cytochrome-c-t and Head-and-Neck-Neoplasms

Cisplatin is among the most important chemotherapeutic agents ever developed. It is a critical component of therapeutic regimens in a broad range of malignancies. However, more than a generation after its clinical introduction, the exact mechanism of cisplatin action on tumor cells is not fully defined. The preponderance of research over the last three decades has focused on cisplatin interactions with nuclear DNA which are felt to lead to apoptotic cell death in sensitive cells. However, recent data have shown that cisplatin may have important direct interactions with mitochondria which can induce apoptosis and may account for a significant portion of the clinical activity associated with this drug. These direct interactions between cisplatin and mitochondria may have critical implications for our understanding of this class of drugs and the development of new therapeutic agents.

Topics: Antineoplastic Agents; Apoptosis; Cell Death; Cisplatin; Cytochromes c; Head and Neck Neoplasms; Humans; Ion Channel Gating; Mitochondria; Voltage-Dependent Anion Channels

Topics: Apoptosis; Cell Line, Tumor; Cytochromes c; Flavonoids; Flavonols; G2 Phase Cell Cycle Checkpoints; Head and Neck Neoplasms; Humans; Kaempferols; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

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

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

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

The aim of this study was to investigate how head and neck squamous cell carcinoma (HNSCC) tissue biopsies maintained in a pseudo in vivo environment within a bespoke microfluidic device respond to radiation treatment.. Feasibility study.. Tertiary referral center.. Thirty-five patients with HNSCC were recruited, and liver tissue from 5 Wistar rats was obtained. A microfluidic device was used to maintain the tissue biopsy samples in a viable state. Rat liver was used to optimize the methodology. HNSCC was obtained from patients with T1-T3 laryngeal or oropharyngeal SCC; N1-N2 metastatic cervical lymph nodes were also obtained. Irradiation consisted of single doses of between 2 Gy and 40 Gy and a fractionated course of 5×2 Gy. Cell death was assessed in the tissue effluent using the soluble markers lactate dehydrogenase (LDH) and cytochrome c and in the tissue by immunohistochemical detection of cleaved cytokeratin18 (M30 antibody).. A significant surge in LDH release was demonstrated in the rat liver after a single dose of 20 Gy; in HNSCC, it was seen after 40 Gy compared with the control. There was no significant difference in cytochrome c release after 5 Gy or 10 Gy. M30 demonstrated a dose-dependent increase in apoptotic index for a given increase in single-dose radiotherapy. There was a significant increase in apoptotic index between 1×2 Gy and 5×2 Gy.. M30 is a superior method compared with soluble markers in detecting low-dose radiation-induced cell death. This microfluidic technique can be used to assess radiation-induced cell death in HNSCC and therefore has the potential to be used to predict radiation response.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Death; Cytochromes c; Feasibility Studies; Head and Neck Neoplasms; Humans; Immunohistochemistry; Keratin-18; L-Lactate Dehydrogenase; Laryngeal Neoplasms; Liver; Microfluidic Analytical Techniques; Oropharyngeal Neoplasms; Radiotherapy Dosage; Rats; Rats, Wistar

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

Photodynamic therapy in combination with different treatment modalities has been evaluated to study the mechanism of cellular cytotoxicity and apoptosis in various forms of cancer. In the present study, human head and neck cancer cells were treated with radachlorin mediated photodynamic therapy and the chemotherapy drug, carboplatin singly or in combination. Several parameters were studied to check the enhanced cytotoxicity of combination therapy at different time interval. From the cell viability study by MTT assay, a 22% decrease in cell viability was observed in combination treatment. This enhanced activity of combination treatment was confirmed by cell migration assay and Hoechst PI staining. Generation of reactive oxygen species was observed and found to be higher than that of individual treatments. Cytochrome c was found to be released from mitochondria that also induced the enhance efficacy in combination treatment. The expression of other proteins like EGFR and PARP was also modulated with the time of incubation after treatment. In the tumor xenograft study in nude mouse model, the carboplatin treated group did not show any noticeable changes in tumor volume whereas tumor volume was reduced in PDT and the combination group. Though the difference in the reduction of the tumor size was not significant between PDT and combination group, there was a difference in the expression of EGFR between these two groups. Histologic study of the inhibition in tumor growth was also performed. Therefore, this study may provide an avenue of combating head and neck cancer by a combination of conventional chemotherapy and PDT.

