h-89 and Carcinoma--Squamous-Cell

To investigate the mechanism by which the human papillomavirus (HPV) E5 protein contributes to the carcinogenesis of uterine cervical cancer, we studied the effect of HPV E5 on apoptosis of cervical cancer cells and its underlying mechanism. Expression of HPV16 E5 protein inhibited hydrogen peroxide-induced apoptosis in C-33A cervical cancer cells. E5 decreased the expression of Bax protein, and exogenous expression of Bax abolished the anti-apoptotic effect of E5. Knockdown of E5 by small interfering RNA sensitized CaSki cervical cancer cells to hydrogen peroxide-induced apoptosis with concurrent increase in Bax expression. Transient expression of E5 significantly increased the degradation rate of Bax protein by inducing the ubiquitination. The E5-induced decrease in Bax expression was inhibited by a cyclooxygenase-2 (COX-2) inhibitor, prostaglandin E2 (PGE(2)) receptor antagonists and cyclic adenosine monophosphate-dependent protein kinase (PKA) inhibitor. Treatment with PGE(2) decreased the expression of Bax and inhibited hydrogen peroxide-induced apoptosis of C-33A cells. We concluded that HPV16 E5 protein inhibits hydrogen peroxide-induced apoptosis of cervical cancer cells by stimulating the ubiquitin-proteasome-mediated degradation of Bax protein, and the pathway involves COX-2, PGE(2) and PKA. This finding suggests the possibility that HPV 16 E5 protein contributes to cervical carcinogenesis by inhibiting apoptosis of transformed cervical epithelial cells.

Topics: Androstadienes; Apoptosis; bcl-2-Associated X Protein; Biphenyl Compounds; Carcinoma, Squamous Cell; Cell Line, Transformed; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Female; Humans; Hydrogen Peroxide; Isoquinolines; Neoplasm Proteins; Oncogene Proteins, Viral; Proteasome Endopeptidase Complex; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP2 Subtype; Receptors, Prostaglandin E, EP4 Subtype; RNA, Small Interfering; Sulfonamides; Ubiquitin; Uterine Cervical Neoplasms; Wortmannin; Xanthones

Aberrant nuclear activation and phosphorylation of the canonical NF-kappaB subunit RELA/p65 at Serine-536 by inhibitor kappaB kinase is prevalent in head and neck squamous cell carcinoma (HNSCC), but the role of other kinases in NF-kappaB activation has not been well defined. Here, we investigated the prevalence and function of p65-Ser276 phosphorylation by protein kinase A (PKA) in the malignant phenotype and gene transactivation, and studied p65-Ser276 as a potential target for therapy.. Phospho and total p65 protein expression and localization were determined in HNSCC tissue array and in cell lines. The effects of the PKA inhibitor H-89 on NF-kappaB activation, downstream gene expression, cell proliferation and cell cycle were examined. Knockdown of PKA by specific siRNA confirmed the specificity.. NF-kappaB p65 phosphorylated at Ser276 was prevalent in HNSCC and adjacent dysplastic mucosa, but localized to the cytoplasm in normal mucosa. In HNSCC lines, tumor necrosis factor-alpha (TNF-alpha) significantly increased, whereas H-89 inhibited constitutive and TNF-alpha-induced nuclear p65 (Ser276) phosphorylation, and significantly suppressed NF-kappaB and target gene IL-8 reporter activity. Knockdown of PKA by small interfering RNA inhibited NF-kappaB, IL-8, and BCL-XL reporter gene activities. H-89 suppressed cell proliferation, induced cell death, and blocked the cell cycle in G(1)-S phase. Consistent with its biological effects, H-89 down-modulated expression of NF-kappaB-related genes Cyclin D1, BCL2, BCL-XL, COX2, IL-8, and VEGF, as well as induced cell cycle inhibitor p21(CIP1/WAF1), while suppressing proliferative marker Ki67.. NF-kappaB p65 (Ser276) phosphorylation by PKA promotes the malignant phenotype and holds potential as a therapeutic target in HNSCC.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Nucleus; Cell Proliferation; Cyclic AMP-Dependent Protein Kinases; Head and Neck Neoplasms; Humans; Isoquinolines; Mucous Membrane; Phenotype; Phosphorylation; Protein Kinase Inhibitors; Protein Transport; RNA, Small Interfering; Serine; Sulfonamides; Transcription Factor RelA; Tumor Cells, Cultured

Binding of integrins to the extracellular matrix (ECM) activates various signal transduction pathways and regulates gene expression in many cell types. Integrin-dependent cytoplasmic protein/protein interactions are necessary for activation of those signal transduction cascades. In our studies we investigated a possible association of pp125FAK, an adhesion involved tyrosine kinase, with the integrin beta 1 subunit. Further we wanted to know to which extent protein tyrosine phosphorylation affects cell adhesion to the ECM and the possible beta 1 integrin/pp125FAK complex. We were able to show that in HaCaT cells (a human keratinocyte derived cell line) the integrin beta 1 subunit is associated with tyrosine kinase pp125FAK. This association was observed in ECM-adherent cells and nonadherent cells and is independent of tyrosine phosphorylation. However, cell adhesion of HaCaT cells to specific substrates requires tyrosine phosphorylation since genistein treatment that blocks phosphorylation of many cellular proteins as pp125FAK led to a reduced substrate adhesion.

Topics: Carcinoma, Squamous Cell; Cell Adhesion; Cell Adhesion Molecules; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Extracellular Matrix; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Genistein; Humans; Integrin beta1; Isoquinolines; Keratinocytes; Macromolecular Substances; Neoplasm Proteins; Phosphorylation; Polyhydroxyethyl Methacrylate; Polylysine; Protein Kinase C; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Signal Transduction; Skin Neoplasms; Sulfonamides; Tumor Cells, Cultured