Metabolic characteristics of granulosa cell tumor: role of PPARγ signaling†.

Granulosa cell tumors are relatively rare, posing challenges for comprehension and therapeutic development due to limited cases and preclinical models. Metabolic reprogramming, a hallmark of cancer, manifests in granulosa cell tumors with notable lipid accumulation and increased expression of peroxisome proliferator-activated receptor gamma (PPARγ), a key lipid metabolism regulator. The roles of these features, however, remain unclear. In our previous work, we established a granulosa cell tumor model in mice by introducing a constitutively active Pik3ca mutant in oocytes, enabling the study of predictable tumor patterns from postnatal day 50. In this study, we characterized metabolic alterations during tumorigenesis (postnatal day 8 to day 50) and tumor growth (day 50 to day 65) in this model and explored the impact of PPARγ antagonism on human granulosa cell tumor proliferation. The tumor exhibited significant lipid accumulation, with PPARγ and the proliferation marker Ki67 co-localizing at postnatal day 65. Transcriptome analysis demonstrates that pathways for lipid metabolism and mitochondrial oxidation are promoted during tumorigenesis and tumor growth, respectively. Overlappingly upregulated genes during tumorigenesis and tumor growth are associated with lipid metabolism pathways. Correspondingly, mouse granulosa cell tumor shows overexpression of peroxisome proliferator-activated receptor gamma and DGAT2 proteins at postnatal day 65. Furthermore, GW9662 reduces the proliferation of KGN human granulosa cell tumor cells and decreases the phosphorylation of AKT and SMAD3. Our findings identify metabolic abnormalities in ooPIK3CA* granulosa cell tumor model and suggest peroxisome proliferator-activated receptor gamma as a potential driver for primary granulosa cell tumor growth.

Uncovering Tumor-Promoting Roles of Activin A in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with high incidence rates of metastasis and cachexia. High circulating activin A, a homodimer of inhibin ßA subunits that are encoded by INHBA gene, predicts poor survival among PDAC patients. However, it still raises the question of whether activin A suppression renders favorable PDAC outcomes. Here, the authors demonstrate that activin A is abundantly detected in tumor and stromal cells on PDAC tissue microarray and mouse PDAC sections. In orthotopic male mice, activin A suppression, which is acquired by tumor-targeted Inhba siRNA using cholesterol-modified polymeric nanoparticles, retards tumor growth/metastasis and cachexia and improves survival when compared to scramble siRNA-treated group. Histologically, activin A suppression coincides with decreased expression of proliferation marker Ki67 but increased accumulation of α-SMAhigh fibroblasts and cytotoxic T cells in the tumors. In vitro data demonstrate that activin A promotes KPC cell proliferation and induces the downregulation of α-SMA and upregulation of IL-6 in pancreatic stellate cells (PSC) in the SMAD3-dependent mechanism. Moreover, conditioned media from activin A-stimulated PSC promoted KPC cell growth. Collectively, our data provide a mechanistic basis for tumor-promoting roles of activin A and support therapeutic potentials of tumor activin A suppression for PDAC.

TAp63 determines the fate of oocytes against DNA damage.

Cyclophosphamide and doxorubicin lead to premature ovarian insufficiency as an off-target effect. However, their oocyte death pathway has been debated. Here, we clarified the precise mechanism of ovarian depletion induced by cyclophosphamide and doxorubicin. Dormant oocytes instead of activated oocytes with high PI3K activity were more sensitive to cyclophosphamide. Checkpoint kinase 2 (CHK2) inhibitor rather than GNF2 protected oocytes from cyclophosphamide and doxorubicin, as cyclophosphamide up-regulated p-CHK2 and depleted primordial follicles in Abl1 knockout mice. Contrary to previous reports, TAp63 is pivotal in cyclophosphamide and doxorubicin-induced oocyte death. Oocyte-specific Trp63 knockout mice prevented primordial follicle loss and maintained reproductive function from cyclophosphamide and doxorubicin, indicated by undetectable levels of BAX and cPARP. Here, we demonstrated that TAp63 is fundamental in determining the signaling of oocyte death against DNA damage. This study establishes the role of TAp63 as a target molecule of adjuvant therapies to protect the ovarian reserve from different classes of chemotherapy.

