cytochrome-c-t and Mouth-Neoplasms

During the transition from human oral mucosal epithelial cells (HOMEC) to oral squamous cell carcinoma cells (Cal27), the cells must have undergone a precancerous state. To explore the malignant rule of HOMEC, plv-HOMEC was used as a model cell for the precancerous state to investigate plv-HOMEC's apoptosis by comparing human oral mucosal epithelial cells established by Lentivirus-mediated hTERT (plv-HOMEC) with HOMEC and human Cal27. The lentiviral particles overexpressing hTERT were packaged and transfected into primary HOMEC to obtain plv-HOMEC. Expression levels of NF-κB were detected in the cytoplasm and nucleus of Cal27, plv-HOMEC and HOMEC. The level of intracellular reactive oxygen species was measured to verify the endoplasmic reticulum pathway, cytochrome C expression was detected to verify the mitochondrial pathway, and FasL gene expression was detected to verify the death receptor apoptosis pathway. The total expression of NF-κB in plv-HOMEC increased, mainly due to the greater nuclear import of NF-κB, but it was still much lower than Cal27. The endoplasmic reticulum apoptosis pathway of plv-HOMEC was not significantly affected, and there were no significant differences between them and the HOMEC cells; the mitochondrial apoptosis pathway of plv-HOMEC was inhibited, and the expression of Cyt C was very close to that of Cal27, indicating that the characteristics of plv-HOMEC are so familiar with cancer cells; the death receptor apoptosis pathway of plv-HOMEC was also inhibited, and in this apoptotic pathway, plv-HOMEC were more similar to cancer cells than to HOMEC cells. The present data suggest that NF-κB nucleation may increase in the early stage of healthy cells' carcinogenesis, followed by inhibition of the mitochondrial pathway and the death receptor apoptotic pathway.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Epithelial Cells; Humans; Lentivirus; Mitochondria; Mouth Mucosa; Mouth Neoplasms; NF-kappa B; Reactive Oxygen Species; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Telomerase; Transcription Factor RelA

The main aim of this study was to investigate the effects of phloretin loaded chitosan nanoparticles (PhCsNPs) on 7,12-dimethylbenz[a]anthracene (DMBA) induced experimental cancer in hamsters. Oral squamous cell carcinoma (OSCC) was induced in male golden Syrian hamsters by painting with 0.5% DMBA three times a week for 14 weeks. Varying concentration of PhCsNPs (5, 10, and 20 mg/kg b.wt.) was orally administered on alternative days to evaluate the optimum dose. The experiment design was terminated at the end of the 14th week. The development of OSCC was confirmed by histopathological and biochemical analysis (lipid peroxidation, antioxidant profile, and detoxification enzymes) in plasma, erythrocyte, buccal, and liver tissues. Significant increases in oxidation and lipid peroxidation were noticed in DMBA-painted hamsters. Oral administration of PhCsNPs in various doses on alternate days reversed the deleterious effects induced by DMBA. In addition, immunoblot analyses of PhCsNPs treatment enhanced the release of Bcl-2 associated X protein (Bax), cytochrome c, caspase-3, 9 and suppressed the B-cell lymphoma 2 (Bcl-2) expression, which the use of PhCsNPs for mitochondrial-mediated apoptosis. These findings suggest biofabricated PhCsNPs may act as a potent antioxidant and anti-carcinogenic in DMBA induced oral cancer in experimental animals.

Topics: Administration, Oral; Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Squamous Cell; Caspase 3; Chitosan; Cricetinae; Cytochrome P-450 Enzyme System; Cytochromes c; Down-Regulation; Lipid Peroxidation; Male; Mouth Neoplasms; Nanoparticles; Phloretin; Proto-Oncogene Proteins c-bcl-2

Silymarin, a standardized extract from milk thistle fruits has been found to exhibit anti-cancer effects against various cancers. Here, we explored the anti-cancer activity of silymarin and its molecular target in human oral cancer in vitro and in vivo. Silymarin dose-dependently inhibited the proliferation of HSC-4 oral cancer cells and promoted caspase-dependent apoptosis. A human apoptosis protein array kit showed that death receptor 5 may be involved in silymarin-induced apoptosis, which was also shown through western blotting, immunocytochemistry, and reverse transcription-polymerase chain reaction. Silymarin increased cleaved caspase-8 and truncated Bid, leading to accumulation of cytochrome c. In addition, silymarin activated death receptor 5/caspase-8 to induce apoptotic cell death in two other oral cancer cell lines (YD15 and Ca9.22). Silymarin also suppressed tumor growth and volume without any hepatic or renal toxicity in vivo. Taken together, these results provide in vitro and in vivo evidence supporting the anti-cancer effect of silymarin and death receptor 5, and caspase-8 may be essential players in silymarin-mediated apoptosis in oral cancer.

Topics: Antineoplastic Agents; Apoptosis; Caspase 8; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Humans; Mouth Neoplasms; Receptors, TNF-Related Apoptosis-Inducing Ligand; Silymarin

The present study is aimed to investigate the apoptosis-inducing effect of icaritin in human oral squamous cell carcinoma (OSCC) cells and the associated mechanisms.. KB and SCC9 cell lines were used as model cell lines. Effect of icaritin on apoptosis was analyzed by flow cytometry. The effect of icaritin on mitochondrial apoptotic pathway was demonstrated by loss of mitochondrial membrane potential and release of cytocrome C from mitochondria. MiR-124 mimic and miR-124 inhibitor were used to manipulate the expression of miR-124 in OSCC cells. SiRNA targeting Sp1 and DNMT1 as well as Sp1 and DNMT1 overexpressing vector were utilized to confirm their roles in the apoptosis-inducing effect of icaritin in OSCC cells. Activation of relevant signaling pathway by icaritin and effect of icaritin on expression of targeting molecules were determined by western blots or qRT-PCR.. Our results showed that icaritin inhibited tumor cell viability in a dose- and time-dependent manner, and induced cell apoptosis via intrinsic mitochondrial pathway by upregulating miR-124. Moreover, our results showed that the icaritin exerted regulatory effect on miR-124 through suppressing Sp1/DNMT1 signaling.. Our data provide the first experimental evidence that icaritin induces mitochondrial apoptosis in OSCC cells by upregulating miR-124 and suggest a new mechanism to explain its anti-tumor effects.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Dose-Response Relationship, Drug; Flavonoids; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Membrane Potential, Mitochondrial; MicroRNAs; Mitochondria; Mouth Neoplasms; RNA Interference; Signal Transduction; Sp1 Transcription Factor; Squamous Cell Carcinoma of Head and Neck; Time Factors; Transfection; Up-Regulation

