benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Carcinoma

Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk) inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5) expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Carcinoma; Caspase Inhibitors; Cell Line, Tumor; Down-Regulation; Flavonoids; Flavonols; Humans; Kidney Neoplasms; Mice; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Transcription, Genetic; Tumor Suppressor Protein p53; Up-Regulation

Cordycepin, an active ingredient of the insect fungus Cordyceps spp., shows strong antioxidant and anticancer activities. Several molecular mechanisms have been attributed to its inhibitory effects on a wide range of tumor cells; however, the mechanism causing cancer cell death is still obscure. For the current study, we further investigated the mechanism responsible for targeting cordycepin-induced cell death and its association with autophagy in human prostate carcinoma LNCaP cells. Our results show that cordycepin resulted in significant reduction in LNCaP cell survival by inducing apoptotic cell death. Cordycepin treatment caused a dose-dependent increase of pro-apoptotic Bax and decrease of anti-apoptotic Bcl-2, triggering collapse of the mitochondrial membrane potential and activation of caspase-9 and -3. Cordycepin-induced cell death was also associated with induction of Fas and death receptor 5, activation of caspase-8, and truncation of Bid (tBid), suggesting that tBid might serve to connect activation of both the mitochondrial-mediated intrinsic and death receptor-mediated extrinsic apoptotic pathways. The general caspase inhibitor, z-VAD-fmk, completely abolished cordycepin-induced cell death, demonstrating that cordycepin-induced apoptosis was dependent on the activation of caspases. Cordycepin also stimulated autophagy, which was evidenced by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3-II, and elevation of autophagic flux; however, blockage of autophagic flux by the autophagic inhibitor bafilomycin A1 promoted cell-switching to apoptotic cell death. These findings suggest that cordycepin-induced autophagy functions as a survival mechanism and that autophagy is a potential strategy for treating prostate cancer that is resistant to pro-apoptotic therapeutics.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Autophagy; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carcinoma; Caspase Inhibitors; Caspases; Cell Line, Tumor; Deoxyadenosines; Humans; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2

Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and a key element in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Moreover, it is a negative regulator of autophagy and acts as a central regulator in cell growth. For the treatment of cancer, mTOR is a novel and validated therapeutic target. Previous studies have shown that Akt is frequently activated in nasopharyngeal carcinoma (NPC) tissues; thus, the inhibition of mTOR may be a treatment strategy for this tumor type. To evaluate the effect of the mTOR inhibitor RAD001 on NPC cell lines, we performed 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assays, lactate dehydrogenase (LDH) assays, western blotting and flow cytometry to evaluate the mechanisms of cell death. The growth of both CNE-1 and HONE-1 cells was inhibited in a time- and dose-dependent manner. CNE-1 was more sensitive, with a 50% growth inhibition (GI50) of 30.0±1.0 µM compared to HONE-1, cells which had a GI50 of 56.9±13.1 µM. RAD001 induced apoptosis and autophagy in both cell lines. RAD001 induced a significant increase in growth inhibition in the two cell lines when used in combination with the autophagy inhibitor, 3-methyladenine; however, the percentages of apoptotic cells decreased when RAD001 was combined with the caspase inhibitor, z-VAD-fmk. In conclusion, the main mechanism of the mTOR inhibitor RAD001 in these two NPC cells was apoptotic, not autophagic cell death. The combination of RAD001 with autophagy inhibitors may be a useful therapeutic strategy for nasopharyngeal carcinoma.

Topics: Amino Acid Chloromethyl Ketones; Analysis of Variance; Antineoplastic Agents; Apoptosis; Autophagy; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cell Shape; Everolimus; Flow Cytometry; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Sirolimus; TOR Serine-Threonine Kinases

Longikaurin A is a natural ent-kaurene diterpenoid isolated from Isodon genus. The ent-kaurene diterpenoids isolated from medicinal plants have been shown to have anti-disease effects. The present study was designed to examine the anti-tumour effects of longikaurin A (LK-A) in nasopharyngeal carcinoma in vitro and in vivo.. Apoptosis and cell cycle arrest were determined by flow cytometry analysis of the cells treated with Longikaurin A. The proteins of apoptosis signaling pathway were detected by western blotting analysis. Finally, we examined whether LK-A exhibits anti-tumour activity in xenograft models.. Longikaurin A inhibited the cell growth by inducing apoptosis and cell cycle arrest. At low concentrations, longikaurin A induced S phase arrest and at higher concentrations, longikaurin A induced caspase-dependent apoptosis by regulating apoptotic molecules. Finally, longikaurin A significantly inhibited the tumour growth of CNE2 xenografts in vivo and showed no obvious effect on the body weights of the mice.. Our results suggest that Longikaurin A exhibited anti-tumour activity in nasopharyngeal carcinoma in vitro and in vivo.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Diterpenes, Kaurane; Mice; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Poly(ADP-ribose) Polymerases; S Phase; Signal Transduction; Tumor Stem Cell Assay; Up-Regulation; Xenograft Model Antitumor Assays

