benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Head-and-Neck-Neoplasms

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

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

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

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

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

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

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

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

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

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