Topics: Animals; Carboplatin; Cell Line, Tumor; Cell Movement; Cell Survival; Combined Modality Therapy; Cytochromes c; Drug Synergism; ErbB Receptors; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Mitochondria; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Transplantation, Heterologous

Tumors are heterogeneous masses of cells characterized pathologically by their size and spread. Their chaotic biology makes treatment of malignancies hard to generalize. We present a robust and reproducible glass microfluidic system, for the maintenance and "interrogation" of head and neck squamous cell carcinoma (HNSCC) tumor biopsies, which enables continuous media perfusion and waste removal, recreating in vivo laminar flow and diffusion-driven conditions. Primary HNSCC or metastatic lymph samples were subsequently treated with 5-fluorouracil and cisplatin, alone and in combination, and were monitored for viability and apoptotic biomarker release 'off-chip' over 7 days. The concentration of lactate dehydrogenase was initially high but rapidly dropped to minimally detectable levels in all tumor samples; conversely, effluent concentration of WST-1 (cell proliferation) increased over 7 days: both factors demonstrating cell viability. Addition of cell lysis reagent resulted in increased cell death and reduction in cell proliferation. An apoptotic biomarker, cytochrome c, was analyzed and all the treated samples showed higher levels than the control, with the combination therapy showing the greatest effect. Hematoxylin- and Eosin-stained sections from the biopsy, before and after maintenance, demonstrated the preservation of tissue architecture. This device offers a novel method of studying the tumor environment, and offers a pre-clinical model for creating personalized treatment regimens.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biopsy; Carcinoma, Squamous Cell; Cell Proliferation; Cisplatin; Cytochromes c; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Hydro-Lyases; Lymphatic Metastasis; Male; Microfluidic Analytical Techniques; Neoplasm Proteins; Tumor Cells, Cultured

Overexpression of inhibitors of apoptosis proteins (IAP) contributes to therapeutic resistance. Second mitochondria-derived activator of caspase (Smac) promotes caspase activation by binding to IAPs upon release from the mitochondria. IAP antagonists, also called SMAC mimetics, are promising anticancer agents modeled after this mechanism. We investigated the role and mechanisms of Smac- and Smac mimetic-mediated chemosensitization in head and neck squamous cell carcinoma (HNSCC) cells.. The effects of SMAC knockdown, SMAC overexpression, and a small molecule Smac mimetic on the chemosensitivities of HNSCC cells were determined. The mechanisms of Smac- and Smac mimetic-mediated chemosensitization were investigated by analyzing growth suppression, the mitochondrial apoptotic pathway, caspase activation, and IAP proteins. The therapeutic responses of HNSCC cells with different levels of Smac were compared in xenograft models.. We found that Smac mediates apoptosis induced by several classes of therapeutic agents through the mitochondrial pathway. SMAC knockdown led to impaired caspase activation, mitochondrial membrane depolarization, and release of cytochrome c. A small molecule Smac mimetic, at nanomolar concentrations, significantly sensitized HNSCC cells to gemcitabine-induced apoptosis and restored gemcitabine sensitivity in SMAC knockdown cells, through caspase activation, X-linked IAP dissociation, and mitochondria-associated events, but not the TNF-α pathway. Furthermore, Smac levels modulated the therapeutic response of HNSCC cells to gemcitabine in xenograft models.. Our results establish a critical role of Smac in mediating therapeutic responses of HNSCC cells and provide a strong rationale for combining Smac mimetics with other anticancer agents to treat HNSCC.

Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Biomimetic Materials; Blotting, Western; Caspase 3; Cell Line, Tumor; Cytochromes c; Deoxycytidine; Drug Synergism; Enzyme Activation; Female; Gemcitabine; Head and Neck Neoplasms; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Nude; Mitochondria; Mitochondrial Proteins; RNA Interference; Signal Transduction; Xenograft Model Antitumor Assays

Treatment of head and neck cancers is still rather poor and worldwide new treatment options are sought. Sensitizing radioresistant tumours by combining irradiation with other therapeutics to induce apoptosis are widely investigated. We examined whether chicken anaemia virus-derived apoptin protein would have a beneficial effect on irradiation of radiosensitive SCC61 and radioresistant SQD9 human head and neck squamous carcinoma cell lines. In both cell lines, concurrent exposure to irradiation and apoptin resulted in analysed mitochondrial cytochrome c release and in cleavage of caspase-3, whereas irradiation alone of SQD9 cells under identical conditions did not. Moreover, in comparison with the irradiation, only the synchronized treatment of apoptin and irradiation resulted in increased cell death in especially the radioresistant SQD9 cells, as measured by means of a colony survival assay. Our data reveal that apoptin treatment represents an effective way for enhancing radiotherapy of tumours responding poorly to radiotherapy.

Topics: Capsid Proteins; Carcinoma, Squamous Cell; Caspase 3; Cell Death; Cell Line, Tumor; Combined Modality Therapy; Cytochromes c; Head and Neck Neoplasms; Humans; Mitochondria; Radiation-Sensitizing Agents

Cyclin-dependent kinases (CDKs) are involved in the regulation of the cell cycle and the growth of tumor cells. In this study, we investigated the antitumor effect and differentially expressed genes (DEGs) in head and neck cancer cells treated by a novel CDK inhibitor, 2-[1,1'-biphenyl]- 4-yl-N-[5-(1,1-dioxo-1lambda(6)-isothiazolidin-2-yl)-1H-indazol-3-yl] acetamide (BAI).. Cell growth was measured by XTT assay. Cell cycle and apoptosis were determined using flow cytometry. GeneFishing PCR was utilized to identify DEGs. Protein expression was analyzed by Western blot.. Exposure to BAI of 2 different head and neck cancer cell lines, AMC-HN4 and AMC-HN6, induced apoptosis in association with growth inhibition, cell cycle arrest, caspase-3 activation and cytochrome c release. Significantly, data from GeneFishing PCR experiments demonstrated 10 DEGs in AMC-HN6 cells treated with BAI. Some of these DEGs turned out to encode proteins with functions related to key cellular processes.. These results indicate that BAI has strong anticancer activities on head and neck cancer cells, and the DEGs induced by BAI may become involved in BAI-induced cancer cell death.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cyclin-Dependent Kinases; Cytochromes c; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Head and Neck Neoplasms; Humans; Indazoles; Protein Kinase Inhibitors; Thiazolidines; Type C Phospholipases

MicroRNAs (mirs) are small noncoding RNA molecules (~22 nucleotides) that regulate posttranscriptional gene expression. Currently, there has not been a comprehensive study of their role in primary head and neck squamous cell carcinoma (HNSCC). To determine the role of mirs in HNSCC, we screened for altered microRNA expression in HNSCC primary tissue and cell lines. We then further tested the functional impact of alterations of specific mirs. An initial screening of 4 primary HNSCC, 4 normal mucosal controls and 4 HNSCC cell lines was analyzed for mature microRNA expression by microarray. Significance was determined using significance analysis of microarrays (SAM). Nine microRNAs were found by SAM to be upregulated or downregulated in tumor tissue including mir-21, let-7, 18, 29c, 142-3p, 155, 146b (overexpressed) and 494 (underexpressed). Mir-21 was validated by qRT-PCR. Functional validation by growth assays was performed, further validating mir-21. Transfection of mir-21 into JHU-011 and JHU-012 cell lines showed a 39% increase in cell growth at 72 hr relative to controls (p < 0.05). Transfection of the inhibitor into JHU-O12 cell lines showed a 92% decrease in cell growth relative to controls at 72 hr (p < 0.05). In addition, flow cytometry analysis of JHU-012 cells 48 hr after mir-21 inhibitor transfection showed a statistically significant increase in cytochrome c release and increased apoptosis. These differentially expressed microRNAs may be of interest as potential novel oncogenes and tumor suppressor genes in HNSCC. Mir-21 is a putative oncogenic microRNA in head and neck cancer.

