Ovarian cancer is currently the most fatal gynecologic cancer and the fifth leading cause of fatal cancer in women overall. As compared to the better-characterized malignancies, such as such as prostate, breast and colorectal cancers, there have been no major changes in methods of detection or treatment of ovarian cancers since the 1970's. As a result, the incidence and age-adjusted death rates for this disease have improved only marginally since that time. The molecular changes required for ovarian cancer pathogenesis remain poorly defined. Lysophosphatidic acid (LPA) has emerged as a biomarker present in the ascitic fluid and serum of ovarian cancer patients. Subsequent studies have identified LPA as an agonist for G protein coupled receptors (GPCRs). LPA has been well characterized as a pro-migratory factor in ovarian cancer and other cell systems. However, the role of LPA in mediating a proliferative response in ovarian cancer cells has yet to be fully characterized. In addition, the identity of the G protein pathways involved in this proliferative response remains a major unresolved question in the field. To investigate the mitogenic role of LPA in ovarian cancers, a panel of representative human ovarian cancer cells was assembled. A series of immunoblot and RT-PCR analyses was used to profile the LPA receptors and Gα-subunits expressed in these cells. In addition to verifying the migratory effect of LPA in these cells, a series of proliferation assays were used to investigate the potential role for LPA as a mitogen. The results indicate that stimulation with LPA results in a robust and statistically significant proliferative response. This response was quantified using multiple approaches. In addition, the proliferative response was observed in three independent ovarian cancer cell lines using concentrations of LPA within the range found in vivo in the ascitic fluid of ovarian cancer patients. Taken together, these data for the first time validate the role of LPA as a mitogen in ovarian cancer cells. To gain further insight into the oncogenic signaling response stimulated by LPA, activation of the mitogen activated protein kinase (MAPK) modules was determined. Using a series of immunoblot analyses and kinase assays, LPA was found to stimulate ERK as well as JNK modules. To investigate the functional roles of these pathways, a series of proliferation assays were carried out using inhibitors of ERK and JNK signaling. Consistent with the role of ERK as a crucial regulator of growth-factor induced proliferation in other cell systems, the results demonstrated a significantly attenuated growth response to LPA with ERK inhibition. Moreover, additional studies demonstrated for the first time that inhibition of JNK signaling significantly attenuates the proliferative response to LPA. In order to investigate the potential role of Gα12 in mediating the oncogenic response to LPA, the activation status of Gα12 was monitored in ovarian cancer cells stimulated with LPA. These studies demonstrate rapid activation of Gα12 with LPA stimulation. Finally to investigate the functional role of LPA-Gα12 signaling, a series of cell lines was established which express a dominant negative form of Gα12. Expression of this construct induced complete inhibition of Gα12 activation by LPA. These cells were then used to determine the effects of Gα12 inhibition on the oncogenic response to LPA. Consistent with the role of G12 family members in mediating cell migration, these cells demonstrated an attenuated migratory response to LPA. In addition, inhibition of Gα12 resulted in an attenuated proliferative response to serum. Finally, to investigate the role of Gα12 in mediating the proliferative response to LPA, a series of proliferation assays was carried out. The results indicated a significant > 50% inhibition in multiple ovarian cancer cell lines. Taken together, these results, presented here for the first time, establish that LPA is a potent mitogen that induces a proliferative response in human ovarian carcinoma cells. Although LPA had previously been shown to induce a proliferative response in multiple other cell types, it had not been known if LPA activates specific oncogenic pathways. This thesis tested the hypothesis that LPA, which is crucially involved in the pathophysiology of ovarian carcinoma, induces the activation of Gα12. In this context, the data presented here demonstrating a novel role for Gα12- which has been defined as the gep oncogene - in mediating this proliferative response in ovarian carcinoma, represents a major finding in the field. / Molecular Biology and Genetics

No description available.

