Vitamin-k-3 induces cell-death via apoptosis in human cervical-carcinoma tsgh8302 cells

Vitamin K-3 (VK3) exhibits antitumor activity in rodent and human cancer cells. The relationship between VK3-induced cytotoxicity, and morphological changes in human cervical carcinoma TSGH8302 cells were studied. Cell viability was analyzed by sulforhodamine B protein binding and clonogenic assays. Inhibition of cell growth by VK3 was cell density dependent as measured by IC50 values, which were 17 mu M at 0.5 x 10(4) cells/well and 36 mu M at 1.0 x 10(4) cells/well. Treatment of 10(6) cells with VK3 (5-100 mu M) for 1 h followed by recovery for 24 h caused depletion of the reduced glutathione pool. Under light, fluorescence and scanning electron microscopes, cells showed morphological changes after 1-h treatment with 25 mu M VK3, followed by a 4-h or 12-h recovery. The cells appeared retracted with blebs but no surface microvilli. They exhibited chromatin condensation and DNA fragmentation. Since these phenomena are characteristics of apoptosis, VK3-induced cell death appears to be mediated by apoptosis.     

神经酰胺诱导人乳腺癌细胞凋亡的研究

目的 :观察神经酰胺对人乳腺癌细胞株MCF 7及耐多柔比星细胞株MCF 7/ADR的诱导凋亡作用及其对bcl 2基因表达的影响 ,探讨神经酰胺诱导凋亡的机制。方法 :采用MTT法检测细胞生存率 ,用流式细胞术检测凋亡率 ,运用流式细胞术、RT PCR方法检测bcl 2基因表达。结果 :神经酰胺对MCF 7、MCF 7/ADR细胞有明显的抑制生长作用。 2 5 μmol/LC6 神经酰胺作用48h后 ,MCF 7及MCF 7/ADR细胞凋亡率分别为( 2 0 70± 2 66) %、( 12 3 9± 1 76) % ,前者凋亡率高于后者 ,P <0 0 1。流式细胞术检测 ,MCF 7、MCF 7/ADR细胞bcl 2蛋白阳性率分别为 ( 2 7 5 9± 2 94) %、( 79 5 6±3 2 0 ) %。MCF 7/ADR细胞经神经酰胺作用后 ,bcl 2蛋白及mRNA水平均降低。结论 :神经酰胺对MCF 7、MCF 7/ADR细胞有抑制生长及诱导凋亡作用 ,神经酰胺诱导凋亡可能是通过下调bcl 2基因表达而实现的     

Induction of apoptosis in human lung cancer cells by curcumin

Curcumin, a phenolic compound from the rhizome of the plant Curcuma longa has anti-inflammatory, antioxidant and anti-cancer activities. Although the precise mode of action of this compound is not yet elucidated, studies have shown that chemo-preventive action of curcumin might be due to its ability to induce apoptosis and to arrest cell cycle. This study investigated the cellular and molecular changes induced by curcumin leading to the induction of apoptosis in human lung cancer cell lines-A549 and H1299. A549 is p53 proficient and H1299 is p53 null mutant. The lung cancer cells were treated with curcumin (0-160 microM) for 12-72 h. Curcumin inhibited the growth of both the cell lines in a concentration dependent manner. Growth inhibition of H1299 cell lines was both time and concentration dependent. Curcumin induced apoptosis in both the lung cancer cell lines. A decrease in expression of p53, bcl-2, and bcl-X(L) was observed after 12 h exposure of 40 microM curcumin. Bak and Caspase genes remained unchanged up to 60 microM curcumin but showed decrease in expression levels at 80-160 microM. The data also suggest a p53 independent induction of apoptosis in lung cancer cells.     

