Withaferin A ameliorates ovarian cancer-induced renal damage through the regulation of expression of inflammatory cytokines.

BACKGROUND: Cachexia a multifactorial syndrome is a common sequala in patients with cancer. It varies from 42 to 80% depending upon the oncological stage and is directly responsible for 30% of deaths in these patients. Previous research from our laboratory demonstrated that peritoneal ovarian cancer generated in NSG mice resulted in skeletal and cardiac muscle atrophy - leading to loss of skeletal muscle mass and strength, and cardiac dysfunction (cachexia). Treatment of mice bearing i.p. tumors with withaferin A (WFA) showed reversal of skeletal muscle and cardiac cachexia. The present study is focused on determining effects of peritoneal ovarian tumors on kidney damage and effects of WFA treatment on ameliorating kidney damage. METHODS: We generated intraperitoneal ovarian cancer by injecting female NSG mice with ovarian cancer cell line (A2780). After one week of injecting cancer cells, mice were treated with WFA (4 mg/kg) every third day, for three weeks. After 4 weeks of injection of cancer cells, the mice were sacrificed and various tissues including kidney and blood were collected, snap-frozen in liquid nitrogen, and stored at -800C. The presence of kidney biomarker creatinine, was measured in the plasma by an ELISA. The mRNA was isolated from mouse kidneys and was used to examine the expression levels of signaling proteins, inflammatory cytokines, and genes responsible for inducing cachexia (IL-1ß, IL-6, TNF-α, TGF-ß, GDF-15, and MYD88). RESULTS: Our results showed a significant increase in levels of expression of inflammatory cytokine IL-1 ß (p < 0.01), IL-6 (p < 0.001), TNF-α (p < 0.001), and other related genes including TRAF6 (p < 0.01), MYD88 (p < 0.01), and GDF-15 (p = 0.005) in tumor-bearing mice compared to controls. Treatment of mice bearing tumors with WFA attenuated the increase in expression of each gene. In addition, our results showed a significant increase in creatinine levels in circulation in tumor-bearing mice compared to control mice. Treatment of tumor-bearing mice with WFA resulted in a significant decrease in plasma creatinine levels compared to tumor-bearing mice. CONCLUSIONS: Our results conclude that ovarian tumors in NSG mice caused kidney damage and renal dysfunction, which was effectively ameliorated by WFA treatment, suggesting a protective effect of WFA on kidney injury induced by ovarian cancer.

Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia.

In our previous studies, we showed that the generation of ovarian tumors in NSG mice (immune-compromised) resulted in the induction of muscle and cardiac cachexia, and treatment with withaferin A (WFA; a steroidal lactone) attenuated both muscle and cardiac cachexia. However, our studies could not address if these restorations by WFA were mediated by its anti-tumorigenic properties that might, in turn, reduce the tumor burden or WFA's direct, inherent anti-cachectic properties. To address this important issue, in our present study, we used a cachectic model induced by the continuous infusion of Ang II by implanting osmotic pumps in immunocompetent C57BL/6 mice. The continuous infusion of Ang II resulted in the loss of the normal functions of the left ventricle (LV) (both systolic and diastolic), including a significant reduction in fractional shortening, an increase in heart weight and LV wall thickness, and the development of cardiac hypertrophy. The infusion of Ang II also resulted in the development of cardiac fibrosis, and significant increases in the expression levels of genes (ANP, BNP, and MHCß) associated with cardiac hypertrophy and the chemical staining of the collagen abundance as an indication of fibrosis. In addition, Ang II caused a significant increase in expression levels of inflammatory cytokines (IL-6, IL-17, MIP-2, and IFNγ), NLRP3 inflammasomes, AT1 receptor, and a decrease in AT2 receptor. Treatment with WFA rescued the LV functions and heart hypertrophy and fibrosis. Our results demonstrated, for the first time, that, while WFA has anti-tumorigenic properties, it also ameliorates the cardiac dysfunction induced by Ang II, suggesting that it could be an anticachectic agent that induces direct effects on cardiac muscles.

Cardiac Cachexia: Unaddressed Aspect in Cancer Patients.

