The HIV protease inhibitor saquinavir induces endoplasmic reticulum stress, autophagy, and apoptosis in ovarian cancer cells.

OBJECTIVE: HIV patients taking antiretroviral protease inhibitors have a lower incidence of infection-associated malignancies, leading to the hypothesis that these drugs have antineoplastic activity. Given the need for novel treatment approaches in ovarian cancer, we sought to determine whether the protease inhibitor saquinavir has antineoplastic activity in ovarian cancer cell lines, and to elucidate the mechanism through which this occurs. METHODS: A panel of ovarian cancer cell lines was treated with saquinavir. The effect of saquinavir on cell growth, viability, apoptotic and non-apoptotic cell death was determined. Stimulation of endoplasmic reticulum stress (ERS) response was assessed by immunoblotting for ERS regulators GRP78 and ATF6. Induction of autophagy was assessed using transmission electron microscopy (TEM), and confocal microscopy was performed to demonstrate changes in green fluorescent protein-labeled LC3 expression patterns. RESULTS: Saquinavir induced cell death in chemosensitive and chemoresistant ovarian cancer cells in a time- and dose-dependent manner. Saquinavir treatment resulted in caspase-dependent apoptosis and caspase-independent cell death characterized by induction of ERS and autophagy. Cellular morphology assessed by TEM revealed apoptotic, autophagic, and necrotic cell death. CONCLUSIONS: Saquinavir is an FDA-approved agent for the treatment of HIV, and our data suggest that it may also have clinical application in the treatment of ovarian cancer. Saquinavir induces endoplasmic reticulum stress, autophagy, and apoptosis in ovarian cancer cells. Given the challenges of chemoresistance in ovarian cancer, saquinavir may have particular benefit in the treatment of chemoresistant tumors that may respond to the induction of caspase-independent cell death by mechanisms such as autophagy.

Merlin is required for coordinating proliferation of two stem cell lineages in the Drosophila testis.

Although the mechanisms that balance self-renewal and differentiation of a stem cell lineage have been extensively studied, it remains poorly understood how tissues that contain multiple stem cell lineages maintain balanced proliferation among distinct lineages: when stem cells of a particular lineage proliferate, how do the other lineages respond to maintain the correct ratio of cells among linages? Here, we show that Merlin (Mer), a homolog of the human tumor suppressor neurofibromatosis 2, is required to coordinate proliferation of germline stem cells (GSCs) and somatic cyst stem cells (CySCs) in the Drosophila testis. Mer mutant CySCs fail to coordinate their proliferation with that of GSCs in multiple settings, and can be triggered to undergo tumorous overproliferation. Mer executes its function by stabilizing adherens junctions. Given the known role of Mer in contact-dependent inhibition of proliferation, we propose that the proliferation of CySCs are regulated by crowdedness, or confluency, of cells in their lineage with respect to that of germline, thereby coordinating the proliferation of two lineages.

Resveratrol-induced autophagocytosis in ovarian cancer cells.

Resveratrol (3,5,4-trihydroxystilbene), a natural phytoalexin present in grapes, nuts, and red wine, has antineoplastic activities. Several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo. In the present study, the response of ovarian cancer cells to resveratrol is explored. Resveratrol inhibited growth and induced death in a panel of five human ovarian carcinoma cell lines. The response was associated with mitochondrial release of cytochrome c, formation of the apoptosome complex, and caspase activation. Surprisingly, even with these molecular features of apoptosis, analysis of resveratrol-treated cells by light and electron microscopy revealed morphology and ultrastructural changes indicative of autophagocytic, rather than apoptotic, death. This suggests that resveratrol can induce cell death through two distinct pathways. Consistent with resveratrol's ability to kill cells via nonapoptotic processes, cells transfected to express high levels of the antiapoptotic proteins Bcl-x(L) and Bcl-2 are equally sensitive as control cells to resveratrol. Together, these findings show that resveratrol induces cell death in ovarian cancer cells through a mechanism distinct from apoptosis, therefore suggesting that it may provide leverage to treat ovarian cancer that is chemoresistant on the basis of ineffective apoptosis.

Mice lacking sodium channel beta1 subunits display defects in neuronal excitability, sodium channel expression, and nodal architecture.