Topics: Apoptosis; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Flow Cytometry; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; MicroRNAs; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Ubiquitin-Protein Ligases; Up-Regulation

Cisplatin is a chemotherapeutic agent that is widely used to treat cancers such as head and neck squamous cell carcinoma (HNSCC). Previously, we have reported that cisplatin induced an early caspase-dependent apoptosis (8 hr) in a HNSCC cell, HN4. In this study, we examined a late caspase-independent apoptosis as well as an early caspase-dependent apoptosis in cisplatin-treated HN4 cells. While z-VAD-fmk, a pan-caspase inhibitor, blocked the caspase activities and protected cells from the early apoptosis, it did not provide protection against delayed apoptosis occurring after extended exposure (16 hr) to cisplatin, suggesting that the delayed apoptotic response in the presence of z-VAD-fmk was caspase-independent. Cisplatin treatment induced reactive oxygen species (ROS) generation, loss of the mitochondrial membrane potential (MMP) and nuclear translocation of endonuclease G (EndoG). Small interfering RNA mediated-knockdown of EndoG significantly protected cells from the delayed apoptosis induced by cisplatin in the presence of z-VAD-fmk. Overexpression of Bcl-2 in HN4 cells prevented loss of MMP, nuclear translocation of EndoG and protected cells from the delayed apoptosis induced by cisplatin in the presence of z-VAD-fmk. Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, prevented both ROS generation, loss of the MMP and nuclear translocation of EndoG. Together, our data indicate that cisplatin treatment induced ROS-mediated loss of the MMP, and, then, the nuclear translocation of EndoG, which played a crucial role in caspase-independent apoptosis of HN4 cells in the presence of z-VAD-fmk. This is the first report about the involvement of EndoG in cisplatin-induced caspase-independent apoptosis of cells.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caspases; Cisplatin; Cytochromes c; Endodeoxyribonucleases; Head and Neck Neoplasms; Humans; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Mitochondria; Protein Transport; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Subcellular Fractions

Mitochondrial dysfunction has been linked to defects in the apoptotic pathway, and solid tumors, including head and neck squamous cell carcinoma (HNSCC), exhibit defects in apoptosis. Loss of mitochondrial membrane potential (DeltaPsim) is an early initiating event in the mitochondrial apoptotic pathway. We investigated the apoptotic response of 3 head and neck cancer cell lines treated with a mitochondrial-membrane-depolarizing agent, valinomycin, and studied the ability of depolarization to induce release of cytochrome c in these cell lines.. HNSCC cell lines JHU-011, -012 and -019, and a leukemia control cell line HL-60 were assayed for DeltaPsim after valinomycin treatment by staining with mitochondrial-membrane-potential-specific probe JC-1 and stained with apoptosis-specific probe annexin-V to measure their rate of apoptosis by FACS. Western blotting was also applied to detect cytoplasmic cytochrome c release.. DeltaPsim in head and neck cell lines started to show slight loss of DeltaPsim, while HL-60 showed significant loss of DeltaPsim after 30 min of treatment. All cell lines demonstrated complete mitochondrial depolarization within 24 h, however, only the control cell line HL-60 underwent apoptosis. In addition, HNSCC cell lines did not demonstrate cytoplasmic cytochrome c release despite significant mitochondrial membrane depolarization, while HL-60 cell initiated apoptosis and cytochcrome c release after 24 h of treatment.. Head and neck cancer cell lines exhibit defects in mitochondrial-membrane-depolarization-induced apoptosis as well as impaired release of cytochrome c despite significant mitochondrial membrane depolarization. Proximal defects in the mitochondrial apoptosis pathway are a feature of HNSCC.