Induction of apoptosis by thiosulfinates in primary human prostate cancer cells

Thiosulfinates, a substance of Allium tuberosum L., is a known folk medicine that has been extensively used in diet to treat diseases. In the present study, we have evaluated the effect of thiosulfinates from Allium tumberosum L. on proliferation of metastasis (DU145) and primary malignant tumor (RC-58T/h/SA#4)-derived human prostate cancer cells. Thiosulfinates decrease viable cell numbers in a dose- and time-dependent manner and induce apoptosis. The apoptosis induced by thiosulfinates is associated with the activation of initiator caspase-8, and -9, and the effector caspase-3. Thiosulfinates stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates decreased the expression of the anti-apoptotic protein Bcl-2, and increased the expression of the pro-apoptotic protein Bax. Thiosulfinates also increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, in RC-58T/h/#4 cells and induced DNA fragmentation and chromatin condensation. These results indicate that thiosulfinates from Allium tuberosum L. inhibit cell proliferation by inducing apoptosis in RC-58T/h/#4 cells which may be mediated via both caspase-dependent and caspase-independent pathways.     

Induction of apoptosis by capsaicin in A172 human glioblastoma cells

Capsaicin induced apoptosis of A172 human glioblastoma cells in a time- and dose-dependent manner. Neither capsazepine, a vanilloid receptor antagonist, nor bis-(o-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM), an intracellular Ca(2+) chelator, significantly inhibited the capsaicin-induced apoptosis, although capsaicin increased intracellular Ca(2+) level. Capsaicin markedly reduced the basal generation of reactive oxygen species (ROS) and lipid peroxidation. Exogenous application of H(2)O(2) significantly prevented the cells from the apoptosis by capsaicin. Treatment with N-acetyl cysteine alone induced both reduction of the basal production of ROS and apoptosis. Taken together, these results suggest that capsaicin induced apoptosis in A172 cells and that vanilloid receptors and intracellular Ca(2+) may not be involved in the apoptotic mechanism of capsaicin. Reduction of the basal generation of ROS may play a role in the induction of apoptosis by capsaicin.     

INDUCTION OF APOPTOSIS OF HUMAN LEUKEMIA CELLS BY α-ANORDRIN

AnordrinisacontraceptiveagentwhichwasfirstdevelopedinChina.Previousstudyshowedthataisomerofanordrin(ANO)exhibitedantitumoreffectbothinvilroandinvivo.l'2Itwasalsofoundthatatsmalldoses,ANQcouldinducedifferentiationofhumanpromyelocyticleukemia.HL-60cells.3However,themechanismofitsaniitumoractivitywasstillnuclear.Inthiswork,wefurtherinvestigatedthetumor-inhibitoryeffectofANOtoexplorethepossiblemechanismsofitsaction.MATERIALSANDMETHODSDrugANOwasproducedbyShanghaiNo.19PharmaceuticalF…     

Induction of apoptosis by morphine in human tumor cell lines in vitro

Most previous studies of the induction of tumor cell apoptosis by morphine have been conducted with concentrations very much higher than those used clinically. An investigation of the ability of morphine to induce apoptosis at its clinical concentration (10(-8) M) was therefore undertaken. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, induction of early apoptosis and necrosis by fluorescence-activated cell sorter (FACS) analysis with Annexin V and propidium iodide (PI), activation of caspase -2, -3, -8 and -9 by cleavage of specific substrates, DNA fragmentation by agarose gel electrophoresis, radical intensity and O2- scavenging activity by ESR spectroscopy. Millimolar concentrations of morphine showed higher cytotoxicity against human tumor cell lines (HL-60, A549, MCF7) than against normal human cells (HGF, HPC, HPLF). The clinical concentration of morphine produced early apoptotic markers in HL-60 and A549 cells whereas it induced higher numbers of necrotic cells in MCF7 cells, both in a naloxone-sensitive manner. The clinical concentration of morphine failed to activate any caspase species and induced only trace amounts of internucleosomal DNA fragmentation, in contrast to cytotoxic concentrations of morphine. Morphine, with a C-3 hydroxyl group, showed higher cytotoxicity and O2- scavenging activity than codeine, in which the hydroxyl group at C-3 was replaced with a methoxy group, suggesting the involvement of a radical-mediated reaction. The present report may offer new strategies for treatment and prevention of cancer using a clinical concentration of morphine not only as an anti-nociceptive, but also as an apoptosis or necrosis inducer.     

