High Glucose Promotes Epithelial-Mesenchymal Transition of Uterus Endometrial Cancer Cells by Increasing ER/GLUT4-Mediated VEGF Secretion.

BACKGROUND/AIMS: Uterus endometrial cancer (UEC) is the common malignancy among gynecologic cancers, and most of them are type I estrogen-dependent UEC. Diabetes is well-known risk factor for the development of UEC. However, the underlying link between high glucose (HG) and the estrogen receptor in UEC remains unclear. Epithelial-mesenchymal transition (EMT) has also been shown to occur during the initiation of metastasis in cancer progression. The aim of this study was to determine the relationships and roles of HG, estrogen receptor and EMT in the growth and migration of UEC. METHODS: The expression of glucose transport protein 4 (GLUT4) in the control endometrium and UEC tissues was detected by immunohistochemistry (IHC); the cell viability and invasion were analyzed through CCK-8 and Matrigel invasion assays; the transcriptional level of EMT-related genes was evaluated through real-time PCR; and the effect of HG and / or GLUT4 on estrogen receptors, vascular endothelial growth factor (VEGF) and its receptor VEGFR was analyzed through western blotting, ELISA and flow cytometry (FCM) assay, respectively. In addition, Ishikawa-xenografted nude mice were constructed and were used to analyze the effect of estrogen and GLUT4 on the growth of UEC in vivo. RESULTS: Here, we found that exposure to HG led to a high level of viability and invasion of UEC cell lines (UECC, Ishikawa and RL95-2 cells). Compared with the normal endometrium, a higher level of GLUT4 was observed in UEC tissues. Silencing GLUT4 obviously inhibited the HG-promoted viability, invasion and expression of EMT-related genes (TWIST, SNAIL and CTNNB1) of UECC promoted by HG. Further analysis showed that HG and GLUT4 promoted the secretion of VEGF and expression of VEGFR in UECC. Treatment with HG led to the increase of estrogen receptor α (ERα) and ß (ERß) in UECC, blocking ERα or ERß resulted in the decreases in GLUT4 expression, TWIST, SNAIL and CTNNB1 transcription, and VEGF and VEGFR expression in UECC. Treatment with anti-human VEGF neutralizing antibody restricted the viability and invasion of UECC that was induced by HG and estrogen. Exposure to estrogen accelerated growth, VEGF production, and TWIST and CTNNB1 expression in UEC in Ishikawa-xenografted nude mice, and silencing GLUT4 restricted these effects. CONCLUSION: These data suggest that HG increases GLUT4 and VEGF/VEGFR expression, further promotes EMT process and accelerates the development of UEC by up-regulating ER.

Indoleamine 2,3-dioxygenase suppresses the cytotoxicity of 1 NK cells in response to ectopic endometrial stromal cells in endometriosis.

It has been reported that the impaired cytotoxicity of natural killer (NK) cells and abnormal cytokines that are changed by the interaction between ectopic endometrial cells and immune cells is indispensable for the initiation and development of endometriosis (EMS). However, the mechanism of NK cells dysfunction in EMS remains largely unclear. Here, we found that NK cells in peritoneal fluid from women with EMS highly expressed indoleamine 2,3-dioxygenase (IDO). Furthermore, IDO+NK cells possessed lower NKp46 and NKG2D but higher IL-10 than that of IDO-NK. Co-culture with endometrial stromal cells (nESCs) from healthy control or ectopic ESCs (eESCs) from women with EMS led to a significant increase in the IDO level in NK cells from peripheral blood, particularly eESCs, and an anti-TGF-ß neutralizing antibody suppressed these effects in vitro. NK cells co-cultured with ESC more preferentially inhibited the viability of nESCs than eESCs did, and pretreating with 1-methyl-tryptophan (1-MT), an IDO inhibitor, reversed the inhibitory effect of NK cells on eESC viability. These data suggest that ESCs induce IDO+NK cells differentiation partly by TGF-ß, and that IDO further restricts the cytotoxicity of NK cells in response to eESCs, which provides a potential therapeutic strategy for EMS patients, particularly those with a high number of impaired cytotoxic IDO+NK cells.

The IFN-γ-IDO1-kynureine pathway-induced autophagy in cervical cancer cell promotes phagocytosis of macrophage.

