (CA-4) is a tubulin-binding compound currently in phase II trial as a tumor vascular-targeting agent. The present study evaluates the anti-tumor activities and establishes the mechanism of the action of 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-amine(XN0502), a novel synthesized CA-4 analogue, in an effort towards finding the favorable therapeutics of CA-4 derivatives. XN0502 is characterized by its more potent anti-proliferative activities against non-small cell lung cancer A549 cells (IC₅₀: 1.8 ± 0.6 μM), than that on the normal human liver HL-7702 cells (IC₅₀: 9.1 ± 0.4 μM). Of note, using tubulin polymerization assay, western blot and immuofluorescence analyses, XN0502 was showed to inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further studies indicated that XN0502 induced time- and dose-dependent G2/M arrest, accompanying with the reduction of CDC2/p34 expression and the downregulation of CDK7. The protein level alteration and the nuclear translocation of cyclinB1 were observed, denoting the M phase arrest in XN0502-treated cells. Moreover, XN0502 caused caspase-mediated apoptosis, as indicated by the cleavage of PARP, the reduction of procaspase-3 and procaspase-9, and the down-regulation of XIAP. Taken together, the current study demonstrates that the novel CA-4 analogue XN0502 is a promising anti-cancer agent with potent G2/M arrest- and apoptotic-inducing activities via targeting tubulin deserving further research and development, and helps provide data for exploiting new CA-4 analogues.     

A new Phaseolus vulgaris lectin induces selective toxicity on human liver carcinoma Hep G2 cells

We describe here the purification and characterization of a new Phaseolus vulgaris lectin that exhibits selective toxicity to human hepatoma Hep G2 cells and lacks significant toxicity on normal liver WRL 68 cells. This polygalacturonic acid?Cspecific lectin (termed BTKL) was purified from seeds of P. vulgaris cv. Blue tiger king by liquid chromatography techniques. The 60-kDa dimeric lectin showed strong and broad-spectrum hemagglutinating activity toward human, rabbit, rat, and mouse erythrocytes. Bioinformatic analysis unveils substantial N-terminal sequence similarity of BTKL to other Phaseolus lectins. Among a number of tumor cells tested, BTKL exhibits potent anti-Hep G2 activity which is associated with (1) induction of DNA fragmentation, (2) production of apoptotic bodies and chromatin condensation, (3) triggering of cell apoptosis and necrosis, and (4) depolarization of mitochondrial membrane (low ????m). Furthermore, BTKL could induce inducible nitric oxide synthase (iNOS) expression and subsequent nitric oxide production in vitro in mouse macrophages, which may contribute to its antitumor activity. In addition, BTKL could bring about a significant dose-dependent increase in the production of mRNAs of proinflammatory cytokines including interleukin-1 beta, interleukin-2, tumor necrosis factor alpha, and interferon-gamma. In sum, the antitumor activity and mechanism of BTKL provided here suggest that it has potential therapeutic value for human liver cancer.     

Novel podophyllotoxin and benzothiazole derivative induces transitional morphological and functional changes in HaCaT cells

Podophyllotoxin (PPT) is an antimitotic drug used topically in the treatment of anogenital warts. Due to its toxicity it cannot be administered systemically as an anticancer agent. However, modified PPT derivatives such as etoposide and teniposide are used clinically as systemic agents. Thus, we invented novel PPT derivative KL3 that was synthesized by photocyclization. Earlier we have shown that KL3 has an anticancer effect in various cell lines. Here we compared the toxicity of KL3 vs PPT on non-cancerous normal human keratinocytes (HaCaT) and peripheral blood mononuclear cells (PBMC) showing that KL3 is less toxic than PPT to non-cancerous cells. At concentrations that neither induced cell death, nor affected cell cycle, KL3 in HaCaT cells evoked transient ultrastructural features of ER stress, swelling of mitochondria and elongation of cytoplasmic processes. Those changes partially reversed with prolonged incubation while features of autophagy were induced. PPT in equivalent concentrations induced HaCaT cell death by cell cycle arrest, intrinsic apoptosis and finally disintegration of cell membranes followed by secondary necrosis. In conclusion, we show that the KL3 derivative of PPT in contrast to PPT allows repair of normal keratinocytes and triggers mechanisms that restore non-tumor cell homeostasis     

A novel structural derivative of natural alkaloid ellipticine,MDPSQ, induces necrosis in leukemic cells

DNA intercalating molecules are promising chemotherapeutic agents. In the present study, a novel DNA intercalating compound of pyrimido[4′,5′:4,5]selenolo(2,3-b)quinoline series having 8-methyl-4-(3 diethylaminopropylamino) side chain is studied for its chemotherapeutic properties. Our results showed that 8-methyl-4-(3 diethylaminopropylamino) pyrimido [4′,5′:4,5] selenolo(2,3-b)quinoline (MDPSQ) induces cytotoxicity in a time- and concentration-dependent manner on leukemic cell lines. Both cell cycle analysis and tritiated thymidine assays revealed that MDPSQ affects DNA replication. Treatment with MDPSQ resulted in both elevated levels of DNA strand breaks and repair proteins, further indicating its cytotoxic effects. Besides, Annexin V/PI staining revealed that MDPSQ induces cell death by triggering necrosis rather than apoptosis.     