Induction of apoptosis in human ovarian epithelial cancer cells by antisurvivin oligonucleotides

Survivin, an anti-apoptosis gene that is abnormally overexpressed in a variety of human tumors, may play an important role in the carcinogenesis and drug resistance of cancer. This study was designed to explore the effects of liposome-survivin antisense oligonucleotide (Lip-ASODN) on the growth and apoptosis of human ovarian cancer cell lines, A2780 and SKOV3. To investigate the use of survivin as a therapeutic target on ovarian cancer, we carried out transfections with Lip-ASODN to induce apoptosis in ovarian cancer cell lines, A2780 and SKOV3. The expression of survivin mRNA and relative protein were evaluated separately by quantitative real-time RT-PCR and Western blot analysis. Cell proliferation inhibition was determined by methyl thiazolyl tetrazolium (MTT) assay, and the induced cell apoptosis was examined using flow cytometry (FCM) after Lip-ASODN transfection. Our results showed that the overexpression of survivin led to infinite carcino-proliferation, and survivin expression in the survivin-positive ovarian cancer cell line A2780 and SKOV3 cells was significantly and gradually reduced when transfected with Lip-ASODN at concentrations of 200, 400 and 600 nM by degrees. Lip-ASODN transfection induced greater apoptosis rates in the human ovarian cancer cell lines A2780 and SKOV3 (p<0.05). The growth inhibition and apoptotic rates of tumor cells change when treated with different concentrations of Lip-ASODN. The cell growth inhibition peak rate was reached when increasing Lip-ASODN concentration to 600 nM. Furthermore, time course evaluation showed that survivin protein expression was inhibited by Lip-ASODN within 12 h after transfection. We concluded that down-regulation of survivin by a targeted antisense oligonucleotide appears to be an effective gene therapy approach in the treatment of ovarian cancer.     

Induction of apoptosis in human salivary gland tumor cells by anti-NCAM antibody

Neural cell adhesion molecule (NCAM) is a type of cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant salivary gland tumors such as adenoid cystic carcinoma. We have previously demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG, in vitro. In the present study, we have aimed to clarify the hypothesis that NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding and involves the prevention of signal transduction for perineural invasion using HSG cells. NCAM mRNA and protein expression was found to decrease in a dose-dependent manner upon treatment with the anti-NCAM antibody (MAb NCAM) for 24 h. The MTT assay showed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells underwent apoptosis after treatment with MAb NCAM. The activation of caspases 3, 7 and 9 was observed in HSG cells after treatment with MAb NCAM, thus confirming that apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with MAb NCAM. The up-regulation of TGF-beta1-mediated NCAM expression appeared to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined using an immunohistochemical method. NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC. These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism, but also with tumorigenesis, including growth, development and perineural invasion in human salivary gland tumors.     

Protooncogene expression in human glioma derived cell-lines

Growth factor/ligand interactions have been shown to play an important role in various malignancies. Expression of two differentially spliced forms of SCF was detected in 20 of 24 glioma cell lines by RT-PCR. Northern blot analysis revealed expression of the corresponding receptor, c-kit (11/24), as well as PDGF alpha-receptor (22/25 glioma cell lines), PDGF beta-receptor (22/25), TGF-alpha (13/24 ) and PDGF B/c-sis (7/16) expression. As determined by FACS analysis, expression of EGFR and p185HER2 was detected in 21 of 21 and 15 of 21 glioma cell lines, respectively. Four cell lines showed moderate EGFR overexpression (>90,000 receptors/cell) and in one cell line p 185HER2 expression exceeded EGFR levels. Loss of EGFR gene amplification during in vitro culturing was observed in 3 of 18 investigated cell lines by differential PCR. In summary, our work suggests the simultaneous activation of several different growth factor/receptor systems in human glioma cell lines.     