Background: Cervical cancer is a common malignant disease in female patients accompanied by activation of autophagy in tumor cells. However, the exact regulatory factors of autophagy and its effects on the immune response remain unknown. Methods: The induction of autophagy in HeLa and SiHa cells treated with IFN-γ, tryptophan depletion, kynurenine and epacadostat was detected by western blot analysis and by an autophagy detection kit. Following co-culture with pre-treated HeLa and SiHa cells, U937 cells were analyzed by flow cytometry to detect CD80, CD86, CD163 and CD206 expression and the induction of phagocytosis. Results: IFN-γ caused a significant increase in the autophagy levels of HeLa and SiHa cells by promoting indoleamine-2,3-dioxygenase-1 (IDO1) expression. The induction of phagocytosis in HeLa and SiHa cells and the expression levels of CD80 and CD86 in U937 cells were increased significantly following treatment with recombinant human IFN-γ. This effect was associated with the induction of tumor cell autophagy. IFN-γ treatment and IDO1 overexpression promoted tryptophan depletion and kynurenine accumulation in cervical cancer cells. The latter was more potent in inducing autophagy of cervical cancer cells and promoting phagocytosis of macrophages. In vivo, IDO1 overexpression restricted tumor growth in C57 mice and enhanced the induction of phagocytosis in macrophages. Conclusions: IFN-γ promoted induction of autophagy and macrophage phagocytosis in cervical cancer cells possibly via IDO1 expression and kynurenine metabolism.

Melatonin restricts the viability and angiogenesis of vascular endothelial cells by suppressing HIF-1α/ROS/VEGF.

Angiogenesis is an essential process involved in various physiological, including placentation, and pathological, including cancer and endometriosis, processes. Melatonin (MLT), a well­known natural hormone secreted primarily in the pineal gland, is involved in regulating neoangiogenesis and inhibiting the development of a variety of cancer types, including lung and breast cancer. However, the specific mechanism of its anti­angiogenesis activity has not been systematically elucidated. In the present study, the effect of MLT on viability and angiogenesis of human umbilical vein endothelial cells (HUVECs), and the production of vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS), under normoxia or hypoxia was analyzed using Cell Counting kit 8, tube formation, flow cytometry, ELISA and western blot assays. It was determined that the secretion of VEGF by HUVECs was significantly increased under hypoxia, while MLT selectively obstructed VEGF release as well as the production of ROS under hypoxia. Furthermore, MLT inhibited the viability of HUVECs in a dose­dependent manner and reversed the increase in cell viability and tube formation that was induced by hypoxia/VEGF/H2O2. Additionally, treatment with an inhibitor of hypoxia inducible factor (HIF)­1α (KC7F2) and MLT synergistically reduced the release of ROS and VEGF, and inhibited cell viability and tube formation of HUVECs. These observations demonstrate that MLT may serve dual roles in the inhibition of angiogenesis, as an antioxidant and a free radical scavenging agent. MLT suppresses the viability and angiogenesis of HUVECs through the downregulation of HIF­1α/ROS/VEGF. In summary, the present data indicate that MLT may be a potential anticancer agent in solid tumors with abundant blood vessels, particularly combined with KC7F2.

Pleiotropic roles of melatonin in endometriosis, recurrent spontaneous abortion, and polycystic ovary syndrome.

Melatonin is a neurohormone synthesized from the aromatic amino acid tryptophan mainly by the pineal gland of mammals. Melatonin acts as a broad-spectrum antioxidant, powerful free radical scavenger, anti-inflammatory agent, anticarcinogenic factor, sleep inducer and regulator of the circadian rhythm, and potential immunoregulator. Melatonin and reproductive system are interrelated under both physiological and pathological conditions. Oxidative stress, inflammation, and immune dysregulation are associated with the pathogenesis of the female reproductive system which causes endometriosis (EMS), recurrent spontaneous abortion (RSA), and polycystic ovary syndrome (PCOS). Accumulating studies have indicated that melatonin plays pleiotropic and essential roles in these obstetrical and gynecological disorders and would be a candidate therapeutic drug to regulate inflammation and immune function and protect special cells or organs. Here, we systematically review the pleiotropic roles of melatonin in EMS, RSA, and PCOS to explore its pathological implications and treatment potential.

The ginsenoside PPD exerts anti-endometriosis effects by suppressing estrogen receptor-mediated inhibition of endometrial stromal cell autophagy and NK cell cytotoxicity.

Endometriosis (EMS) is an estrogen-dependent gynecological disease with a low autophagy level of ectopic endometrial stromal cells (eESCs). Impaired NK cell cytotoxic activity is involved in the clearance obstruction of the ectopic endometrial tissue in the abdominopelvic cavity. Protopanaxadiol (PPD) and protopanaxatriol (PPT) are two metabolites of ginsenosides, which have profound biological functions, such as anti-cancer activities. However, the role and mechanism of ginsenosides and metabolites in endometriosis are completely unknown. Here, we found that the compounds PPD, PPT, ginsenoside-Rg3 (G-Rg3), ginsenoside-Rh2 (G-Rh2), and esculentoside A (EsA) led to significant decreases in the viability of eESCs, particularly PPD (IC50 = 30.64 µM). In vitro and in vivo experiments showed that PPD promoted the expression of progesterone receptor (PR) and downregulated the expression of estrogen receptor α (ERα) in eESCs. Treatment with PPD obviously induced the autophagy of eESCs and reversed the inhibitory effect of estrogen on eESC autophagy. In addition, eESCs pretreated with PPD enhanced the cytotoxic activity of NK cells in response to eESCs. PPD decreased the numbers and suppressed the growth of ectopic lesions in a mouse EMS model. These results suggest that PPD plays a role in anti-EMS activation, possibly by restricting estrogen-mediated autophagy regulation and enhancing the cytotoxicity of NK cells. This result provides a scientific basis for potential therapeutic strategies to treat EMS by PPD or further structural modification.

