Zinc deficiency is associated with the development of ovarian endometrial cysts.

Ovarian cancers derived from endometrial cysts, also known as endometriosis in ovaries, are widespread histological types in Japan. Several studies suggest that zinc deficiency plays a role in endometriosis; however, the biological mechanism of zinc deficiency and endometrial cyst remains unknown. Thus, we investigated the association between zinc status and endometrial cysts. We measured the serum zinc levels in patients who had undergone surgery for endometrial cysts (n=19) and non-endometrial benign cysts (n=36). We analyzed cell proliferation, microarray data, and gene expression using N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a zinc chelator, in human immortalized endometrial epithelial cells (EMosis). The endometrial cyst group had considerably lower serum zinc levels than the non-endometrial benign cyst group. After adjusting for age, body mass index, alcohol consumption, smoking, and supplement use, endometrial cysts were markedly associated with serum zinc levels. EMosis cells treated with 5 µM TPEN demonstrated extensively increased proliferation compared to untreated cells. In the microarray analysis of EMosis cells treated with 5 µM TPEN, the enriched cellular components contained nucleoplasm, nuclear parts, and nuclear lumen. The upregulated biological processes included responses to hypoxia and decreased oxygen levels. The upregulated Kyoto Encyclopedia of Genes and Genomes pathway included the hypoxia-inducible factor-1 signaling pathway. EMosis cells treated with 5 µM TPEN demonstrated increased activator 1 (SRA1) expression and decreased AT-rich interaction domain 1A (ARID1A) expression. Protein-protein interaction network analysis indicated that ARID1A and SRA1 were associated with SMARCD1 and ATF1 among the differentially expressed genes in the microarray. EMosis cells treated with 5 µM TPEN revealed increased SRA1 mRNA levels and decreased ARID1A mRNA expression, whereas EMosis cells treated with 5 µM TPEN together with 10 µM zinc did not reveal changes in the mRNA levels of SRA1 or ARID1A compared with those without TPEN. These results suggest that zinc deficiency contributes to endometrial cyst development. Accordingly, zinc supplementation may suppress endometrial cyst development.

Copper content in ascitic fluid is associated with angiogenesis and progression in ovarian cancer.

BACKGROUND: Ascites is associated with the poor prognosis of malignant tumors. The biological importance of the changes in the content of trace elements in the ascitic fluid is unknown. Herein, we analyzed trace elements in the ascitic fluid of patients with ovarian tumors and used cultured cells to determine the copper (Cu)-induced changes in gene expression in ovarian cancer. METHODS: Inductively coupled plasma mass spectrometry (ICP-MS) was used to compare ascitic fluid trace element levels in patients with benign ovarian tumors (n = 22) and borderline/malignant tumors (n = 5) for primary screening. Cu levels were validated using atomic absorption spectrometry (AAS) in 88 benign, 11 borderline, and 25 malignant ovarian tumor patients. To confirm Cu-induced gene expression changes, microarray analysis was performed for Cu-treated OVCAR3, A2780, and Met5A cells. The vascular endothelial growth factor (VEGF) concentration in the cell supernatant or ascitic fluid (ovarian cancer samples) was measured using ELISA. RESULTS: ICP-MS showed that Co, Ni, Cu, Zn, As, Se, and Mo levels significantly increased in patients with malignant/borderline ovarian tumors compared to those in patients with benign ovarian tumors. AAS showed that malignant ovarian tumors were independently associated with elevated levels of Cu in ascites adjusted for age, body mass index, alcohol, smoking, and supplement use (p < 0.001). Microarray analysis of both Cu-treated ovarian cancer cell lines OVCAR3 and A2780 and the mesothelial cell line Met-5A revealed the upregulation of the angiogenesis biological process. Real-time polymerase chain reaction and ELISA demonstrated that an increased Cu content significantly enhanced VEGF mRNA expression and protein secretion in OVCAR3, A2780, and Met-5A cells. VEGF levels and clinical stages of the tumors correlated with the ascitic fluid Cu content in patients with malignant ovarian tumors (correlation coefficient 0.445, 95 % confidence interval [CI]: 0.069-0.710, p = 0.023 and correlation coefficient 0.406, 95 % CI: 0.022-0.686, p = 0.040, respectively). CONCLUSION: Cu levels significantly increased in patients with malignant ovarian cancer. Cu induced angiogenic effects in ovarian cancer and mesothelial cells, which affected ascites fluid production. This study clarifies the link between elevated Cu in ascites and malignant ovarian tumor progression. Strategies to decrease Cu levels in the ascitic fluid may help downregulate VEGF expression, thereby improving the prognosis of ovarian malignancies.

Hyperthermic intraperitoneal chemotherapy (HIPEC) for gynecological cancer.