重组人TRAIL促卵巢癌细胞凋亡作用的研究

目的:分析重组人可溶性肿瘤坏死因子相关的凋亡诱导配体(TRAIL)分子诱导卵巢癌细胞的凋亡。方法:观察重组人可溶性TRAIL对卵巢癌细胞直接细胞毒作用,MTT法分析不同浓度TRAIL对卵巢癌细胞3AO、HO-8910的生长抑制率,采用流式细胞技术观察TRAIL作用于3AO后的凋亡率的变化。结果:重组人可溶性TRAIL蛋白可抑制卵巢癌细胞生长,诱导卵巢癌细胞发生凋亡;倒置显微镜下观察细胞呈现圆形固缩形态,细胞凋亡之后存活细胞减少。结论:重组人可溶性TRAIL具有明显诱导卵巢癌细胞凋亡的活性,并具有量效性及时效性。其抑制卵巢癌细胞机制可能与其促凋亡作用有关。     

Characterization of protease expression in human prostate-cancer cell-lines

To better understand the biochemical mechanisms necessary for prostate cancer invasion and metastases, we studied the expression and interaction of proteolytic enzymes cathepsin D, cathepsin B, urokinase and collagenase IV in human prostate cell lines LNCAP (hormone sensitive) and DU145 (hormone refractory). Cellular fractionation and immunoblotting revealed that both cell lines expressed similar amounts of the 34 kD form of cathepsin D, 72 kD form of collagenase IV and the 55 kD form of urokinase. However, DU145 expressed an increased amount of the 28 kD form of cathepsin B. When E64, a cysteine protease inhibitor was added, a decreased amount of the active cathepsin D was expressed. Furthermore, when cathepsin B was added to concentrated plasma membrane homogenates, urokinase was processed to its active form at 33 kD. E64 inhibited the ability of both cell lines to degrade fibronectin. An in vitro Boyden chamber demonstrated that DU145 was more motile than LNCAP and that preincubation with E64 could decrease motility of both cell lines. We suggest that cathepsin B may promote tumor invasion not only by proteolysis of basement membrane components, but also by activating other proteases.     

Radiosensitization by Cisplatin treatment in Cisplatin-resistant and sensitive human ovarian-carcinoma cell-lines

The responses of cisplatin resistant (A2780(cp)) and cisplatin sensitive (A2780) ovarian carcinoma cell lines to radiation, cisplatin and cisplatin plus radiation have been studied. The cisplatin resistant cell line showed cross resistance to radiation. When cells were exposed to 2 mu g/ml of cisplatin treatment for one hour radiosensitization was achieved. The degree of radiosensitization was treatment sequence dependent. Irradiation followed by cisplatin treatment resulted in synergistic interaction with dose modifying factors (DMFs) as high as 1.5 in the resistant line and 1.1 in the sensitive line. The reverse sequence resulted in antagonistic interaction with DMFs of 0.8 to 0.9 at the 10% survival level. At lower survival levels the interaction remained antagonistic for the resistant cell line. Increasing the cisplatin concentration to 4 mu g/ml did not increase the degree of cisplatin radiosensitization. Simultaneous treatment of irradiation during the middle of a one or two hour cisplatin treatment did not increase the degree of radiosensitization. The cisplatin resistant cell line had a much greater cisplatin radiosensitization effect than the sensitive parental cell line with maximum DMFs for the resistant cell line ranging from 0.9-1.5 and for the sensitive cell line from 0.8-1.1. These results indicate that cisplatin may be an effective radiosensitizer especially in cisplatin resistant cell lines. This effect may be related to the inhibition of repair of radiation damage which may be elevated as a mechanism of resistance in the resistant cell line.     

Induction of apoptosis in human neuroblastoma cells by abrogation of integrin-mediated cell adhesion

