leupeptins and Head-and-Neck-Neoplasms

Human papilloma virus (HPV) is the main culprit in cancers of the cervix, penis, anus, skin, eye and head and neck. Current treatments for HPV cancers have not altered survival outcomes for 30 years and there is a significant lack of targeted therapeutic agents in the management of advanced HPV-related HNSCC. Here we show that survival and maintenance of HPV-positive HNC cells relies on the continuous expression of the major HPV oncogene, E7, and that Aurora kinases are critical for survival of high-risk HPV-positive HNC cells.. To assess the role of HPV E7 on HNC cell survival, RNA interference (RNAi) of the E7 gene was initially performed. Using an Aurora kinase inhibitor, Alisertib, the role of Aurora kinases in the carcinogenesis of HPV E7 positive HNC tumour lines was then investigated. An in vivo HNC xenograft model was also utilised to assess loss of tumour volume in response to RNAi E7 gene silencing and Alisertib treatment.. RNAi silencing of the HPV E7 gene inhibited the growth of HPV-positive HNC cells and in vivo tumour load. We show that HPV E7 oncogene expression confers sensitivity to Alisertib on HNC cells where Alisertib-mediated loss in in vitro cell viability and in vivo tumour load is dependent on E7 expression. Moreover, Aurora kinase inhibition induced degradation of MCL-1 in HPV E7-expressing HNC cells.. Overall, we show that Aurora kinases are a novel therapeutic target for HPV-positive HNCs. It might be feasible to combine Aurora kinase and MCL-1 inhibitors for future HNC therapies.

Topics: Animals; Apoptosis; Aurora Kinase A; Aurora Kinase B; Azepines; Female; Head and Neck Neoplasms; Human papillomavirus 16; Humans; Leupeptins; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Papillomavirus E7 Proteins; Papillomavirus Infections; Proteolysis; Pyrimidines; RNA Interference; Xenograft Model Antitumor Assays

Head and neck squamous cell carcinoma (HNSCC) is often resistant to conventional chemotherapy and thus requires novel treatment regimens. Here, we investigated the effects of the proteasome inhibitor MG132 in combination with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAIL receptor 1 (DR4)-specific monoclonal antibody, AY4, on sensitization of TRAIL- and AY4-resistant human HNSCC cell lines. Combination treatment of HNSCC cells synergistically induced apoptotic cell death accompanied by caspase-8, caspase-9, and caspase-3 activation and Bid cleavage into truncated Bid (tBid). Generation and accumulation of tBid through the cooperative action of MG132 with TRAIL or AY4 and Bik accumulation through MG132-mediated proteasome inhibition are critical to the synergistic apoptosis. In HNSCC cells, Bak was constrained by Mcl-1 and Bcl-X(L), but not by Bcl-2. Conversely, Bax did not interact with Mcl-1, Bcl-X(L), or Bcl-2. Importantly, tBid plays a major role in Bax activation, and Bik indirectly activates Bak by displacing it from Mcl-1 and Bcl-X(L), pointing to the synergistic mechanism of the combination treatment. In addition, knockdown of both Mcl-1 and Bcl-X(L) significantly sensitized HNSCC cells to TRAIL and AY4 as a single agent, suggesting that Bak constraint by Mcl-1 and Bcl-X(L) is an important resistance mechanism of TRAIL receptor-mediated apoptotic cell death. Our results provide a novel molecular mechanism for the potent synergy between MG132 proteasome inhibitor and TRAIL receptor agonists in HNSCC cells, suggesting that the combination of these agents may offer a new therapeutic strategy for HNSCC treatment.

Topics: Antibodies, Monoclonal; Apoptosis; Apoptosis Regulatory Proteins; Base Sequence; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Carcinoma, Squamous Cell; Cell Line, Tumor; Drug Synergism; Gene Knockdown Techniques; Head and Neck Neoplasms; Humans; Leupeptins; Membrane Proteins; Mitochondrial Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Proteasome Inhibitors; Protein Stability; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Receptors, TNF-Related Apoptosis-Inducing Ligand; RNA Interference; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand

To determine whether proteasome inhibition could reverse E6-mediated p53 degradation, cause selective growth inhibition, and induce apoptosis in human papillomavirus E6-transformed primary tonsil epithelial cells.. Primary human and mouse tonsil epithelial cell lines were transformed with a retrovirus containing human papillomavirus 16 oncogenes. MG132 was used to inhibit proteasome degradation in vitro and in vivo, and biochemical assays regarding p53 and apoptosis were performed.. In cells that express E6, proteasome inhibition with MG132 restored p53 protein levels and decreased proliferation in a dose-dependent fashion that was significantly more pronounced compared with controls. However, inhibition of proliferation occurred at a lower concentration than restoration of p53 protein expression. Also, wild-type and p53 knockout mouse tonsil epithelial cells that express E6 had near-identical inhibition of growth, suggesting that growth inhibition was p53 independent. In vivo studies did not demonstrate any growth inhibition.. The findings suggest that proteasome inhibition preferentially inhibits proliferation in cells expressing E6 through a p53-independent mechanism.

Topics: Animals; Apoptosis; Caspase 3; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Dose-Response Relationship, Drug; Epithelial Cells; Head and Neck Neoplasms; Humans; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Knockout; Oncogene Proteins, Viral; Palatine Tonsil; Protease Inhibitors; Proteasome Inhibitors; Repressor Proteins; Tumor Suppressor Protein p53

We have isolated a new human head and neck carcinoma cell line (C-10E) that is highly resistant to BLM (40-fold) when compared to the parental (A-253) cell line. Consonant with BLM resistance in the C-10E cell line, we found that this cell line accumulated 2- to 3-fold less BLM A2 than A-253 cells. Kinetic analyses of BLM A2 association revealed a decreased Vmax for C-10E cells with little change in Ka. Furthermore, the BLM-resistant cell line (C-10E) metabolized BLM A2 to a greater extent than its sensitive counterpart (A-253). Thus, compared to A-253 cells, the C-10E cells exhibited both decreased cellular association and increased metabolism of BLM. Synergistic cytotoxicity was seen when BLM was combined with either E-64 or leupeptin, cysteine proteinase inhibitors known to block BLM metabolism in vitro. E-64 inhibited the metabolism of BLM A2 in both C-10E and A-253 cells, and cellular accumulation of radiolabeled BLM A2 was increased by leupeptin or E-64 in only A-253 cells. These results suggest that both inhibition of drug metabolism and increased drug accumulation contribute to this synergism.

Topics: Bleomycin; Carcinoma, Squamous Cell; Cell Division; Cell Survival; Cysteine Proteinase Inhibitors; Drug Resistance; Drug Synergism; Head and Neck Neoplasms; Leucine; Leupeptins; Tumor Cells, Cultured