Topics: Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cytochromes c; Flow Cytometry; Head and Neck Neoplasms; Humans; Membrane Potential, Mitochondrial; Mitochondria

The Bcl-2 oncoprotein is a potent inhibitor of apoptosis and is overexpressed in a wide variety of malignancies. Until recently, it was generally accepted that Bcl-2 primarily mediates its antiapoptotic function by regulating cytochrome c release from mitochondria. However, more recent studies have shown that Bcl-2 is present on several intracellular membranes and mitochondria may not be the only site where Bcl-2 exercises its survival function. In this study, we investigated if Bcl-2 can protect endothelial cells against gamma-radiation by a cytochrome c-independent signaling pathway. Human dermal microvascular endothelial cells (HDMEC), when exposed to gamma-radiation, exhibited a time-dependent activation of caspase-3 that was associated with increased cytochrome c release from mitochondria. Bcl-2 expression in endothelial cells (HDMEC-Bcl-2) significantly inhibited irradiation-induced caspase-3 activation. However, Bcl-2-mediated inhibition of caspase-3 was significantly reversed by inhibition of the Raf-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway. Interestingly, caspase-3 activation in HDMEC-Bcl-2 cells was not associated with cytochrome c release. We also observed that endothelial cell Bcl-2 expression significantly increased the expression of survivin and murine double minute-2 (Mdm2) via the Raf-MEK-ERK pathway. Endothelial cells expressing Bcl-2 also inhibited gamma-radiation-induced activation of p38 MAPK and p53 accumulation. Inhibition of p53 accumulation in HDMEC-Bcl-2 could be due to the enhanced expression of Mdm2 in these cells. Taken together, these results show three mechanisms by which Bcl-2 may mediate endothelial cell cytoprotection independently of cytochrome c release: (a) increased survivin expression, (b) inhibition of p53 accumulation, and (c) inhibition of p38 MAPK.

Topics: Apoptosis; Caspase 3; Caspase Inhibitors; Cytochromes c; Endothelial Cells; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gamma Rays; Head and Neck Neoplasms; Humans; Inhibitor of Apoptosis Proteins; MAP Kinase Signaling System; Microtubule-Associated Proteins; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Proto-Oncogene Proteins c-raf; Radiation Tolerance; RNA, Small Interfering; Survivin; Tumor Suppressor Protein p53; Up-Regulation

N-(4-hydroxyphenyl)retinamide (4HPR), which has shown efficacy in cancer chemopreventionand therapy, induces the mitochondrial apoptosis pathway via increased generation of reactive oxygen species (ROS). ROS is also known to be able to induce an endoplasmic reticulum (ER) stress response, which can contribute to apoptosis but may also antagonize it. Therefore, we used human head and neck squamous cell carcinoma (HNSCC) cells to determine whether 4HPR affects ER stress. Different experimental approaches have indicated that 4HPR induces ER stress response: electron microscopy, which showed extensive ER dilation; splicing of the X-box binding protein 1 (XBP-1), a marker of unfolded protein response (UPR) activation; and quantitative real-time PCR and immunoblotting, which revealed the upregulation of several ER-stress associated mRNAs and proteins, including the chaperone heat shock protein HSPA1A. Most of these effects of 4HPR were abrogated by cotreatment of cells with the antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) indicating that they were downstream of the increase in ROS. Furthermore, siRNA-mediated silencing and chemical inhibition of HSPA1A, which exerts either pro- or anti-apoptotic effects, decreased 4HPR-induced apoptosis. These results demonstrate that 4HPR induces ER stress and uncovered a pro-apoptotic role for HSPA1A in 4HPR-induced apoptosis.

