benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Carcinoma--Squamous-Cell

Combining photodynamic therapy (PDT) with another anticancer treatment modality is an important strategy for improved efficacy. PDT with Pc4, a silicon phthalocyanine photosensitizer, was combined with C6-pyridinium ceramide (LCL29) to determine their potential to promote death of SCC17B human head and neck squamous cell carcinoma cells. PDT+LCL29-induced enhanced cell death was inhibited by zVAD-fmk, a pan-caspase inhibitor, and fumonisin B1 (FB), a ceramide synthase inhibitor. Quantitative confocal microscopy showed that combining PDT with LCL29 enhanced FB-sensitive ceramide accumulation in the mitochondria. Furthermore, PDT+LCL29 induced enhanced FB-sensitive redistribution of cytochrome c and caspase-3 activation. Overall, the data indicate that PDT+LCL29 enhanced cell death via FB-sensitive, mitochondrial ceramide accumulation and apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; Ceramides; Cytochromes c; Drug Synergism; Enzyme Activation; Fumonisins; Head and Neck Neoplasms; Humans; Indoles; Mitochondria; Organosilicon Compounds; Photochemotherapy; Photosensitizing Agents; Protein Transport; Pyridinium Compounds

The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Squamous Cell; Cell Line, Tumor; Ceramides; Cytochromes c; Endoplasmic Reticulum; Enzyme Inhibitors; Fumonisins; Head and Neck Neoplasms; Humans; Indoles; Mass Spectrometry; Mitochondria; Organosilicon Compounds; Oxidoreductases; Photochemotherapy

Non-melanoma skin cancers (NMSCs), one of the most common neoplasms, cause serious morbidity and mortality. Therefore, identification of non-toxic phytochemicals for prevention/treatment of NMSCs is highly desirable. Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid, present in fruits and vegetables possesses anti-oxidant and antiproliferative properties. The aim of this study was to investigate the chemotherapeutic potential of fisetin in cultured human epidermoid carcinoma A431 cells. Treatment of A431 cells with fisetin (5-80 μm) resulted in a significant decrease in cell viability in a dose- and time-dependent manner. Employing clonogenic assay, we found that fisetin treatment significantly reduced colony formation in A431 cells. Fisetin treatment of A431 cells resulted in G₂ /M arrest and induction of apoptosis. Furthermore, treatment of A431 cells with fisetin resulted in (i) decreased expression of anti-apoptotic proteins (Bcl2; Bcl-xL and Mcl-1); (ii) increased expression of pro-apoptotic proteins (Bax, Bak and Bad); (iii) disruption of mitochondrial potential; (iv) release of cytochrome c and Smac/DIABLO from mitochondria; (v) activation of caspases; and (vi) cleavage of Poly(ADP-ribose) polymerase (PARP) protein. Pretreatment of A431 cells with the pan-caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced cleavage of caspases and PARP. Taken together, these data provide evidence that fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells. Overall, these results suggest that fisetin could be developed as a novel therapeutic agent for the management of NMSCs.

Topics: Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; Carcinoma, Squamous Cell; Caspase Inhibitors; Caspases; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Flavonoids; Flavonols; G2 Phase; Humans; Membrane Potential, Mitochondrial; Proto-Oncogene Proteins c-bcl-2; Treatment Outcome

Ever since protoporphyrin IX (PpIX) was discovered to accumulate preferentially in cancer cells after 5-aminolevulinic acid (ALA) treatment, photodynamic treatment or therapy (PDT) has been developed as an exciting new treatment option for cancer patients. However, the level of PpIX accumulation in oral cancer is fairly low and insufficient for PDT. Ferrochelatase (FECH) and ATP-binding cassette transporter G2 (ABCG2) are known to regulate PpIX accumulation. In addition, serum enhances PpIX export by ABCG2. We investigated here whether and how inhibitors of FECH and ABCG2 and their combination could improve PpIX accumulation and PDT efficacy in an oral cancer cell line in serum-containing medium. ABCG2 inhibitor and the combination of ABCG2 and FECH inhibitors increased PpIX in the presence of fetal bovine serum (FBS) in an oral cancer cell line. Analysis of ABCG2 gene silencing also revealed the involvement of ABCG2 in the regulation of PpIX accumulation. Inhibitors of FECH and ABCG2, and their combination increased the efficiency of ALA-PDT even in the presence of FBS. ALA-PDT-induced cell death was accompanied by apoptotic events and lipid peroxidation. These results suggest that accumulation of PpIX is determined by the activities of ABCG2 and FECH and that treatment with a combination of their inhibitors improves the efficacy of PDT for oral cancer, especially in the presence of serum.