The aim of this study was to determine the apoptotic activity of methanol extract of Ashwagandha (MEAG) and in human head and neck squamous cell carcinoma (HNSCC) cells and to investigate the underlying mechanisms.. We investigated the effects of MEAG on programmed cell death in HNSCC cells using a Live/Dead assay, detection of nuclear morphologic changes, Mitotracker, siRNA knockdown, and RT-PCR.. Treatment with MEAG showed dose-dependent growth-inhibitory activity that attribute to caspase-dependent apoptosis. Loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase 9 suggested that MEAG leads to activation of mitochondria-mediated apoptosis. MEAG selectively upregulated the expression of Bim protein at the transcriptional level and induced the translocation of Bim into the mitochondria. Knockdown of Bim by siRNA partially blocked MEAG-mediated apoptosis. MEAG also caused an increase in truncated Bid (t-Bid), cleaved caspase-8, and death receptor 5 (DR5). Interestingly, withaferin A (WA), a bioactive component of MEAG, clearly induced apoptosis accompanied by upregulation of Bim, t-Bid, caspase-8, and DR5 similar to the effects of MEAG.. These suggest that MEAG and WA may be potential natural materials for the treatment of HNSCC.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carcinoma, Squamous Cell; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Head and Neck Neoplasms; Humans; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Mouth Neoplasms; Plant Extracts; Receptors, TNF-Related Apoptosis-Inducing Ligand; Squamous Cell Carcinoma of Head and Neck; Up-Regulation; Withanolides

Goniothalamin is a natural occurring styryl-lactone compound isolated from Goniothalamus macrophyllus. It had been demonstrated to process promising anticancer activity on various cancer cell lines. However, little study has been carried out on oral cancer. The aim of this study was to determine the cytotoxic effects of goniothalamin against H400 oral cancer cells and its underlying molecular pathways. Results from MTT assay demonstrated that goniothalamin exhibited selective cytotoxicity as well as inhibited cells growth of H400 in dose and time-dependent manner. This was achieved primarily via apoptosis where apoptotic bodies and membrane blebbing were observed using AO/PI and DAPI/Annexin V-FITC fluorescence double staining. In order to understand the apoptosis mechanisms induced by goniothalamin, apoptosis assessment based on mitochondrial membrane potential assay and cytochrome c enzyme-linked immunosorbent assay were carried out. Results demonstrated that the depolarization of mitochondrial transmembrane potential facilitated the release of mitochondrial cytochrome c into cytosol. Caspases assays revealed the activation of initiator caspase-9 and executioner caspase-3/7 in dose-dependent manners. This form of apoptosis was closely associated with the regulation on Bcl-2 family proteins, cell cycle arrest at S phase and inhibition of NF-κβ translocation from cytoplasm to nucleus. Conclusion, goniothalamin has the potential to act as an anticancer agent against human oral squamous cell carcinoma (H400 cells).

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Cytosol; Down-Regulation; Enzyme Induction; Head and Neck Neoplasms; Humans; Membrane Potential, Mitochondrial; Metabolic Networks and Pathways; Mitochondria; Mouth Neoplasms; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Pyrones; S Phase; Squamous Cell Carcinoma of Head and Neck

Dracaena cinnabari Balf.f. is a red resin endemic to Socotra Island, Yemen. Although there have been several reports on its therapeutic properties, information on its cytotoxicity and anticancer effects is very limited. This study utilized a bioassay-guided fractionation approach to determine the cytotoxic and apoptosis-inducing effects of D. cinnabari on human oral squamous cell carcinoma (OSCC). The cytotoxic effects of D. cinnabari crude extract were observed in a panel of OSCC cell lines and were most pronounced in H400. Only fractions DCc and DCd were active on H400 cells; subfractions DCc15 and DCd16 exhibited the greatest cytotoxicity against H400 cells and D. cinnabari inhibited cells proliferation in a time-dependent manner. This was achieved primarily via apoptosis where externalization of phospholipid phosphatidylserine was observed using DAPI/Annexin V fluorescence double staining mechanism studied through mitochondrial membrane potential assay cytochrome c enzyme-linked immunosorbent and caspases activities revealed depolarization of mitochondrial membrane potential (MMP) and significant activation of caspases 9 and 3/7, concomitant with S phase arrest. Apoptotic proteins array suggested that MMP was regulated by Bcl-2 proteins family as results demonstrated an upregulation of Bax, Bad, and Bid as well as downregulation of Bcl-2. Hence, D. cinnabari has the potential to be developed as an anticancer agent.

Topics: Apoptosis; Biological Assay; Caspases; Cell Cycle Checkpoints; Cell Proliferation; Cell Shape; Chemical Fractionation; Cytochromes c; DNA Fragmentation; Dracaena; Fibroblasts; Humans; Inhibitory Concentration 50; Membrane Potential, Mitochondrial; Mitochondria; Mouth Neoplasms; Neoplasms, Squamous Cell; Nucleosomes; Phosphatidylserines; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; S Phase; Signal Transduction

Direct antitumor effects of bisphosphonates (BPs) have been demonstrated in various cancer cells in vitro. However, the effective concentrations of BPs are typically much higher than their clinically relevant concentrations. Oral cancers frequently invade jawbone and may lead to the release of Ca(2+) in primary lesions. We investigated the effects of the combined application of zoledronic acid (ZA) and Ca(2+) on proliferation and apoptosis of oral cancer cells. Human oral cancer cells, breast cancer cells, and colon cancer cells were treated with ZA at a wide range of concentrations in different Ca(2+) concentration environments. Under a standard Ca(2+) concentration (0.6mM), micromolar concentrations of ZA were required to inhibit oral cancer cell proliferation. Increasing extracellular Ca(2+) concentrations greatly enhanced the potency of the ZA cytocidal effect. The ability of Ca(2+) to enhance the cytocidal effects of ZA was negated by the Ca(2+)-selective chelator EGTA. In contrast, the cytocidal effect of ZA was less pronounced in breast and colon cancer cells regardless of whether extracellular Ca(2+) was elevated. In oral cancer cells incubated with 1.6mM Ca(2+), ZA up-regulated mitochondrial Bax expression and increased mitochondrial Ca(2+) uptake. This was associated with decreased mitochondrial membrane potential and increased release of cytochrome c. We suggest that ZA can specifically produce potent cytocidal activity in oral cancer cells in an extracellular Ca(2+)-dependent manner, implying that BPs may be useful for treatment of oral squamous cell carcinoma with jawbone invasion leading to the hypercalcemic state.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Calcium; Calcium Chelating Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Diphosphonates; Dose-Response Relationship, Drug; Head and Neck Neoplasms; Humans; Imidazoles; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck; Zoledronic Acid