Adipose Tissue Wasting as a Determinant of Pancreatic Cancer-Related Cachexia.

Pancreatic cancer (PC) is the third leading cause of cancer-related death in the US, and its 5-year survival rate is approximately 10%. The low survival rates largely stem from diagnostic delay and the presence of significant adipose tissue and muscle wasting, commonly referred to as cachexia. Cachexia is present in nearly 80% of PC patients and is a key cause of poor response to treatment and about 20% of death in PC patients. However, there are few clinical interventions proven to be effective against PC-related cachexia. Different cancer types feature distinct secretome profiles and functional characteristics which would lead to cachexia development differently. Therefore, here we discuss affected tissues and potential mechanisms leading to cachexia in PC. We postulate that the most affected tissue during the development of PC-related cachexia is adipose tissue, historically and still thought to be just an inert repository for excess energy in relation to cancer-related cachexia. Adipose tissue loss is considerably greater than muscle loss in quantity and shows a correlation with poor survival in PC patients. Moreover, we suggest that PC mediates adipose atrophy by accelerating adipocyte lipid turnover and fibroblast infiltration.

Oocyte CTR1 is not essential for cisplatin-induced oocyte death of primordial follicle.

Accumulated evidence indicates that cisplatin, a platinum-based alkylating agent, causes preferential DNA damage to oocytes of primordial follicles (PFs) in the ovary, suggesting oocyte-favored accumulation of cisplatin. Copper transporter 1 (CTR1; Slc31a1 ) is implicated in facilitating cisplatin uptake in cells. Here we found that oocytes of PFs had constitutively higher expression of CTR1 than other cell types in mouse ovary. However, oocyte-specific Slc31a1 knockout was not sufficient to prevent cisplatin-induced depletion of PFs in vitro . Our data indicate that CTR1 would not be the only route for cisplatin to be transported inside the oocytes of PFs in the ovary.

3D bioprinted white adipose model forin vitrostudy of cancer-associated cachexia induced adipose tissue remodeling.

Cancer-associated cachexia (CAC) is a complex metabolic and behavioral syndrome with multiple manifestations that involve systemic inflammation, weight loss, and adipose lipolysis. It impacts the quality of life of patients and is the direct cause of death in 20%-30% of cancer patients. The severity of fat loss and adipose tissue remodeling negatively correlate with patients' survival outcomes. To address the mechanism of fat loss and design potential approaches to prevent the process, it will be essential to understand CAC pathophysiology through white adipose tissue models. In the present study, an engineered human white adipose tissue (eWAT) model based on three-dimensional (3D) bioprinting was developed and induced with pancreatic cancer cell-conditioned medium (CM) to mimic the status of CACin vitro. We found that the CM induction significantly increased the lipolysis and accumulation of the extracellular matrix (ECM). The 3D eWATs were further vascularized to study the influence of vascularization on lipolysis and CAC progression, which was largely unknown. Results demonstrated that CM induction improved the angiogenesis of vascularized eWATs (veWATs), and veWATs demonstrated decreased glycerol release but increasedUCP1expression, compared to eWATs. Many unique inflammatory cytokines (IL-8, CXCL-1, GM-CSF, etc) from the CM were detected and supposed to contribute to eWAT lipolysis,UCP1up-regulation, and ECM development. In response to CM induction, eWATs also secreted inflammatory adipokines related to the metastatic ability of cancer, muscle atrophy, and vascularization (NGAL, CD54, IGFBP-2, etc). Our work demonstrated that the eWAT is a robust model for studying cachectic fat loss and the accompanying remodeling of adipose tissue. It is therefore a useful tool for future research exploring CAC physiologies and developing potential therapies.

