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Endometrial cancer is the most common gynecologic cancer in the United States and its incidence and mortality has been rising over the past decade. Few treatment options are available for patients with advanced and recurring endometrial cancers. Novel therapies, which are frequently toxic, are difficult to establish in this patient population which tends to be older and plagued by comorbidities such as diabetes mellitus and hypertension. Therefore, novel, non-toxic therapies are urgently needed. Megestrol acetate is a frequently used drug in endometrial cancer patients. However, its response rate is only 20-30%. To enhance the activity of megestrol acetate in endometrial cancer patients, we explored the potential of combining natural supplements with megestrol acetate and found that the addition of the natural phenolic compound, pterostilbene, to megestrol acetate resulted in a synergistic inhibition of cancer cell growth in vitro and an enhanced reduction of tumor growth in a xenograft mouse model. In addition, dual treatment led to attenuation of signaling pathways, as well as cell cycle and survival pathways. Our results demonstrated for the first time that the anti-tumor activity of megestrol acetate can be enhanced by combining with pterostilbene, providing an insight into the potential application of pterostilbene and megestrol acetate combination for the treatment of endometrial cancer.

Resistance to progestin treatment is a major hurdle in the treatment of advanced and reoccurring endometrial cancer. Fenretinide is a synthetic retinoid that has been evaluated in clinical trials as a cancer therapeutic and chemo-preventive agent. Fenretinide has been established to be cytotoxic to many kinds of cancer cells. In the present study, we demonstrate that fenretinide decreased cell viability and induced apoptosis in Ishikawa cells, which are an endometrial cancer cell line, in dose dependent manner in-vitro. This effect was found to be independent of retinoic acid nuclear receptor signaling pathway. Further, we have shown that this induction of apoptosis by fenretinide may be caused by increased retinol uptake via STRA6. Silencing of STRA6 was shown to decrease apoptosis which was inhibited by knockdown of STRA6 expression in Ishikawa cells. Results of an in-vivo study demonstrated that intraperitoneal injections of fenretinide in endometrial cancer tumors (created using Ishikawa cells) in mice inhibited tumor growth effectively. Immunohistochemistry of mice tumors showed a decrease in Ki67 expression and an increase in cleaved caspase-3 staining after fenretinide treatment when compared to vehicle treated mice. Collectively, our results are the first to establish the efficacy of fenretinide as an antitumor agent for endometrial cancer both in-vitro and in-vivo, providing a valuable rationale for initiating more preclinical studies and clinical trials using fenretinide for the treatment of endometrial cancer.

Background: The number of endometrial cancer (EC) cases is escalating rapidly, with no evident improvements in survival rates. The downregulation of progesterone receptor, resulting in progestin resistance, is presently a major problem regarding the therapeutic aspect. On the basis of this, we can focus more on the downstream signaling pathways that are controlled by progesterone. Lipid biosynthesis mediated by sterol regulatory element-binding protein-1/fatty acid synthase (SREBP-1/FASN) is of utmost importance to the growth and the proliferation of EC cells, so we hypothesize that SREBP-1/FASN might be involved in suppressing the proliferation and promoting apoptosis in EC cells through the effects induced by progesterone., Material and Methods: The Cell Counting Kit-8 was used to analyze the growth inhibition ratio of Ishikawa cells upon treatment with megestrol acetate (MA; MA is a progesterone derivative, also known as 17α-acetoxy-6-dehydro-6-methylprogesterone) and to determine the 50% inhibitory concentration. Apoptosis ratio was analyzed by treatment of the cells with MA at 50% inhibitory concentration at different time intervals using Annexin V-FITC/propidium iodide. The protein and messenger RNA levels of SREBP-1 and FASN were compared between the experimental and control groups (MA-treated Ishikawa cells were considered to be the experimental group)., Results: The experimental group showed obvious growth inhibition that was time and concentration dependent. The apoptosis ratio was also significantly higher in the experimental group compared with the control group (P < 0.01). The protein and messenger RNA levels of SREBP-1 and FASN were significantly reduced by MA too., Conclusions: Sterol regulatory element-binding protein-1/FASN is involved in the proliferation suppression and apoptosis promotion brought about by MA in Ishikawa cells.

