benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Breast-Neoplasms

LCL161 is a second mitochondrial activator of caspases (SMAC) mimetic and inhibitor of apoptosis protein (IAP) antagonist that has oral bioavailability, exhibits anti-tumor effects and improves the chemical sensitivity of many cancers. The aim of this study was to ascertain the effects and mechanisms of the SMAC analog LCL161 on breast cancer drug-resistant cells after undergoing caspase inhibition. This was achieved through use of colony formation and CCK-8 assays to detect cell proliferation. Flow cytometry, Western blot analysis, ATP assay, immunofluorescence and siRNA transfection were used to study the molecular mechanisms of LCL161-induced death of cisplatin-resistant MCF-7 cells after caspase inhibition. LCL161 exhibited an inhibitory effect on MCF-7/DDP cells including after inhibition of caspase. However, LCL161 could not on its own induce a necroptosis effect on MCF-7/DDP cells (P < 0.01 or P < 0.001). When used jointly with the caspase inhibitor z-VAD-fmk, it significantly decreased intracellular ATP levels (P < 0.01 or P < 0.05). This induction of necroptosis occurred through the activation of the RIP1-RIP3-MLKL programmed cell necrosis cascade. Knockdown of RIP3 using siRNA protected against the combined LCL161 / z-VAD-fmk-induced cell death (P < 0.01 or P < 0.001). These findings support the hypothesis that LCL161 combined with caspase inhibition can induce a necroptosis effect on MCF-7/DDP cells, suggesting that it has potential to be an effective treatment for breast cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Gene Knockdown Techniques; Humans; MCF-7 Cells; Mitochondria; Necroptosis; Receptor-Interacting Protein Serine-Threonine Kinases; RNA, Small Interfering; Thiazoles

There is an urgent requirement for the development of novel targeted therapies to treat breast cancer, which is the most comment type of malignancy among women. The evasion of apoptosis is a hallmark of cancer, and is often due to the upregulation of inhibitor of apoptosis proteins (IAPs) in tumor cells. Second mitochondrial‑derived activator of caspase/direct IAP‑binding protein with low PI is a natural IAP antagonist, which is found in the mitochondrion; this protein has a motif, which binds to a surface groove on the baculovirus IAP repeat domains of the IAPs. In the present study, the effects of the LCL161 Smac mimetic, a small molecule IAP antagonist, on breast cell lines was examined. The results from MTT and colony formation assays demonstrated that LCL161 markedly inhibited the proliferation and induced the apoptosis of MDA‑MB‑231 and MCF‑7 cell lines. As determined by western blotting, cIAP1 was degraded in the breast cancer cells, which occurred in an LCL161‑dependent manner. Upon caspase activation, LCL161 treatment induced necroptosis, another form of programmed cell death. The downregulation of receptor‑interacting protein kinase‑1 via small interfering RNA protected the cells from LCL161‑induced necroptosis. Taken together, the results of the present study showed that LCL161 can induce multiple forms of programmed cell death in breast cancer cells, and may thus offer promise as an anticancer agent in diverse genotypic backgrounds.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Breast Neoplasms; Caspase Inhibitors; Caspases; Cell Proliferation; Female; Humans; Inhibitor of Apoptosis Proteins; MCF-7 Cells; Membrane Potential, Mitochondrial; Necrosis; Nuclear Pore Complex Proteins; Proteolysis; RNA-Binding Proteins; Thiazoles

Furanodiene is a natural product isolated from Rhizoma curcumae, and exhibits broad-spectrum anti-cancer activities in vitro and in vivo. Our previous study proved that furanodiene could increase growth inhibition of steroidal agent in ERα-positive breast cancer cells, but whether furanodiene can influence ER status is not clear. In this study, we confirmed that furanodiene down-regulated the ERα protein expression level and inhibited E2-induced estrogen response element (ERE)-driven reporter plasmid activity in ERα-positive MCF-7 cells. Actually, ERα-knockdown cells were more sensitive than ERα positive cells to furanodiene on the cytotoxicity effect. So the anti-cancer effects of furanodiene and non-steroidal agent in breast cancer cells still requires further investigation. Our results showed that furanodiene exposure could enhance growth inhibitory effects of doxorubicin in ERα-negative MDA-MB-231 cells and ERα-low expression 4T1 cells. However, furanodiene did not increase the cytotoxicity of doxorubicin in ERα-positive breast cancer cells, non-tumorigenic breast epithelial cells, macrophage cells, hepatocytes cells, pheochromocytoma cells and cardiac myoblasts cells. Furanodiene enhances the anti-cancer effects of doxorubicin in ERα-negative breast cancer cells through suppressing cell viability via inducing apoptosis in mitochondria-caspases-dependent and reactive oxygen species-independent manners. These results indicate that furanodiene may be a promising and safety natural agent for cancer adjuvant therapy in the future.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Breast; Breast Neoplasms; Doxorubicin; Drug Synergism; Epithelial Cells; Estrogen Receptor alpha; Furans; Gene Expression Regulation, Neoplastic; Hepatocytes; Heterocyclic Compounds, 2-Ring; Humans; Macrophages; MCF-7 Cells; Myoblasts; Pheochromocytoma; Reactive Oxygen Species

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Breast Neoplasms; Caspase 8; Cell Adhesion Molecules; Cell Line, Tumor; Cell Survival; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Mice; Mice, Nude; MicroRNAs; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; RNA-Binding Proteins; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Of more than one million global cases of breast cancer diagnosed each year, a high percentage are characterized as triple-negative, lacking the oestrogen, progesterone and Her2/neu receptors. The incidence exceeds the incidence of malignancies like CML by far. Lack of effective therapies, younger age at onset and early metastatic spread have contributed to the poor prognosis and outcomes associated with these malignancies.. Here, we investigate the ability of the PI3K/AKT inhibitor AEZS 126 to selectively target the triple-negative breast cancer (TNBC) cell proliferation and survival in vitro by MTT-assays and FACS-based analysis. Furthermore, the mechanism of cytotoxicity is analysed by FACS-based assays and Western blots.. AEZS 126 showed good anti-tumour activity in in vitro models of TNBC as well as in MCF-7 cells. Main mechanism of cytotoxicity seems to be programmed cell death after an incubation time of 72 h, which could be abrogated by co-incubation with z-VAD-fmk in MCF-7 and MDA-MB468 cells. In HCC1806 cells, addition of necrostatin-1 has only slightly protective effects, but in HCC1937 cells, the addition of necrostatin-1 has the same protective effect as co-incubation with z-VAD-fmk, and this observation argues for cell death caused by apoptosis and necroptosis in this cell line.. We demonstrated the highly efficient anti-tumour activity of AEZS 126 in in vitro models of TNBC. Due to the good anti-tumour activity and the expected favourable toxicity profile, AEZS 126 in combination with chemotherapy seems to be a promising candidate for clinical testing in TNBC especially in the basal-like subgroup of TNBC.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Female; Flow Cytometry; Humans; Imidazoles; Indoles; MCF-7 Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone

E-cadherin is a transmembrane protein that serves as a cell adhesion molecule component of the adherens junction. We previously showed that cadmium induced γ-secretase-dependent E-cadherin cleavage via oxidative stress. In this study, we report that staurosporine (STS)-induced apoptosis induces caspase-2 and/or -8-dependent E-cadherin cleavage. STS increased γ-secretase-dependent cleavage of E-cadherin in breast cancer cells through caspase activation. The ability of the γ-secretase inhibitor DAPT and the caspase inhibitor zVAD-FMK to block E-cadherin cleavage provided support for these results. The cleavage of E-cadherin was blocked by caspase-2 and -8 inhibitors. Immunofluorescence analysis confirmed that, along with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulated in the cytosol. In the presence of an inhibitor of γ-secretase or caspase, the cleavage of E-cadherin was partially blocked. Our findings suggest that activation of caspase-2/-8 stimulated the disruption of cadherin-mediated cell-cell contacts in apoptotic cells via γ-secretase activation.