Visceral adipose tissue remodeling in pancreatic ductal adenocarcinoma cachexia: the role of activin A signaling.

Pancreatic ductal adenocarcinoma (PDAC) patients display distinct phenotypes of cachexia development, with either adipose tissue loss preceding skeletal muscle wasting or loss of only adipose tissue. Activin A levels were measured in serum and analyzed in tumor specimens of both a cohort of Stage IV PDAC patients and the genetically engineered KPC mouse model. Our data revealed that serum activin A levels were significantly elevated in Stage IV PDAC patients in comparison to age-matched non-cancer patients. Little is known about the role of activin A in adipose tissue wasting in the setting of PDAC cancer cachexia. We established a correlation between elevated activin A and remodeling of visceral adipose tissue. Atrophy and fibrosis of visceral adipose tissue was examined in omental adipose tissue of Stage IV PDAC patients and gonadal adipose tissue of an orthotopic mouse model of PDAC. Remarkably, white visceral adipose tissue from both PDAC patients and mice exhibited decreased adipocyte diameter and increased fibrotic deposition. Strikingly, expression of thermogenic marker UCP1 in visceral adipose tissues of PDAC patients and mice remained unchanged. Thus, we propose that activin A signaling could be relevant to the acceleration of visceral adipose tissue wasting in PDAC-associated cachexia.

Development of ovarian tumour causes significant loss of muscle and adipose tissue: a novel mouse model for cancer cachexia study.

BACKGROUND: Cancer-associated cachexia (CAC) is a complex syndrome of progressive muscle wasting and adipose loss with metabolic dysfunction, severely increasing the morbidity and mortality risk in cancer patients. However, there are limited studies focused on the underlying mechanisms of the progression of CAC due to the complexity of this syndrome and the lack of preclinical models that mimics its stagewise progression. METHODS: We characterized the initiation and progression of CAC in transgenic female mice with ovarian tumours. We measured proposed CAC biomarkers (activin A, GDF15, IL-6, IL-1ß, and TNF-α) in sera (n = 6) of this mouse model. The changes of activin A and GDF15 (n = 6) were correlated with the decline of bodyweight over time. Morphometry and signalling markers of muscle atrophy (n ≥ 6) and adipose tissue wasting (n ≥ 7) were assessed during CAC progression. RESULTS: Cancer-associated cachexia symptoms of the transgenic mice model used in this study mimic the progression of CAC seen in humans, including drastic body weight loss, skeletal muscle atrophy, and adipose tissue wasting. Serum levels of two cachexia biomarkers, activin A and GDF15, increased significantly during cachexia progression (76-folds and 10-folds, respectively). Overactivation of proteolytic activity was detected in skeletal muscle through up-regulating muscle-specific E3 ligases Atrogin-1 and Murf-1 (16-folds and 14-folds, respectively) with decreasing cross-sectional area of muscle fibres (P < 0.001). Muscle wasting mechanisms related with p-p38 MAPK, FOXO3, and p-AMPKα were highly activated in concurrence with an elevation in serum activin A. Dramatic fat loss was also observed in this mouse model with decreased fat mass (n ≥ 6) and white adipocytes sizes (n = 6) (P < 0.0001). The adipose tissue wasting was based on thermogenesis, supported by the up-regulation of uncoupling protein 1 (UCP1). Fibrosis in adipose tissue was also observed in concurrence with adipose tissue loss (n ≥ 13) (p < 0.0001). CONCLUSIONS: Our novel preclinical CAC mouse model mimics human CAC phenotypes and serum biomarkers. The mouse model in this study showed proteolysis in muscle atrophy, browning in adipose tissue wasting, elevation of serum activin A and GDF15, and atrophy of pancreas and liver. This mouse line would be the best preclinical model to aid in clarifying molecular mediators of CAC and dissecting metabolic dysfunction and tissue atrophy during the progression of CAC.