Acacetin (5,7-dihydroxy-40-methoxyflavone), present in safflower seeds, plants, flowers, Cirisium rhinoceros Nakai, has been reported to be able to exert anti-peroxidative, anti-inflammatory, anti-plasmodial, and anti-proliferative activities by inducing apoptosis and blocking the progression of cell cycles.. The objective of this study is to investigate the mechanism of acacetin-induced apoptosis of oral squamous cell carcinoma cell line (HSC-3).. Acacetin caused 50% growth inhibition (IC50) of HSC-3 cells at 25μg/mL over 24h in the MTT assay. Apoptosis was characterized by DNA fragmentation and increase of sub-G1 cells and involved activation of caspase-3 and PARP (poly-ADP-ribose polymerase). Maximum caspase-3 activity was observed with 100μg/mL of acacetin for 24h. Caspase-8 and -9 activation cascades mediated the activation of caspase-3. Acacetin caused reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c into the cytoplasm. Pretreatment with casapse-3 (Z-DEVD-FMK), -8 (Z-IETD-FMK), and 9 inhibitor (z-LEHD-fmk) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c. The mitogen-activated protein kinases (MAPKs) were activated by acacetin. Moreover, pretreating the cells with each of the caspase inhibitor or MAPKs specific inhibitors apparently inhibited acacetin-induced cytotoxicity of HSC-3 cells.. In conclusion, acacetin induce the apoptosis of oral squamous cell carcinoma cell line, which is closely related to its ability to activate the MAPK-mediated signaling pathways with the subsequent induction of a mitochondria- and caspase-dependent mechanism. These results strongly suggest that acacetin might have cancer inhibition and therapeutic potential.

Topics: Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caspase 3; Caspase 8; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Flavones; Flow Cytometry; Humans; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinases; Mouth Neoplasms; Oligopeptides; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Isocudraxanthone K (IK) is a novel, natural compound from a methanol extract of the root bark of Cudrania tricuspidata. It has not been shown previously that IK possessed antitumor activity. We investigated the antitumor effects and molecular mechanism of IK and related signal transduction pathway(s) in oral squamous cell carcinoma cells (OSCCCs). The MTT assay revealed that IK had an antiproliferative effect on OSCCCs, in a dose- and time-dependent manner. IK induced apoptosis in OSCCCs, as identified by a cell-cycle analysis, annexin V-FITC and propidium iodide staining, and the nuclear morphology in cell death. IK caused time-dependent phosphorylation of Akt, p38, and ERK (extracellular signal-regulated kinase). In addition, IK increased the cytosolic to nuclear translocation of nuclear factor-κB (NF-κB) p65 and the degradation and phosphorylation of IκB-α in HN4 and HN12 cells. Furthermore, IK treatment downregulated hypoxia-inducible factor 1α (HIF-1α) and its target gene, vascular endothelial growth factor (VEGF). Cobalt chloride (CoCl2), a HIF-1α activator, attenuated the IK-induced growth-inhibiting and apoptosis-inducing effects, and blocked IK-induced expression of apoptosis regulatory proteins, such as Bax, Bcl-2, caspase-3, caspase-8, and caspase-9, and cytochrome c. Collectively, these data provide the first evidence of antiproliferative and apoptosis-inducing effects of IK as a HIF-1α inhibitor and suggest it may be a drug candidate for chemotherapy against oral cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Drug Screening Assays, Antitumor; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; I-kappa B Proteins; Moraceae; Mouth Neoplasms; Plant Bark; Plant Extracts; Plant Roots; Proto-Oncogene Proteins c-bcl-2; Transcription Factor RelA; Xanthones

Oral squamous cell carcinoma (OSCC) accounts for 5.8% of all malignancies in Taiwan and the incidence of OSCC is on the rise. OSCC is also a common malignancy worldwide and the five-year survival rate remains poor. Therefore, new and effective treatments are needed to control OSCC. In the present study we have investigated the efficacy and associated mechanisms of polyenylpyrroles and their analogs in both in vitro cell culture and in vivo nude mice xenografts. Auxarconjugatin B (compound 1a) resulted in cell cycle arrest in the G2/M phase and caspase-dependent apoptosis in OEC-M1 and HSC-3 cells by activating DNA damage and mitochondria dysfunction through the loss of mitochondrial membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein level, and decrease in B-cell lymphoma-2 level. Compound 1a-induced generation of intracellular reactive oxygen species through cytochrome P450 1A1 was identified as a major mechanism of its effect for DNA damage, mitochondria dysfunction and apoptosis, which was reversed by antioxidant N-acetylcysteine as well as cytochrome P450 1A1 inhibitor and specific siRNA. Furthermore, compound 1a-treated nude mice showed a reduction in the OEC-M1 xenograft tumor growth and an increase in the caspase-3 activation in xenograft tissue. These results provide promising insights as to how compound 1a mediates cytotoxicity and may prove to be a molecular rationale for its translation into a potential therapeutic against OSCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Cell Cycle Checkpoints; Cell Death; Cell Division; Cell Line, Tumor; Cytochromes c; DNA Damage; Female; G2 Phase; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Mouth Neoplasms; Oxidative Stress; Pyrones; Pyrroles; Reactive Oxygen Species; Taiwan

Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino. The authors investigated the effects of Gyp on cell morphology, viability, cell cycle distribution, and induction of apoptosis in human oral cancer SAS cells and the determination of murine SAS xenograft model in vivo.. Flow cytometry was used to quantify the percentage of viable cells; cell cycle distribution; sub-G1 phase (apoptosis); caspase-3, -8, and -9 activity; reactive oxygen species (ROS) production, intracellular Ca(2+) determination; and the level of mitochondrial membrane potential (ΔΨ(m)). Western blotting was used to examine levels of apoptosis-associated proteins, and confocal laser microscopy was used to examine the translocation of proteins in cells.. Gyp induced morphological changes, decreased the percentage of viable cells, caused G0/G1 phase arrest, and triggered apoptotic cell death in SAS cells. Cell cycle arrest induced by Gyp was associated with apoptosis. The production of ROS, increased intracellular Ca(2+) levels, and the depolarization of ΔΨ(m) were observed. Gyp increased levels of the proapoptotic protein Bax but inhibited the levels of the antiapoptotic proteins Bcl-2 and Bcl-xl. Gyp also stimulated the release of cytochrome c and Endo G. Translocation of GADD153 to the nucleus was stimulated by Gyp. Gyp in vivo attenuated the size and volume of solid tumors in a murine xenograft model of oral cancer.. Gyp-induced cell death occurs through caspase-dependent and caspase-independent apoptotic signaling pathways, and the compound reduced tumor size in a xenograft nu/nu mouse model of oral cancer.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Calcium; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Cytochromes c; G1 Phase; Gynostemma; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Plant Extracts; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Resting Phase, Cell Cycle; Signal Transduction; Transcription Factor CHOP