Cisplatin inhibits the growth, migration and invasion of cervical cancer cells by down-regulating IL-17E/IL-17RB.

Interleukin (IL)-17E mainly produced by immune cells, is a distinct member of the IL-17 cytokine family, which has multifarious immunomodulatory activities. As a potent anticancer drug, cisplatin is commonly used against various types of solid tumors. The present study was performed to investigate whether cisplatin regulates the expression of IL-17E and it receptor IL-17RB, and the role of IL17E in cervical cancer cells in vitro. The expression of IL-17E and IL-17RB in cervical cancer cells was detected by flow cytometry and ELISA. The viability, apoptosis, migration and invasion of cervical cancer cells were analyzed by CCK8, Annexin V-7AAD apoptosis, transwell migration, wound healing, and matrigel invasion assays. Here, we found that cervical cancer cells co-expressed IL-17E and IL-17RB, especially HeLa and SiHa cells. Recombinant human IL-17E protein (rhIL-17E) enhanced the viability, migration and invasion of HeLa and SiHa cells, and blocking IL-17E with anti-human IL-17RE neutralizing antibody promoted the apoptosis of HeLa and SiHa cells. Cisplatin significantly down-regulated the expression of IL-17E and IL-17RB, and further reversed the regulatory effects of rhIL-17E on viability, apoptosis, migration and invasion of HeLa and SiHa cells. The results suggest that cisplatin inhibits the viability, migration, invasion, and promotes the apoptosis of cervical cancer cells possibly by down-regulating IL-17E/17RB signaling. Cisplatin may be the first choice for cervical cancer patients with abnormally high IL-17E expression.

Protopanaxadiol and metformin synergistically inhibit estrogen-mediated proliferation and anti-autophagy effects in endometrial cancer cells.

Metformin is commonly used for treating type II diabetes and has recently been reported to possess anti-proliferative properties that can be exploited for the prevention and treatment of a variety of cancers. Ginsenosides are the main effective biological components of ginseng. It has been reported that ginsenoside-Rb2 inhibit the invasiveness of endometrial cancer cells (ECC). The aim of this study was to investigate whether protopanaxadiol (PPD, a metabolite of ginsenosides) and metformin could synergistically regulate the biological behavior of ECC and analyze its possible mechanism. We here found that either metformin or PPD treatment led to a decreased viability and increased apoptosis and autophagy levels in ECC lines (Ishikawa and RL95-2 cells), and combination of PPD and metformin could enhance these effects induced by metformin or PPD in vitro. PPD and metformin significantly decreased the expression of estrogen receptor alpha (ERα) in Ishikawa and RL95-2 cells. Estrogen promoted the viability and restricted the apoptosis and autophagy of Ishikawa and RL95-2 cells, and PPD and metformin reversed these effects. In vivo trials showed that combination of PPD and metformin had the strongest activity of anti-tumor growth compared with PPD alone and metformin alone. These data suggest that PPD and metformin can be used together to play a more powerful anti-EC effect. Our study provides a scientific basis for the clinical application of PPD and metformin in the treatment of EC, especially in estrogen-dependent patients.

The cross talk between cervical carcinoma cells and vascular endothelial cells mediated by IL-27 restrains angiogenesis.

PROBLEM: To explore whether cervical carcinoma cell-derived interleukin-27 (IL-27) modulates the angiogenesis of vascular endothelial cells. METHOD OF STUDY: The expression of IL-27 in cervical cancer tissues and cervical cell lines was analyzed by immunohistochemistry, ELISA and flow cytometry. Then, the effects of IL-27 on the proliferation and apoptosis-related molecules and angiogenesis in vitro of human umbilical vein endothelial cells (HUVECs) were investigated. Finally, in vivo experiment was performed to further confirm the effects of IL-27. RESULTS: Compared with cervicitis, the cervical cancer tissues highly expressed IL-27. Both HeLa and CaSki cells secreted IL-27, and HUVECs expressed low levels of IL-27 receptors (IL-27R). However, the co-culture of cervical cell lines and HUVECs led to a significant elevation of IL-27R on HUVECs. Co-culturing with IL-27-overexpressed HeLa cells downregulated Ki-67 and Bcl-2 and upregulated Fas expression in HUVECs. In addition, overexpression of IL-27 in HeLa cells and CasKi cells secreted less IL-8 and could further restrict angiogenesis compared with control cells in vitro. In the subcutaneous tumorous model of C57/BL6 mouse, there were decreased vessel density and tumor volume when inoculation with IL-27-overexpressed TC-1 cells. CONCLUSION: This study indicates that IL-27 secreted by cervical carcinoma cells restricts the angiogenesis in a paracrine manner in the pathogenesis of cervical cancer.