Septins are members of the GTPase superfamily, which has been implicated in diverse cellular functions including cytokinesis and morphogenesis. Septin 12 (SEPT12) is a testis-specific gene critical for the terminal differentiation of male germ cells. We report the identification of two missense SEPT12 mutations, c.266C>T/p.Thr89Met and c.589G>A/p.Asp197Asn, in infertile men. Both mutations are located inside the GTPase domain and may alter the protein structure as suggested by in silico modeling. The p.Thr89Met mutation significantly reduced guanosine-5'-triphosphate (GTP) hydrolytic activity, and the p.Asp197Asn mutation (SEPT12(D197N)) interfered with GTP binding. Both mutant SEPT12 proteins restricted the filament formation of the wild-type SEPT12 in a dose-dependent manner. The patient carrying SEPT12(D197N) presented with oligoasthenozoospermia, whereas the SEPT12(T89M) patient had asthenoteratozoospermia. The characteristic sperm pathology of the SEPT12(D197N) patient included defective annulus with bent tail and loss of SEPT12 from the annulus of abnormal sperm. Our finding suggests loss-of-function mutations in SEPT12 disrupted sperm structural integrity by perturbing septin filament formation.

Cathepsins have long been considered as housekeeping molecules. However, specific functions have also been attributed to each of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wortmannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an antiapoptotic role through anti-cat L activity.

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Purpose: Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway frequently accounts for the tumorigenesis in head and neck cancer. To develop a new treatment, we investigated the effect of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in head and neck cancer cells.Experimental Design: The in vitro antitumor effect of BGT226 was determined in various cancer cell lines. Animal models were also applied to examine drug potency. The inhibitory ability of BGT226 on the PI3K/AKT/mTOR signaling pathway was analyzed.Results: The growth inhibition assay revealed that BGT226 was active against all tested cancer cell lines. Cross-resistance was not observed in the cisplatin-resistant cell line. The activation of the AKT/mTOR signal cascade was suppressed by BGT226 in a concentration-and time-dependent manner. Flow cytometric analysis revealed an accumulation of cells in the G 0 -G 1 phase with concomitant loss in the S-phase. Results of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and the analysis of caspase 3/7 and PARP indicated that BGT226 induced cancer cell death through an apoptosisindependent pathway. BGT226 induced autophagy as indicated by the aggregation and upregulation of the microtubule-associated protein light chain 3B-II, and p62 degradation. Gene silencing of Beclin1 or cotreatment of the autophagosome inhibitor, 3-methyladenine, inhibited the BGT226-induced autophagy and led to the retrieval of colony survival. In a xenografted animal model, BGT226 significantly delayed tumor growth in a dose-dependent manner, along with suppressed cytoplasmic expression of p-p70 S6 kinase and the presence of autophagosome formation.Conclusions: These data indicate that BGT226 is a potential drug in the treatment of head and neck cancer. Clin Cancer Res; 17(22); 7116-26. Ó2011 AACR.