Evidence suggests anti-tumor activities of glucosamine-hydrochloride (GS-HCl). In the present study, we investigated anti-proliferative, growth suppressive and/or pro-apoptotic effects of GS-HCl on YD-8 human oral squamous cell carcinoma (OSCC) cells. Fundamentally, treatment with GS-HCl strongly inhibited proliferation and induced apoptosis in YD-8 cells, as determined by MTS and DNA fragmentation analyses. Of further note, as measured by Western analyses, GS-HCl treatment led to activation of caspase-3, cytosolic accumulation of cytochrome c, down-regulation of Mcl-1 and HIF-1α, up-regulation of GRP78, an indicator of ER stress, and generation of ROS in YD-8 cells. Importantly, results of pharmacological inhibition studies showed that treatment with z-VAD-fmk, a pan-caspase inhibitor, but not with vitamin E, an anti-oxidant strongly blocked the GS-HCl-induced apoptosis in YD-8 cells. Analyses of additional cell culture works further revealed that GS-HCl had a strong growth suppressive effect on not only YD-8 but also YD-10B and YD-38, two other human OSCC cell lines. These findings collectively demonstrate that GS-HCl has anti-proliferative, anti-survival, and pro-apoptotic effects on YD-8 cells and the effects appear to be mediated via mechanisms associated with the mitochondrial-dependent activation of caspases, down-regulation of Mcl-1, and induction of ER stress. Considering HIF-1α as a tumor angiogenic transcription factor, the ability of GS-HCl to down-regulate HIF-1α in YD-8 cells may further support its anti-cancer property. It is thus suggested that GS-HCl may be used as a potential anti-cancer drug against human OSCC.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Down-Regulation; Endoplasmic Reticulum Chaperone BiP; Glucosamine; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mouth Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction

Photodynamic therapy (PDT) with several photosensitizers is a promising modality for the treatment of cancer. In this study, the therapeutic effect of PDT using the synthetic photosensitizer pheophorbide a (Pa-PDT) was examined in AT-84 murine oral squamous cell carcinoma (OSCC) cells. The MTT assay revealed that Pa-PDT induced cell growth inhibition in a dose- and time-dependent manner. Pa-PDT treatment significantly induced intracellular ROS generation, which is critical for cell death induced by Pa-PDT. Cell cycle analysis showed the increased sub-G1 proportion of cells in Pa-PDT-treated cells. Induction of apoptotic cell death was confirmed by DAPI staining and the reduction of mitochondrial membrane potential (ΔΨm) on Pa-PDT-treated cells. The changes in apoptosis-related molecules were next examined using western blotting. Cytochrome c release and cleavage of caspase-3 and PAPR were observed in AT-84 cells, whereas Bcl-2 protein levels were decreased. To determine the therapeutic effect of Pa-PDT in vivo, a murine OSCC animal model was used. Treatment of mice with Pa-PDT significantly inhibited tumor growth, especially PDT with Pa intravenous administration (i.v. Pa-PDT), and increased proliferative cell nuclear antigen (PCNA) levels and TUNEL-stained apoptotic cells compared to vehicle-treated controls. The data demonstrate that the in vitro effects of Pa-PDT on the inhibition of tumor cell proliferation and induction of apoptosis correlate to the anticancer activity of Pa-PDT in vivo. Our findings suggest the therapeutic potential of Pa-PDT in OSCC.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Cytochromes c; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C3H; Mitochondria; Mouth Neoplasms; Photochemotherapy; Photosensitizing Agents; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

In this study, a new lanostane triterpene glycoside (fomitoside-K) having biologically active molecules was isolated from a mushroom Fomitopsis nigra to test its anticancer activity on human oral squamous cell carcinomas (YD-10B). We focused on the effect of fomitoside-K on apoptosis, the mitochondria-mediated death pathway and the accumulation of reactive oxygen species (ROS) in YD-10B cells. Fomitoside-K could induce a dose and time-dependent apoptosis in YD-10B cells as characterized by cell morphology, cell cycle arrest, inhibition of survivin, activation of poly(ADP-ribose) polymerase (PARP), caspase-3, -9 and an increased expression ratio of Bax/Bcl-2. The mitochondria membrane potential loss and cytochrome c (Cyt C) release from mitochondria to cytosol were observed during the induction. Moreover, fomitoside-K caused dose-dependent elevation of intracellular ROS level and increase phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in YD-10B cells. To further investigate the mechanisms, we examined the effects of ROS scavenger N-acetyl-L-cysteine (NAC) and selective inhibitors for mitogen activated protein kinase (MAPK) pathways on the cell death. The fomitoside-K induced cell death by ROS was significantly inhibited by NAC, ERK (PD98059) and JNK inhibitor (SP600125). In addition, fomitoside-K has a synergistic effect with adriamycin in suppressing the growth of YD-10B cells. These data suggest that fomitoside-K induces apoptosis in YD-10B cells through the ROS-dependent mitochondrial dysfunction pathway and provides a mechanistic framework for further exploring the use of fomitoside-K against the proliferation of human oral cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Squamous Cell; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Coriolaceae; Cyclin D1; Cytochromes c; Glycosides; Humans; Mitochondria; Mitogen-Activated Protein Kinases; Mouth Neoplasms; Reactive Oxygen Species; Signal Transduction; Triterpenes

PUMA (a p53 up-regulated modulator of apoptosis) is induced by p53 tumor suppressor and other apoptotic stimuli. It was found to be a principal mediator of cell death in response to diverse apoptotic signals, implicating PUMA as a likely tumor suppressor.. In this study, we examined the efficacy of targeted PUMA gene therapy in human oral cancer (SAS) cells using polyethylenimine (PEI)-mediated transfection for gene delivery.. Exogenous expression of PUMA in SAS cells resulted in apoptosis with cytochrome c release, activation of caspase-3 and -9, and cleavage of PARP. Gene delivery of PEI/PUMA in SAS xenografts induced apoptosis and resulted in significant reductions (∼60%) of tumor growth in vivo. Furthermore, we have shown that PEI-mediated PUMA gene therapy prolonged survival of animals with orthotopic SAS oral cancers.. Taken together, these results indicated that PUMA gene therapy via PEI delivery could be a promising method for the treatment of oral squamous cell carcinoma.