Tumor-derived cachectic factors such as proinflammatory cytokines and neuromodulators not only affect skeletal muscle but also affect other organs, including the heart, in the form of cardiac muscle atrophy, fibrosis, and eventual cardiac dysfunction, resulting in poor quality of life and reduced survival. This article reviews the holistic approaches of existing diagnostic, pathophysiological, and multimodal therapeutic interventions targeting the molecular mechanisms that are responsible for cancer-induced cardiac cachexia. The major drivers of cardiac muscle wasting in cancer patients are autophagy activation by the cytokine-NFkB, TGF ß-SMAD3, and angiotensin II-SOCE-STIM-Ca2+ pathways. A lack of diagnostic markers and standard treatment protocols hinder the early diagnosis of cardiac dysfunction and the initiation of preventive measures. However, some novel therapeutic strategies, including the use of Withaferin A, have shown promising results in experimental models, but Withaferin A's effectiveness in human remains to be verified. The combined efforts of cardiologists and oncologists would help to identify cost effective and feasible solutions to restore cardiac function and to increase the survival potential of cancer patients.

Overview of follicle stimulating hormone and its receptors in reproduction and in stem cells and cancer stem cells.

Follicle stimulating hormone (FSH) and its receptor (FSHR) have been reported to be responsible for several physiological functions and cancers. The responsiveness of stem cells and cancer stem cells towards the FSH-FSHR system make the function of FSH and its receptors more interesting in the context of cancer biology. This review is comprised of comprehensive information on FSH-FSHR signaling in normal physiology, gonadal stem cells, cancer cells, and potential options of utilizing FSH-FSHR system as an anti-cancer therapeutic target.

Ovarian Cancer Stem Cells: Characterization and Role in Tumorigenesis.

Ovarian cancer is a heterogenous disease with variable clinicopathological and molecular mechanisms being responsible for tumorigenesis. Despite substantial technological improvement, lack of early diagnosis contributes to its highest mortality. Ovarian cancer is considered to be the most lethal female gynaecological cancer across the world. Conventional treatment modules with platinum- and Taxane-based chemotherapy can cause an initial satisfactory improvement in ovarian cancer patients. However, approximately 75-80% patients of advanced stage ovarian cancer, experience relapse and nearly 40% have overall poor survival rate. It has been observed that a subpopulation of cells referred as cancer stem cells (CSCs), having self renewal property, escape the conventional chemotherapy because of their quiescent nature. Later, these CSCs following its interaction with microenvironment and release of various inflammatory cytokines, chemokines and matrix metalloproteinases, induce invasion and propagation to distant organs of the body mainly peritoneal cavity. These CSCs can be enriched by their specific surface markers such as CD44, CD117, CD133 and intracellular enzyme such as aldehyde dehydrogenase. This tumorigenicity is further aggravated by the epithelial to mesenchymal transition of CSCs and neovascularisation via epigenetic reprogramming and over-expression of various signalling cascades such as Wnt/ß-catenin, NOTCH, Hedgehog, etc. to name a few. Hence, a comprehensive understanding of various cellular events involving interaction between cancer cells and cancer stem cells as well as its surrounding micro environmental components would be of unmet need to achieve the ultimate goal of better management of ovarian cancer patients. This chapter deals with the impact of ovarian cancer stem cells in tumorigenesis which would help in the implementation of basic research into the clinical field in the form of translational research in order to reduce the morbidity and mortality in ovarian cancer patients through amelioration of diagnosis and impoverishment of therapeutic resistance.

Application of Stem Cell Therapy for Infertility.

Infertility creates an immense impact on the psychosocial wellbeing of affected couples, leading to poor quality of life. Infertility is now considered to be a global health issue affecting approximately 15% of couples worldwide. It may arise from factors related to the male (30%), including varicocele, undescended testes, testicular cancer, and azoospermia; the female (30%), including premature ovarian failure and uterine disorders; or both partners (30%). With the recent advancement in assisted reproduction technology (ART), many affected couples (80%) could find a solution. However, a substantial number of couples cannot conceive even after ART. Stem cells are now increasingly being investigated as promising alternative therapeutics in translational research of regenerative medicine. Tremendous headway has been made to understand the biology and function of stem cells. Considering the minimum ethical concern and easily available abundant resources, extensive research is being conducted on induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSC) for their potential application in reproductive medicine, especially in cases of infertility resulting from azoospermia and premature ovarian insufficiency. However, most of these investigations have been carried out in animal models. Evolutionary divergence observed in pluripotency among animals and humans requires caution when extrapolating the data obtained from murine models to safely apply them to clinical applications in humans. Hence, more clinical trials based on larger populations need to be carried out to investigate the relevance of stem cell therapy, including its safety and efficacy, in translational infertility medicine.