Topics: Amino Acid Chloromethyl Ketones; Aminolevulinic Acid; Apoptosis; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blood Proteins; Carcinoma, Squamous Cell; Caspase Inhibitors; Cell Line, Tumor; Deferoxamine; Ferrochelatase; Gene Silencing; Humans; Lipid Peroxidation; Mouth Neoplasms; Neoplasm Proteins; Photochemotherapy; Photosensitizing Agents; Protoporphyrins; Siderophores

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1- and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

Topics: Abdominal Neoplasms; Acid Ceramidase; Amino Acid Chloromethyl Ketones; Animals; Annexin A5; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspase 9; Cell Line, Tumor; Ceramides; Dasatinib; Enzyme Activation; Head and Neck Neoplasms; Indoles; Mice; Mice, Inbred C3H; Mitochondria; Oxidoreductases; Photochemotherapy; Propidium; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrimidines; RNA, Messenger; Sphingosine; Thiazoles

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55-60%) and calcium-dependent (40-45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Calpain; Carcinoma, Squamous Cell; Caspases; Cell Line, Tumor; Cell Membrane; Cytochromes c; Egtazic Acid; Electric Stimulation; Electricity; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Protease Inhibitors; Reactive Oxygen Species; Time Factors

Chemoradiation is the treatment of choice for locally advanced head and neck squamous cell carcinoma (HNSCC). However, radioresistance, which contributes to local recurrence, remains a significant therapeutic problem. In this study, we characterized SM-164, a small second mitochondria-derived activator of caspase -mimetic compound that promotes degradation of cellular inhibitor of apoptosis-1(cIAP-1; also known as baculoviral IAP repeat-containing protein 2, BIRC2) and releases active caspases from the X-linked inhibitor of apoptosis inhibitory binding as a radiosensitizing agent in HNSCC cells. We found that SM-164 at nanomolar concentrations induced radiosensitization in some HNSCC cell lines in a manner dependent on intrinsic sensitivity to caspase activation and apoptosis induction. Blockage of caspase activation via short interfering RNA knockdown or a pan-caspase inhibitor, z-VAD-fmk, largely abrogated SM-164 radiosensitization. On the other hand, the resistant lines with a high level of Bcl-2 that blocks caspase activation and apoptosis induction became sensitive to radiation on Bcl-2 knockdown. Mechanistic studies revealed that SM-164 radiosensitization in sensitive cells was associated with NF-κB activation and TNFα secretion, followed by activation of caspase-8 and -9, leading to enhanced apoptosis. Finally, SM-164 also radiosensitized human tumor xenograft while causing minimal toxicity. Thus, SM-164 is a potent radiosensitizer via a mechanism involving caspase activation and holds promise for future clinical development as a novel class of radiosensitizer for the treatment of a subset of head and neck cancer patients.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Baculoviral IAP Repeat-Containing 3 Protein; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Squamous Cell; Caspase Inhibitors; Caspases; Cell Line, Tumor; Combined Modality Therapy; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; Head and Neck Neoplasms; Humans; Inhibitor of Apoptosis Proteins; Mice; Mice, Nude; Molecular Structure; Proto-Oncogene Proteins c-bcl-2; Radiation-Sensitizing Agents; Radiotherapy; RNA Interference; Triazoles; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