Topics: Analysis of Variance; Animals; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Squamous Cell; Caspase 3; Cytochromes c; Disease Models, Animal; Genetic Therapy; Genetic Vectors; Humans; In Situ Nick-End Labeling; In Vitro Techniques; Kaplan-Meier Estimate; Mice; Mice, SCID; Mouth Neoplasms; Neoplasms, Experimental; Polyethyleneimine; Random Allocation; Sensitivity and Specificity; Statistics, Nonparametric; Survival Rate; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Suppressor Proteins

Phytochemicals have been used as potential chemopreventive or chemotherapeutic agents. However, there are data suggesting a mutagenic effect of some phytochemicals. We hypothesized that safrole would have anticancer effects on human oral squamous cell carcinoma HSC-3 cells. Safrole decreased the percentage of viable HSC-3 cells via induction of apoptosis by an increased level of cytosolic Ca(2+) and a reduction in the mitochondrial membrane potential (ΔΨ(m)). Changes in the membrane potential were associated with changes in the Bax, release of cytochrome c from mitochondria, and activation of downstream caspases-9 and -3, resulting in apoptotic cell death. In vivo studies also showed that safrole reduced the size and volume of an HSC-3 solid tumor on a xenograft athymic nu/nu mouse model. Western blotting and flow cytometric analysis studies confirmed that safrole-mediated apoptotic cell death of HSC-3 cells is regulated by cytosolic Ca(2+) and by mitochondria- and Fas-dependent pathways.

Topics: Analysis of Variance; Animals; Apoptosis; Calcium; Carcinoma, Squamous Cell; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cytochromes c; DNA Damage; Fas Ligand Protein; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mouth Neoplasms; Neoplasm Transplantation; Safrole; Statistics, Nonparametric

Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exerts an anti-tumor effect. This study was performed to elucidate whether the epidermal growth factor (EGF) receptor and phosphatidylinositol-3-kinase (PI3K) signaling pathways are involved in NFD-induced apoptosis of oral squamous cell carcinoma (OSCC). Immunoblot showed that NFD suppressed the phosphorylation of EGF receptor and activation of PI3K/Akt, downstream molecules of EGF receptor signaling pathway, in Ca9-22 cells. The levels of downstream targets of Akt, including phospho-glycogen synthase kinase-3beta (p-GSK-3beta), GSK-3beta, forkhead transcription factor (FKHR), and cyclin D1, were also reduced after NFD treatment. Moreover, inactivation of nuclear factor-kappaB (NF kappaB), modulation of I kappa K beta and I kappaB alpha, up-regulation of Bad, and down-regulation of anti-apoptotic proteins including phospho-Bad, Bcl-X(L), myeloid cell leukemia-1(Mcl-1), and XIAP were found in NFD-treated cells. In addition, NFD treatment disrupted mitochondrial membrane potential (Delta Psi m), resulted in release of cytochrome c, and activation of both caspases-9 and caspase-3. Taken together, these results indicate that NFD induces apoptosis in Ca9-22 cells via inactivation of the EGF receptor-mediated survival pathway.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Enzyme Activation; ErbB Receptors; Humans; Inhibitor of Apoptosis Proteins; Membrane Potential, Mitochondrial; Mitochondria; Mouth Neoplasms; Naphthoquinones; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction

The protein kinase C (PKC) inhibitor safingol increased rounding and detachment of human oral squamous cell carcinoma (SCC) cells in monolayer cultures. When dissociated cells were incubated in the presence of safingol, cell adhesion was prevented and cell viability was lost gradually, while most cells survived in the absence of safingol even if their attachment was blocked by coating the culture plates with polyhydroxyethyl methacrylate. Flow cytometric analysis and agarose gel electrophoresis of cellular DNA revealed an increase in the proportion of sub-G(1) cells and DNA fragmentation, indicating that safingol induced apoptosis of dissociated cells. During the induction of apoptosis in cell suspensions by safingol, there was an increase of the pro-apoptotic BH-3 only protein Bim and decrease of pro-survival Bcl-2 family proteins Bcl-xL and mitochondrial pro-apoptogenic factor endonuclease G translocated to the nucleus. The level of phosphorylated focal adhesion kinase (FAK) required for cell survival also rapidly decreased, followed by a decrease in the protein level. The introduction of siRNA against PKCalpha into SAS cells resulted in an increase of Bim, a decrease of Bcl-xL, the translocation of endonuclease G, and a decrease in the phosphorylation of FAK. These results suggest that Bim, Bcl-xL, FAK and endonuclease G are involved in safingol-induced apoptosis of detached oral SCC cells. Safingol can be used to induce apoptosis with cell detachment, anoikis, of oral SCC cells.

Topics: Anoikis; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Carcinoma, Squamous Cell; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Survival; Cytochromes c; DNA Fragmentation; Endodeoxyribonucleases; Focal Adhesion Protein-Tyrosine Kinases; Humans; Membrane Proteins; Mouth Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins; RNA, Small Interfering; Sphingosine

Although N(1)-guanyl-1,7,-diamineoheptane (GC7), an inhibitor of deoxyhypusine synthase, has been shown to inhibit cell growth, the mechanism of its action is not completely understood. In this study, we investigated the mechanisms of the effects of GC7 on cell growth, differentiation and apoptosis in relation to adenosine monophosphate-activated protein kinase (AMPK) activation, as AMPK is known to be a possible target for cancer treatment.. The effects of GC7 on the growth of immortalized human oral keratinocytes (IHOK) and primary oral cancer cells (HN4), was investigated using MTT assay, Western blotting, cell cycle analysis, DNA fragmentation and expression of apoptotic pathway proteins.. N(1)-guanyl-1,7,-diamineoheptane inhibited cell proliferation in a time- and dose-dependent manner in IHOK and HN4 cells. GC7 treatment decreased the expression of differentiation markers, such as involucrin, CK13 and CK19. The major mechanism of growth inhibition by GC7 treatment was induction of apoptosis, which is supported by sub-G(1) phase arrest, annexin V-FITC staining and DNA fragmentation analysis. GC7 treatment increased the cytosolic level of cytochrome c and resulted in caspase-3 activation. GC7 treatment also resulted in a strong activation of AMPK. Furthermore, specific AMPK activator blocked the GC7-induced growth inhibition effect, as well as apoptosis.. These results demonstrate that GC7 blocks immortalized and malignant keratinocyte cell proliferation and differentiation by inducing apoptosis through the mitochondrial and AMPK pathways. On the basis of these observations, we propose that a strategy combining GC7 and AMPK inhibition could be developed into a novel chemotherapeutic modality in oral cancer.

