Protein restriction during puberty alters nutritional parameters and affects ovarian and uterine histomorphometry in adulthood in rats.

In a large part of the population inefficient ingestion of proteins, whether for cultural, aesthetic or economic reasons, is a global concern. Low-protein diets can cause severe functional complications, mainly during the development and maturation of organs and systems, including the female reproductive system. The present study investigated the effect of nutritional protein restriction during puberty on the oestrous cycle and expression of sex steroid receptors (AR, ERα e ERß) in ovarian and uterine tissues of adult rats. Protein restriction promoted lower body weight gain, feed efficiency and higher caloric intake. There was an increase in the oestrus phase arrest without changing the total length of the oestrous cycle. The consumption of low-protein diet also reduced the thickness of the uterine endometrium (uterine epithelium and endometrial stroma) in addition to increasing the number of primary and atretic follicles in the ovaries. Furthermore, the low-protein diet reduced the levels of androgen receptor (AR) and increased the oestrogen receptor ß (ERß) in the ovary, while no significant changes were observed in the uterus. Our study reinforces the importance of adequate protein intake during puberty, since physiological changes in this developmental period interfere with the histomorphometry of the ovaries and uteri, possibly resulting in impaired folliculogenesis and fertility in the reproductive period.

Maternal protein restriction impairs nutrition and ovarian histomorphometry without changing p38MAPK and PI3K-AKT-mTOR signaling in adult rat ovaries.

AIMS: Because an adequate protein supply is detrimental for the maintenance of folliculogenesis and ovulation, we evaluated the impact of maternal low protein diet on nutritional parameters, estrous cycle, ovarian histomorphometry, and on the expression of metabolic and survival signaling molecules in different follicular stages. MAIN METHODS: Twenty Wistar pregnant rats were divided into two groups: the normoprotein (NP) group, composed of animals that received 17% protein, and a low-protein (LP) group, composed of animals that received 6% protein during gestation and lactation period. After weaning, female rats were fed with standard diet until the 120-days-old. KEY FINDINGS: LP animals showed reduced body mass index, total body weight, energy intake, feed efficiency, and visceral fat. The ovarian tissue presented vascular congestion and fat accumulation in the medulla, followed by a significant reduction in the amount of primordial and primary follicles. In addition, the number of atretic follicles was higher in LP than in NP animals. Maternal undernutrition also resulted in increased levels of estradiol (E2) and progesterone (P4) while testosterone (T) was unchanged in the offspring. Although discrete changes in p38MAPK and in PI3K-AKT-mTOR immunostaining were observed in the ovarian follicles and corpus luteum in LP, no differences were found at their protein levels. SIGNIFICANCE: Maternal protein restriction alters estrous cycle and histomorphometry of the offspring's ovary without changing the levels of intracellular regulatory molecules in adulthood. These morphofunctional changes may alter reproductive performance in female offspring, highlighting maternal dietary conditions as an important factor for offspring reproductive health.

P-MAPA activates TLR2 and TLR4 signaling while its combination with IL-12 stimulates CD4+ and CD8+ effector T cells in ovarian cancer.

AIMS: Ovarian cancer (OC) is the most lethal gynecological malignancies and many women develop chemoresistance associated with the inflammatory process. We investigated the effects of P-MAPA and IL-12 on the inflammatory and immune responses in a chemically-induced OC model. MAIN METHODS: OCs were induced with 7,12-dimethylbenz(a)anthracene into the ovarian bursa, and the animals were given P-MAPA (5 mg/kg bw., i.p., twice a week), or IL-12 (300 ng/kg bw., i.p., one a week) for 60 days, or both P-MAPA and IL-12. Immunohistochemistry, western blot, flow cytometry, and multiplex assay were used to examine the effectiveness of immunotherapies in OC. KEY FINDINGS: The combinatory therapy improved the general OC features, reducing inflammatory cells and adipocyte accumulation, in addition to revealing a soft and mobile tissue with no adherences and peritoneal implants. P-MAPA treatment increased the levels of TLR2, TLR4 and TRIF in OCs while decreasing the number of regulatory T (Treg) cells. Additionally, the association of P-MAPA with IL-12 significantly increased the number of CD4+ and CD8+ T effector cells in draining lymph nodes. Regarding the inflammatory mediators, P-MAPA enhanced the levels of the pro-inflammatory cytokine IL-17 while P-MAPA+IL-12 increased the levels of IL-1ß. Treatment with IL-12 enhanced the cytokine levels of IL-17, TNF-α, IL-1ß, and IL-2 in addition to the chemokine MIP-1α. SIGNIFICANCE: We conclude that P-MAPA upregulated TLR2 and TLR4 signaling, possibly activating the non-canonical pathway, while attenuating the tumor immunosuppression. Also, the combination of P-MAPA with IL-12 improves the antitumor immunoresponse, opening a new therapeutic approach for fighting OC.