Serous ovarian cancer is the 5th leading cause of cancer-related deaths in women, with a 30% survival rate when spread into the highly hypoxic and visceral peritoneal cavity. Despite efforts to treat this highly metastatic disease, traditional chemotherapeutic and cytoreductive therapies are unable to diminish or induce cell death of circulating metastases from colonizing secondary sites due to their genetic and histologic heterogeneity and development of drug resistance. The dissemination route for primary metastasis, however, is most often conserved to the peritoneal cavity, which is low in nutrients and hypoxic (1-2% O2). Cells exfoliated from the primary tumor will aggregate during migration, which elicits a survival signal to maintain viability in this environment. The underlying cellular and molecular changes involved with aggregation have yet to be determined. We have previously found that aggregation of murine ovarian surface epithelial (MOSE) cells present a more suppressed metabolic phenotype upon aggregation. My research sought to identify how the mitochondria were internally regulated to support malignant transformation, migration, and invasion through modulation of quality control, mitochondrial dynamics, mitophagy, and mitobiogenesis. We have shown that aggregation of cancer cells supports increased mitochondrial fragmentation localized to the hypoxic core of our spheroid models. Further, aggregation supports enhanced viability through an upregulation of cancer genetic pathways associated with cell death, proliferation, stemness, and epithelial mesenchymal transition (EMT). Nutrient deprivation during migration further enhanced mitochondrial fragmentation and induction of mitophagy to prevent activation of apoptosis. Additionally, we have identified a phenotypic switch from enhanced mitophagy during peritoneal dissemination that supports survival of ovarian cancer cell aggregates to mitochondrial biogenesis during secondary tissue colonization that enables proliferation upon invasion. We have associated these changes with an increased bioenergetic proliferative niche through inhibition of proliferation, migration, and mitochondrial translation. This research has contributed to the understanding for the role of mitophagy as a survival rather than apoptotic signal in cancer cells as adaptation to nutrient-deprived environments, while also identifying how these processes can be reversed upon adhesion to support invasion and metastatic capacity during secondary colonization. This research is significant because it will identify molecular adaptations associated with the viability of disseminating cancer metastases as well as promote novel preventative therapeutics that can be used to limit the mortality of highly aggressive ovarian cancer in women. / Doctor of Philosophy / Ovarian cancer continues to be one of the highest contributors of gynecologic cancer-related deaths in women. This is due to limited symptomology, biomarker availability, and screenings for patients. Women are mostly diagnosed when the disease has already spread throughout the abdominal cavity which makes treatment much more difficult and, accordingly, the survival rate is much lower. Ovarian metastases mostly spread throughout the peritoneal cavity. Interestingly, this cavity has been identified to being limited in nutrients and oxygen that are essential for survival thus suggesting that these cancer cells must adapt to these harsh conditions to remain viable. We have previously observed that the cancer cells are able to clump together, and form 3D structures known as spheroids which have drastically reduced their proliferation and appear highly resistant tor treatment than single cells. In this project, we wanted to determine how the mitochondria (primary energy producers) were structurally changing in response to the formation of these spheroids and in nutrient- and oxygen-starved conditions. We have found that these organelles become much smaller and circular in low-oxygen conditions, especially in the center of the spheroids. Further, we found changes in cancer- and mitochondrial-related pathways during spheroid formation which could further support survival. Finally, we found that key functions related to the mitochondrial quality control and enhanced mitochondrial content and activity are switched when changing nutrient availability from low oxygen and nutrient conditions to oxygenated and nutrient-rich conditions and generate conditions that allow the spheroids to attach to abdominal organs and form secondary tumors. This research is important because it suggests new possible markers that can be used as therapeutic targets to prevent these aggressive functions associated with more terminally staged disease.