Topics: Amino Acid Chloromethyl Ketones; Amyloid Precursor Protein Secretases; Apoptosis; Breast Neoplasms; Cadherins; Caspase 2; Caspase 8; Caspase Inhibitors; Cell Line, Tumor; Enzyme Inhibitors; Female; Humans; Microscopy, Fluorescence; Staurosporine

Metformin appears to interfere directly with cell proliferation and apoptosis in cancer cells in a non-insulin-mediated manner. One of the key mechanisms of metformin's action is the activation of adenosine monophosphate activated protein kinase (AMPK). AMPK is linked with the phosphatidylinositol 3-kinase (PI3K)/ phosphatase and tensin homolog (PTEN)/protein kinase B (AKT) pathway and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) cascades--all known for being frequently dysregulated in breast cancer. Therefore, simultaneously targeting AMPK through metformin and the PI3K/AKT/mTOR pathway by an mTOR inhibitor could become a therapeutic approach. The aim of this study was to evaluate the anticancer effect of metformin alone and in combination with chemotherapeutic drugs and the mTOR inhibitor RAD001.. The proliferation of breast cancer cells was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; and the cell apoptosis with enzyme-linked immunosorbent assay (ELISA). Gene expression at the protein level was determined by western blot.. We tested metformin alone and in combination with RAD001 and/or chemotherapeutic agents (carboplatin, paclitaxel and doxorubicin, respectively) on several human breast cancer cell lines with respect to cell proliferation, apoptosis and autophagy. Metformin alone inhibited cell proliferation and induced apoptosis in different breast cancer cell lines (ERα-positive, HER2-positive, and triple-negative). The cytotoxic effect of metformin was more remarkable in triple-negative breast cancer cell lines than in other cell lines. The cell apoptosis induced by metformin is, at least partly, caspase-dependent and apoptosis inducing factor (AIF)-dependent. Interestingly, we demonstrated that metformin induced cell autophagy. Inhibiting autophagy with chloroquine, enhanced the treatment efficacy of metformin, indicating that autophagy induced by metformin may protect breast cancer cells from apoptosis. We further demonstrated that co-administration of metformin with chemotherapeutic agents and RAD001 intensified the inhibition of cell proliferation. The analysis of cell cycle-regulating proteins cyclin D, cyclin E and p27 by western blot indicated that the synergistic inhibition of G1 phase of the cell cycle by the combination treatment of metformin, chemotherapeutic drugs and/or RAD001 contributed to the synergistic inhibition of cell proliferation.. Our investigation provides a rationale for the clinical application of metformin within treatment regimens for breast cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Autophagy; Breast Neoplasms; Carboplatin; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Everolimus; Female; Humans; Metformin; Sirolimus; TOR Serine-Threonine Kinases

Previous studies have reported that the Curcuma wenyujin Y.H. Chen et C. Ling extract, which has a high furanodiene content, showed anti-cancer effects in breast cancer cells in vitro. The present study was designed to evaluate the in vitro and in vivo anti-cancer activity of furanodiene.. The in vitro effects of furanodiene were examined on two human breast cancer cell lines, MCF-7 and MDA-MB-231 cells. Assays of proliferation, LDH release, mitochondrial membrane potential (ΔΨm), cell cycle distribution, apoptosis and relevant signaling pathways were performed. The in vivo effect was determined with MCF7 tumor xenograft model in nude mice.. Furanodiene significantly inhibited the proliferation and increased the LDH release in both cell lines in a dose-dependent manner. ΔΨm depolarization, chromatin condensation, and DNA fragmentation were also observed after furanodiene treatment. Furanodiene dose-dependently induced cell cycle arrest at the G0/G1 phase. The protein expressions of p-cyclin D1, total cyclin D1, p-CDK2, total CDK2, p-Rb, total Rb, Bcl-xL, and Akt were significantly inhibited by furanodiene, whereas the protein expressions of Bad and Bax, and the proteolytic cleavage of caspase-9, caspase-7, and poly-ADP-ribose polymerase (PARP) were dramatically increased. Furthermore, the z-VAD-fmk markedly reversed the furanodiene-induced cell cytotoxicity, the proteolytic cleavage of caspase-9, and DNA fragmentation but did not affect the proteolytic cleavage of PARP, whereas the Akt inhibitor VIII increased the furanodiene-induced cytotoxicity and PARP cleavage. In addition, furanodiene dose-dependently suppressed the tumor growth in vivo, achieving 32% and 54% inhibition rates after intraperitoneal injection of 15 mg/kg and 30 mg/kg, respectively.. Taken together, we concluded that furanodiene suppresses breast cancer cell growth both in vitro and in vivo and could be a new lead compound for breast cancer chemotherapy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; Breast Neoplasms; Caspase 7; Caspase 9; Cell Line, Tumor; Cell Proliferation; Curcuma; DNA Fragmentation; Female; Furans; G1 Phase Cell Cycle Checkpoints; Heterocyclic Compounds, 2-Ring; Humans; L-Lactate Dehydrogenase; MCF-7 Cells; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Transplantation, Heterologous

Ursolic acid (3-hydroxy-urs-12-en-28-oic acid) is a pentacyclic triterpenoid derived from leaves, berries, fruits, and flowers of medicinal plants, such as Rosemarinus officinalis. Ursolic acid has been shown to inhibit tumorigenesis, tumor promotion, and suppress angiogenesis. In our present study, we found that ursolic acid decreased cell proliferation rate and induce apoptosis in human breast cancer cell line, MDA-MB-231. When we checked the expression levels of proteins associated with apoptosis signal by using immunoblotting, we found that ursolic acid induces various apoptotic molecules related to either extrinsic or intrinsic apoptosis signal pathway in MDA-MB-231 cells. In our study, we found that ursolic acid induced the appearance of Fas receptor and cleavage of caspase-8, -3 and PARP. We also found that ursolic acid induced Bax up-regulation and Bcl-2 down-regulation and release of cytochrome C to the cytosol from mitochondria. Moreover, ursolic acid cleaved caspase-9 and decreased mitochondrial membrane potential (ΔΨm) as shown with JC-1 staining. These data indicate that ursolic acid induce apoptosis through both mitochondrial death pathway and extrinsic death receptor dependent pathway in MDA-MB-231 cells. Our data clearly indicate that ursolic acid could be used as a potential anticancer drug for breast cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; Down-Regulation; Drug Evaluation, Preclinical; Drug Screening Assays, Antitumor; Female; Humans; Membrane Potential, Mitochondrial; Mitochondria; Poly(ADP-ribose) Polymerases; Receptors, Death Domain; Signal Transduction; Triterpenes; Up-Regulation; Ursolic Acid

A novel series of methylene-substituted DIMs (C-DIMs), namely 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes containing t-butyl (DIM-C-pPhtBu) and phenyl (DIM-C-pPhC6H5) groups inhibit proliferation of invasive estrogen receptor-negative MDA-MB-231 and MDA-MB-453 human breast cancer cell lines with IC50 values between 1-5 uM. The main purpose of this study was to investigate the pathways of C-DIM-induced cell death.. The effects of the C-DIMs on apoptotic, necrotic and autophagic cell death were determined using caspase inhibitors, measurement of lactate dehydrogenase release, and several markers of autophagy including Beclin and light chain associated protein 3 expression (LC3).. The C-DIM compounds did not induce apoptosis and only DIM-C-pPhCF3 exhibited necrotic effects. However, treatment of MDA-MB-231 and MDA-MB-453 cells with C-DIMs resulted in accumulation of LC3-II compared to LC3-I protein, a characteristic marker of autophagy, and transient transfection of green fluorescent protein-LC3 also revealed that treatment with C-DIMs induced a redistribution of LC3 to autophagosomes after C-DIM treatment. In addition, the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker, accumulated in vacuoles after C-DIM treatment, and western blot analysis of lysates from cells treated with C-DIMs showed that the Beclin 1/Bcl-2 protein ratio increased.. The results suggest that C-DIM compounds may represent a new mechanism-based agent for treating drug-resistant ER-negative breast tumors through induction of autophagy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Female; Humans; Immunohistochemistry; Indoles; L-Lactate Dehydrogenase; Macrolides; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Microtubule-Associated Proteins; Necrosis; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen; Recombinant Fusion Proteins; Time Factors; Transfection; Xenograft Model Antitumor Assays