Topics: Anticarcinogenic Agents; Antioxidants; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caspases; Cytochromes c; DNA-Binding Proteins; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Fenretinide; Head and Neck Neoplasms; HSP70 Heat-Shock Proteins; Humans; Nuclear Proteins; Oxidative Stress; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Regulatory Factor X Transcription Factors; Reverse Transcriptase Polymerase Chain Reaction; RNA Splicing; RNA, Messenger; RNA, Small Interfering; Transcription Factors; Tumor Cells, Cultured; X-Box Binding Protein 1

N-(4-hydroxyphenyl)retinamide (4HPR), a synthetic retinoid effective in cancer chemoprevention and therapy, is thought to act via apoptosis induction resulting from increased reactive oxygen species (ROS) generation. As ROS can activate MAP kinases and protein kinase C (PKC), we examined the role of such enzymes in 4HPR-induced apoptosis in HNSCC UMSCC22B cells. 4HPR increased ROS level within 1 h and induced activation of caspase 3 and PARP cleavage within 24 h. Activation of MKK3/6 and MKK4, JNK, p38 and ERK was detected between 6 and 12 h, increased up to 24 h and preceded apoptosis. 4HPR-induced activation of these kinases was abrogated by the antioxidants BHA and vitamin C. SP600125, a JNK inhibitor, suppressed 4HPR-induced c-Jun phosphorylation, cytochrome c release from mitochondria and apoptosis. Suppression of JNK1 and JNK2 using siRNA decreased, whereas overexpression of wild type-JNK1 enhanced 4HPR-induced apoptosis. PD169316, a p38, inhibitor suppressed phosphorylation of Hsp27 and apoptosis. PD98059, an MEK1/2 inhibitor, also suppressed ERK1/2 activation and apoptosis induced by 4HPR. Likewise, PKC inhibitor GF109203X suppressed ERK and p38 phosphorylation and PARP cleavage. These data indicate that 4HPR-induced apoptosis is triggered by ROS increase, leading to the activation of the mitogen-activated protein serine/threonine kinases JNK, p38, PKC and ERK, and subsequent apoptosis.

Topics: Anticarcinogenic Agents; Antioxidants; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cytochromes c; Enzyme Activation; Enzyme Inhibitors; Fenretinide; Head and Neck Neoplasms; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase C; Proto-Oncogene Proteins c-jun; Reactive Oxygen Species; Tumor Cells, Cultured

Cisplatin adducts to nuclear DNA (nDNA) are felt to be the molecular lesions that trigger apoptosis, but the mechanism linking nDNA adduct formation and cell death is unclear. Some literature in the last decade has suggested a possible direct effect of cisplatin on mitochondria independent of nDNA interaction. In this study, we define separately the sequelae of cisplatin interactions with nDNA and with mitochondria in head and neck squamous cell carcinoma (HNSCC) cell lines.. Cisplatin binding to mitochondrial DNA (mtDNA) and proteins was analyzed by atomic absorption spectroscopy and other methods.. Following 1 hour of exposure to cisplatin, platinum adducts to mtDNA were 300- to 500-fold more abundant than adducts to nDNA; these differences were not due to differences in rates of adduct repair. Whereas HNSCC cell cytoplasts free of nDNA retained the same dose-dependent cisplatin sensitivity as parental cells, HNSCC rho(0) cells free of mtDNA were 4- to 5-fold more resistant to cisplatin than parental cells. Isolated mitochondria released cytochrome c within minutes of exposure to cisplatin, and ultrastructural analysis of intact HNSCC cells by electron microscopy showed marked mitochondrial disruption after 4 hours of cisplatin treatment, whereas the nucleus and other cellular structures remain intact. The very prompt release of cytochrome c from isolated mitochondria implies that apoptosis does not require alteration in mitochondrial gene transcription. Further, cisplatin binds preferentially to mitochondrial membrane proteins, particularly the voltage-dependent anion channel.. Cisplatin binding to nDNA is not necessary for induction of apoptosis in HNSCC, which can result from direct action of cisplatin on mitochondria.