Topics: AMP-Activated Protein Kinases; Annexin A5; Apoptosis; Carcinoma; Caspase 3; Cell Cycle Proteins; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; G1 Phase; Guanine; Humans; Keratin-13; Keratin-19; Keratinocytes; Mitochondria; Mouth Mucosa; Mouth Neoplasms; Oxidoreductases Acting on CH-NH Group Donors; Protein Precursors

Although hydrogen peroxide may play an important role in the development of cancer, it can be an efficient inducer of apoptosis in cancer cells; the exact mechanism by which this action occurs is not completely understood in oral cancer cells.. In this study, the mechanisms by which H(2)O(2) inhibited growth and induced apoptosis were differentially investigated using HPV-immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4).. H(2)O(2) treatment sensitively and dose-dependently induced growth inhibition and typical apoptosis in IHOK and HN4 cells, as demonstrated by a decreased level of cell viability, an increased population of cells in the sub-G(0)/G(1) phase, ladder formation of the genomic DNA, chromatin condensation and accumulation of Annexin V(+)/PI(+) cells. Furthermore, the expression of Bax, p53 and p21(WAF1/CIP1) increased, whereas the expression of Bcl-2 decreased in immortalized and malignant keratinocytes that were treated with H(2)O(2). In addition, cytochrome-c from the mitochondria was observed in H(2)O(2)-treated IHOK and oral cancer cells, and this was accompanied by the activation of caspase-3 and -9. Additionally, H(2)O(2) treatment induced upregulation of CHOP, GRP78 and several representative endoplasmic reticulum (ER) stress-responsive proteins, including heme oxygenase-1.. Overall, these results suggest that H(2)O(2) triggers apoptosis via the mitochondrial and ER stress pathway in IHOK and HN4 cells, and that increasing the cellular levels of H(2)O(2) sufficiently may lead to selective killing of oral cancer cells and therefore be therapeutically useful.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Chromatin; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; DNA; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Heme Oxygenase-1; Humans; Hydrogen Peroxide; Keratinocytes; Mitochondria; Molecular Chaperones; Mouth Mucosa; Mouth Neoplasms; Oxidants; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Resting Phase, Cell Cycle; Transcription Factor CHOP; Tumor Suppressor Protein p53

In the present study, we examined whether caspases and their upstream regulators are involved in rotenone-induced cytotoxicity. Rotenone significantly inhibited the proliferation of oral cancer cell lines in a dose-dependent manner compared to normal oral mucosal fibroblasts. Flow cytometric analysis of DNA content showed that rotenone treatment induced apoptosis following G2/M arrest. Western blotting showed activation of both the caspase-8 and caspase-9 pathways, which differed from previous studies conducted in other cell types. Furthermore, p53 protein and its downstream pro-apoptotic target, Bax, were induced in SAS cells after treatment with rotenone. Rotenone-induced apoptosis was inhibited by antioxidants (glutathione, N-acetylcysteine, and tiron). In conclusion, our results demonstrate significant involvement of caspases and their upstream regulators in rotenone-induced cytotoxicity.

Topics: Antineoplastic Agents; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Caspase 8; Caspase 9; Cell Division; Cell Line, Tumor; Cell Proliferation; Cytochromes c; G2 Phase; Humans; Mouth Neoplasms; Rotenone; Tumor Suppressor Protein p53

The green tea polyphenol epigallocatechin-3-gallate (EGCG) regulates gene expression differentially in tumor and normal cells. In normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. In humans, reduced expression of p57 often is associated with advanced tumors, and tumor cells with inactivated p57 undergo apoptosis when exposed to EGCG. The mechanism of p57 induction by EGCG is not well understood. Here, we show that in NHEK, EGCG-induces p57 via the p38 mitogen-activated protein kinase (MAPK) signaling pathway. In p57-negative tumor cells, JNK signaling mediates EGCG-induced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis via inhibition of c-Jun N-terminal kinase (JNK). We also found that restoration of p57 expression in tumor cells significantly reduced tumorigenicity in athymic mice. These results suggest that p57 expression may be an useful indicator for the clinical course of cancers, and could be potentially useful as a target for cancer therapies.

Topics: Animals; Anthracenes; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Catechin; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p57; Cytochromes c; Disease-Free Survival; Enzyme Inhibitors; Female; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Nude; Mouth Neoplasms; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Signal Transduction; Transfection; Xenograft Model Antitumor Assays

Many studies have shown the anti-proliferative effects of iron deprivation on cancer cells, but the effects of iron-chelators on oral cancer have not been clearly elucidated.. To investigate the effects of an iron chelator, desferrioxamine (DFO), on the growth of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12) and human skin keratinocytes (HaCaT) in the MTT assay, three-dimensional (3D) raft cultures, Western blotting, cell cycle analysis, nuclear staining and cytochrome c expression for apoptosis signaling pathway were used.. Desferrioxamine inhibited the growth of immortalized IHOK and HaCaT and malignant HN4 and HN12 keratinocytes in a time- and dose-dependent manner according to the MTT assay. The 3D organotypic culture also revealed that DFO-treated cells showed less epithelial maturation, less surface keratinization and decreased epithelial thickness. The major mechanism of growth inhibition with the micromolar DFO treatment was by the induction of apoptosis, which was supported by nuclear DAPI staining, DNA fragmentation analysis and flow cytometric analysis for sub-G(1) phase arrest and Annexin V-FITC (fluorescein isothiocyanate) staining. Furthermore, Bax expression increased together with p53 and p21(WAF1/CIP1), while the Bcl-2 expression decreased in the immortalized and malignant keratinocytes treated with DFO. Time-dependent cytochrome c from mitochondria was observed in DFO-treated IHOK and oral cancer cells and was accompanied by the activation of caspase-3 in IHOK cells.. These results demonstrate that DFO has growth inhibitory effects on immortalized and malignant oral keratinocytes through the induction of apoptosis and suggest that further evaluation of DFO as a potential therapeutic agent for human oral precancerous lesions is warranted.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Deferoxamine; Enzyme Activation; Flow Cytometry; Humans; Keratinocytes; Mouth Neoplasms; Siderophores; Skin