AIM: Hyperthermic intraperitoneal chemotherapy (HIPEC) is a method of administering anticancer agents directly while heating the abdominal cavity. The aim of this review is to know the current position of HIPEC in ovarian cancer and uterine sarcoma and its future prospects. METHODS: This article reviews the current literature and evidence for the clinical trial of HIPEC in ovarian cancer and uterine sarcoma with consideration of the cases treated in our department. RESULTS: In January 2018, van Driel et al. reported the results of their phase 3, randomized, controlled trial and the usefulness of neoadjuvant chemotherapy followed by interval debulking surgery. With respect to greater than grade 3 complications, such as suture failure, intestinal perforation, postoperative bleeding, wound issues and death, there were no significant differences between the HIPEC group and the no-HIPEC group. In a meta-analysis including two randomized, controlled studies and 11 observational studies in 2019, the addition of HIPEC to cytoreductive surgery significantly improved overall survival of ovarian cancer patients. Moreover, growing evidence of the efficacy of cytoreductive surgery with HIPEC has also been reported in uterine sarcoma with peritoneal sarcomatosis in a multi-institutional study. HIPEC could be one of the new therapeutic strategies for such disseminated peritoneal lesions. CONCLUSION: Since the usage regimen and temperature setting of HIPEC are not standardized, and its effectiveness and adverse events are greatly affected by the time of administration, it is necessary to consider clinical trials for the optimization and establishment of HIPEC in Japan in the future.

Prognostic impact of Dynamin related protein 1 (Drp1) in epithelial ovarian cancer.

BACKGROUND: The mitochondrial fission protein, Dynamin related protein 1 (Drp1), and its upstream protein calcium/calmodulin-dependent protein kinase I (CaMKI) play a critical role in chemoresistance in ovarian cancer (OVCA). Thus, we examined the expression of Drp1, CaMKI and their phosphorylated forms and their prognostic impact in epithelial OVCA patients. METHODS: Expression analysis was performed by immunohistochemistry (IHC) of paraffin-embedded tumor samples from 49 patients with epithelial OVCA. Staining intensity and the percentage of positively stained tumor cells were used to calculate an immunoreactive score (IRS) of 0-12. The expression scores calculated were correlated with clinicopathological parameters and patient survival. RESULTS: High immunoreactivity of phospho-Drp1Ser637 was significantly correlated with high-grade serous carcinoma (HGSC) (p = 0.034), residual postoperative tumor of > 1 cm (p = 0.006), and non-responders to adjuvant chemotherapy (p = 0.007), whereas high expression of CaMKI was significantly correlated with stage III/IV [International Federation of Gynecologists and Obstetricians (FIGO)] (p = 0.011) and platinum-resistant recurrence (p = 0.030). ROC curve analysis showed that Drp1, phospho-Drp1Ser637 and CaMKI could significantly detect tumor progression with 0.710, 0.779, and 0.686 of area under the curve (AUC), respectively. The Kaplan-Meier survival curve showed that patients with high Drp1, phospho-Drp1Ser637 and CaMKI levels had significantly poorer progression free survival (PFS) (p = 0.003, p < 0.001 and p = 0.017, respectively). Using multivariate analyses, phospho-Drp1Ser637 was significantly associated with PFS [p = 0.043, hazard ratio (HR) 3.151, 95% confidence interval (CI) 1.039-9.561]. CONCLUSIONS: Drp1 and CaMKI are novel potential candidates for the detection and prognosis of epithelial OVCA and as such further studies should be performed to exploit their therapeutic significance.

Saikosaponin-d, a calcium mobilizing agent, sensitizes chemoresistant ovarian cancer cells to cisplatin-induced apoptosis by facilitating mitochondrial fission and G2/M arrest.

Cisplatin (CDDP) and its derivatives are first line anti-cancer drugs for ovarian cancer (OVCA). However, chemoresistance due to high incidence of p53 mutations leads to poor clinical prognosis. Saikosaponin-d (Ssd), a saponin from a herbal plant extract, has been shown to induce cell death and sensitize chemoresistant cells to chemotherapeutic agents. Here, we demonstrated that Ssd sensitized chemoresistant OVCA cells with either p53-wt, -mutant and -null to CDDP. The action of Ssd appears to be through induction of mitochondrial fragmentation and G2/M arrest. Ssd is mediated via calcium signaling, up-regulation of the mitochondrial fission proteins Dynamin-related protein 1 (Drp1) and optic atrophy 1 (Opa1), and loss in mitochondrial membrane potential (MMP). Moreover, in the presence of CDDP, Ssd also down-regulates protein phosphatase magnesium-dependent 1 D (PPM1D) and increases the phosphorylation of checkpoint protein kinases (Chk) 1, cell division cycle 25c (Cdc25c) and Cyclin dependent kinase 1 (Cdk1). Our findings suggest that Ssd could sensitize OVCA to CDDP independent of the p53 status through multiple signaling pathways. They support the notion that Ssd may be a novel adjuvant for the treatment of chemoresistant OVCA.