Withaferin A and Ovarian Cancer Antagonistically Regulate Skeletal Muscle Mass.

Cachexia is a complex wasting syndrome that overwhelmingly affects the majority of late-stage cancer patients. Additionally, there are currently no efficacious therapeutic agents to treat the muscle atrophy induced by the cancer. While several preclinical studies have investigated the molecular signals orchestrating cachexia, very little information exists pertaining to ovarian cancer and the associated cachexia. Work from our lab has recently demonstrated that the steroidal lactone Withaferin A (WFA) is capable of attenuating the atrophying effects of ovarian cancer in a preclinical mouse model. However, it remained to be determined whether WFA's effect was in response to its anti-tumorigenic properties, or if it was capable of targeting skeletal muscle directly. The purpose of this study was to uncover whether WFA was capable of regulating muscle mass under tumor-free and tumor-bearing conditions. Treatment with WFA led to an improvement in functional muscle strength and mass under tumor-bearing and naïve conditions. WFA and ovarian cancer were observed to act antagonistically upon critical skeletal muscle regulatory systems, notably myogenic progenitors and proteolytic degradation pathways. Our results demonstrated for the first time that, while WFA has anti-tumorigenic properties, it also exerts hypertrophying effects on skeletal muscle mass, suggesting that it could be an anti-cachectic agent in the settings of ovarian cancer.

An Assessment on Impact of COVID-19 Infection in a Gender Specific Manner.

Coronavirus disease 2019 (COVID-19) is caused by novel coronavirus Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was first time reported in December 2019 in Wuhan, China and thereafter quickly spread across the globe. Till September 19, 2020, COVID-19 has spread to 216 countries and territories. Severe infection of SARS-CoV-2 cause extreme increase in inflammatory chemokines and cytokines that may lead to multi-organ damage and respiratory failure. Currently, no specific treatment and authorized vaccines are available for its treatment. Renin angiotensin system holds a promising role in human physiological system specifically in regulation of blood pressure and electrolyte and fluid balance. SARS-CoV-2 interacts with Renin angiotensin system by utilizing angiotensin-converting enzyme 2 (ACE2) as a receptor for its cellular entry. This interaction hampers the protective action of ACE2 in the cells and causes injuries to organs due to persistent angiotensin II (Ang-II) level. Patients with certain comorbidities like hypertension, diabetes, and cardiovascular disease are under the high risk of COVID-19 infection and mortality. Moreover, evidence obtained from several reports also suggests higher susceptibility of male patients for COVID-19 mortality and other acute viral infections compared to females. Analysis of severe acute respiratory syndrome coronavirus (SARS) and Middle East respiratory syndrome coronavirus (MERS) epidemiological data also indicate a gender-based preference in disease consequences. The current review addresses the possible mechanisms responsible for higher COVID-19 mortality among male patients. The major underlying aspects that was looked into includes smoking, genetic factors, and the impact of reproductive hormones on immune systems and inflammatory responses. Detailed investigations of this gender disparity could provide insight into the development of patient tailored therapeutic approach which would be helpful in improving the poor outcomes of COVID-19. Graphical abstract.

Physiological Relevance of Angiotensin Converting Enzyme 2 As a Metabolic Linker and Therapeutic Implication of Mesenchymal Stem Cells in COVID-19 and Hypertension.