The survival, proliferation and differentiation of neuroblastoma (NB) cells are largely dependent on adhesion to extra-cellular matrix (ECM) proteins. Integrin occupancy seems to play a primary role. To elucidate the role of integrin heterodimers during neuronal cell death, we have analysed the changes in integrin expression in 2 human NB cell lines which represent different stages of neuronal maturation. Retinoic acid (RA) had different effects on the 2 NB cell lines: on LAN-5 cells it acted as a differentiation-promoting agent, while it had an anti-proliferative effect on GI-LI-N cells, driving them driving them to apoptosis. Indeed, this occurrence was evidenced by the visualization of a “DNA ladder” on gel electrophoresis, by propidium iodine staining, and by DNA flow cytofluorimetric analysis. RA treatment rapidly and drastically decreased integrin expression and cell adhesion on GI-LI-N cells. These findings were also obtained by treating both NB cell lines with the apoptotic agent fenretinide. Furthermore, treatment of NB cells with anti-sense oligonucleotides to β, integrin chain specifically induced chromatin condensation and nucleosomal DNA laddering. Moreover, blocking cell-matrix interations by means of perturbing antibody against β, subunit resulted in the induction of typical features of apoptotic cells. In conclusion, these findings incidate that abrogation of cell adhesion through down-modulation of integrin receptors plays a crucial role in the induction of neuroblastoma programmed cell death. Int. J. Cancer 70:688–698, 1997. © 1997 Wiley-Liss, Inc.     

Induction of apoptosis or necrosis in human endometrial carcinoma cells by 2-methoxyestradiol

BACKGROUND: We investigated the effects of 2methoxyestradiol (2-ME), an endogenous estrogenic metabolite, on human endometrial cancer HEC-1-A and RL-95-2 cell lines. MATERIALS AND METHODS: After exposure of HEC-1-A and RL-95-2 cells to 2-ME, the morphological changes were evaluated by acridine orange staining and transmission electron microscopy. Cell cycle progress, apoptosis and necrosis were assessed by flow cytometry, DNA fragmentation and Western blot. RESULTS: 2-ME inhibited cell growth by blocking the S- and G2/M-phase in both cell lines, by inducing apoptosis in HEC-1-A cells and by causing necrosis in RL-95-2 cells. Apoptosis, on HEC-1-A cells, was accompanied by an increased expression of iNOS and STAT1. This apoptotic effect was prevented by the iNOS inhibitor 1400W and eliminated by the caspase inhibitor Z-VAD-FMK. Necrosis, on RL-95-2 cells, was due to a severe disruption of the mitochondrial membrane potential. 2-ME had no significant effect on normal human endometrial cells. CONCLUSION: The data suggest that 2-ME has an antitumor effect on human endometrial carcinoma cells (HEC-1-A and RL-95-2) and may contribute as a new therapeutic agent for endometrial cancer patients.     

吡柔比星诱导结肠癌细胞株SW-260的凋亡

目的：观察吡柔比星（pirarubicin,ＴＨＰ）对结肠癌细胞株ＳＷ－２６０凋亡的影响。 方法：将ＴＨＰ加入ＳＷ－２６０细胞培养液,Ｈoechst３３２５８荧光染色、透射电镜观察ＳＷ－２６０细胞形态学变化;ＤＮＡ抽提琼脂糖电泳观察ＤＮＡ的“梯状”条带;并测定细胞ＤＮＡ断裂百分率,以探讨ＴＨＰ能否引起人结肠癌细胞株ＳＷ－２６０发生凋亡。结果：不同浓度ＴＨＰ（５,１０,１５umol/Ｌ）作用于ＳＷ－２６０细胞２４小时及１     

Induction of apoptosis by 7-piperazinethylchrysin in HCT-116 human colon cancer cells

The antitumor activity of 7-piperazinethylchrysin (7-PEC) was investigated in HCT-116 human colon cancer cells. MTT assay revealed that the IC50 of 7-PEC in HCT-116 cells was 1.5 μM after 72 h of treatment, much lower than that of chrysin (>100 μM). The data showed that 7-PEC was able to inhibit the growth of HCT-116 cells in a concentration- and time-dependent manner. Topical morphological changes of apoptotic body formation after 7-PEC treatment were observed by Hoechst?33258 staining. 7-PEC reduced mitochondrial membrane potential (?Ψm) of cells in a concentration-dependent manner and increased the production of intracellular reactive oxygen species (ROS). After treatment with 7-PEC, a significant increase of Bax protein expression and decrease of Bcl-2 protein expression were observed at the same time. These events paralleled with activation of p53, caspase-3 and?-9 and the release of cytochrome?c (cyt?c), as well as poly(ADP-ribose) polymerase-1 (PARP1) cleavage and downregulation of p-Akt. However, the apoptosis induced by 7-PEC was blocked by Ac-DEVD-CHO, a caspase-3 inhibitor. These results demonstrate that 7-PEC-induced mitochondrial dysfunction in HCT-116 human colon cancer cells triggers events responsible for caspase-dependent apoptosis pathways, and the elevated ratio of Bax/Bcl-2 is likely involved in this effect.     