We previously reported that treatment with P-glycoprotein (P-gp) substrates promotes in vitro invasion in multidrug-resistant (MDR) breast cancer cells. This effect is initiated by the P-gp pump function and mediated by interaction of P-gp with some unknown component(s). However, the underlying mechanism(s) remains poorly understood. Here we confirm a novel physical interaction between P-gp and cellular prion protein (PrP(c)). Blocking P-gp activity or depletion of PrP(c) inhibited paclitaxel (P-gp substrate)- induced invasion. Paclitaxel further facilitated the formation of P-gp/PrP(c) clusters residing in caveolar domains and promoted the association of P-gp with caveolin-1. Both caveolin-1 and the integrity of caveolae were required for the drug-induced invasion. In addition, the P-gp/PrP(c) complex also played an important role in anti-apoptotic activity of MCF7/Adr cells.These data provide new insights into the mode by which MDR breast cancers evade cytotoxic attacks from P-gp substrates and also suggest a role for P-gp/ PrP(c) interaction in this process.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Caveolae; Caveolin 1; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; ErbB Receptors; Female; Humans; Paclitaxel; PrPC Proteins

To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro.. The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated DNase (ICAD) protein expressions were detected by Western blot analysis.. Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of DeltaPhimit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by caspase-3.. DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; Diterpenes, Kaurane; Drugs, Chinese Herbal; Female; HSP90 Heat-Shock Proteins; Humans; Medicine, Chinese Traditional; Mitochondria; Poly(ADP-ribose) Polymerases; Signal Transduction; Tumor Suppressor Protein p53

23,24-Dihydrocucurbitacin B (DHCB), a cucurbitacin-derived compound known to posses anticancer and anti-inflammatory activities. In this study, DHCB, isolated from roots of Trichosanthes kirilowli which is a traditional Chinese herb medicine used as treatments for cancer and other diseases, has been found to inhibit the proliferation of human cancer cell lines Bcap37, HeLa, SW620, SMMC-7721, K562 and MCF-7 in a dose- and time-dependent manner, and induce apoptosis in human breast cancer cell line Bcap37 at low concentration. DHCB-induced Bcap37 apoptosis was characterized with the changes in nuclear morphology, DNA fragmentation, activation of caspase-like activities, poly(ADP-ribose) polymerase cleavage, release of cytochrome c into cytosol. The cell death was partly prevented by a caspase-family inhibitor Z-VAD-FMK. The results suggest that DHCB-induced Bcap37 apoptosis through mitochondrial dependent pathway. Flow cytometric analysis revealed that at the lower dose of 1.8 and 3.6muM, DHCB-induced cancer cell lines death via an apoptotic process rather than necrotic one; whereas, the higher dose of 8.9, 17.9 and 35.7muM induced cell death via the necrotic process. Cell-cycle analysis demonstrated DHCB induction of G(2)/M phase cell-cycle arrest and apoptosis. The overall results suggest that DHCB might have the therapeutic value against human cancer cell lines, especially the breast cancer cell lines.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Caspases; Cell Division; Cell Membrane; Cell Proliferation; Cell Shape; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Female; G2 Phase; HeLa Cells; Humans; K562 Cells; Mitochondria; Necrosis; Plant Roots; Poly(ADP-ribose) Polymerases; Prohibitins; Trichosanthes; Triterpenes

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient in a variety of red peppers of the genus Capsicum, is a type of vanilloid. It has been shown to induce apoptosis in many cell types. The effects of vanilloids on apoptosis induction are thought to be correlated with the length and degree of the unsaturation of the fatty acyl chains. In this study, we compared the effect of capsaicin and its docosahexaenoic acid (DHA, C22:6) analog (we named as dohevanil) on human breast cancer MCF-7 cells, which do not express caspase-3. Dohevanil, which was synthesized from DHA and vanillylamine, has longer and highly unsaturated fatty acyl chain than capsaicin. We showed that both vanilloids exhibit effects of growth inhibition and DNA fragmentation induction in MCF-7 cells. These effects of dohevanil were more potent than capsaicin. Because these effects were inhibited by z-VAD-fmk, a broad-spectrum caspase inhibitor, the vanilloids induced the apoptosis via caspase-dependent pathway not involving caspase-3. In conclusion, dohevanil has a more potent effect on apoptosis induction in MCF-7 cells than capsaicin.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Breast Neoplasms; Capsaicin; Caspase 3; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Female; Humans

Apoptin, a chicken anemia virus-derived protein, selectively induces apoptosis in transformed but not in normal cells, thus making it a promising candidate as a novel anticancer therapeutic. The mechanism of apoptin-induced apoptosis is largely unknown. Here, we report that contrary to previous assumptions, Bcl-2 and Bcl-xL inhibit apoptin-induced cell death in several tumor cell lines. In contrast, deficiency of Bax conferred resistance, whereas Bax expression sensitized cells to apoptin-induced death. Cell death induction by apoptin was associated with cytochrome c release from mitochondria as well as with caspase-3 and -7 activation. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad spectrum caspase inhibitor, was highly protective against apoptin-induced cell death. Apoptosis induced by apoptin required Apaf-1, as immortalized Apaf-1-deficient fibroblasts as well as tumor cells devoid of Apaf-1 were strongly protected. Thus, our data indicate that apoptin-induced apoptosis is not only Bcl-2- and caspase dependent, but also engages an Apaf-1 apoptosome-mediated mitochondrial death pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-X Protein; Breast Neoplasms; Capsid Proteins; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Activation; Female; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; Lymphoma, B-Cell; Male; Mitochondria; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Tumor Cells, Cultured

Fatty acid synthase is overexpressed in cancer especially in tumors with a poor prognosis. The specific fatty acid synthase inhibitor cerulenin can induce apoptosis in cancer cells. Likewise, phosphatidylinositol 3-kinase (PI3K)/Akt kinase activities are elevated in primary tumors and cancer cell lines. Here, we tested whether inhibition of PI3K/Akt pathway would sensitize cancer cells to cerulenin-induced apoptosis. We show that LY294002, an inhibitor of PI3K, sensitized MDA-MB468 breast cancer cells to cerulenin-induced apoptosis. In MDA-MB468 cells, cerulenin- and LY294002-mediated apoptosis was associated with caspase-3 activation and the release of cytochrome c from mitochondria to cytosol. In addition, we observed additional species of Bak in mitochondria, suggesting a possible Bak activation. Treatment of cells with cerulenin and LY294002 down-regulated the protein levels of X chromosome-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis 1 (cIAP-1), and Akt, whereas the levels of mitogen-activated protein/extracellular signal-regulated kinase kinase and other antiapoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xl) did not change. Interestingly, the nonspecific caspase inhibitor, z-VAD-FMK, inhibited the down-regulation of Akt, XIAP, and cIAP-1 in cerulenin- and LY294002-treated cells. In conclusion, these studies show that inhibition of PI3K can sensitize cerulenin-induced apoptosis in MBA-MB468 breast cancer cells via activation of caspases, down-regulation of antiapoptotic proteins, such as XIAP, cIAP-1 and Akt, and possibly, activation of Bak in mitochondria.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-X Protein; Breast Neoplasms; Caspase 3; Caspase Inhibitors; Caspases; Cerulenin; Chromones; Cytochromes c; Cytosol; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; Inhibitor of Apoptosis Proteins; Mitochondria; Morpholines; Oncogene Protein v-akt; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2