Sodium channel beta1 subunits modulate alpha subunit gating and cell surface expression and participate in cell adhesive interactions in vitro. beta1-/- mice appear ataxic and display spontaneous generalized seizures. In the optic nerve, the fastest components of the compound action potential are slowed and the number of mature nodes of Ranvier is reduced, but Na(v)1.6, contactin, caspr 1, and K(v)1 channels are all localized normally at nodes. At the ultrastructural level, the paranodal septate-like junctions immediately adjacent to the node are missing in a subset of axons, suggesting that beta1 may participate in axo-glial communication at the periphery of the nodal gap. Sodium currents in dissociated hippocampal neurons are normal, but Na(v)1.1 expression is reduced and Na(v)1.3 expression is increased in a subset of pyramidal neurons in the CA2/CA3 region, suggesting a basis for the epileptic phenotype. Our results show that beta1 subunits play important roles in the regulation of sodium channel density and localization, are involved in axo-glial communication at nodes of Ranvier, and are required for normal action potential conduction and control of excitability in vivo.

Prothrombotic effects of thrombolytic therapy in a rat (Rattus norvegicus) model of venous thrombolysis.

The use of thrombolytic agents has greatly improved patient outcomes, but the prothrombotic response to these drugs in vivo is unknown. Approximately 24 h after we induced thrombosis in male Sprague-Dawley rats, we placed an infusion line in the inferior vena cava and administered either saline or a thrombolytic agent (tissue plasminogen activator [tPA] or plasmin) for 30 min. Blood was drawn immediately after infusion; rats were euthanized 24 h after infusion for collection of blood and tissue (inferior vena cava and thrombus). Thrombus size was decreased in the tPA-treated rats but not in those that received saline or plasmin; this change correlated with the significant rise in D-dimer levels noted immediately after infusion in the tPA-treated rats. Plasma soluble P-selectin, a prothrombotic marker, was elevated at 24 h in the plasmin group compared with the other treatment groups. There were no significant differences in plasma C3a, C5a, or C5b9 levels or in thrombus C3 levels between groups. According to ultrastructural analysis, thrombus structure and vein wall effects did not differ between groups. Local tPA did not induce a prothrombotic state during acute DVT or after thrombolytic therapy in a rodent model of venous thrombolysis. Conversely, levels of the prothrombotic marker plasma soluble P-selectin increased when plasmin was administered.

Efficient cellular release of Rift Valley fever virus requires genomic RNA.

The Rift Valley fever virus is responsible for periodic, explosive epizootics throughout sub-Saharan Africa. The development of therapeutics targeting this virus is difficult due to a limited understanding of the viral replicative cycle. Utilizing a virus-like particle system, we have established roles for each of the viral structural components in assembly, release, and virus infectivity. The envelope glycoprotein, Gn, was discovered to be necessary and sufficient for packaging of the genome, nucleocapsid protein and the RNA-dependent RNA polymerase into virus particles. Additionally, packaging of the genome was found to be necessary for the efficient release of particles, revealing a novel mechanism for the efficient generation of infectious virus. Our results identify possible conserved targets for development of anti-phlebovirus therapies.

Site-specific alteration of actin assembly visualized in living renal epithelial cells during ATP depletion.

Disruption of normal actin organization in renal tubular epithelial cells is an important element of renal injury induced by ischemia. Studies of fixed cells indicate that the cytoskeleton is disrupted by both ischemia and ATP depletion in a site-specific manner. However, few studies have examined these effects in living cells, and the relationship between the time course of ATP reduction and alteration of the cytoskeleton remains unclear. Here, time-lapse video images of cultured renal epithelial cells expressing an enhanced green fluorescent protein (EGFP)-actin fusion protein were obtained, and the kinetics of fluorescence actin distribution before and during ATP depletion is quantified and compared with measured ATP levels. This study found that assembly of lamellar actin is inhibited rapidly as cellular ATP levels are reduced, whereas disruption of actin in stress fibers is more gradual and persistent. Actin associated with focal adhesions is largely resistant to ATP depletion in these experiments, and, consistent with previous studies, particulate aggregates of actin were formed within the cytoplasm of ATP-depleted cells. Most surprisingly, time-lapse imaging of EGFP-actin distribution, quantitative fluorescence imaging of phalloidin-stained cells, and ultrastructural studies indicate that assembly of actin filaments occurs at sites of epithelial cell-cell attachment in ATP-depleted cells. This assembly is initiated early during ATP depletion and continues after ATP levels are maximally reduced. Assembly of actin at sites of cell-cell attachment may be an element of the pathology of injury induced by ischemia, or alternatively, could reflect the function of a protective mechanism. These studies directly demonstrate site-specific alteration of actin assembly in living epithelial cells during ATP depletion. The results also reveal that actin reorganization continues after ATP levels are maximally decreased and that epithelial cell-cell attachments are sites of actin assembly in ATP-depleted cells.