Severe acute respiratory syndrome corona virus - 2 (SARS-CoV-2) is a single stranded RNA virus and responsible for infecting human being. In many cases the individual may remain asymptomatic. Some recently reported studies revealed that individuals of elderly age group and with pre-existing medical conditions such as hypertension, diabetes mellitus had severe consequences, even may lead to death. However, it is not clearly delineated whether hypertension itself or associated comorbidities or antihypertensive therapy contributes to the grave prognosis of COVID-19 infections. This review is aimed to decipher the exact mechanisms involved at molecular level from existing evidence and as reported. It has been reported that SARS-CoV-2 enters into the host cell through interaction between conserved residues of viral spike protein and angiotensin converting enzyme 2 (ACE2) receptor which is highly expressed in host's cardiac and pulmonary cells and finally transmembrane protease, serine-2 (TMPRSS2), helps in priming of the surface protein. Subsequently, symptom related to multi organ involvement is primarily contributed by cytokine storm. Although various clinical trials are being conducted on renin- angiotensin- system inhibitor, till to date there is no standard treatment protocol approved for critically ill COVID-19 positive cases with pre-existing hypertension. Recently, several studies are carried out to document the safety and efficacy outcome of mesenchymal stem cell transplantation based on its immunomodulatory and regenerative properties. Therefore, identification of future novel therapeutics in the form of mesenchymal stem cell either alone or in combination with pharmacological approach could be recommended for combating SARS-CoV-2 which might be dreadful to debilitating elderly people. Graphical Abstract.

Correction: Withaferin A (WFA) inhibits tumor growth and metastasis by targeting ovarian cancer stem cells.

[This corrects the article DOI: 10.18632/oncotarget.20170.].

Withaferin A attenuates ovarian cancer-induced cardiac cachexia.

Cachexia is a common multifactorial syndrome in the advanced stages of cancer and accounts for approximately 20-30% of all cancer-related fatalities. In addition to the progressive loss of skeletal muscle mass, cancer results in impairments in cardiac function. We recently demonstrated that WFA attenuates the cachectic skeletal muscle phenotype induced by ovarian cancer. The purpose of this study was to investigate whether ovarian cancer induces cardiac cachexia, the possible pathway involved, and whether WFA attenuates cardiac cachexia. Xenografting of ovarian cancer induced cardiac cachexia, leading to the loss of normal heart functions. Treatment with WFA rescued the heart weight. Further, ovarian cancer induced systolic dysfunction and diastolic dysfunction Treatment with WFA preserved systolic function in tumor-bearing mice, but diastolic dysfunction was partially improved. In addition, WFA abrogated the ovarian cancer-induced reduction in cardiomyocyte cross-sectional area. Finally, treatment with WFA ameliorated fibrotic deposition in the hearts of tumor-bearing animals. We observed a tumor-induced MHC isoform switching from the adult MHCα to the embryonic MHCß isoform, which was prevented by WFA treatment. Circulating Ang II level was increased significantly in the tumor-bearing, which was lowered by WFA treatment. Our results clearly demonstrated the induction of cardiac cachexia in response to ovarian tumors in female NSG mice. Further, we observed induction of proinflammatory markers through the AT1R pathway, which was ameliorated by WFA, in addition to amelioration of the cachectic phenotype, suggesting WFA as a potential therapeutic agent for cardiac cachexia in oncological paradigms.

Withaferin A: a potential therapeutic agent against COVID-19 infection.

The outbreak and continued spread of the novel coronavirus disease 2019 (COVID-19) is a preeminent global health threat that has resulted in the infection of over 11.5 million people worldwide. In addition, the pandemic has claimed the lives of over 530,000 people worldwide. Age and the presence of underlying comorbid conditions have been found to be key determinants of patient mortality. One such comorbidity is the presence of an oncological malignancy, with cancer patients exhibiting an approximate two-fold increase in mortality rate. Due to a lack of data, no consensus has been reached about the best practices for the diagnosis and treatment of cancer patients. Interestingly, two independent research groups have discovered that Withaferin A (WFA), a steroidal lactone with anti-inflammatory and anti-tumorigenic properties, may bind to the viral spike (S-) protein of SARS-CoV-2. Further, preliminary data from our research group has demonstrated that WFA does not alter expression of ACE2 in the lungs of tumor-bearing female mice. Downregulation of ACE2 has recently been demonstrated to increase the severity of COVID-19. Therefore, WFA demonstrates real potential as a therapeutic agent to treat or prevent the spread of COVID-19 due to the reported interference in viral S-protein to host receptor binding and its lack of effect on ACE2 expression in the lungs.