The role of Toll-like receptor 4 signaling pathway in ovarian, cervical, and endometrial cancers.

Toll-like receptors (TLRs) are critical sensors related to inflammation and tumorigenesis. Among all subtypes, the TLR4 is a highly described transmembrane protein involved in the inflammatory process. The TLR4/myeloid differentiation factor 88 (MyD88) signaling pathway has been implicated in oncogenic events in several tissues and is associated with survival of patients. Through activation, TLR4 recruits adaptor proteins, i.e., MyD88 or TRIF, to triggers canonical and non-canonical signaling pathways that result in distinct immune responses. In most cancer cells, uncontrolled TLR4 signaling modifies the tumor microenvironment to proliferate and evade immune surveillance. By contrast, TLR4 activation can produce antitumor activities, thereby inhibiting tumor growth and enhancing the proper immune response. We review herein recent approaches on the role of the TLR4 signaling pathway and discuss potential candidates for gynecological cancer therapies; among these agents, natural and synthetic compounds have been tested both in vitro and in vivo. Since TLR4 ligands have been investigated as effective immune-adjuvants in the context of these aggressive malignancies, we described how TLR4 signaling controls part of the tumor-related inflammatory process and which are the new targeting molecules implicated in the regulation of tumorigenicity in ovarian, cervical, and endometrial cancers.

P-MAPA and Interleukin-12 Reduce Cell Migration/Invasion and Attenuate the Toll-Like Receptor-Mediated Inflammatory Response in Ovarian Cancer SKOV-3 Cells: A Preliminary Study.

Immunotherapies have emerged as promising complementary treatments for ovarian cancer (OC), but its effective and direct role on OC cells is unclear. This study examined the combinatory effects of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride, known as P-MAPA, and the human recombinant interleukin-12 (hrIL-12) on cell migration/invasion, apoptosis, toll-like receptor (TLR)-mediated inflammation, and cytokine/chemokine profile in human OC cell line SKOV-3. P-MAPA and IL-12 showed cancer cell toxicity under low doses after 48 h. Although apoptosis/necrosis and the cell cycle were unchanged by the treatments, P-MAPA enhanced the sensitivity to paclitaxel (PTX) and P-MAPA associated with IL-12 significantly reduced the migratory potential and invasion capacity of SKOV-3 cells. P-MAPA therapy reduced TLR2 immunostaining and the myeloid differentiation factor 88 (MyD88), but not the TLR4 levels. Moreover, the combination of P-MAPA with IL-12 attenuated the levels of MyD88, interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-kB p65). The IL-12 levels were increased and P-MAPA stimulated the secretion of cytokines IL-3, IL-9, IL-10, and chemokines MDC/CCL22 and, regulated on activation, normal T cells expressed and secreted (RANTES)/CCL5. Conversely, combination therapy reduced the levels of IL-3, IL-9, IL-10, MDC/CCL22, and RANTES/CCL5. Collectively, P-MAPA and IL-12 reduce cell dynamics and effectively target the TLR-related downstream molecules, eliciting a protective effect against chemoresistance. P-MAPA also stimulates the secretion of anti-inflammatory molecules, possibly having an immune response in the OC microenvironment.

Mimicking the tumor microenvironment: Fibroblasts reduce miR-29b expression and increase the motility of ovarian cancer cells in a co-culture model.