According to the American Cancer Society, Cancer is the second most common cause of death in the United States, only exceeded by heart disease. Over the past decade, deciphering the complex structure of individual cells and understanding the symptoms of cancer disease has been a highly emphasized research area. The exact cause of Cancer and the genetic heterogeneity that determines the severity of the disease and its response to treatment has been a great challenge. Researchers from the engineering discipline have increasingly made use of recent technological innovations, namely the Atomic Force Microscope (AFM), to better understand cell physics and provide a means for cell biomechanical profiling. The presented work's research objective is to establish a fundamental framework for the development of novel biosensors for cell separation and disease diagnosis. By using AFM nanoindentation, several studies were conducted to identify key distinctions in the trends of cell viscoelasticity between healthy, nontumorigenic cells and their malignant, highly tumorigenic counterparts. The possibility of identifying useful 'biomarkers' was also investigated. Due to the lack of an available human ovarian cell line, experiments were done on a recently developed mouse ovarian surface epithelial (MOSE) cell line, which resembles to human cell characteristics and represents early, intermediate, and late stages of the ovarian cancer. Material properties were extracted via Hertz model contact theory. The experimental results illustrate that the elasticity of late stage MOSE cells were 50% less than that of the early stage. Cell viscosity also decreased by 65% from early to late stage, indicating that the increase in cell deformability directly correlates with increasing levels of malignancy. Various cancer treatment and component-specific drugs were used to identify the causes for the changes in cell biomechanical behavior, depicting that the decrease in the concentration levels of cell structural components, predominantly the actin filament framework, is directly associated with the changes in cell biomechanical property. The investigation of MOSE cells being subject to multiple mechanical loads illustrated that healthy cells react to shear forces by stiffening up to 25% of their original state. On the other hand, cancerous cells are void of such response and at times show signs of decreasing rigidity. Finally, deformation studies on MOSE cancer stem cells have shown that these cells carry a unique elasticity profile among other cell stage phenotypes that could allow for their detection. The results herein carry great potential into contributing to cell separation methods and analysis, furthering the understanding of cell mechanism dynamics. While prior literature emphasizes on the elastic modulus of cells, the study of cell viscosity and other key material properties holds a critical place in the realistic modeling of these complex microstructures. A comprehensive study of individual cells holds a great amount of promise in the development of effective clinical research in the fight against cancer. / Master of Science

Bakgrund: Äggstockscancer är den dödligaste formen av gynekologisk cancer med ungefär 150 000 dödsfall per år, ofta på grund av sen diagnos. Utöver den höga dödssiffran medför äggstockscancer både psykiska och fysiska problem hos kvinnorna. Syfte: Syftet med litteraturstudien var att beskriva kvinnors erfarenheter av att leva med äggstockscancer. Metod: I databaserna PubMed och CINAHL valdes åtta studier ut till denna kvalitativa litteraturstudie. Fribergs femstegsmodell användes vid analys av studierna. Resultat: Analysen av de åtta studierna visade att äggstockscancer på olika sätt innebar förändrat liv hos alla kvinnor, även efter friskförklarande. Fyra huvudkategorier och elva subkategorier utvecklades. Huvudkategorierna var: Förändrat vardagsliv, Kroppen känns annorlunda, Leva med en oviss framtid och Hantera det överhängande hotet. Konklusion: Sjuksköterskan ska erhålla goda kunskaper om äggstockscancer och identifiera kvinnornas olika behov för att ge bästa möjliga vård. Att samtala om det svåra, lyssna och stötta både kvinnan och dem i hennes närhet är tre viktiga faktorer för att lyckas med detta. Vidare krävs utbildning av vårdpersonal och den allmänna befolkningen för att öka medvetenheten kring äggstockscancer och dess påverkan. / Background: Ovarian cancer is a big killer among other forms of gynecologicalcancer with almost 150 000 deaths a year, often caused from late diagnosis. Beyondthe high death-rate ovarian cancer also results in both physical and psychicalproblems for the women. Aim: The aim of this study was to describe women's experiences of living withovarian cancer. Methods: To this qualitative literature study eight studies were taken out fromPubMed and CINAHL. These studies were analyzed using Friberg’s five-step model. Results: The analysis showed that ovarian cancer changed the lives for all women indifferent ways, even after being declared as healthy. Four categories and elevensubcategories were developed. The categories were: Changed everyday life, Feelingsof a different body, Live in uncertainty and Coping with the imminent threat. Conclusion: The nurse should possess knowledge about ovarian cancer and thewomen's different needs to achieve the best possible care. Three important factors areto talk about the difficulties and give support to the women and those close to her.Education for healthcare professionals and the general population is required to raiseawareness about ovarian cancer and its outcomes.