We have shown previously that our 425.3PE immunotoxin inhibits protein synthesis and induces apoptosis in human breast cancer cells. In attempts to further elucidate the intracellular pathways implicated in its cellular effects, we found that the immunotoxin induced an initial stress response, which rapidly caused an imbalance in the cellular energy status with an increase in reactive oxygen species. The AMP-activated protein kinase (AMPK), a sensor of increased cellular AMP/ATP ratio, was activated by 425.3PE. An immunotoxin-induced activation of c-Jun NH2-terminal kinase (JNK) preceded and overlapped caspase-mediated cleavage of the alpha-subunit of AMPK in a time- and dose-dependent manner. The JNK activation occurred already at a dose level too low to induce any detectable changes in the apoptotic machinery or protein synthesis. In contrast, cycloheximide, even at a concentration causing a 90% inhibition of protein synthesis, did neither affect the ATP level nor activate JNK and AMPK. Pretreatment of the cells with the specific AMPK inhibitor compound C and JNK inhibitor SP600125 blocked activation of AMPK and JNK, respectively, and subsequently sensitized the cells to 425.3PE-induced cell death. Whereas the antioxidant N-acetyl-l-cysteine blocked the generation of reactive oxygen species and activation of JNK and AMPK, it did not block immunotoxin-induced apoptosis. Together, the results show that 425.3PE induces several parallel signaling events, observed initially as an early activation of survival pathways, protecting the cells against the toxic effects of the immunotoxin, followed by subsequent apoptosis induction and protein synthesis inhibition. Conceivably, therapeutic manipulation of the signaling intermediates AMPK and JNK might provide a means to maximize the anticancer effects of the 425.3 immunotoxin.

Topics: Adenosine Triphosphate; ADP Ribose Transferases; Amino Acid Chloromethyl Ketones; AMP-Activated Protein Kinases; Antibodies; Apoptosis; Bacterial Toxins; Breast Neoplasms; Caspases; Cell Death; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Enzyme Activation; ErbB Receptors; Exotoxins; Humans; MAP Kinase Kinase 4; Multienzyme Complexes; Protein Serine-Threonine Kinases; Pseudomonas aeruginosa Exotoxin A; Virulence Factors

Pristimerin, a naturally occurring triterpenoid, has been shown to cause cytotoxicity in several cancer cell lines. However, the mechanism for the cytotoxic effect of pristimerin was never explored. In the present study, human breast cancer MDA-MB-231 cells treated with pristimerin (1 and 3 micromol/L) showed rapid induction of apoptosis, as indicated by caspase activation, DNA fragmentation, and morphologic changes. Pretreatment of a pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) completely prevented pristimerin-induced apoptosis. Treatment of tumor cells with pristimerin resulted in a rapid release of cytochrome c from mitochondria, which preceded caspase activation and the decrease of mitochondrial membrane potential. In addition, neither benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone nor permeability transition pore inhibitor cyclosporin A markedly prevented pristimerin-induced mitochondrial cytochrome c release. Pristimerin did not significantly alter the protein level of Bcl-2 family members (Bcl-2, Bcl-X(L), and Bax), nor did it induce Bax translocation. Moreover, Bcl-2 overexpression fails to prevent pristimerin-induced apoptosis. The generation of reactive oxygen species in MDA-MB-231 cells was also not affected by pristimerin. In a cell-free system, pristimerin induced cytochrome c release from isolated mitochondria. Taken together, these results suggested that pristimerin is a novel mitochondria-targeted compound and may be further evaluated as a chemotherapeutic agent for human cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase Inhibitors; Caspases; Cytochromes c; Female; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Pentacyclic Triterpenes; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Triterpenes; Tumor Cells, Cultured

Apoptotic breakdown of cellular structures is largely mediated by caspases. One target of degradation is a proteinaceous framework of the nucleus termed the nuclear matrix. We compared the apoptotic changes of the nuclear matrix in staurosporine-treated caspase-3-deficient MCF-7 cells transfected with intact CASP-3 gene (MCF-7c3) or an empty vector (MCF-7v) as a control. Nuclear Mitotic Apparatus protein (NuMA), lamin A/C and lamin B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. In both cell lines, staurosporine induced rapid cytoplasmic shrinkage and partial chromatin condensation. MCF-7c3 cells formed apoptotic bodies, whereas MCF-7v cells did not. NuMA and lamins were actively cleaved in MCF-7c3 cells following caspase-3 activation, but only minimal or no cleavage was detected in MCF-7v cells. Interestingly, lamin B but not lamin A/C was relocated into cytoplasmic granules in apoptotic MCF-7v cells. Pancaspase inhibitor, z-VAD-fmk, prevented the apoptotic changes, while caspase-3 inhibitor, z-DEVD-fmk, induced lamin B granules in both cell lines. These results show that caspase-3 is involved in the cleavage of NuMA and lamins either directly or by activating other proteases. This may be essential for disintegration of the nuclear structure during apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Antigens, Nuclear; Apoptosis; Breast Neoplasms; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle Proteins; Cell Nucleus; Chromatin; Cysteine Proteinase Inhibitors; Cytoplasm; Enzyme Activation; Enzyme Inhibitors; Female; Fluorescent Antibody Technique; Humans; Lamin Type A; Lamin Type B; Nuclear Matrix-Associated Proteins; Nuclear Proteins; Oligopeptides; Staurosporine; Tumor Cells, Cultured

Paclitaxel is a microtubule-stabilizing and apoptosis-inducing drug that is commonly used to treat metastatic breast cancer, although the mechanism of paclitaxel-induced apoptosis remains incompletely understood. Furthermore, adhesion molecule expression is attenuated on mouse mastocytoma and human leukemia cells that survive short-term culture in the presence of paclitaxel. In the present study we show that MDA-MB-435 human breast carcinoma cells that survived culture for 72 h in the presence of submaximal cytotoxic concentrations of paclitaxel (0.02 and 0.01 microg/ml) showed decreased expression of the adhesion molecule ICAM-1. Paclitaxel treatment of MDA-MB-435 cells was associated with the generation of reactive oxygen species (ROS), dissipation of mitochondrial transmembrane potential, and the activation of caspase-3. The antioxidant glutathione protected MDA-MB-435 cells from paclitaxel-induced cytotoxicity and reduced ICAM-1 expression. In addition, a selective inhibitor of caspase-3 (Z-DEVD-FMK), as well as a pan-caspase inhibitor (Z-VAD-FMK), partially prevented the decrease in ICAM-1 expression observed following paclitaxel treatment, but did not protect against paclitaxel-induced cytotoxicity. We conclude that the paclitaxel-induced reduction in ICAM-1 expression by MDA-MB-435 breast carcinoma cells is both ROS- and caspase-dependent, whereas paclitaxel-induced cytotoxicity is ROS-dependent and does not involve caspases. Decreased ICAM-1 expression by breast carcinoma cells that survive paclitaxel treatment may negatively impact on cytotoxic lymphocyte-mediated destruction of paclitaxel-resistant breast cancer cells in the context of chemo-immunotherapy or chemo-adoptive immunotherapy.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Activation; Fibronectins; Humans; Intercellular Adhesion Molecule-1; Intracellular Membranes; Membrane Potentials; Mitochondria; Oligopeptides; Paclitaxel; Reactive Oxygen Species; Time Factors