Fascin induces membrane protrusions and cell motility. Fascin overexpression was associated with poor prognosis, and its downregulation reduces cell motility and invasiveness in esophageal squamous cell carcinoma (ESCC). Using a stable knockdown cell line, we revealed the effect of fascin on cell growth, cell adhesion and tumor formation.. We examined whether fascin is a potential target in ESCC using in vitro and in vivo studies utilizing a specific siRNA. We established a stable transfectant with downregulated fascin from KYSE170 cell line.. The fascin downregulated cell lines showed a slower growth pattern by 40.3% (p < 0.01) and detachment from collagen-coated plates by 53.6% (p < 0.01), compared to mock cells, suggesting that fascin plays a role in cell growth by maintaining cell adhesion to the extracellular matrix. In vivo, the tumor size was significantly smaller in the tumor with fascin knockdown cells than in mock cells by 95% at 30 days after inoculation.. These findings suggest that fascin overexpression plays a role in tumor growth and progression in ESCC and that cell death caused by its downregulation might be induced by cell adhesion loss. This indicates that targeting fascin pathway could be a novel therapeutic strategy for the human ESCC.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Carcinoma, Squamous Cell; Carrier Proteins; Caspase Inhibitors; Caspases; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Collagen; Cysteine Proteinase Inhibitors; Esophageal Neoplasms; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microfilament Proteins; RNA Interference; RNA, Small Interfering; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Tumor-derived membranous vesicles (MV) isolated from sera of the patients with squamous cell carcinomas of the head and neck (HNSCC) induce apoptosis of activated CD8(+) T cells. We tested if MV molecular profile and activity correlate with disease progression.. CD8(+) Jurkat cells were incubated with MAGE 3/6(+), FasL(+), MHC class I(+) MV isolated from sera of 60 patients with HNSCC and 25 normal controls by exclusion chromatography and ultracentrifugation. Z-VAD-FITC binding to Jurkat was measured and correlated with clinical data.. MV from patients' sera, but not from sera of normal controls, induced Jurkat cell apoptosis. Forty-five percent T cells+MV from patients with N(1)-N(3) disease were apoptotic versus 18% T cells+MV from patients with N(0) disease (p < .008). MV from patients with active disease (AD) expressed higher FasL levels than MV from patients with no evident disease (NED) or normal controls (p

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Chloromethyl Ketones; Antigens, Neoplasm; Apoptosis; Carcinoma, Squamous Cell; Case-Control Studies; Caspases; CD8-Positive T-Lymphocytes; Cysteine Proteinase Inhibitors; Cytoplasmic Vesicles; Disease Progression; Fas Ligand Protein; Female; Head and Neck Neoplasms; Humans; Jurkat Cells; Lymphatic Metastasis; Major Histocompatibility Complex; Male; Middle Aged

Dietary grape seed proanthocyanidins (GSPs) prevent photocarcinogenesis in mice. Here, we report that in vitro treatment of human epidermoid carcinoma A431 cells with GSPs inhibited cellular proliferation (13-89%) and induced cell death (1-48%) in a dose (5-100 mug/ml)- and time (24, 48 and 72 h)-dependent manner. GSP-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest at 24 h, which was mediated through the inhibition of cyclin-dependent kinases (Cdk) Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and simultaneous increase in protein expression of cyclin-dependent kinase inhibitors (Cdki), Cip1/p21 and Kip1/p27, and enhanced binding of Cdki-Cdk. The treatment of A431 cells with GSPs (20-80 mug/ml) resulted in a dose-dependent increase in apoptotic cell death (26-58%), which was associated with an increased protein expression of proapoptotic Bax, decreased expression of antiapoptotic Bcl-2 and Bcl-xl, loss of mitochondrial membrane potential, and cleavage of caspase-9, caspase-3 and PARP. Pretreatment with the pan-caspase inhibitor (z-VAD-fmk) blocked the GSP-induced apoptosis in A431 cells suggesting that GSP-induced apoptosis is associated primarily with the caspase-3-dependent pathway. Together, our study suggests that GSPs possess chemotherapeutic potential against human epidermoid carcinoma cells in vitro, further in vivo mechanistic studies are required to verify the chemotherapeutic effect of GSPs in skin cancers.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinase Inhibitor Proteins; Cyclin-Dependent Kinases; Cyclins; Cysteine Proteinase Inhibitors; Enzyme Activation; G1 Phase; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potential, Mitochondrial; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proanthocyanidins; Signal Transduction; Skin Neoplasms; Vitis