Topics: Antineoplastic Agents; Apoptosis; Cell Nucleus; Cisplatin; Cytochromes c; DNA Adducts; DNA Repair; DNA, Mitochondrial; Drug Resistance, Neoplasm; Head and Neck Neoplasms; Humans; Mitochondrial Membranes; Neoplasms, Squamous Cell; Tumor Cells, Cultured; Voltage-Dependent Anion Channels

Gap junction intercellular channels are required for metabolic cooperation between cells and regulate normal tissue homeostasis by means of the transfer of small molecules between contacting cells. Not surprisingly, the gap junction phenotype is frequently lost during carcinogenesis in human tissues (including those of the upper aerodigestive tract), freeing individual cancer cells from the growth control signals of normal surrounding tissues and less aggressive adjacent cancer cells. We hypothesized that gap junctional intercellular communication (GJIC) could mediate a bystander effect (apoptotic cell death) in squamous cell carcinoma of the head and neck (SCCHN) cells adjacent to individually targeted SCCHN cells.. Single-cell microinjection of cytochrome c was used to induce apoptosis in target SCCHN cells with endogenous GJIC activity and in an SCCHN cell line with exogenously introduced GJIC activity. Apoptosis was followed in target and surrounding bystander cells through light and time course microscopic characterization. All of the preceding experiments were carried out in the absence and presence of 18-beta-glycerretinic acid, a pharmacologic inhibitor of GJIC.. When cytochrome c was introduced into SCCHN cells with endogenous GJIC activity through single-cell microinjection, bystander effects (apoptosis of nontarget cells) were observed. When GJIC activity was blocked with the specific pharmacologic inhibitor of gap junctions, 18-beta-glycerretinic acid, a bystander effect was never seen in GJIC active SCCHN cell lines.. Gap junction intercellular channels can mediate a bystander effect in SCCHN. Inconsistencies in our data will be discussed in the context of recent advances in this field, as well as our future research directions.

Topics: Apoptosis; Bystander Effect; Carcinoma, Squamous Cell; Cell Line; Connexin 43; Cytochromes c; Fluorescent Dyes; Gap Junctions; Glycyrrhetinic Acid; Head and Neck Neoplasms; Humans; Isoquinolines; Microinjections; Microscopy; Transfection; Video Recording

The aim of this study was to develop novel and less toxic therapy for human head and neck squamous cell carcinoma (HNSCCs) and to investigate the mechanism of quercetin-induced apoptosis in human laryngeal HeP2 cells and its effect on cisplatin induced apoptosis. Priming the cells with quercetin (40 microM) increased the apoptosis induced by cisplatin alone from 18.7% to 42.2% in HeP2 cells. Quercetin induced apoptosis via inhibition of Akt/PKB phosphorylation, an upstream kinase of pro-survival protein kinase cascade. Inhibition of Akt phosphorylation was coupled with a significant decrease of anti-apoptotic Bcl-2 and Bcl-XL. Quercetin caused a downregulation of Cu-Zn Superoxide Dismutase which perhaps led to an increase of reactive oxidative stress (ROS). The decrease of Bcl-2 and Bcl-XL along with this oxidative stress caused release of mitochondrial cytochrome c into the cytosol and subsequent induction of pro-caspase-9 processing. Inhibition of heat shock proteins may be another mechanism for the pro-apoptotic activity of quercetin. Cisplatin induced apoptosis appears to be partly due to induction of JNK activity which leads to the activation of endonucleases. Increased JNK activity led to increased phosphorylation of c-Fos. Cisplatin additionally appears to induce apoptosis by down-regulating the enzyme Nitric Oxide Synthase (NOS). Cisplatin also acts by increasing pro-apoptotic Bax concentration in the cells thereby leading to caspase-9 activation via the mitochondrial pathway. These results support the fact that quercetin and cisplatin act by separate pathways and demonstrate interactions between the pathways that result in synergistic actions. Possibly of greater potential value is the interaction of a conventional cytotoxic drug (cisplatin) and a nontoxic chemopreventive agent (quercetin) thereby allowing the use of less toxic doses of chemotherapy for treatment of HNSCCs.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Squamous Cell; Caspase 9; Caspases; Cell Line, Tumor; Cisplatin; Cytochromes c; Cytosol; Enzyme Induction; Head and Neck Neoplasms; Humans; JNK Mitogen-Activated Protein Kinases; Mitochondria; Nitric Oxide Synthase; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fos; Quercetin; Superoxide Dismutase