Iron is essential for neoplastic cell growth, and iron chelators have been tested for potential anti-proliferative and anti-cancer effects, but the effects of iron chelators on oral cancer have not been clearly elucidated. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during iron chelator-induced apoptosis and differentiation of immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4). The iron chelator deferoxamine (DFO) exerted potent time- and dose-dependent inhibitory effects on the growth and apoptosis of IHOK and HN4 cells. DFO strongly activates p38 MAP kinase and extracellular signal-regulated kinase (ERK), but does not activate c-Jun N-terminal kinase/stress-activated protein kinase. Of the three MAP kinase blockers used, the selective p38 MAP kinase inhibitor SB203580 and ERK inhibitor PD98059 protected IHOK and HN4 cells against iron chelator-induced cell death, which indicates that the p38 and ERK MAP kinase is a major mediator of apoptosis induced by this iron chelator. Interestingly, treatment of IHOK and HN4 cells with SB203580 and PD98059 abolished cytochrome c release, as well as the activation of caspase-3 and caspase-8. DFO suppressed the expression of epithelial differentiation markers such as involucrin, CK6, and CK19, and this suppression was blocked by p38 and ERK MAP kinase inhibitors. Collectively, these data suggested that p38 and ERK MAP kinase plays an important role in iron chelator-mediated cell death and in the suppression of differentiation of oral immortalized and malignant keratinocytes, by activating a downstream apoptotic cascade that executes the cell death pathway.

Topics: Apoptosis; Caspase 3; Caspase 8; Caspases; Cell Differentiation; Cell Proliferation; Cytochromes c; Deferoxamine; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Ferric Compounds; Flavonoids; Humans; Imidazoles; Iron Chelating Agents; Keratinocytes; Mouth Neoplasms; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Protein Precursors; Pyridines

Nitric oxide (NO) is known to act cytostatically on several tumor cell when functioning as an effector molecule of activated macrophages, but the differential effects of NO on immortalized and malignant oral keratinocytes have not been examined.. We investigated the influence of NO on the proliferation, cell cycle, apoptosis, and differentiation of immortalized human oral keratinocytes (IHOK) and primary oral cancer cells (HN4) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, sulforhodamine B (SRB) assay, flow cytometry, nuclear DNA staining, and Western blotting.. The MTT and SRB assays indicated inhibited growth of IHOK and HN4 cells that were treated with sodium nitroprusside (SNP) at concentrations higher than 1 mM but not at lower SNP concentrations. The higher concentrations of SNP up-regulated the apoptosis-related protein expression, which is consistent with the analyses of sub-G(1) phase arrest, annexin V-FITC (fluorescein isothiocynate) staining, nuclear staining, and DNA fragmentation. On the other hand, the lower concentrations of SNP enhanced the expression of keratinocyte differentiation markers in IHOK and HN4 cells.. These data suggest that high concentrations of NO can inhibit the growth of IHOK and HN4 cells through the induction of apoptosis, while low concentrations of NO can induce cytodifferentiation. The dual effects of NO, namely, the induction of apoptosis or cytodifferentiation, have important implications for the possible anti-oral cancer treatment.

Topics: Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Differentiation; Cell Line, Transformed; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Keratinocytes; Mouth Mucosa; Mouth Neoplasms; Nitric Oxide

Norcantharidin (NCTD), the demethylated analogue of cantharidin, has been used to treat human cancers in China since 1984. It was recently found to be capable of inducing apoptosis in human colon carcinoma, hepatoma and glioblastoma cells by way of an elusive mechanism. In this study, we demonstrated that NCTD also induces apoptosis in human oral cancer cell lines SAS (p53 wild-type phenotype) and Ca9-22 (p53 mutant) as evidenced by nuclear condensation, TUNEL labeling, DNA fragmentation and cleavage of PARP. Apoptosis induced by NCTD was both dose- and time-dependent. We found NCTD did not induce Fas and FasL, implying that it activated other apoptosis pathways. Our data showed that NCTD caused accumulation of cytosolic cytochrome c and activation of caspase-9, suggesting that apoptosis occurred via the mitochondria mediated pathway. NCTD enhanced the expression of Bax in SAS cells consistent with their p53 status. Moreover, we showed that NCTD downregulated the expression of Bcl-2 in Ca9-22 and Bcl-XL in SAS. Our results suggest that NCTD-induced apoptosis in oral cancer cells may be mediated by an increase in the ratios of proapoptotic to antiapoptotic proteins. Since oral cancer cells with mutant p53 or elevated Bcl-XL levels showed resistance to multiple chemotherapeutic agents, NCTD may overcome the chemoresistance of these cells and provide potential new avenues for treatment.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Caspases; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; Humans; In Situ Nick-End Labeling; Mouth Neoplasms; Proto-Oncogene Proteins c-bcl-2

In patients with oral squamous cell carcinoma, a high proportion of T cells in the tumor undergo apoptosis, which correlates with Fas ligand (FasL) expression on tumor cells. The present study was done to identify mechanisms responsible for apoptosis of T cells seen in the peripheral circulation of these patients.. Sera of 27 patients, normal donor sera, and supernatants of cultured normal or tumor cells were fractionated by size exclusion chromatography and ultracentrifugation to isolate microvesicles. The presence of microvesicle-associated FasL was studied by Western blots, blocking with anti-Fas reagents, and immunoelectron microscopy. Biological activities of microvesicles were tested including the ability to induce apoptosis of Jurkat and T-cell blasts. Semiquantitative analysis of FasL in microvesicles was correlated with caspase-3 activity, DNA fragmentation, cytochrome c release, loss of mitochondrial membrane potential, and TCR-zeta chain expression in lymphocytes.. FasL-positive (FasL+) microvesicles were detected in sera of 21 of 27 patients. Microvesicles contained 42 kDa FasL. These microvesicles induced caspase-3 cleavage, cytochrome c release, loss of mitochondrial membrane potential, and reduced TCR-zeta chain expression in target lymphocytes. Biological activity of the FasL+ microvesicles was partially blocked by ZB4 anti-Fas monoclonal antibody. Microvesicle-associated FasL levels correlated with the patients' tumor burden and nodal involvement.. Sera of patients with active oral squamous cell carcinoma contain FasL+ microvesicles, which induce the receptor and mitochondrial apoptotic pathways in Jurkat and activated T cells.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Cytoplasmic Vesicles; Down-Regulation; Fas Ligand Protein; fas Receptor; Female; Flow Cytometry; Humans; Jurkat Cells; Male; Membrane Glycoproteins; Membrane Proteins; Microscopy, Electron; Middle Aged; Mitochondria; Mouth Neoplasms; Receptors, Antigen, T-Cell; T-Lymphocytes

