Melatonin Reverses the Warburg-Type Metabolism and Reduces Mitochondrial Membrane Potential of Ovarian Cancer Cells Independent of MT1 Receptor Activation.

Ovarian cancer (OC) is the most lethal gynecologic malignancy, and melatonin has shown various antitumor properties. Herein, we investigated the influence of melatonin therapy on energy metabolism and mitochondrial integrity in SKOV-3 cells and tested whether its effects depended on MT1 receptor activation. SKOV-3 cells were exposed to different melatonin concentrations, and experimental groups were divided as to the presence of MT1 receptors (melatonin groups) or receptor absence by RNAi silencing (siRNA MT1+melatonin). Intracellular melatonin levels increased after treatment with melatonin independent of the MT1. The mitochondrial membrane potential of SKOV-3 cells decreased in the group treated with the highest melatonin concentration. Melatonin reduced cellular glucose consumption, while MT1 knockdown increased its consumption. Interconversion of lactate to pyruvate increased after treatment with melatonin and was remarkable in siRNA MT1 groups. Moreover, lactate dehydrogenase activity decreased with melatonin and increased after MT1 silencing at all concentrations. The UCSC XenaBrowser tool showed a positive correlation between the human ASMTL gene and the ATP synthase genes, succinate dehydrogenase gene (SDHD), and pyruvate dehydrogenase genes (PDHA and PDHB). We conclude that melatonin changes the glycolytic phenotype and mitochondrial integrity of SKOV-3 cells independent of the MT1 receptor, thus decreasing the survival advantage of OC cells.

The proteomic landscape of ovarian cancer cells in response to melatonin.

Ovarian cancer (OC) is the most lethal gynecological malignancy with a highly negative prognosis. Melatonin is an indoleamine secreted by the pineal gland during darkness and has shown antitumor activity in both in vitro and in vivo experiments. Herein, we investigated the influence of melatonin on the proteome of human ovarian carcinoma cells (SKOV-3 cell line) using the Ultimate 3000 LC Liquid NanoChromatography equipment coupled to a Q-Exactive mass spectrometry. After 48 h of treatment, melatonin induced a significant cytotoxicity especially with the highest melatonin concentration. The proteomic profile revealed 639 proteins in the control group, and 98, 110, and 128 proteins were altered by melatonin at the doses of 0.8, 1.6, and 2.4 mM, respectively. Proteins associated with the immune system and tricarboxylic acid cycle were increased in the three melatonin-exposed groups of cells. Specifically, the dose of 2.4 mM led to a reduction in molecules associated with protein synthesis, especially those of the ribosomal protein family. We also identified 28 potential genes shared between normal ovarian tissue and OC in all experimental groups, and melatonin was predicted to alter genes encoding ribosomal proteins. Notably, the set of proteins changed by melatonin was linked to a better prognosis for OC patients. We conclude that melatonin significantly alters the proteome of SKOV-3 cells by changing proteins involved with the immune response and mitochondrial metabolism. The concentration of 2.4 mM of melatonin promoted the largest number of protein changes. The evidence suggests that melatonin may be an effective therapeutic strategy against OC.

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.

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 and IL-12 Differentially Regulate Proteins Associated with Ovarian Cancer Progression: A Proteomic Study.

To investigate the potential role of immunotherapies in the cellular and molecular mechanisms associated with ovarian cancer (OC), we applied a comparative proteomic toll using protein identification combined with mass spectrometry. Herein, the effects of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride, known as P-MAPA, and the human recombinant interleukin-12 (hrIL-12) were tested alone or in combination in human SKOV-3 cells. The doses and period were defined based on a previous study, which showed that 25 µg/mL P-MAPA and 1 ng/mL IL-12 are sufficient to reduce cell metabolism after 48 h. Indeed, among 2,881 proteins modulated by the treatments, 532 of them were strictly concordant and common. P-MAPA therapy upregulated proteins involved in tight junction, focal adhesion, ribosome constitution, GTP hydrolysis, semaphorin interactions, and expression of SLIT and ROBO, whereas it downregulated ERBB4 signaling, toll-like receptor signaling, regulation of NOTCH 4, and the ubiquitin proteasome pathway. In addition, IL-12 therapy led to upregulation of leukocyte migration, tight junction, and cell signaling, while cell communication, cell metabolism, and Wnt signaling were significantly downregulated in OC cells. A clear majority of proteins that were overexpressed by the combination of P-MAPA with IL-12 are involved in tight junction, focal adhesion, DNA methylation, metabolism of RNA, and ribosomal function; only a small number of downregulated proteins were involved in cell signaling, energy and mitochondrial processes, cell oxidation and senescence, and Wnt signaling. These findings suggest that P-MAPA and IL-12 efficiently regulated important proteins associated with OC progression; these altered proteins may represent potential targets for OC treatment in addition to its immunoadjuvant effects.

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.