Ovarian cancer (OC) is a highly prevalent gynecological malignancy worldwide. Throughout ovarian carcinogenesis, the crosstalk between cellular components of the microenvironment, including tumor cells and fibroblasts, is proposed to play critical roles in cancer progression. The dysregulation of microRNA expression is also a pronounced feature of the OC. The screening of microRNAs, mainly those involved in OC microenvironment, could have diagnostic and/or therapeutic potential for this malignancy. Thus, we assessed the influence of fibroblasts on microRNA expression and the motility of OC cells. To achieve this goal, SKOV-3 cancer cells were co-cultured with human normal fibroblasts derived from primary culture (FP-96). Cell viability, expression of tumor suppressor microRNAs and oncomiRs by RT-qPCR, cell migration by wound healing assay and analysis of MMP-2 activity by zymography were performed in SKOV-3 cells. Moreover, α-smooth muscle actin (α-SMA) expression was evaluated by Western blot in FP-96 fibroblasts. Notably, the co-culture downregulated the tumor suppressor miR-29b and increased migration of SKOV-3 cells. In addition, co-culture increased the activity of MMP-2, which is a miR-29 target, and accounted for extracellular matrix remodeling and augmented cellular motility. Concomitantly, the co-culture system induced α-SMA expression in FP-96 fibroblasts, the commonly expressed marker in cancer-associated fibroblasts (CAFs). Our findings suggest that the potential crosstalk between OC cells and fibroblasts in tumor microenvironment may play a key role in the progression of OC.

Melatonin Promotes Uterine and Placental Health: Potential Molecular Mechanisms.

The development of the endometrium is a cyclic event tightly regulated by hormones and growth factors to coordinate the menstrual cycle while promoting a suitable microenvironment for embryo implantation during the "receptivity window". Many women experience uterine failures that hamper the success of conception, such as endometrium thickness, endometriosis, luteal phase defects, endometrial polyps, adenomyosis, viral infection, and even endometrial cancer; most of these disturbances involve changes in endocrine components or cell damage. The emerging evidence has proven that circadian rhythm deregulation followed by low circulating melatonin is associated with low implantation rates and difficulties to maintain pregnancy. Given that melatonin is a circadian-regulating hormone also involved in the maintenance of uterine homeostasis through regulation of numerous pathways associated with uterine receptivity and gestation, the success of female reproduction may be dependent on the levels and activity of uterine and placental melatonin. Based on the fact that irregular production of maternal and placental melatonin is related to recurrent spontaneous abortion and maternal/fetal disturbances, melatonin replacement may offer an excellent opportunity to restore normal physiological function of the affected tissues. By alleviating oxidative damage in the placenta, melatonin favors nutrient transfer and improves vascular dynamics at the uterine-placental interface. This review focuses on the main in vivo and in vitro functions of melatonin on uterine physiological processes, such as decidualization and implantation, and also on the feto-maternal tissues, and reviews how exogenous melatonin functions from a mechanistic standpoint to preserve the organ health. New insights on the potential signaling pathways whereby melatonin resists preeclampsia and endometriosis are further emphasized in this review.

Mitochondrial functions and melatonin: a tour of the reproductive cancers.

Cancers of the reproductive organs have a strong association with mitochondrial defects, and a deeper understanding of the role of this organelle in preneoplastic-neoplastic changes is important to determine the appropriate therapeutic intervention. Mitochondria are involved in events during cancer development, including metabolic and oxidative status, acquisition of metastatic potential, resistance to chemotherapy, apoptosis, and others. Because of their origin from melatonin-producing bacteria, mitochondria are speculated to produce melatonin and its derivatives at high levels; in addition, exogenously administered melatonin accumulates in the mitochondria against a concentration gradient. Melatonin is transported into tumor cell by GLUT/SLC2A and/or by the PEPT1/2 transporters, and plays beneficial roles in mitochondrial homeostasis, such as influencing oxidative phosphorylation and electron flux, ATP synthesis, bioenergetics, calcium influx, and mitochondrial permeability transition pore. Moreover, melatonin promotes mitochondrial homeostasis by regulating nuclear DNA and mtDNA transcriptional activities. This review focuses on the main functions of melatonin on mitochondrial processes, and reviews from a mechanistic standpoint, how mitochondrial crosstalk evolved in ovarian, endometrial, cervical, breast, and prostate cancers relative to melatonin's known actions. We put emphasis on signaling pathways whereby melatonin interferes within cancer-cell mitochondria after its administration. Depending on subtype and intratumor metabolic heterogeneity, melatonin seems to be helpful in promoting apoptosis, anti-proliferation, pro-oxidation, metabolic shifting, inhibiting neovasculogenesis and controlling inflammation, and restoration of chemosensitivity. This results in attenuation of development, progression, and metastatic potential of reproductive cancers, in addition to lowering the risk of recurrence and improving the life quality of patients.