Apoptosis induced by docetaxel that interferes with microtubule polymerization dynamics and is used clinically to treat advanced cancers, has not been fully defined in squamous cell carcinoma. In this study, apoptotic events involved in docetaxel treatment were investigated. When the human oral squamous cell carcinoma cell line HSC-3 was exposed to docetaxel for 72 h, a dose-dependent effect was observed in apoptosis using the TUNEL method. We observed activation of caspase cascade including activities like caspase-3, -8, and -9. And the pan-caspase inhibitor z-VAD-fmk prevented apoptosis induced by docetaxel (0.1 microM), showing participation of caspases in this process. Since an antagonistic CD95-antibody (ZB4) exerted no effect on docetaxel-induced apoptosis, CD95/CD95L interaction was not involved in this pathway. The caspase-8-like activity was inhibited not only by IETD-fmk (caspase-8) but also by DEVD-fmk (caspase-3). The results indicate that the caspase-8-like activation occurred downstream of DEVDase. Docetaxel promoted the formation of reactive oxygen species (ROS) in mitochondria, and preincubation of cells with anti-oxidants such as N-acetyl cysteine and pyrrolidine dithiocarbamate, protected against apoptosis mediated by docetaxel. Furthermore, treatment with docetaxel elicited reduction of mitochondrial membrane potential, and release of cytochrome c to cytosol, after 48 h of treatment. We observed binding activity to NF-kappaB consensus site and interference with the mitochondrial function via NF-kappaB after docetaxel treatment. Preventing pro-apoptotic property of NF-kappaB inhibited docetaxel-induced apoptosis. Thus, these results suggest that, following the activation of NF-kappaB by docetaxel, apoptosis is elicited through a mitochondria-dependent pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Squamous Cell; Caspase 8; Caspases; Cytochromes c; Docetaxel; Humans; Membrane Potentials; Mitochondria; Mouth Neoplasms; NF-kappa B; Reactive Oxygen Species; Taxoids; Tumor Cells, Cultured

The transcriptional factor hypoxia-inducible factor-1 (HIF-1) plays an important role in solid tumor cell growth and survival. Overexpression of HIF-1alpha has been demonstrated in many human tumors and predicts a poor response to chemoradiotherapy. We examined the HIF-1alpha-induced survival pathways in human oral squamous cell carcinoma cell (OSCC) lines. The results showed that forced expression of HIF-1alpha suppressed hypoxia-induced apoptosis of OSCC lines by inhibiting cytochrome c release from mitochondria. Overexpression of HIF-1alpha inhibited the generation of reactive oxygen species (ROS), elevation of intracellular Ca(2+) concentration, reduction of mitochondrial membrane potential, and cytosolic accumulation of cytochrome c, which resulted in the inactivation of caspase-9 and caspase-3. In addition, antiapoptotic Bcl-2 and Bcl-X(L) levels were increased and pro-apoptotic Bax and Bak levels were decreased in the HIF-1alpha-overexpressing OSCC line. Overexpression of HIF-1alpha also increased the levels of phosphorylation of Akt and extracellular signal-regulated kinases (ERK). These findings indicate that HIF-1alpha prevents apoptotic cell death through two mechanisms, including inhibition of cytochrome c release and activation of Akt and ERK.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; Calcium; Carcinoma, Squamous Cell; Cell Line, Tumor; Cytochromes c; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Proteins; Mouth Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Transcription Factors; Up-Regulation

We examined the mechanism involved in the induction of apoptosis in human oral cancer (B88) cells with 5-fluorouracil (5-FU) and cisplatin (CDDP) combination. Three different combination treatment sequences were evaluated: i) 5-FU administered simultaneously with CDDP for 72 h (sequence I); ii) CDDP administered for 24 h before 5-FU treatment for 48 h (sequence II); and iii) 5-FU administered for 24 h before CDDP treatment for 48 h (sequence III). When combining the two drugs at doses 40% of their respective cytotoxicity, the growth-suppressing ratios were 49, 55, and 40% in sequences I, II, and III, respectively. Caspase 3 was significantly activated in sequence II as compared to its level of activation in sequence I or III. The mitochondrial release of cytochrome c was much greater in sequence II than in sequence III. In addition, activation of caspase 8 was most strongly activated in sequence II as compared with sequences I and III. Activation of caspase-9 was also observed in sequence II, and, to a lesser extent, in sequence III. Finally, although Bcl-2 was reduced in sequence II, no significant change was observed in sequence III. Accordingly, the data presented here demonstrate that sequence II showing a significant increase in the induction of apoptosis of cancer cells, is superior to sequences I and III.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Squamous Cell; Caspases; Cisplatin; Cytochromes c; Enzyme Activation; Fluorouracil; Humans; Mitochondria; Mouth Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

We are attempting to elucidate the mechanism of apoptotic cell death induced by hypoxia in oral cancer cells. Since hypoxia can render solid tumors more resistant to radiation and chemotherapy, understanding the pathways involved in hypoxia-induced apoptosis of oral cancer cells would be of significant therapeutic value.. Here we showed that oral cancer cells from primary tumor and lymph node metastasis undergo apoptosis after 24 to 48 h of hypoxia. During hypoxic growth, an increase in caspase-3 proteolytic activity was observed, accompanied by the cleavage of PARP (poly (ADP-ribose) polymerase) indicative of caspase activity. In addition, hypoxic stress also lead to activation of caspase-8, -9, and -10 but not -1, elicited the release of cytochrome C into the cytosol, and resulted in internucleosomal DNA fragmentation.. These results show that hypoxia-induced apoptosis in oral carcinoma cell lines relies on both intrinsic (mitochondrial) and extrinsic (cell death receptor mediated) pathways. This novel evidence will assist in designing more efficient combination chemotherapy approaches as promising strategy for the treatment of oral cancers.

Topics: Aged; Apoptosis; Carcinoma; Caspases; Cell Hypoxia; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Cytochromes c; DNA Fragmentation; Humans; Male; Middle Aged; Mouth Neoplasms; Poly(ADP-ribose) Polymerases; Signal Transduction