Nonfunctional p53 and especially upregulation of Bcl-x(L) result in advanced disease and poor prognosis of patients suffering head and neck squamous cell carcinoma (HNSCC). Aberrancies of Bcl-x(L) and/or p53 in HNSCC lead to inability of anticancer drugs to induce apoptosis. Bcl-x(L) and/or mutated p53 inhibit the apoptotic process by preventing the mitochondrial release of cytochrome c and/or activation of execution caspases. Here, we report that expression of the avian virus-derived apoptin protein resulted in induction of apoptosis in the HNSCC-derived cell line UMSSC-14B despite the presence of nonfunctional p53. Apoptin activated the execution caspase 3 and induced the release of mitochondrial cytochrome c. Upregulation of Bcl-x(L) in UMSCC-14B cells did not interfere with the apoptin-induced apoptosis, whereas it clearly negatively affected the p53-induced one. Bcl-x(L) significantly decreased the p53-induced cytochrome c release, but not the apoptin-triggered one. Our data demonstrate that apoptin induces apoptosis independent of Bcl-x(L) and p53 and may constitute a potential therapeutic agent for treatment of HNSCC.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Blotting, Western; Capsid Proteins; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Fluorescent Antibody Technique, Indirect; Genes, p53; Head and Neck Neoplasms; Humans; Microscopy, Fluorescence; Mitochondria; Mutation; Plasmids; Prognosis; Proto-Oncogene Proteins c-bcl-2; Subcellular Fractions; Time Factors; Transfection; Tumor Suppressor Protein p53; Up-Regulation

This study evaluated the combined effect of Low Dose Fractionated Radiation (LDFRT) and Taxotere (TXT) therapy on the growth of SCCHN (squamous cell carcinoma of head and neck; SQ-20B, a p53 mutant SCCHN cell line) tumors in a nude mouse model to exploit the increased hyper radiation sensitivity (HRS) phenomenon present in G(2)/M cell cycle phase when induced by low doses of radiation that was demonstrated in in vitro settings. Seventy-eight animals were randomized into one control group and 5 treatment groups (treatments were administered weekly for six weeks). Tumor regression was observed in all the groups, however, tumor regression was not significant in 2 Gy or TXT or 2 Gy plus TXT treated groups when compared to control group. The tumor regression was significant in both the LDFRT group (p < 0.0043) and LDFRT + TXT group (p < 0.0006) when compared to other groups. A significantly prolonged tumor growth delay was observed in LDFRT group (p < 0.0081). Importantly, in combination of TXT and LDFRT, no tumor regrowth was observed in 12 out of 13 mice since LDFRT + TXT treatment caused a sustained regression of tumors for 9 weeks. Molecular analysis of resected tumor specimens demonstrated that Bax levels were elevated with concomitant increase in cytochrome c release to the cytosol of the treatment Group VI. These findings strongly suggest that LDFRT can be used in combination with TXT to potentiate the effects of drug on tumor regression through an apoptotic mode of death. Furthermore, the G(2)/M cell cycle arrest by TXT appears to be an important component of the enhanced apoptotic effect of TXT + LDFRT combined treatment.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Line, Tumor; Combined Modality Therapy; Cytochromes c; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; G2 Phase; Head and Neck Neoplasms; Immunohistochemistry; In Situ Nick-End Labeling; Kinetics; Mice; Mice, Nude; Microscopy, Fluorescence; Mitosis; Neoplasm Transplantation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Radiation-Sensitizing Agents; Taxoids; Time Factors; Up-Regulation