Protective effect of dienogest on chemotherapy-induced reduced fertility in female rats.

Reduced fertility is one of the main long-term consequences of chemotherapy given for lymphoma, leukemia, and other malignancies in young women. We examined with a female rat model whether and how dienogest, a fourth-generation progestin, modulates reduced fertility following exposure to gonadotoxic chemotherapy. Female rats were administered cyclophosphamide with or without GnRH agonist and different concentrations of dienogest for 20 days. Animals were sacrificed on Day 29, and the numbers of follicle at primordial, preantral and antral stage in the ovaries were counted histologically. Rats treated with sterile saline solution (as control), cyclophosphamide, cyclophosphamide plus GnRH agonist, and cyclophosphamide plus dienogest were also mated with male rats to evaluate their fertility and pregnancy outcomes. Cyclophosphamide significantly reduced the number of primordial follicles, whereas dienogest suppressed depletion of primordial follicle pool induced by chemotherapy. Although the rats exposed to cyclophosphamide alone failed to deliver live births, co-treatment with dienogest improved the pregnancy outcomes of treated rats. The protective effect of dienogest on chemotherapy-induced ovarian damage and reduced fertility was comparable to that of GnRH agonist. The present results suggest that the co-administration of dienogest and chemotherapy may be a useful strategy in preserving ovarian function and fertility in premenopausal women facing gonadotoxic chemotherapy.

Laminin-1-derived scrambled peptide AG73T disaggregates laminin-1-induced ovarian cancer cell spheroids and improves the efficacy of cisplatin.

We found previously that the laminin-1-derived synthetic peptide AG73 (LQVQLSIR) promoted ovarian cancer cell metastasis in vivo. We have now studied the role of this metastasis-promoting peptide in vitro using TAC3 ovarian cancer cells, which display anchorage-independent growth and form multicellular spheroids. Our goal is to better understand how this peptide can regulate metastasis in vivo. We found that the exogenous addition of either laminin-1 or peptide AG73 stimulated the formation and growth of the spheroids. Western blot analysis indicated that laminin-1 enhanced the expression of integrin beta1, and that AG73 peptide enhanced expression of syndecan-1 and downstream effectors, including mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase (ERK), and also phosphatidylinositol (PI)-3 kinase/AKT activity signaling. The soluble peptide AG73T, which is a scramble peptide of AG73, was able to disaggregate the laminin-1-induced spheroids. Furthermore, the disaggregated cells were twice as sensitive to cisplatin as the intact spheroids. The AG73T peptide in the presence of laminin-1 suppressed expression of integrin beta1 and its downstream effectors, including MAPK/ERK and PI3/AKT activity signaling. The MEK inhibitor U0126 reduced TAC3 cell growth more effectively in the presence of both laminin-1 and AG73T than in the presence of laminin-1 alone. Inhibition of the PI3-K cascade with LY294002 was also more effective in the presence of laminin-1 and AG73T. The increased sensitivity to cisplatin in the presence of AG73T may be due to the greater bioavailability of the drug to the free-floating cells over the spheroids. These findings suggest a novel function of AG73T in ovarian cancer and help to define mechanisms important in ovarian cancer spheroid formation and spread.

Efficacy of intraperitoneal continuous hyperthermic chemotherapy as consolidation therapy in patients with advanced epithelial ovarian cancer: a long-term follow-up.

This trial was performed to determine the efficacy and progression-free and overall survivals of patients with advanced ovarian cancer who had been treated with intraperitoneal hyperthermic chemotherapy (IPHC). Ten patients with advanced ovarian cancer participated in this trial and were treated with IPHC. The median progression-free and overall survival rates for all patients treated in this study were 41.2 and 70.2 months, respectively. Two of ten patients received optimal primary cytoreduction surgery followed by IPHC; four of ten, optimal interval debulking surgery followed by IPHC; and four of ten, negative second-look operation followed by IPHC. The groups had 5 and 14.5, 17.75 and 38, and 82.75 and 130.25 months median progression-free and overall survival rates, respectively. Grades 3-4 toxicity included myelosuppresion, and nephropathy was detected. One patient required blood transfusions due to grade 4 anemia and thrombocytopenia. Another patient developed grade 3 nephrotoxicity but did not require continuous hemodialysis. IPHC was feasible, produced manageable toxicity, and showed promise for the treatment of advanced ovarian cancer. Negative second-look laparotomy followed by IPHC was especially effective when consolidation intraperitoneal chemotherapy had been indicated. It produced excellent median progression-free and overall survival rates.