To study the molecular mechanism of TAp63gamma-induced cell apoptosis.. Transcription and protein expression of apoptosis inducing factor and p63 were investigated by immunohistochemistry and RT-PCR in human esophageal squamous carcinoma cell line EC9706 respectively. Twenty-four hours after transfection with pcDNA3.1-TAp63gamma, the apoptosis and translocation of apoptosis inducing factor in EC9706 cells were studied by flow cytometry, laser confocal microscopy and mitochondrial/cytosol/nuclear extraction analysis respectively. Down-regulation of apoptosis inducing factor protein was achieved by RNAi and pretreatment with caspase inhibitor zVAD.fmk of EC9706 cells.. Presence of protein expressions of apoptosis inducing factor and absence of TAp63gamma was observed in the cytoplasm of untransfected cells. RT-PCR verified the subtype of p63 in EC9706 cells was DeltaNp63. After 24 hours of transfection, both nuclear and cytoplasmic expression of apoptosis inducing factor protein were observed in cells transfected with TAp63gamma and p53 expression vectors, but not in cells transfected with control vector. Cell apoptosis rates were 1.37%, 13.64%, 4.52%, 4.03% and 1.91% in the pcDNA3.1 transfection group, pcDNA3.1-TAp63gamma transfection group, apoptosis inducing factor siRNA and pcDNA3.1-TAp63gamma transfection group, zVAD.fmk treatment group, and the group receiving apoptosis inducing factor siRNA, plus zVAD.fmk treatment and pcDNA3.1-TAp63gamma transfection, respectively.. Apoptosis inducing factor of EC9706 cells is released from mitochondria into both the cytoplasm and nucleus during TAp63gamma induced apoptosis. Down-regulation of apoptosis inducing factor inhibits TAp63gamma-induced apoptosis. Overall, TAp63gamma-induced apoptosis is dependent on the expression of apoptosis inducing factor and caspase.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Inducing Factor; Carcinoma, Squamous Cell; Caspase Inhibitors; Cell Line, Tumor; Cell Nucleus; Cytoplasm; Down-Regulation; Esophageal Neoplasms; Humans; Mitochondria; Plasmids; Protein Transport; RNA Interference; RNA, Small Interfering; Trans-Activators; Transcription Factors; Transfection; Tumor Suppressor Proteins

Chemotherapeutic approach using non-toxic botanicals may be one of the strategies for the management of the skin cancers. Here we report that in vitro treatment of human epidermoid carcinoma A431 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability (3-77%, P < 0.05-0.001) and induced cell death (3-51%, P < 0.01-0.001) in a dose (5-75 microM)- and time (12-72 h)-dependent manner, which was associated with an increase in G(1) arrest. G(0)/G(1) phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin kinase inhibitors (Cdki) and cyclins. Our western blot analysis showed that berberine-induced G(1) cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), a simultaneous decrease in Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and enhanced binding of Cdki-Cdk. In additional studies, treatment of A431 cells with berberine (15-75 microM) for 72 h resulted in a significant dose-dependent increase in apoptosis (31-60%, P < 0.05-0.001) than non-berberine-treated control (11.7%), which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase. Pretreatment of A431 cells with the pan-caspase inhibitor (z-VAD-fmk) significantly blocked the berberine-induced apoptosis in A431 cells confirmed that berberine-induced apoptosis is mediated through activation of caspase 3-dependent pathway. Together, this study for the first time identified berberine as a chemotherapeutic agent against human epidermoid carcinoma A431 cells in vitro, further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of non-melanoma skin cancers.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Berberine; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Cytochromes c; G1 Phase; Humans; Keratinocytes; Membrane Potentials; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms

These results demonstrate that sulindac sulfide can induce cell death in maxillary cancer cells, and that sulindac sulfide-induced apoptosis is related to the extracellular signal-regulated kinase/p38 MAPK-caspase 3 signaling pathway.. Head and neck cancer is the sixth commonest cancer in the human body. Squamous cell carcinoma accounts for most sinonasal cancers. However, little is known regarding the biochemical mechanism(s) of cell death in sinonasal cancers. Recently, human epidemiological and clinical intervention studies have indicated that sulindac, a non-steroidal anti-inflammatory drug, exhibits chemopreventive activity in colorectal cancer. In this study, we aimed to investigate whether sulindac sulfide can induce apoptosis in sinonasal cancer cells and what type of molecular mechanisms induces the death of sinonasal cancer cells.. Sinonasal cancer cells (Asan Medical Center Head and Neck Cancer 5) were treated with various concentrations of sulindac sulfide. The degree of cell death was determined by means of a fluorescence-activated cell scan and the signal transduction pathway for cell death was examined.. Human nasal cavity cancer cells treated with sulindac sulfide underwent cell death, and the induction of apoptosis occurred in a dose-dependent manner. Moreover, sulindac sulfide-induced apoptosis was abolished by treatment with the caspase inhibitor Z-VAD-fmk and the mitogen-activated protein kinase (MAPK) inhibitors PD98059 and SB203580.