Withaferin A ameliorates ovarian cancer-induced cachexia and proinflammatory signaling.

BACKGROUND: Ovarian cancer is the fifth leading cause of cancer-related deaths amongst women in the United States. Cachexia is the primary cause of death in approximately 30% of cancer patients, and is often evidenced in ovarian cancer patients. We tested the steroidal lactone Withaferin A to examine if it could ameliorate ovarian cancer-induced cachexia. METHODS: Six-week-old severely immunodeficient female mice were xenografted with the ovarian cancer cell line A2780 followed by treatment with Withaferin A or vehicle. Changes in functional grip strength were assessed on a weekly basis. Postmortem, H&E staining was performed on skeletal muscle sections and immunofluorescent immunohistochemistry was performed on skeletal muscle and tumor sections. The levels of NF-κB-related proinflammatory cytokines were assessed in the xenografted tumors and in resident host skeletal muscle. RESULTS: Xenografting of the A2780 cell line resulted in a significant rate of mortality, which was attenuated by a therapeutic dosage of Withaferin A. Mice that received vehicle treatment following xenografting exhibited functional muscle decline over the course of the study. The therapeutic dosage Withaferin A treatment attenuated this reduction in grip strength, whereas the supratherapeutic dosage of Withaferin A was found to be toxic/lethal and demonstrated a further decline in functional muscle strength and an increased rate of mortality on par with vehicle treatment. At a histological level, the vehicle treated tumor-bearing mice exhibited a profound reduction in myofibrillar cross-sectional area compared to the vehicle treated tumor-free control group. The atrophic changes induced by the xenografted tumor were significantly ameliorated by treatment with Withaferin A. The combination of functional muscle weakening and induction of myofibrillar atrophy corroborate a cachectic phenotype, which was functionally rescued by Withaferin A. Further, treatment completely abolished the slow-to-fast myofiber type conversion observed in the settings of cancer-induced cachexia. In both host resident skeletal muscle and the xenografted tumors, we report an increase in NF-κB-related proinflammatory cytokines that was reversed by Withaferin A treatment. Finally, we demonstrated that Withaferin A significantly downregulates cytosolic and nuclear levels of phospho-p65, the active canonical NF-κB transcription factor, in xenografted tumors. CONCLUSIONS: Cumulatively, our results demonstrate a previously overlooked role of Withaferin A in a xenograft model of ovarian cancer. We propose mechanisms by which Withaferin A reduces NF-κB-dependent pro-inflammatory cytokine production leading to an attenuation of the cachectic phenotype in an i.p. xenograft model of ovarian cancer.

PTTG1: a Unique Regulator of Stem/Cancer Stem Cells in the Ovary and Ovarian Cancer.

Origin of cancer stem cells (CSCs) and mechanisms by which oncogene PTTG1 contributes to tumor progression via CSCs is not known. Ovarian CSCs exhibit characteristics of self-renewal, tumor-initiation, growth, differentiation, drug resistance, and tumor relapse. A common location of putative origin, namely the ovarian surface epithelium, is shared between the normal stem and CSC compartments. Existence of ovarian stem cells and their co-expression with CSC signatures suggests a strong correlation between origin of epithelial cancer and CSCs. We hereby explored a putative oncogene PTTG1 (Securin), reported to be overexpressed in various tumors, including ovarian. We report a previously overlooked role of PTTG1 as a marker of CSCs thereby modulating CSC, germline, and stemness-related genes. We further characterized PTTG1's ability to regulate (cancer) stem cell-associated self-renewal and epithelial-mesenchymal transition pathways. Collectively, the data sheds light on a potential target expressed during ovarian tumorigenesis and metastatically disseminated ascites CSCs in the peritoneal cavity. Present study highlights this unconventional, under-explored role of PTTG1 in regulation of stem and CSC compartments in ovary, ovarian cancer and ascites and highlights it as a potential candidate for developing CSC specific targeted therapeutics.

Targeting of Lung Cancer Stem Cell Self-Renewal Pathway by a Small Molecule Verrucarin J.