Many anticancer drugs kill cancer cells by inducing apoptosis. Despite the progress in understanding apoptosis, how to harness the cellular death machinery to selectively deliver tumor-specific cytotoxicity (while minimizing damage to other cells) remains an important challenge. We report here that expression of the NRIF3 family of transcriptional coregulators in a variety of breast cancer cell lines induces rapid and profound apoptosis (nearly 100% cell death within 24 h). A novel death domain (DD1) was mapped to a short 30-amino-acid region common to all members of the NRIF3 family. Mechanistic studies showed that DD1-induced apoptosis occurs through a novel caspase 2-mediated pathway that involves mitochondrial membrane permeabilization but does not require other caspases. Interestingly, the cytotoxicity of NRIF3 and DD1 appears to be cell type specific, as they selectively kill breast cancer or related cells but not other examined cells of different origins. Our study demonstrates the feasibility of selectively inducing cytotoxicity in a specific cancer and suggests that breast cancer cells contain a novel "death switch" that can be specifically triggered by NRIF3 or DD1. Strategies utilizing NRIF3 and/or DD1 and/or targeting this death switch may lead to the development of novel and more selective therapeutics against breast cancer.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Breast Neoplasms; Caspase 2; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Female; Humans; In Situ Nick-End Labeling; Matrix Metalloproteinases; Nuclear Proteins; Protein Structure, Tertiary; Recombinant Fusion Proteins; RNA, Small Interfering; Transfection

LIGHT is a new member of the tumor necrosis factor superfamily, which binds to lymphotoxin beta receptor, herpes virus entry mediator, or TR6. This work was carried out to elucidate the molecular mechanism of LIGHT-sensitized, interferon gamma (IFNgamma)-mediated apoptosis of MDA-MB-231 cells. It was revealed that LIGHT treatment resulted in down-regulation of anti-apoptosis Bcl-2 family member: Bcl-2, Bcl-X(L), Bag-1, and Mcl-1; up-regulation of pro-apoptosis Bcl-2 family member: Bak and Ser (112)-phosphor-Bad; down-regulation of pro-apoptosis Bcl-2 member Bax; the other pro-apoptosis member Bid remains unaltered. LIGHT treatment also resulted in activation of caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, DFF45, and PARP. However, caspase activation and caspase activity, especially caspase-3 activity, is not required for LIGHT-induced apoptosis of MDA-MB-231 cells, since caspase-3 inhibitor, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, and a broad range caspase inhibitor, benzyloxycarbonyl-val-ala-asp-fluoromethylketone failed to block the apoptosis induced by LIGHT and IFNgamma in MDA-MB-231 cells. In summary, LIGHT-sensitized IFNgamma-mediated apoptosis of MDA-MB-231 cells is probably through down-regulation of anti-apoptosis Bcl-2 family members; it could be caspase (especially caspase-3)-independent, even though extensive caspase activation was observed.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carrier Proteins; Caspases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Interferon-gamma; Membrane Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Oligopeptides; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transcription Factors; Tumor Cells, Cultured; Tumor Necrosis Factor Ligand Superfamily Member 14; Tumor Necrosis Factor-alpha

The role of insulin-like growth factor-binding protein (IGFBP)-5 in human breast cancer cell growth is unclear. We determined the effects of IGFBP-5 expression on the growth of human breast cancer cell lines in vivo and in vitro. Expression of IGFBP-5, both by stable transfection and adenoviral-mediated infection, was inhibitory to the growth of MDA-MB-231 and Hs578T human breast cancer cells over a 13-day period. IGFBP-5 expression resulted in a G2/M cell cycle arrest and the induction of apoptosis in both cell lines, an effect that was abrogated in the presence of the broad-spectrum caspase inhibitor, z-VAD-fmk. IGFBP-5-induced apoptosis was associated with a transcriptional increase in expression of the proapoptotic regulator bax and decrease in the anti-apoptotic bcl-2 compared with vector controls. Secreted IGFBP-5 when added exogenously to breast cancer cells was not internalized and had no effect on cell growth or apoptosis, suggesting that IGFBP-5 may elicit its inhibitory effects via a novel, intracrine mechanism. In athymic nude mice, stable expression of IGFBP-5 significantly inhibited both the formation and growth of tumors derived from MDA-MB-231 cells. IGFBP-5-expressing tumors also had a significantly elevated level of bax mRNA and decreased levels of bcl-2 mRNA compared with vector tumors. These data suggest that IGFBP-5 is a potent growth inhibitor and proapoptotic agent in human breast cancer cells via modulation of cell cycle regulation and apoptotic mediators.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Northern; Breast Neoplasms; Cell Cycle; Cell Division; Culture Media, Conditioned; DNA Fragmentation; Dose-Response Relationship, Radiation; Enzyme Inhibitors; Flow Cytometry; Genetic Vectors; Humans; Immunoblotting; Insulin-Like Growth Factor Binding Protein 5; Ligands; Mice; Mice, Nude; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Thymidine; Time Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Kahalalide F (KF) is a novel antitumor drug of marine origin under clinical investigation. KF showed a potent cytotoxic activity against a panel of human prostate and breast cancer cell lines, with IC(50) ranging from 0.07 micro M (PC3) to 0.28 micro M (DU145, LNCaP, SKBR-3, BT474, MCF7). Importantly, nontumor human cells (MCF10A, HUVEC, HMEC-1, IMR90) were 5-40 times less sensitive to the drug (IC(50) = 1.6-3.1 micro M). KF cytotoxicity did not correlate with the expression level of the multidrug resistance MDR1 and of the tyrosine kinase HER2/NEU, and only slightly by the anti-apoptotic BCL-2 protein. KF action was triggered rapidly by short pulse treatments (15 min caused 50% maximum cytotoxicity). Neither a general caspase inhibitor (Z-VAD-fmk) nor transcription or translation inhibitors (actinomycin D, cycloheximide) blocked KF action. Flow cytometry analysis revealed that KF induced neither cell-cycle arrest nor apoptotic hypodiploid peak. Using mitochondrial (JC-1)- and lysosomal (LysoTracker Green, Acridine Orange)-specific fluorophores, we detected loss of mitochondrial membrane potential and of lysosomal integrity following KF treatment. Confocal laser and electron microscopy revealed that KF-treated cells underwent a series of profound alterations including severe cytoplasmic swelling and vacuolization, dilation and vesiculation of the endoplasmic reticulum, mitochondrial damage, and plasma membrane rupture. In contrast, the cell nucleus showed irregular clumping of chromatin into small, condensed masses, while chromatin disappeared from other nuclear domains, but the nuclear envelope was preserved and no DNA degradation was detected. Together, these data indicate that KF induces cell death via oncosis preferentially in tumor cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Breast Neoplasms; Caspase Inhibitors; Caspases; Cell Cycle; Cell Nucleus; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Depsipeptides; Female; Flow Cytometry; Humans; Lysosomes; Male; Mollusk Venoms; Oligopeptides; Peptides; Prostatic Neoplasms; Tumor Cells, Cultured