Objective: To detect the expression change of claudin-4 in Ishikawa endometrial adenocarcinoma cell line in response to progesterone. To determine whether claudin-4 is involved in the anticancer effect of progesterone., Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the 50% inhibitory concentration (IC50) of megestrol acetate (MA) in treating Ishikawa cells. After the Ishikawa cells were treated with MA at IC50, cell apoptosis was examined by flow cytometry and transmission electron microscopy. The messenger RNA and protein expression levels of claudin-4 were further quantified by real-time polymerase chain reaction and Western blot. The localization of claudin-4 was examined by immunofluorescent staining., Results: The IC50 of MA on Ishikawa cells was 15 mg/L incubated for 72 hours. Apoptosis percentage was elevated from 0.07% ± 0.02% to 3.93% ± 0.81% after MA treatment. The expression of claudin-4 at both protein and messenger RNA levels was significantly decreased after the treatment of MA (P < 0.05). The localization of claudin-4 transferred from cytomembrane to cytoplasm and nucleus., Conclusion: Megestrol acetate can inhibit the growth of Ishikawa cells. It may work through decreasing claudin-4 expression and cell apoptosis. The localization change of claudin-4 may also be involved in the anticancer effect of progesterone.

Objective: To explore the regulation of claudin-4 expression in endometrial adenocarcinoma cell lines by progesterone., Methods: Ishikawa cells were treated with various concentrations of megestrol acetate (MA: 2, 5, 10, 15, 20 mg/L). After cultured for 24, 48 and 72 hours, cells growth were measured by methyl thiazolyl tetrazolium (MTT). The group of Ishikawa cells incubated with MA at the 50% inhibitory concentration (IC(50)) was selected for cell apoptosis assay by using transmission electron microscopy and flow cytometry method. Real-time PCR and western blot were used for detecting the mRNA and protein expression levels of claudin-4. The localization of claudin-4 was examined by immunofluorescent staining., Results: The inhibitory effects of megestrol acetate on the growth of Ishikawa cells were dose-dependent and time-dependent. IC(50) of MA on Ishikawa cells was 15 mg/L after incubated for 72 hours. After MA treatment, Ishikawa cells showed shrinkage, nuclear chromatin condensation, fractures of nuclear membrane and endoplasmic reticulum expansion, even round apoptotic bodies were found. The apoptosis rate of cells before MA treatment was (0.076 ± 0.024)%, and the rate was (3.934 ± 0.816)% by MA treated for 72 hours, in which there were signicant difference (P < 0.05). The relative quantification of claudin-4 mRNA and protein of the cells before MA treatment were 0.64 ± 0.20 and 0.94 ± 0.18, while they were 0.47 ± 0.15 and 0.62 ± 0.15 after MA treated. The expression of claudin-4 was significantly decreased after MA treatment (P < 0.05). The localization of claudin-4 transferred from cytomembrane to cytoplasm and nucleus after MA treatment., Conclusions: MA could inhibite the growth of Ishikawa cells, in which the mechanism may be decrease the expression of claudin-4 and the apoptosis of cells. The distribution change of claudin-4 may be related to the anti-cancer effect of progesterone.

Objectives: The Ishikawa endometrial cancer cell line is hormonally responsive, expressing estrogen and progesterone receptors (ER, PR) when grown in traditional monolayer culture. The purpose of this paper is to demonstrate a three-dimensional spheroid culture system for cancer cells. We used this system to determine the response of the Ishikawa cell line to estradiol-17 beta (E), tamoxifen (T), megestrol acetate (MA), and progesterone (P)., Methods: Ishikawa cells were incubated in polyurethane culture bags using phenol red-free media containing ethanol (0.1%, controls), E (1 mumol, or 1 nmol), T (1 mumol, or 10 nmol), MA (1 mumol, or 10 nmol), or P (1 mumol). Cellular morphology was assessed by hematoxylin and eosin staining, and expression of estrogen and progesterone receptors was determined immunohistochemically using an immunoperoxidase technique., Results: Cells in control cultures demonstrated minimal organization and lacked hormone receptors. In contrast, cells exposed to either E or T displayed significant glandular formation, with multicellular, microvilli-rich, columnar epithelia exhibiting polarized nuclear arrangements. Within 4 weeks, E- and T-treated cultures showed upregulated nuclear staining for PR, with little ER present. Cells treated with MA or P showed less glandular organization but expressed ER with PR downregulation., Conclusions: These data support the use of this novel three-dimensional culture system to study the modulation of tumor cell biologic activity in response to hormonal agents. Future applications of this model include examining in vitro responsiveness of cancer cell lines to additional biologic agents and chemotherapeutic regimens.