Metastasis remains the largest contributor for ovarian cancer mortality. The five-year survival rate decreases dramatically as the disease advances from the primary tumor site to other organ sites within the peritoneal cavity. Thus, characterizing the mechanisms behind this metastatic potential may better elucidate the molecular mechanisms of ovarian cancer progression and may reveal novel targets for preventative and therapeutic treatments. Sphingosine-1-phosphate (S1P) is a critical secondary messenger responsible for many pro-cancer signals, e.g., proliferation, angiogenesis, inflammation, anti-apoptosis, and others. While S1P's role in the aggressive profile of many other cancers is well defined, its function in ovarian cancer development is less understood. The concentration of S1P is significantly increased in the ascites of women with malignant ovarian cancer, suggesting a role in ovarian cancer progression. This study aims to understand the importance of S1P in ovarian cancer metastasis. Using our well-characterized murine cell model for progressive ovarian cancer, we investigate the impact of S1P on ovarian cells and their interactions with the stromal vascular fraction recruited from the adipose tissue in culture conditions that mimic the physiologic environment of the peritoneal cavity. These studies will provide a mechanistic link of obesity, inflammation, and the increased risk of obese women to develop and die from ovarian cancer and identify signaling events as targets for interventions. / Master of Science / The mortality rate of women diagnosed with ovarian cancer increases significantly as the disease metastasizes to other regions. Understanding the progression of this disease can create better detection and treatment methods, improving the outcome of women diagnosed with ovarian cancer. Sphingosine-1-phosphate (S1P) is a lipid molecule that has been implicated in many pro-tumorigenic properties in cancer cells; however, its role in ovarian cancer is less known. Stromal cells excrete high levels of S1P and are recruited into tumors for support and many other functions. Elucidating the role stromal cell incorporation into tumors and the role of S1P in ovarian cancer aggressiveness may highlight key pathways that can be targeted for screening methods and therapeutic treatments. This paper aims to understand the connections between S1P, stromal cells, and ovarian cancer as it progresses from a primary site to a metastatic, highly aggressive disease.

Yes / The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3–6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3–6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3–6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.

Many human tumors harbor persistently-active Signal Transducer and Activator of Transcription (ST AT)3 protein. There is substantial evidence that aberrantly-active STAT3 is a master regulator of events that promote carcinogenesis and human tumor formation. Abnormal STAT3 activity induces uncontrolled growth and survival of cells, thereby contributing to neoplastic transformation and progression. Cisplatin is a major chemotherapeutic modality for ovarian cancer, but is frequently challenged by drug resistance. Given that STAT3 is aberrantly-active in many human tumors, including ovarian cancer, there is the potential that it contributes to the development of Cisplatin resistance, a problem ripe for investigation. This study was conducted to explore the potential that the aberrant STAT3 present in ovarian cancer cells contributes to the decreased sensitivity to Cisplatin observed for ovarian cancer cells. The investigation revealed that STAT3 is aberrantly activated in cancer cell lines resistant to Cisplatin, but not in sensitive cells. Inhibition of aberrant STAT3 activity by the small-molecule STAT3 inhibitor, NSC 74859, increased growth inhibition induced by Cisplatin in resistant ovarian cancer cells. Furthermore, NSC 74859 enhanced apoptosis induced by Cisplatin in resistant cells in vitro by nearly 52%. Collectively, these observations indicate that inhibition of hyperactive STAT3 increases Cisplatin sensitivity, and therefore effectiveness, in resistant cells. Thus, STAT3 represents a viable target for enhancing the sensitivity of ovarian cancer cells to Cisplatin.