We analyzed the anti-tumor effect and the mechanism of action of perifosine, an orally active alkylphospholipid AKT inhibitor using in vitro models of human ovarian cancer.. Ovarian cancer cells OAW42, PA-1, SKOV3, and A2780 as well as platinum resistant A2780cis cells were incubated with increasing concentrations of perifosine, with and without multi-caspase inhibitor zVAD-FMK. The effect of a combined treatment with cisplatin and perifosine was investigated in OAW42, SKOV3, A2780 and A2780cis cells. Cytotoxic effects of perifosine were analyzed using crystal violet staining, FACS analysis of DNA content as well as Annexin V/propidium iodide-double staining. The effect of perifosine on AKT phosphorylation was determined by Western blotting.. Perifosine displayed anti-tumor activity in all five cell lines, which increased time-dependently. While IC(50) values at 24 h were >40 μM, IC(50) values after 72 h decreased to 10 μM in OAW42 and 25 μM in PA-1 and 30 μm in SKOV3 cells. In platinum resistant A2780cis cells perifosine showed good antiproliferative activity (IC(50) = 3 μm). At adequate doses, perifosine increased cytotoxic effects of cisplatin in OAW42, A2780 and A2780cis cell. Anti-tumor activity of perifosine was not confined to a specific phase of the cell cycle and could not be decreased by the pan-caspase inhibitor zVAD-FMK. AnnexinV/propidium iodide-double staining after treatment with perifosine was not indicative of classical apoptosis. AKT phosphorylation was dose-dependently inhibited by perifosine.. Perifosine showed substantial cytotoxic effects in various in vitro models of ovarian cancer. Since anti-tumor effects were not confined to platinum-sensitive cells perifosine seems to be a good candidate for clinical studies in patients especially with platinum resistant ovarian cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma; Cell Line, Tumor; Cisplatin; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Female; Humans; Ovarian Neoplasms; Phosphorylcholine; Proto-Oncogene Proteins c-akt

Cervical carcinomas are most frequently associated with human papillomaviruses (HPV), whose E6 and E7 oncogenes products induce cellular immortalization. The papillomavirus E2 protein is a transcription factor, which represses the expression of the viral oncogenes, and activates viral DNA replication during the vegetative viral cycle. This protein is specifically inactivated in HPV18-associated carcinoma cells, suggesting that E2 functions prevent carcinogenic progression. Indeed, ectopic expression of E2 in cervical carcinoma cells strongly inhibits cell proliferation. Here we show that above a threshold level of expression, the E2 protein induces apoptosis, independently of other viral functions. The amino-terminal domain is responsible for this apoptotic activity, but surprisingly with no involvement of its transcriptional functions. The death pathway triggered by E2 relies on activation of the initiator caspase 8, specific of the extrinsic pathway of apoptosis. E2 itself is cleaved by caspases during cell death, providing an example of an apoptotic inducer that is itself a target for caspase processing. The autonomous proapoptotic activity of HPV18 E2 described here may counteract the proliferative functions of viral oncogenes, and renders the inactivation of E2 crucial for carcinogenic progression.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Female; Humans; Mutation; Oncogene Proteins, Viral; Protein Structure, Tertiary; Recombinant Proteins; Threshold Limit Values; Trans-Activators; Transcription, Genetic; Uterine Cervical Neoplasms

Bovine seminal ribonuclease (BS-RNase), a natural dimeric homolog of bovine pancreatic RNase (RNase A), and HHP2-RNase, an engineered dimeric form of human pancreatic RNase (HP-RNase), are endowed with powerful antitumor effects. Here we show that BS- and HHP2-RNases, but not monomeric RNase A, induce apoptosis of human thyroid carcinoma cell lines. RNase-induced apoptosis was associated with activation of initiation caspase-8 and -9. This was followed by activation of executioner caspase-3, leading to the proteolytic cleavage of poly(ADP-ribose) polymerase. The caspase inhibitor Z-Val-Ala-Asp-(OMe)-fluoromethylketone protected thyroid cancer cells from BS-RNase-induced apoptosis. RNase-triggered apoptosis and caspase activation were accompanied by reduced phosphorylation of Akt/protein kinase B (PKB), a serine-threonine kinase that when phosphorylated is able to deliver survival signals to cancer cells. BS-RNase antitumor effects in nude mice were accompanied by caspase activation and apoptosis. Because of the high selectivity of apoptotic effects for malignant cells, BS- and HHP2-RNase are promising tools for the treatment of aggressive thyroid cancer.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Carcinoma; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Endoribonucleases; Humans; Mice; Mice, Nude; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Ribonuclease, Pancreatic; Thyroid Neoplasms; Tumor Cells, Cultured

