Dorothy R. Sorenson

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-xL 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 β1 Subunits Display Defects in Neuronal Excitability, Sodium Channel Expression, and Nodal Architecture

Sodium channel β1 subunits modulate α subunit gating and cell surface expression and participate in cell adhesive interactions in vitro. β1(-/-) 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 Nav1.6, contactin, caspr 1, and Kv1 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 β1 may participate in axo-glial communication at the periphery of the nodal gap. Sodium currents in dissociated hippocampal neurons are normal, but Nav1.1 expression is reduced and Nav1.3 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 β1 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.

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.

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.

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.

Histological Observation of Islet Hemorrhage Induced by Diagnostic Ultrasound with Contrast Agent in Rat Pancreas

Contrast enhanced diagnostic ultrasound CEDUS has been shown to induce capillary hemorrhage in heart and kidney. This study characterized the capillary hemorrhage induced in rat pancreas. The pancreata of anesthetized hairless rats were accessed by laparotomy. A 1.5 MHz diagnostic ultrasound probe with 2.3 MPa peak rarefactional pressure amplitude and 1 s intermittent trigger was used to scan the pancreas, located at the focus (3.8 cm), through saline coupling. The probe was swept to expose the entire organ in 5 min during infusion of Definity® contrast agent at 10 µL/kg/min, and this was repeated in a reverse sweep. The entire pancreas was removed, spread flat for fixation and histological slides were prepared from the mid-plane. Slides were scored blind for islet hemorrhage over the entire area of the organ. Intra-islet microlesions were evident and hemorrhage surrounded many islets. The hemorrhage often impacted nearby acini, and expanded into inter-lobular septa. In CEDUS pancreata removed soon after scanning, 76.2±11.8% (n = 6) of islets had evidence of hemorrhage and/or islet microlesions compared to 1.1±2.5% (n = 5) for sham CEDUS (P<0.001). In pancreata removed after 4 hr, fibrin formation was detected by immunohistology in the hemorrhage and intra-islet microlesions. Diagnostic ultrasound with contrast agent induced substantial capillary hemorrhage in rat pancreas, concentrated particularly in the islets.

Abstract 1586: Oral administration of HIV-protease inhibitors reduces ovarian tumor growth in vivo

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL INTRODUCTION: Ritonavir boosted lopinavir (LPV/r) is an FDA approved HIV-protease inhibitor combination that is a well tolerated and preferred treatment for HIV patients. Ritonavir is a potent irreversible inhibitor of cytochrome P450 isoform 3A (CYP3A) which can substantially increase bioavailability of lopinavir in vivo. This combination of lopinavir and ritonavir has been shown to increase the efficacy of reducing viral load in AIDS patients. HIV patients taking antiretroviral protease inhibitors such as LPV/r have a lower incidence of HIV associated malignancies such as non-Hodgkins lymphoma, cervical cancer and Kaposis sarcoma, leading to the hypothesis that these drugs have antineoplastic activity. Given the need for novel treatment approaches in ovarian cancer, we are investigating the antineoplastic effects of LPV/r, in vivo. METHODS: A xenograph model of ovarian cancer is used to assess the antineoplastic effect of LPV/r in vivo. Oral doses of LPV/r dissolved in corn oil are administered to mice over the course of 2-3 weeks. Tumor area and mouse weight are monitored. After 2-3 weeks tumors are harvested and analyzed. RESULTS: Preliminary results indicate that LPV/r administered orally in combination with a high fat diet inhibits tumor growth in vivo without inducing weight loss. In contrast, the dose of intraperitoneal cisplatin required to attain a similar antineoplastic effect resulted in significant weight loss and morbidity. CONCLUSIONS: Because protease inhibitors can induce both apoptotic and non-apoptotic ovarian cancer cell death in vitro and in vivo, these drugs may circumvent chemoresistance due to alterations in apoptotic response. Our data suggests that LPV/r may also have clinical application in the treatment of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1586. doi:10.1158/1538-7445.AM2011-1586

Abstract 3259: HIV protease inhibitors induce apoptotic and non-apoptotic cell death in ovarian cancer cells in vitro and in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: HIV patients taking antiretroviral protease inhibitors have a lower incidence of HIV associated malignancies, leading to the hypothesis that these drugs have antineoplastic activity. Given the need for novel treatment approaches in ovarian cancer, we investigated the antineoplastic effects of the HIV protease inhibitors saquinavir and nelfinavir in vitro and in vivo. Methods: A panel of ovarian cancer cell lines was treated with saquinavir and nelfinavir. The effect of protease inhibitors 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. A xenograph model of ovarian cancer was used to assess the antineoplastic effect of nelfinavir in vivo. Results: HIV protease inhibitors induced cell death in chemosensitive and chemoresistant ovarian cancer cells in a time- and dose-dependent manner. Treatment with protease inhibitors 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. Preliminary results indicate that nelfinavir administer intraperitoneally inhibits tumor growth in vivo without inducing weight loss. In contrast, the dose of intraperitoneal cisplatin required to attain a similar antineoplastic effect resulted in significant weight loss and morbidity. Conclusions: Because protease inhibitors can induce both apoptotic and non-apoptotic cell death, these drugs may circumvent chemoresistance due to alterations in apoptotic response. Saquinavir and Nelfinavir are FDA-approved agents for the treatment of HIV, and our data suggest that these drugs may also have clinical application in the treatment of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3259.

The Fine Structure of the Sperm of the Round Goby (Neogobius melanostomus)

Abstract The fine structural details of the spermatozoon of the round goby are presented for the first time in this study. Scanning and transmission electron microscopic examination of testis reveals an anacrosomal spermatozoon with a slightly elongate head and uniformly compacted chromatin. The midpiece contains a single, spherical mitochondrion. Two perpendicularly oriented centrioles lie in a deep, eccentric nuclear fossa with no regularly observed connection to the nucleus. The flagellum develops bilateral fins soon after emerging from the fossa; each extends approximately 1 μm from the axoneme and persists nearly the length of the flagellum.