Despite considerable advances made in understanding of lung cancer biology, there has been meek improvement in lung cancer treatment outcome with 4% to 5% increase in 5-year survival rates in the last four decades. Underlying problem of lung cancer recurrence and poor prognosis is attributed to the presence of cancer stem cells (CSCs) which possess the potential to differentiate, proliferate and trigger chemo-resistance, tumor progression and metastasis, despite initial elimination of the tumor. To address specific targeting of CSCs, we investigated the effects of a small molecule Verrucarin J (VJ) on lung cancer cell lines A549 and H1793. VJ significantly inhibited cell proliferation of both cell lines, with IC50 values of approximately 10 nM for A549 and 20 nM for H1793 respectively after 48 h of treatment. A549 cell line when treated with VJ, induced cell apoptosis with concomitant down regulation of key CSC specific genes- ALDH1, LGR5, OCT4 and CD133 in a dose-dependent manner. To delineate the molecular mechanism by which VJ targets lung cancer cells and CSCs, we determined the effects of VJ on CSC self-renewal pathways Wnt1/ß-catenin and Notch1. Treatment of A549 cell line with VJ inhibited significantly both the signalling pathways, suggesting inhibition of expression of CSC genes by VJ through the inhibition of CSC self-renewal signalling pathways. Taken together, our results suggest that VJ may serve as a potent anticancer drug to target cancer cells and CSCs.

Bioactive Phospholipids Enhance Migration and Adhesion of Human Leukemic Cells by Inhibiting Heme Oxygenase 1 (HO-1) and Inducible Nitric Oxygenase Synthase (iNOS) in a p38 MAPK-Dependent Manner.

Bioactive phospholipids, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), lysophosphatidylcholine (LPC), and its derivative lysophosphatidic acid (LPA), have emerged as important mediators regulating the trafficking of normal and cancer cells. While the role of S1P in regulating migration of hematopoietic cells is well established, in this work we compared its biological effects to the effects of C1P, LPC, and LPA. We employed 10 human myeloid and lymphoid cell lines as well as blasts from AML patients. We observed that human leukemic cells express functional receptors for phospholipids and respond to stimulation by phosphorylation of p42/44 MAPK and AKT. We also found that bioactive phospholipids enhanced cell migration and adhesion of leukemic cells by downregulating expression of HO-1 and iNOS in a p38 MAPK-dependent manner but did not affect cell proliferation. By contrast, downregulation of p38 MAPK by SB203580 enhanced expression of HO-1 and iNOS and decreased migration of leukemic cells in vitro and their seeding efficiency to vital organs in vivo after injection into immunodeficient mice. Based on these findings, we demonstrate that, besides S1P, human leukemic cells also respond to C1P, LPC, and LPA. Since the prometastatic effects of bioactive phospholipids in vivo were mediated, at least in part, by downregulating HO-1 and iNOS expression in a p38 MAPK-dependent manner, we propose that inhibitors of p38 MAPK or stimulators of HO-1 activity will find application in inhibiting the spread of leukemic cells in response to bioactive phospholipids.

Novel view of the adult stem cell compartment - a developmental story of germline and parental imprinting.

Evidence has accumulated that postnatal tissues contain developmentally early stem cells that remain in a dormant state as well as stem cells that are more proliferative, supplying tissue-specific progenitor cells and thus playing a more active role in the turnover of adult tissues. The most primitive, dormant, postnatal tissue-derived stem cells, called very small embryonic like stem cells (VSELs), are regulated by epigenetic changes in the expression of certain parentally imprinted genes, a molecular phenomenon previously described for maintaining primordial germ cells (PGCs) in a quiescent state. Specifically, they show erasure of parental imprinting at the Igf2-H19 locus, which keeps them in a quiescent state in a similar manner as migrating PGCs. To date, the presence of these cells in adult postnatal tissues have been demonstrated by at least 25 independent laboratories. We envision that similar changes in expression of parentally imprinted genes may also play a role in the quiescence of dormant VSELs present in other non-hematopoietic tissues. Recent data indicate that VSELs expand in vivo and in vitro after reestablishment of somatic imprinting at the Igf2-H19 locus by nicotinamide treatment in response to stimulation by pituitary gonadotrophins (follicle stimulating factor, luteinizing hormone and prolactin) and gonadal androgens and estrogens. These cells could be also successfully expanded ex vivo in the presence of the small molecule UM177.