Effects of PD-1 blockade on ovarian follicles in a prepubertal female mouse.

Immunotherapy has emerged at the forefront of cancer treatment. Checkpoint inhibitor pembrolizumab (KEYTRUDA), a chimeric antibody which targets programmed cell death protein 1 (PD-1), has been approved by the Food and Drug Administration (FDA) for use in pediatric patients with relapsed or refractory classical Hodgkin's lymphoma. However, there is currently no published data regarding the effects of pembrolizumab on the ovary of female pediatric patients. In this study, prepubertal immunocompetent and immunodeficient female mice were injected with pembrolizumab or anti-mouse PD-1 antibody. The number of primordial follicles significantly decreased post-injection of both pembrolizumab and anti-mouse PD-1 antibody in immunocompetent mice. However, no changes in follicle numbers were observed in immunodeficient nude mice. Superovulation test and vaginal opening experiments suggest that there is no difference in the number of cumulus-oocyte complexes (COCs) and the timing of puberty onset between the control and anti-mouse PD-1 antibody treatment groups, indicating that there is no effect on short-term fertility. Elevation of pro-inflammatory cytokine TNF-α following COX-2 upregulation was observed in the ovary. CD3+ T-cell infiltration was detected within some ovarian follicles and between stromal cells of the ovaries in mice following treatment with anti-mouse PD-1 antibody. Thus, PD-1 immune checkpoint blockade affects the ovarian reserve through a mechanism possibly involving inflammation following CD3+ T-cell infiltration.

The Role of Mutant p63 in Female Fertility.

The transcription factor p63, one of the p53 family members, plays an essential role in regulating maternal reproduction and genomic integrity as well as epidermal development. TP63 (human)/Trp63 (mouse) produces multiple isoforms: TAp63 and ΔNp63, which possess a different N-terminus depending on two different promoters, and p63a, p63b, p63g, p63δ, and p63ε as products of alternative splicing at the C-terminus. TAp63 expression turns on in the nuclei of primordial germ cells in females and is maintained mainly in the oocyte nuclei of immature follicles. It has been established that TAp63 is the genomic guardian in oocytes of the female ovaries and plays a central role in determining the oocyte fate upon oocyte damage. Lately, there is increasing evidence that TP63 mutations are connected with female infertility, including isolated premature ovarian insufficiency (POI) and syndromic POI. Here, we review the biological functions of p63 in females and discuss the consequences of p63 mutations, which result in infertility in human patients.

Anthocyanin-Rich Aronia Berry Extract Mitigates High-Fat and High-Sucrose Diet-Induced Adipose Tissue Inflammation by Inhibiting Nuclear Factor-κB Activation.

Obesity-induced inflammation in adipose tissue (AT) promotes the development of metabolic dysregulations by increasing macrophage recruitment in the stromal vascular fraction (SVF). The activation of nuclear factor-κB (NF-κB) signaling in macrophages serves as a pivotal mediator of AT inflammatory responses by increasing the expression of proinflammatory genes in obesity. Given the purported anti-inflammatory effects of berry consumption in humans, we evaluated if anthocyanin-rich aronia berry extract (ARN) can prevent obesity-induced AT inflammation in vivo. We also examined whether ARN suppresses lipopolysaccharide (LPS)-induced NF-κB activation in RAW 264.7 macrophages and mouse bone marrow-derived macrophages (BMDMs). Male C57BL/6J mice were fed a low-fat diet, a high-fat (HF), and high-sucrose (HS) diet or HF/HS diet supplemented with 0.2% ARN (HF/HS + ARN) for 14 weeks. Compared to HF-/HS-fed mice, ARN supplementation tended to decrease fasting serum glucose (P = .07). Furthermore, ARN supplementation significantly inhibited the phosphorylation of NF-κB p65 in epididymal AT with a concomitant decrease in the expression of Cd11b and Tnfα mRNAs in epididymal SVF isolated, compared with those from HF-/HS-fed mice. Consistent with these in vivo findings, ARN treatment significantly decreased the phosphorylation of p65 in LPS-stimulated RAW 264.7 macrophages and BMDMs. Moreover, ARN suppressed LPS-induced mRNA expression of inflammation mediators (iNos, Cox-2, Tnfα, Mcp-1, and Il-6) and glycolysis markers (Glut1, G6pdh, and Hk1) in both cell types. Taken together, our in vivo and in vitro results suggest that ARN supplementation may attenuate obesity-induced AT inflammation by inhibiting NF-κB signaling and glycolytic pathway in macrophages.