Topics: Amino Acid Chloromethyl Ketones; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Carcinoma, Squamous Cell; Caspase Inhibitors; Flavonoids; Fluorescence; Humans; Imidazoles; Mitogen-Activated Protein Kinases; Paranasal Sinus Neoplasms; Pyridines; Signal Transduction; Sulindac

Green tea constituent (-) epigallocatechin-3-gallate (EGCG) has shown remarkable cancer-preventive and some cancer-therapeutic effects. This is partially because of its ability to induce apoptosis in cancer cells without affecting normal cells. Previous studies from our laboratory have shown the involvement of NF-kappa B pathway in EGCG-mediated cell-cycle deregulation and apoptosis of human epidermoid carcinoma A431 cells. Here we show the essential role of caspases in EGCG-mediated inhibition of NF-kappa B and its subsequent apoptosis. Treatment of A431 cells with EGCG (10-40 microg/ml) resulted in dose-dependent inhibition of NF-kappa B/p65, induction of DNA breaks, cleavage of poly(ADP-ribose) polymerase (PARP) and morphological changes consistent with apoptosis. EGCG treatment of cells also resulted in significant activation of caspases, as shown by the dose- and time-dependent increase in DEVDase activity, and protein expression of caspase-3, -8 and -9. EGCG-mediated caspase activation induces proteolytic cleavage of NF-kappa B/p65 subunit, leading to the loss of transactivation domains, and driving the cells towards apoptosis. EGCG-mediated induction of apoptosis was significantly blocked by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-FMK), and moderately blocked by the specific caspase-3 inhibitor Z-DEVD-FMK. Further, pretreatment of cells with Z-VAD-FMK was found to suppress the cleavage of NF-kappa B/p65 subunit, thereby increasing nuclear translocation, DNA binding and transcriptional activity, thus protecting the cells from EGCG-induced apoptosis. Taken together, these studies for the first time demonstrate that EGCG-mediated activation of caspases is critical, at least in part, for inhibition of NF-kappa B and subsequent apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Squamous Cell; Caspase Inhibitors; Caspases; Catechin; Cell Line, Tumor; Cell Nucleus; Cell Survival; DNA; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Male; Models, Biological; NF-kappa B; Oligopeptides; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein Structure, Tertiary; Time Factors; Transcription, Genetic

15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ(2)) and troglitazone have been shown to induce apoptosis in several carcinoma cell lines. However, apoptotic signaling pathways of these agents are poorly understood. We tested the hypothesis that peroxisome proliferator-activated receptor-gamma ligands such as these two agents will induce caspase-mediated apoptosis in human oral squamous cell carcinomas (SCC). Treatment of these cell lines with 15-d-PGJ(2) or troglitazone decreased cell viability in a time- and dose-dependent manner. 15-d-PGJ(2), but not troglitazone, induced apoptosis, and this effect was time-dependent. Exposure of cells to 20 micro M of 15-d-PGJ(2) initiated early cytochrome c release, followed by late caspase activation. Furthermore, co-treatment with caspase inhibitors such as Z-VAD-FMK or Z-DEVD-FMK of oral SCC cells that had been treated with 20 micro M of 15-d-PGJ(2) blocked apoptosis. Our study demonstrates that treatment with 15-d-PGJ(2), but not troglitazone, induces apoptosis in human SCC cell lines, and 15-d-PGJ(2) appears to work through cytochrome c release and caspase activation.

Topics: Amino Acid Chloromethyl Ketones; Analysis of Variance; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspase Inhibitors; Caspases; Cell Survival; Chromans; Cysteine Proteinase Inhibitors; Cytochrome c Group; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Immunologic Factors; Mouth Neoplasms; Oligopeptides; Prostaglandin D2; Signal Transduction; Thiazoles; Thiazolidinediones; Time Factors; Troglitazone; 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