Effect of aspirin on the induction of apoptosis in colorectal adenocarcinoma cell-lines

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit experimental carcinogenesis and their use in humans has been related epidemiologically to a reduced risk of colorectal polyps and cancer, although the mechanism involved is not known. We found that aspirin triggered the death of SW948 human colorectal adenocarcinoma cells through activation of an apoptotic pathway. Exposure of SW480 and SW948 cells to 25 mu M aspirin for 5 h resulted in the detatchment of cells from the monolayer culture at 48 h. SW948 cells with continuous exposure to 25 mu M aspirin exhibited various morphological and biochemical characteristics of apoptosis, including compact patches of condensed nuclear chromatin, and DNA fragmentation. These in vitro data suggest that apoptosis may play a role in the antitumor effect of aspirin and other NSAIDs and that the induction of apoptosis may provide an attractive therapeutic target in colorectal carcinogenesis.     

Induction of apoptosis by abrogation of HSP70 expression in human oral cancer cells

We have reported differential expression of 70-kDa heat shock protein (HSP70) in human oral tumorigenesis. The functional significance of elevated levels of HSP70 protein in oral squamous cell carcinomas (SCC) remains to be elucidated. The present study was designed to investigate the role of HSP70 protein in the proliferation and survival of oral tumour cells. Abrogation of HSP70 expression by antisense HSP70 oligonucleotides treatment of human oral carcinoma cells isolated from primary tumours or HSC-2 cells triggered cell death with several characteristic features, including DNA laddering, chromatin condensation and fragmentation. Flow-cytometric analysis showed a hypodiploid DNA peak of propidium iodide-stained nuclei in the antisense oligomer-treated cells. This response was accompanied by a decrease in the percentage of cells in the S phase of the cell cycle, suggesting inhibition of cell proliferation. Treatment of oral cancer cells with HSP70 antisense oligomers resulted in decreased expression of anti-apoptotic signal protein bcl-2. Our results suggest that HSP70 antisense oligomer treatment abrogates the expression of HSP70 protein that may disrupt HSP70-bcl-2 interactions, in turn inhibiting cell proliferation and inducing apoptosis. Conversely, the data suggest that HSP70 is required for proliferation and survival of oral tumour cells.     

Induction of apoptosis by bestatin (ubenimex) in human leukemic cell lines.

We investigated the growth inhibitory activity of bestatin, an inhibitor of aminopeptidase N (CD13), on six human leukemic cell lines. Proliferation of all the cell lines except KG1 was inhibited by bestatin. P39/TSU, HL60 and U937 were highly sensitive, with 50% growth inhibitory concentrations (IC50) close to the maximum serum concentration when bestatin was orally administered at 30 mg in clinical application. All cell lines except for K562 highly expressed CD13, but a clear correlation between the sensitivity to bestatin and expression of CD13 was not observed. Other aminopeptidase inhibitors such as amastatin A, arphamenine B and WM15 antibody showed no growth inhibitory effects. To confirm the growth inhibitory effects of bestatin, we quantitatively examined DNA fragmentation in five bestatin-sensitive cell lines. Bestatin dose-dependently induced DNA fragmentation in those cell lines. In case of U937, bestatin induced DNA fragmentation quantitatively and DNA ladder and enhanced caspase-3 activity. Furthermore, the growth inhibition by bestatin was reduced by the caspase inhibitor Z-Asp-CH2-DCB. These results suggested that bestatin exhibits direct antileukemic effects against human leukemic cell lines through the induction of apoptosis.