Antidiabetic effect of chitosan oligosaccharide (GO2KA1) is mediated via inhibition of intestinal alpha-glucosidase and glucose transporters and PPARγ expression.

We have previously reported that administration of low molecular weight chitosan oligosaccharide (GO2KA1) significantly suppressed postprandial blood glucose rise with increased plasma adiponectin and HbA1c levels in animals and humans. However, the cellular mechanisms whereby GO2KA1 exerts antihyperglycemic effects still remain to be determined. Using intestinal Caco-2 cells and 3T3-L1 cells, here we show that GO2KA1 has dual modes of antidiabetic action by (1) inhibiting intestinal α-glucosidase as well as glucose transporters SGLT1 and GLUT2 that were distinct from the acarbose effect; (2) enhancing adipocyte differentiation, PPARγ expression and its target genes, such as FABP4, adiponectin, and GLUT4, whereas the effects were abolished by co-treatment with BADGE, a PPARγ antagonist. Moreover, GO2KA1 significantly increased glucose uptake, which was reduced in the presence of BADGE. Our data show that GO2KA1 may prevent hyperglycemia by inhibiting intestinal glucose digestion and transport and also enhance glucose uptake, at least in part, by upregulating adiponectin expression through PPARγ in adipocytes. These findings may provide potential molecular modes of action for the antidiabetic effects of chitosan oligosaccharide observed in clinical and animal studies. © 2016 BioFactors, 43(1):90-99, 2017.

Effect of High Temperature- and High Pressure-Treated Red Ginseng on Lipolysis and Lipid Oxidation in C2C12 Myotubes.

Korean red ginseng (KRG), a highly valuable medicinal herb in oriental societies, has biological activity similar to that of Panax ginseng. Recently, it has been discovered that the biological activities of red ginseng can vary according to heating and steaming processes under different conditions that change the principal components of KRG and result in changes in biological activity. This study evaluated and compared the effects of high temperature- and high pressure-treated red ginseng (HRG) and commercial red ginseng (RG) on ß-oxidation in C2C12 myotubes. HRG enhanced the phosphorylation levels of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), but RG did not affect the phosphorylation of AMPK in C2C12 myotubes. HRG also promoted the nuclear translocation of forkhead box protein O1 (FoxO1), and the translocation exerted an increase in the protein expression of adipose triglyceride lipase (ATGL). As a consequence, HRG increased the mRNA expression level of carnitine palmitoyltransferase 1 (CPT-1) compared to the control. Taken together, our results indicated that HRG promotes the lipolysis of triglycerides and mitochondrial ß-oxidation of fatty acids in C2C12 myotubes, suggesting that alterations to the principal components by high temperature and pressure may positively influence the nutraceutical functions of HRG.