We have found that omega-hydroxy palmitic acid (16-hydroxy palmitic acid, omega-HPA) has both cell growth inhibiting and cell death inducing actions on human lung adenosquamous carcinoma cell line H596 and adenocarcinoma cell line A549. Further, these effects were dose- and time-dependent in both cell lines. However, in squamous carcinoma cell line H226, omega-HPA had no cytotoxic effect. On the other hand, in the human small cell lung carcinoma (SCLC) cell line H128, this compound showed weak cytotoxicity. The sensitivity toward omega-HPA was higher in H596 cells than in A549 cells. In both H596 and A549 cells, cell growth was inhibited to 24.4 and 9.4%, respectively, by treatment with 100 microM omega-HPA for 12 h. In the 24 h treatment cells, growth inhibition was increased to 100 and 38.1%, respectively. In cytotoxicity experiments, the number of dead cells increased with incubation times in the presence of omega-HPA: on three days incubation with 100 microM omega-HPA, viability was 0 and 13.5%, respectively, in H596 and A549 cells. Further, the fragmentation of DNA to oligonucleosomal-sized ladder fragments, which is an index of apoptosis, was observed in both cell lines on treatment with omega-HPA. Therefore, it is assumed that these cell deaths induced by omega-HPA, were apoptosis in these cell lines. Since the number of dead cells following treatment with omega-HPA decreased by treatment with omega-HPA in combination with Z-VAD-fmk, a caspase family inhibitor, it is thought that apoptotic cell death was related to caspase activity.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Caspase Inhibitors; Cell Division; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; DNA Fragmentation; DNA, Neoplasm; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Palmitic Acids; Time Factors; 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

A series of in vitro studies were carried out to investigate genistein-induced cell death, and the nature of cell death, in two human prostate cancer cell lines (LNCaP and Du145), and the possible involvement of caspase-3 protease in genistein-induced apoptosis in the target cells. The major findings of these studies are: i) genistein inhibits growth and proliferation of both LNCaP and DU145 cells via apoptosis mainly, and necrosis at higher concentrations; ii) genistein induces activation and expression of caspase-3 (CPP32) in both target cells; iii) genistein-induced apoptosis and CPP32 activation could be significantly inhibited by the caspase-3 inhibitor, z-VAD-fmk (N-benzyloxycarbonyl-Val-Asp-fluoromethyl-ketone), thus confirming a mediator role of CPP32 in the genistein-induced apoptotic pathway in the target cells. The potency of most known chemopreventive drugs for cancer is due to induction of apoptosis in solid tumors (Thompson, Science 267 (1995) 1456; Gurney et al., Science 288 (2000) 283). Inevitably, agents that increase transcription of caspase-3 protease could reinforce cell death via CPP32-mediated apoptosis. In this regard, genistein may find an application in the treatment of human prostate carcinoma, independently of hormone sensitivity.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Bisbenzimidazole; Carcinoma; Caspase 3; Caspases; Cell Division; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Interactions; Fluorescent Dyes; Genistein; Gonadal Steroid Hormones; Humans; In Situ Nick-End Labeling; Male; Prostatic Neoplasms; Signal Transduction; Tumor Cells, Cultured

Activated caspase-3-like proteases promote apoptotic cell death by cleaving cellular substrates. Caspase-3-like activity was measured in colonic carcinomas and in matched normal colonic mucosa from 31 patients and was significantly elevated in 25/ 31 colonic carcinomas and adenomas when compared to normal mucosa (P < 0.0001). Caspase-3-like activity was much higher in normal mucosa and tumours of female subjects than of males (P < 0.0001). No correlation was obtained between caspase-3-like activity and location of the tumour, tumour grade, stage, or patient age. The marked increase in caspase-3-like activity in colorectal carcinomas may reflect an increase in the proportion of cells undergoing spontaneous apoptosis.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma; Caspase 3; Caspase Inhibitors; Caspases; Colorectal Neoplasms; Coumarins; Cysteine Proteinase Inhibitors; Enzyme Precursors; Female; Humans; Intestinal Mucosa; Jurkat Cells; Male; Middle Aged; Oligopeptides