Most women die of ovarian metastasis rather than the effects of the primary tumor. However, little is known about the factors that support the survival and secondary outgrowth of exfoliated ovarian cancer cells. In addition to genetic and molecular factors, the unique environment of the peritoneal cavity exposes ovarian cells to biophysical forces, particularly fluid shear stress (FSS). These biomechanical forces, only recently identified as a hallmark of cancer, induce rapid signaling events in attached and aggregated cells, a process termed mechanotransduction. The cellular responses to these forces and their impact on tumor initiation, progression, and metastasis are not understood. In order to delineate these phenomena, dynamic and syngeneic cell models are needed that represent the development of the disease and can be used in relevant engineered testing platforms. Thus, in an interdisciplinary approach, this work bridges molecular and cancer biology, device engineering, fluid mechanics, and biophysics strategies. The results demonstrated that even a low level of continual FSS significantly and differentially affected the viability of epithelial ovarian cancer cells of various stages of progression over time, and enhanced their aggregation, adhesion, and cellular architecture, traits of more aggressive disease. Furthermore, benign cells that survived FSS displayed phenotypic and genotypic changes resembling more aggressive stages of the disease, suggesting an impact of FSS on early stages of tumor development. After identifying a biological affect, we designed an in vitro testing platform for controlled FSS investigations, and we modeled the system fluid mechanics to understand the platform's performance capability. A cylindrical platform divided into annular sections with lid-driven flow was selected to allow continuous experiments sustainable for long durations. Tuning of the lid speed or fluid height resulted in a wide range of FSS magnitudes (0- 20 N/m2) as confirmed by analytical and numerical modeling. Further, detailed numerical modeling uncovered that FSS magnitudes experienced by cell aggregates were larger than previously observed, suggesting an even larger role of FSS in ovarian cancer. Finally, we built and engineered the designed platform to investigate changes in benign and cancer cells as a function of time and FSS magnitude. Device precision was balanced with biological consistency needs, and a novel platform was built for controlled FSS investigations. This work provides a foundational understanding of the physical environment and its potential links to ovarian cancer progression and metastatic potential. / Ph. D. / Most women die of ovarian metastasis rather than the effects of the primary tumor. However, little is known about the factors that support the survival and secondary outgrowth of exfoliated ovarian cancer cells. In addition to genetic and molecular factors, the unique environment of the peritoneal cavity exposes ovarian cells to biophysical forces, particularly fluid shear stress (FSS). These biomechanical forces, only recently identified as a hallmark of cancer, induce rapid signaling events in attached and aggregated cells, a process termed mechanotransduction. The cellular responses to these forces and their impact on tumor initiation, progression, and metastasis are not understood. In order to delineate these phenomena, dynamic and syngeneic cell models are needed that represent the development of the disease and can be used in relevant engineered testing platforms. Thus, in an interdisciplinary approach, this work bridges molecular and cancer biology, device engineering, fluid mechanics, and biophysics strategies. The results demonstrated that even a low level of continual FSS significantly and differentially affected the viability of epithelial ovarian cancer cells of various stages of progression over time, and enhanced their aggregation, adhesion, and cellular architecture, traits of more aggressive disease. Furthermore, benign cells that survived FSS displayed phenotypic and genotypic changes resembling more aggressive stages of the disease, suggesting an impact of FSS on early stages of tumor development. After identifying a biological affect, we designed an in vitro testing platform for controlled FSS investigations, and we modeled the system fluid mechanics to understand the platform’s performance capability. A cylindrical platform divided into annular sections with lid-driven flow was selected to allow continuous experiments sustainable for long durations. Tuning of the lid speed or fluid height resulted in a wide range of FSS magnitudes (0 − 20 N/m² ) as confirmed by analytical and numerical modeling. Further, detailed numerical modeling uncovered that FSS magnitudes experienced by cell aggregates were larger than previously observed, suggesting an even larger role of FSS in ovarian cancer. Finally, we built and engineered the designed platform to investigate changes in benign and cancer cells as a function of time and FSS magnitude. Device precision was balanced with biological consistency needs, and a novel platform was built for controlled FSS investigations. This work provides a foundational understanding of the physical environment and its potential links to ovarian cancer progression and metastatic potential.

PURPOSE: The anti-tumor activity of glucose analogs 2-deoxy-glucose (2-DG) and D-allose was investigated alone or in combination with p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 or platinum analogs as a strategy to pharmacologically target glycolytic tumor phenotypes. METHODS: Hypoxia inducible factor-1 alpha (HIF-1alpha) protein accumulation in pancreatic cell lines treated with SB202190 alone and in combination with glucose analogs was analyzed by Western blot. HIF-1alpha transcriptional activity was measured in MIA PaCa-2 cells stably transfected with a hypoxia response element luciferase reporter following treatment with glucose analogs alone, and in combination with SB202190. Induction of cleaved poly(ADP-ribose) polymerase (PARP) was measured by Western blot in the MIA PaCa-2 cells. In vitro anti-proliferative activity of 2-DG and D-allose alone, or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines), or with SB202190 were investigated using the MTT assay. RESULTS: SB202190 decreased HIF-1alpha protein accumulation and transcriptional activity. 2-DG demonstrated greater anti-proliferative activity than D-allose. Pre-treatment with SB202190 enhanced activity of both 2-DG and D-allose in MIA PaCa-2, BxPC-3, ASPC-1, and SK-OV-3 cells. The combination of D-allose and platinum agents was additive to moderately synergistic in all but the OVCAR-3 and HEY cells. SB202190 pre-treatment further enhanced activity of D-allose and 2-DG with platinum agents in most cell lines investigated. CONCLUSIONS: SB202190 induced sensitization of tumor cells to 2-DG and D-allose may be partially mediated by inhibition of HIF-1alpha activity. Combining glucose analogs and p38 MAPK inhibitors with chemotherapy may be an effective approach to target glycolytic tumor phenotypes.