Death-associated protein kinase (DAP-kinase) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptosis systems. However, its apoptosis-promoting mechanism is poorly understood. Here, we demonstrate that DAP-kinase suppresses integrin-mediated cell adhesion and signal transduction, whereas dominant-negative interference of this kinase promotes adhesion. This effect of DAP-kinase is neither a consequence of apoptosis nor a result of decreased expression of integrins. Rather, DAP-kinase downregulates integrin activity through an inside-out mechanism. We present evidence indicating that this adhesion-inhibitory effect accounts for a major mechanism of the apoptosis induced by DAP-kinase. First, in growth-arrested fibroblasts, DAP-kinase triggers apoptosis in cells plated on fibronectin, but does not affect the death of cells on poly-l-lysine. Second, in epithelial cells, DAP-kinase induces apoptosis in the anoikis-sensitive MCF10A cells, but not in the anoikis-resistant BT474 cells. Most importantly, the apoptosis-promoting effect of DAP-kinase is completely abolished by enforced activation of integrin-mediated signaling pathways from either integrin itself or its downstream effector, FAK. Finally, we show that integrin or FAK activation blocks the ability of DAP-kinase to upregulate p53. Our results indicate that DAP-kinase exerts apoptotic effects by suppressing integrin functions and integrin-mediated survival signals, thereby activating a p53-dependent apoptotic pathway.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Caspase 3; Caspase Inhibitors; Caspases; Cell Adhesion; Cell Line; Cell Size; Cell Survival; Cysteine Proteinase Inhibitors; Death-Associated Protein Kinases; Epithelial Cells; Extracellular Matrix; Genes, Reporter; Humans; Integrins; Mice; Signal Transduction; Tumor Suppressor Protein p53

To investigate the mechanisms underlying apoptosis in breast cancer cells, staurosporine was used as an apoptotic stimulus in the human breast cancer cell lines MCF-7 and T47D. Staurosporine induced dose and time dependent increases in DNA fragmentation which was abrogated by z-VAD-fmk. MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved. Staurosporine induced DEVDase activity in T47D cells suggesting the involvement of caspase-3 and/or caspase-7, yet there was no DEVDase activity in MCF-7 cells, probably ruling out the involvement caspase-7. However, staurosporine induced the cleavage of pro-caspase-6 in MCF-7 cells, but not in T47D cells. Caspase dependent PARP cleavage was detected in MCF-7 cells at 3 h, whereas only partial PARP cleavage was detected in T47D cells and then only after 24 h. Moreover, staurosporine led to cytochrome c release at 2 h in MCF-7 cells and 6 h in T47D cells. In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c. Translocation of Bax from the cytosol to mitochondria was observed in both cell types, and this preceded cytochrome c release in both T47D and MCF-7 cells. Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Caspase Inhibitors; Caspases; Cytochrome c Group; Enzyme Activation; Enzyme Inhibitors; Humans; In Situ Nick-End Labeling; Membrane Potentials; Membrane Proteins; Mitochondria; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Staurosporine; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Ceramide has been proposed as a second messenger for stress-induced apoptosis. By characterization of murine melanoma cells and their E1A transfectants, we found several lines of evidences against the role of ceramide as a second messenger for ultraviolet (UV)-induced apoptosis. First, although E1A transfected melanoma cells were more sensitive to UV-induced apoptosis than parental cells, the relative endogenous ceramide elevation induced by UV was greater in parental cells than in E1A transfectants. Second, UV-resistant melanoma cells were more sensitive to exogenous ceramide than UV-sensitive E1A transfectants. The differential responses to UV and ceramide by E1A require the same functional CR2 domain of E1A. Third, unlike the action of UV, transient exposure (up to 2 h) of lethal dose of ceramide was not sufficient to cause apoptosis in these cells, and persistent presence of ceramide was required for processing the apoptotic process. Finally, ceramide and UV do not share a common pathway in apoptosis induction. UV-induced apoptosis was blocked by interleukin-1beta-converting enzyme (ICE) inhibitor z-VAD whereas ceramide-induced apoptosis was not. Therefore, we conclude that ceramide is not a general second messenger for UV-induced apoptosis.

Topics: Adenovirus E1A Proteins; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Breast Neoplasms; Caspase 1; Caspase 3; Caspase Inhibitors; Caspases; Ceramides; Enzyme Inhibitors; Female; Humans; Melanoma, Experimental; Mice; Protein Structure, Tertiary; Radiation Tolerance; Recombinant Fusion Proteins; Second Messenger Systems; Sphingosine; Transfection; Tumor Cells, Cultured; Ultraviolet Rays

Tumour Necrosis Factor alpha binding at the cell surface induces a complex series of signaling events culminating in the caspase cascade, which is central to apoptosis. However, recent work from several laboratories has questioned caspase involvement in commitment to cell death. We have therefore investigated the involvement of caspases in the crucial commitment stage of tumour necrosis factor-induced apoptosis in human T-leukaemic CEM-C7 cells and breast carcinoma MCF-7 cells, using both peptide-based and viral caspase inhibitors. Our observations converge on the conclusion that commitment to death in these systems is dependent on caspase activity, e.g. baculovirus p35 produces over 50-fold protection of colony-forming ability, the most stringent criterion of cell survival. These observations strongly support the view that the caspase family is of great biological and medical significance, since caspase dysfunction resulting in failure to commit to cell death after treatment with tumour necrosis factor or other stimuli may contribute to cancer development.

Topics: Acridine Orange; Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cell Division; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Genes, p53; Humans; Time Factors; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Elevated insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) mRNA in senescent human mammary epithelial cells suggested that the IGFBP-3 gene product may inhibit cell proliferation. To test this hypothesis, we used a retroviral vector to express IGFBP-rP1 cDNA in the IGFBP-rP1-deficient MCF-7 breast cancer cell line. Compared with control vector-transduced cells, cumulative cell numbers for IGFBP-rP1-transduced polyclonal or clonal cell cultures were reduced by 39 and 74%, respectively, after 1 week. Medium conditioned by IGFBP-rP1-producing cultures reduced cumulative cell numbers in parental MCF-7 cultures by 20% compared with medium from cultures of a control vector-transduced cell line. Nuclear fragmentation analysis and cell proliferation assays completed in the presence of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone excluded apoptosis as the responsible mechanism. The percentage of cells containing senescence-associated beta-galactosidase activity was doubled compared with control cell cultures. Flow cytometry analysis indicated that twice as many noncycling cells were present in the IGFBP-rP1-transduced MCF-7 cell cultures compared with controls. These findings indicate that IGFBP-rP1 is an inhibitor of MCF-7 breast cancer cell proliferation and may act via a cellular senescence-like mechanism.

Topics: Amino Acid Chloromethyl Ketones; beta-Galactosidase; Breast Neoplasms; Carrier Proteins; Caspase Inhibitors; Cell Division; Cellular Senescence; Cysteine Proteinase Inhibitors; DNA Fragmentation; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor Binding Proteins; Transduction, Genetic; Tumor Cells, Cultured