Curcumin induces apoptosis in tumor necrosis factor-alpha-treated HaCaT cells

Psoriasis is a benign, chronic skin disease characterized by keratinocyte hyperproliferation and abnormal differentiation. Curcumin, a selective phosphorylase kinase inhibitor, is a natural phytochemical present in turmeric. Curcumin has been confirmed to have anti-inflammatory properties as well as the ability to inhibit proliferation and decrease the expression of pro-inflammatory cytokines in psoriatic keratinocytes. However, the pro-apoptotic effect of curcumin in keratinocytes remains unclear. In the present study, we investigated the effect of curcumin on apoptosis induction in TNF-α-treated HaCaT cells. These results show that curcumin exhibited a significant pro-apoptotic effect on HaCaT cells only in the presence of TNF-α and/or TRAIL. The pro-apoptotic effect of curcumin resulted from the increased expression of TRAIL-R1/R2 and the decreased expression of anti-apoptotic proteins. Our results indicate that both curcumin and TNF-α up-regulated the expression of TRAIL-R1/R2. In addition, the expression of anti-apoptotic proteins (IAP1, IAP2, Bcl-X(L)) was up-regulated by TNF-α but suppressed by curcumin in HaCaT cells. Because these proteins are regulated by NF-κB, we examined the role of curcumin in NF-κB activation. As expected, curcumin inhibited TNF-α-induced activation of NF-κB, including NF-κB-P65. Curcumin also inhibited the TNF-α-induced production of IL-6/IL-8 in HaCaT cells. These results imply that curcumin-induced apoptosis of HaCaT cells only occurs when TNF-α or/and TRAIL are present. Therefore, we believe that curcumin is able to reverse the anti-apoptotic function of TNF-α in HaCaT cells and thus expect curcumin to be successful in the treatment of psoriasis.     

Serotonin induces apoptosis in PGT-beta pineal gland tumor cells

Serotonin-induced neuronal cell death has been implicated as a possible cause of neurodegenerative and neuropsychiatric disorders. To investigate the involvement of serotonin-induced apoptosis as a potential mechanism in the pathophysiology of serotonin-related diseases, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, DAPI (4',6-diamidino-2-phenylindole) staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) assay and flow cytometric analysis were performed using the immortalized pineal cell line PGT-beta. Through morphological and biochemical analyses, it was demonstrated that cell death induced by serotonin in PGT-beta cells shows classic apoptotic features. These data suggest that serotonin induces apoptosis in PGT-beta cells.     

Terminal telomere repeats are actually short in telomerase-negative immortal human cells

Telomerase-negative immortal human cells maintained telomere length by a mechanism called alternative lengthening of telomeres (ALT mechanism). These cells (ALT cells) have two prominent characteristics of long telomere repeats at each chromosome end revealed by Southern blotting (terminal restriction fragments: TRF) and the presence extrachromosomal telomere repeat (ECTR) DNA. We report here that the TRF length of ALT cells revealed by the conventional unidirectional (UD) current or pulse-field (PF) current electrophoresis appeared to be over estimated. The TRF length determined by the pulse inverse-field (PIF) current electrophoresis (2-9 kbp depending upon cell lines) was much smaller than that (ca. 23 kbp) by UD or PF current electrophoresis. These results were in consistent with very weak telomere staining in situ at chromosome ends in ALT cells. When a mixture of HinfI-digested genomic DNA of human diploid fibroblasts and synthetic telomere repeat DNA with similar size of ECTR DNA was electrophoresed using a UD current, the apparent TRF size shifted to larger molecular weight, while the size shift did not occur by PIF current electrophoresis. These results together with other data indicate that the unusually long TRF of ALT cells determined by using conventional electrophoresis is an artifact produced by a complex formed by short TRF and short ECTR DNA.     

多胺衍生物NNIspm诱导肝癌细胞HepG2衰老及其分子机制

探讨多胺衍生物NNIspm诱导肝癌细胞HepG2衰老及其分子机制。采用β-半乳糖苷酶染色检测细胞衰老; 应用高内涵活细胞成像系统检测细胞内NNIspm含量, 细胞周期分布以及细胞内活性氧水平; Western blotting检测蛋白的表达水平; 应用高效液相色谱法检测细胞内多胺含量的变化。结果表明: NNIspm可明显诱导HepG2细胞发生衰老, 这与其降低细胞内多胺含量, 诱导细胞周期阻滞于G₀/G₁期、增加细胞内活性氧有关。此外, 检测到衰老标志蛋白p21的表达明显增加; 相反, Cyclin E和CDK2的表达明显减弱。本研究结果表明: NNIspm引起的细胞衰老是其产生抗肿瘤作用的机制之一。