P-MAPA immunotherapy potentiates the effect of cisplatin on serous ovarian carcinoma through targeting TLR4 signaling.

BACKGROUND: Toll-like receptors (TLRs) are transmembrane proteins expressed on the surface of ovarian cancer (OC) and immune cells. Identifying the specific roles of the TLR-mediated signaling pathways in OC cells is important to guide new treatments. Because immunotherapies have emerged as the adjuvant treatment for patients with OC, we investigated the effect of a promising immunotherapeutic strategy based on protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) combined with cisplatin (CIS) on the TLR2 and TLR4 signaling pathways via myeloid differentiation factor 88 (MyD88) and TLR-associated activator of interferon (TRIF) in an in vivo model of OC. METHODS: Tumors were chemically induced by a single injection of 100 µg of 7,12-dimethylbenz(a)anthracene (DMBA) directly under the left ovarian bursa in Fischer 344 rats. After the rats developed serous papillary OC, they were given P-MAPA, CIS or the combination P-MAPA+CIS as therapies. To understand the effects of the treatments, we assessed the tumor size, histopathology, and the TLR2- and TLR4-mediated inflammatory responses. RESULTS: Although CIS therapy was more effective than P-MAPA in reducing the tumor size, P-MAPA immunotherapy significantly increased the expressions of TLR2 and TLR4. More importantly, the combination of P-MAPA with CIS showed a greater survival rate compared to CIS alone, and exhibited a significant reduction in tumor volume compared to P-MAPA alone. The combination therapy also promoted the increase in the levels of the following OC-related proteins: TLR4, MyD88, TRIF, inhibitor of phosphorylated NF-kB alpha (p-IkBα), and nuclear factor kappa B (NF-kB p65) in both cytoplasmic and nuclear sites. While P-MAPA had no apparent effect on tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6, it seems to increase interferon-γ (IFN-γ), which may induce the Thelper (Th1)-mediated immune response. CONCLUSION: Collectively, our results suggest that P-MAPA immunotherapy combined with cisplatin could be considered an important therapeutic strategy against OC cells based on signaling pathways activated by TLR4.

Melatonin as a promising agent to treat ovarian cancer: molecular mechanisms.

Ovarian cancer (OC) has the highest mortality rate of all gynecological cancers, and most patients develop chemoresistance after first-line treatments. Despite recent advances, the 5-year relative survival is ~45% for all OC subtypes, and invasive epithelial OC has only a 17% survival rate when diagnosed at a late stage. Identification of new efficacious molecules or biomarkers represents important opportunities in the treatment of OC. The pharmacological and physiological properties of melatonin indicate this agent could be useful against OC progression and metastasis. In normal cells, melatonin has potent antioxidant and anti-apoptotic actions. Conversely, melatonin has pro-oxidant as well as anti-proliferative, anti-angiogenic and immunomodulatory properties in many cancer types including hormone-dependent cancers. Although melatonin receptors have been identified in OC cells, the exact mechanism by which melatonin induces anticancer activities remains incompletely understood. Clinical studies have reported negative correlation between aggressiveness of OC and serum levels of melatonin, reinforcing the idea that melatonin may be a critical factor determining OC development. In vitro and in vivo studies suggest melatonin differentially regulates multiple signaling pathways in OC cells. This focused review explores the potential mechanisms of action of melatonin on cultured OC cells and in experimental models of OC in an attempt to clarify how melatonin modulates the signaling pathways involved in cancer cell apoptosis, survival, inflammation, proliferation and metabolic processes. Based on the evidence presented, we feel that melatonin, as an agent that controls cellular signals associated with malignancy, may be beneficial in combination with other therapeutics for OC treatment.