Several reports have linked activating protein 2alpha (AP-2alpha) to apoptosis, leading us to hypothesize that AP-2alpha is a substrate for caspases. We tested this hypothesis by examining the effects of tumor necrosis factor alpha (TNF-alpha) on the expression of AP-2 in breast cancer cells. Here, we provide evidence that TNF-alpha downregulates AP-2alpha and AP-2gamma expression posttranscriptionally during TNF-alpha-induced apoptosis. Both a general caspase antagonist (zVADfmk) and a caspase 6-preferred antagonist (zVEIDfmk) inhibited TNF-alpha-induced apoptosis and AP-2alpha downregulation. In vivo tests showed that AP-2alpha was cleaved by caspases ahead of the DNA fragmentation phase of apoptosis. Recombinant caspase 6 cleaved AP-2alpha preferentially, although caspases 1 and 3 also cleaved it, albeit at 50-fold or higher concentrations. Activated caspase 6 was detected in TNF-alpha-treated cells, thus confirming its involvement in AP-2alpha cleavage. All three caspases cleaved AP-2alpha at asp(19) of the sequence asp-arg-his-asp (DRHD(19)). Mutating D(19) to A(19) abrogated AP-2alpha cleavage by all three caspases. TNF-alpha-induced cleavage of AP-2alpha in vivo led to AP-2alpha degradation and loss of DNA-binding activity, both of which were prevented by pretreatment with zVEIDfmk. AP-2alpha degradation but not cleavage was inhibited in vivo by PS-431 (a proteasome antagonist), suggesting that AP-2alpha is degraded subsequent to cleavage by caspase 6 or caspase 6-like enzymes. Cells transfected with green fluorescent protein-tagged mutant AP-2alpha are resistant to TNF-alpha-induced apoptosis, further demonstrating the link between caspase-mediated cleavage of AP-2alpha and apoptosis. This is the first report to demonstrate that degradation of AP-2alpha is a critical event in TNF-alpha-induced apoptosis. Since the DRHD sequence in vertebrate AP-2 is widely conserved, its cleavage by caspases may represent an important mechanism for regulating cell survival, proliferation, differentiation, and apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Animals; Apoptosis; Base Sequence; Blotting, Western; Breast Neoplasms; Caspase 6; Caspases; Cell Death; Cysteine Endopeptidases; DNA; DNA Fragmentation; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Flow Cytometry; Green Fluorescent Proteins; Humans; In Situ Nick-End Labeling; Luminescent Proteins; Molecular Sequence Data; Multienzyme Complexes; Mutagenesis, Site-Directed; Mutation; Peptides; Proteasome Endopeptidase Complex; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Processing, Post-Transcriptional; Time Factors; Transcription Factor AP-2; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The cytokine hepatocyte growth factor/scatter factor (HGF/SF) has been found to protect a variety of epithelial and cancer cell types against cytotoxicity and apoptosis induced by DNA damage, but the specific apoptotic signaling events and the levels at which they are blocked by HGF/SF have not been identified. We found that treatment of MDA-MB-453 human breast cancer cells with adriamycin (also known as doxorubicin, a DNA topoisomerase IIalpha inhibitor) induced a series of time-dependent events, including the mitochondrial release of cytochrome c and apoptosis-inducing factor, mitochondrial membrane depolarization, activation of a set of caspases (caspase-9, -3, -7, -2, and -8), cleavage of poly(ADP-ribose) polymerase (PARP), and up-regulation of expression of the Fas ligand. All of these events were blocked by preincubation of the cells with HGF/SF. In contrast, the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethylketone blocked some of these events (e.g. caspase-3 activation and PARP cleavage) but did not block cytochrome c release or mitochondrial depolarization. These findings suggest that HGF/SF functions, in part, upstream of the mitochondria to block mitochondrial apoptosis signaling, prevent activation of multiple caspases, and protect breast cancer cells against apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Breast Neoplasms; Caspase 3; Caspases; Coloring Agents; Cysteine Proteinase Inhibitors; Cytochrome c Group; Doxorubicin; Enzyme Activation; Enzyme Inhibitors; Fas Ligand Protein; Flavoproteins; Hepatocyte Growth Factor; Humans; Membrane Glycoproteins; Membrane Potentials; Membrane Proteins; Mitochondria; Recombinant Proteins; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors; Tumor Cells, Cultured; Up-Regulation

Despite extensive previous investigation, the events occurring between paclitaxel-induced mitotic arrest and the subsequent onset of apoptosis remain incompletely understood. In the present study, the sequential morphological and biochemical changes that occur after paclitaxel treatment were examined in MDA-MB-468 (p53 mutant) and MCF-7 (p53 wild-type) breast cancer cells. Flow cytometry indicated that paclitaxel induces tetraploidy that persists until the onset of apoptosis in both cell lines. Light and electron microscopy indicated that the cells transiently arrest in mitosis and then enter a multinucleated interphase state characterized by the absence of punctate staining for CENP-F, a G(2) marker, but the presence of cyclin E, a G(1) cyclin, and p21(waf1/cip1), a cyclin-dependent kinase inhibitor. Despite high p21(waf1/cip1) levels, paclitaxel-treated cells incorporated thymidine into DNA. Aphidicolin inhibited this DNA synthesis but not the subsequent onset of apoptosis. Conversely, the broad-spectrum caspase inhibitor benzyloxycarbonyl-val-ala-asp(OMe)-fluoromethylketone inhibited apoptosis and enhanced the number of multinucleated cells but did not facilitate generation of octaploid cells. These results are consistent with a multistep model in which breast cancer cells exposed to paclitaxel undergo an aberrant mitotic exit; proceed through a tetraploid, multinucleated G(1) state; initiate an aphidicolin-suppressible process of DNA repair; and subsequently undergo apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Nucleus; Cell Survival; Cysteine Proteinase Inhibitors; Female; Humans; Paclitaxel; Ploidies; Thymidine; Tumor Cells, Cultured

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either deficient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-x(L) prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less effective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Caspase 8; Caspase 9; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Precursors; Etoposide; fas Receptor; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Mitochondria; Mitomycin; Neoplasm Proteins; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Tamoxifen (TAM) is widely used in the treatment of breast cancer. The cytostatic effects of TAM have been attributed to the antagonism of estrogen receptor (ER) and inhibition of estrogen-dependent proliferative events. However, the mechanism by which TAM is also effective against certain ER-negative breast tumors remains to be elucidated. Here we report that TAM induced the activity of caspase-3-like proteases in ER-negative breast cancer cell lines MDA-MB-231 and BT-20, as evidenced by the cleavage of fluorogenic tetrapeptide substrate and of poly(ADP-ribose) polymerase. The activation of caspase-3-like proteases preceded TAM-induced chromatin condensation and nuclear fragmentation, the typical apoptotic morphologies. Pretreatment of cells with a specific inhibitor of caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde, or with a general inhibitor of caspases, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, prevented TAM-induced apoptosis. TAM also stimulated c-Jun NH2-terminal kinase (JNK) 1 activity, and interfering with the JNK pathway by over-expressing a DN JNK1 mutant attenuated TAM-induced apoptosis. In addition, treatment of cells with a lipid-soluble antioxidant vitamin E blocked TAM-induced caspase-3 and JNK1 activation as well as apoptosis, whereas water-soluble antioxidants N-acetyl L-cysteine and glutathione had little effect. Thus, this study demonstrates that TAM induces apoptosis in ER-negative breast cancer cells through caspase-3 and JNK1 pathways, which are probably initiated at the cell membrane by an oxidative mechanism.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Drug Interactions; Enzyme Activation; Enzyme Induction; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Oligopeptides; Tamoxifen; Tumor Cells, Cultured

Breast cancer frequently spreads to bone and is almost always associated with osteolysis. This tumor-induced osteolysis is caused by increased osteoclastic bone resorption. Bisphosphonates are used successfully to inhibit bone resorption in tumor bone disease and may prevent development of new osteolytic lesions. The classical view is that bisphosphonates only act on bone cells. We investigated their effects on breast cancer cells using three human cell lines, namely, MCF-7, T47D, and MDA.MB.231, and we tested four structurally different bisphosphonates: clodronate, pamidronate, ibandronate, and zoledronate. We performed time course studies for each bisphosphonate at various concentrations and found that all four compounds induced a nonreversible growth inhibition in both MCF-7 and T47D cell lines in a time- and dose-dependent manner. The MDA.MB.231 cell line was less responsive. Bisphosphonates induced apoptosis in MCF-7 and cell necrosis in T47D cells. The inhibition of MCF-7 cell proliferation could be reverted almost completely by the benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk) inhibitor of caspases, suggesting that the apoptotic process observed in the MCF-7 cell line is mediated, at least partly, by the caspase system. Caspase activity was little changed by bisphosphonates in T47D cells and the inhibitor of caspase did not modify bisphosphonates effects. In summary, we found that bisphosphonates inhibit breast cancer cell growth by inducing cell death in vitro. Such effects could contribute to the beneficial role of bisphosphonates in the treatment and the prevention of tumor-induced osteolysis.