Selected tea and tea pomace extracts inhibit intestinal α-glucosidase activity in vitro and postprandial hyperglycemia in vivo.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by postprandial hyperglycemia, which is an early defect of T2DM and thus a primary target for anti-diabetic drugs. A therapeutic approach is to inhibit intestinal α-glucosidase, the key enzyme for dietary carbohydrate digestion, resulting in delayed rate of glucose absorption. Although tea extracts have been reported to have anti-diabetic effects, the potential bioactivity of tea pomace, the main bio waste of tea beverage processing, is largely unknown. We evaluated the anti-diabetic effects of three selected tea water extracts (TWE) and tea pomace extracts (TPE) by determining the relative potency of extracts on rat intestinal α-glucosidase activity in vitro as well as hypoglycemic effects in vivo. Green, oolong, and black tea bags were extracted in hot water and the remaining tea pomace were dried and further extracted in 70% ethanol. The extracts were determined for intestinal rat α-glucosidases activity, radical scavenging activity, and total phenolic content. The postprandial glucose-lowering effects of TWE and TPE of green and black tea were assessed in male Sprague-Dawley (SD) rats and compared to acarbose, a known pharmacological α-glucosidase inhibitor. The IC50 values of all three tea extracts against mammalian α-glucosidase were lower or similar in TPE groups than those of TWE groups. TWE and TPE of green tea exhibited the highest inhibitory effects against α-glucosidase activity with the IC50 of 2.04 ± 0.31 and 1.95 ± 0.37 mg/mL respectively. Among the specific enzymes tested, the IC50 values for TWE (0.16 ± 0.01 mg/mL) and TPE (0.13 ± 0.01 mg/mL) of green tea against sucrase activity were the lowest compared to those on maltase and glucoamylase activities. In the animal study, the blood glucose level at 30 min after oral intake (0.5 g/kg body wt) of TPE and TWE of both green and black tea was significantly reduced compared to the control in sucrose-loaded SD rats. The TPE of all three teas had significantly higher phenolic content than those of the TWE groups, which correlated strongly with the DPPH radical scavenging activity. This is the first report of tea pomace extract significantly inhibits intestinal α-glucosidase, resulting in delayed glucose absorption and thereby suppressed postprandial hyperglycemia. Our data suggest that tea pomace-derived bioactives may have great potential for further development as nutraceutical products and the reuse of otherwise biowaste as valuable bioresources for the industry.

Inhibitory effect of high temperature- and high pressure-treated red ginseng on exercise-induced oxidative stress in ICR mouse.

As previously reported, high temperature- and high pressure-treated red ginseng (HRG) contain higher contents of phenolic compounds and protect C2C12 muscle cells and 3T3-L1 adipocytes against oxidative stress. This study investigated the effect of HRG on oxidative stress using a mouse model. Our results show that the levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, hepatic malondialdehyde in the HRG group were significantly lower than those of the exercise groups supplemented with commercial red ginseng (CRG) or not supplemented. The muscular glycogen level, glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities of the HGR group were higher than that of the CGR group. Furthermore, the HRG treatment group displayed upregulated mRNA expression of Cu/Zn-SOD and muscle regulatory factor 4. These results indicate that HRG may protect oxidative stress induced by exercise as well as improve exercise performance capacity.

Phenolic composition, antioxidant activity and anti-adipogenic effect of hot water extract from safflower (Carthamus tinctorius L.) seed.

This study was to evaluate the phenolic content and composition of Carthamus tinctorius L. seed extract (CSE) and to further assess its antioxidant and anti-adipogenic activities using various radical scavenging systems and 3T3-L1 cells. Our results show that the total phenolic and flavonoid contents of CSE were 126.0 ± 2.4 mg GAE/g and 62.2 ± 1.9 mg QE/g, respectively. The major phenolic compounds in CSE was (-)-epigallocatechin (109.62 mg/g), with a 4-hydroxy benzhydrazide derivative and gallocatechin present at 18.28 mg/g and 17.02 mg/g, respectively. CSE exhibited remarkable radical scavenging activities, FRAP (ferric reducing antioxidant power) and reducing power in a dose-dependent manner. Moreover, the oxygen radical absorbance capacity (ORAC) value of CSE (0.1 mg/mL) was 62.9 ± 4.7 µM TE (trolox equivalent)/g. During adipogenesis, CSE significantly inhibited fat accumulation in 3T3-L1 cells compared with control cells. Overall, these results indicate that CSE might be a valuable source of bioactive compounds that impart functional food and natural antioxidant properties.