Topics: Amino Acid Chloromethyl Ketones; Annexins; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase Inhibitors; Caspases; Cell Death; Cell Nucleus; Clodronic Acid; Cysteine Proteinase Inhibitors; Diphosphonates; Dose-Response Relationship, Drug; Female; Humans; Ibandronic Acid; Imidazoles; Kinetics; Pamidronate; Tumor Cells, Cultured; Zoledronic Acid

In this study we investigated the signalling requirements for TNF-induced cytotoxicity modulated by the methyltransferase inhibitor S-adenosyl-L-homocysteine (AdoHcy) using the TNF-sensitive human breast carcinoma MCF7 cells and its established TNF-resistant clones (R-A1 and clone 1001). Our data indicate that inhibition of methylation reactions by adenosine plus homocysteine, which are known to condense within cells to AdoHcy, markedly potentiated TNF-induced cytotoxicity in MCF7 cells and rendered related TNF-resistant variants, TNF-sensitive by a mechanism independent from the ceramide pathway. We demonstrated that the dominant-negative derivative of FADD (FADD-DN) blocked methylation inhibition/TNF-induced cell death. Moreover, TNF-mediated cytotoxicity modulated by AdoHcy was blocked by the ICE-inhibiting peptide z-VAD-fmk, suggesting that an ICE-like protease is required for the methylation inhibition/TNF-inducible death pathway. In conclusion, these results suggest that the methyltransferase inhibitor AdoHcy potentiates TNF-induced cytotoxicity in MCF7 cells and renders TNF-resistant MCF7 clones, TNF-sensitive via the ceramide independent pathway and that FADD and the ICE-like protease are likely necessary components in transducing methylation inhibition/TNF signals for cell death.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Apoptosis; Arabidopsis Proteins; Breast Neoplasms; Caspase Inhibitors; Ceramides; Cysteine Proteinase Inhibitors; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Drug Resistance; Fatty Acid Desaturases; Female; Humans; Recombinant Fusion Proteins; S-Adenosylhomocysteine; Tumor Necrosis Factor-alpha

The hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3, and its two analogues, EB 1089 and CB 1093, are novel putative anticancer agents with an interesting profile of induction of growth inhibition, differentiation, and apoptosis in tumor cells. To study the signaling pathways mediating these events, we used two human breast cancer cell lines: MCF-7 cells, expressing a wild-type p53 tumor suppressor protein, and T47D cells, lacking a functional p53. Vitamin D compounds induced a growth arrest followed by apoptosis in both cell lines at concentrations ranging from 1 to 100 nM, indicating that p53 is not necessary for growth-inhibitory effects induced by vitamin D compounds. Surprisingly, apoptosis induced by these compounds occurred also independently of known caspases. Inhibition of caspase activation by overexpression of a cowpox-derived caspase inhibitor CrmA or by addition of inhibitory peptides acetyl-Asp-Glu-Val-Asp-aldehyde (200 microM), acetyl-Ile-Glu-Thr-Asp-aldehyde (50 microM), and Z-Val-Ala-D,L-Asp-fluoromethylketone (1 microM) showed no effect on the induction of growth arrest or apoptosis by vitamin D compounds under assay conditions in which apoptosis induced by TNF or staurosporine was effectively inhibited. Moreover, overexpression of caspase-3 in MCF-7 cells had no sensitizing effect to vitamin D compounds, and neither caspase-3-like protease activity nor cleavage of a caspase substrate poly(ADP)ribose polymerase was detected in lysates from apoptotic cells following the treatment with these compounds. Contrary to CrmA, overexpression of an antiapoptotic protein Bcl-2 in MCF-7 cells conferred a nearly complete protection from apoptosis induced by vitamin D compounds. Taken together, these data indicate that vitamin D compounds induce apoptosis via a novel caspase- and p53-independent pathway that can be inhibited by Bcl-2. This may prove useful in the treatment of tumors that are resistant to therapeutic agents that are dependent on the activation of p53 and/or caspases.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calcitriol; Caspase 3; Caspases; Cell Division; Cysteine Proteinase Inhibitors; Female; Humans; In Situ Nick-End Labeling; Kinetics; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Serpins; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Viral Proteins

Immunotoxins composed of antibodies linked to plant or bacterial toxins are being evaluated in the treatment of cancer. It is known that the toxin moieties of immunotoxins, including Pseudomonasexotoxin A (PE), diphtheria toxin, and ricin, are capable of inducing apoptosis. Since the efficiency of induction of apoptosis and the apoptosis pathway may have direct effects on the therapeutic usefulness of immunotoxins, we have studied how B3(Fv)-PE38, a genetically engineered immunotoxin in which the Fv fragment of an antibody is fused to a mutated form of PE, induces apoptosis of the MCF-7 breast cancer cell line. We show for the first time that a PE-containing immunotoxin activates ICE/ced-3 proteases, now termed caspases, and causes characteristic cleavage of the "death substrate" poly(ADP)-ribose polymerase (PARP) to an 89 kDa fragment with a time course of cleavage comparable to that induced by TNFalpha. Also the fluorescent substrate, DEVD-AFC, is cleaved 2-4-fold more rapidly by lysates from B3(Fv)-PE38 treated MCF-7 cells than untreated control cells, suggesting that a CPP32-like caspase is involved in B3(Fv)-PE38-mediated apoptosis. B3(Fv)-PE38-induced PARP cleavage is inhibited by several protease inhibitors known to inhibit caspases (zVAD-fmk, zDEVD-fmk, zIETD-fmk) as well as by overexpression of Bcl-2 providing additional evidence for caspase involvement. zVAD-fmk, a broad spectrum inhibitor of most mammalian caspases, prevents the early morphological changes and loss of cell membrane integrity produced by B3(Fv)-PE38, but not its ability to inhibit protein synthesis, arrest cell growth, and subsequently kill cells. Despite inhibition of apoptosis, the immunotoxin is still capable of selective cell killing, which indicates that B3(Fv)-PE38 kills cells by two mechanisms: one requires caspase activation, and the other is due to the arrest of protein synthesis caused by inactivation of elongation factor 2. The fact that an immunotoxin can specifically kill tumor cells without the need of inducing apoptosis makes such agents especially valuable for the treatment of cancers that are protected against apoptosis, e.g., by overexpression of Bcl-2.

Topics: ADP Ribose Transferases; Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; Bacterial Toxins; Breast Neoplasms; Caspase 1; Caspase 2; Caspase 3; Caspases; Cycloheximide; Enzyme Activation; Exotoxins; Female; Humans; Immunotoxins; Oligopeptides; Peptide Fragments; Poly(ADP-ribose) Polymerases; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Virulence Factors

Programmed cell death, or apoptosis, is inhibited by the antiapoptotic oncogene, Bcl-2, and is mediated by a cascade of aspartate-specific cysteine proteases, or caspases, related to interleukin 1-beta converting enzyme. Depending on cell type, apoptosis can be induced by treatment with thapsigargin (TG); a selective inhibitor of the endoplasmic reticulum-associated calcium-ATPase. The role of caspases in mediating TG-induced apoptosis was investigated in the Bcl-2-negative human breast cancer cell line, MDA-MB-468. Apoptosis developed in MDA-MB-468 cells over a period of 24-72 h following treatment with 100 nM TG, and was prevented by Bcl-2 overexpression. TG-induced apoptosis was associated with activation of caspase-3 and was inhibited by stable expression of the baculovirus p35 protein, an inhibitor of caspase activity. Also, TG-induced apoptosis was inhibited by treating cells with Z-VAD-fmk, a cell-permeable fluoromethylketone inhibitor of caspases. These findings indicate that TG-induced apoptosis of MDA-MB-468 breast cancer cells is subject to inhibition by Bcl-2 and is mediated by caspase activity. This model system should be useful for further investigation directed toward understanding the role of calcium in signaling apoptosis, and its relationship to Bcl-2 and the caspase proteolytic cascade.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Breast Neoplasms; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Inhibitor of Apoptosis Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Thapsigargin; Tumor Cells, Cultured; Viral Proteins