Insulin-like growth factor-I receptor inhibition by specific tyrosine kinase inhibitor NVP-AEW541 in endometrioid and serous papillary endometrial cancer cell lines

Purpose

The role of the insulin-like growth factor (IGF) system in endometrial cancer has been well established. The IGF-I receptor (IGF-IR) emerged as a promising therapeutic target in a number of cancers. NVP-AEW541 (Novartis Pharma) is a pyrrolo(2,3-d)pyrimidine derivative with specific IGF-IR tyrosine kinase inhibitory activity. NVP-AEW541 has been shown to abrogate IGF-I-mediated IGF-IR autophosphorylation and to reduce activation of the IGF-IR signaling pathways. The aim of the present study was to investigate the anti-proliferative activity of NVP-AEW541 in Type I (endometrioid) and Type II (uterine serous papillary endometrial carcinoma, USPC) endometrial cancer cell lines.

Methods

Type I (ECC-1, Ishikawa) and Type II (USPC-1, USPC-2) endometrial cancer cell lines were treated with NVP-AEW541 in the presence of IGF-I, and the following parameters were measured: IGF-IR, AKT and ERK phosphorylation, apoptosis, proliferation, cell cycle progression and IGF-IR internalization.

Results

Results obtained showed that NVP-AEW541 abolished the IGF-I stimulated IGF-IR phosphorylation in all of the cell lines investigated, whereas it abolished AKT and ERK phosphorylation preferentially in ECC-1 and USPC-1 cells. Furthermore, the inhibitor prevented from IGF-I from exerting its antiapoptotic effect in ECC-1, USPC-1 and USPC-2 cells. In addition, proliferation assays showed that NVP-AEW541 caused a decrease in proliferation rate in all of the cell lines. NVP-AEW541 had no major effect on the insulin receptor.

Conclusion

Our results suggest that specific IGF-IR inhibition by NVP-AEW541 might be a promising therapeutic tool in endometrial cancer.     

7 Methyl indole ethyl isothiocyanate causes ROS mediated apoptosis and cell cycle arrest in endometrial cancer cells

Objective

Chemotherapy options for advanced endometrial cancer are limited and newer therapeutic agents are urgently needed. This study describes the therapeutic potential of 7 Methyl-indole ethyl isothiocyanate (7Me-IEITC) in endometrial cancer cell lines.

Methods

7Me-IEITC was synthesized in our laboratory. The cell viability of 7Me-IEITC treated ECC-1 and KLE endometrial cancer cell was determined by MTS assay. Morphology and apoptosis were further confirmed by DAPI-staining and TUNEL assay. The measurement of reactive oxygen species (ROS), mitochondrial transmembrane depolarization potential (ΔΨm) and cell cycle phase was determined by FACS analysis. Expression of proteins involved in apoptosis, survival and cell-cycle progression was analyzed by Western blotting.

Results

7Me-IEITC reduced the viability of the ECC-1 and KLE cancer cell-lines (IC₅₀ ~ 2.5-10 μM) in a dose dependent fashion. 7Me-IEITC treatment caused mitochondrial transmembrane potential reduction, elevated the production of ROS, leading to activation of apoptosis in endometrial cancer KLE and ECC-1 cells. 7Me-IEITC treatment activated Bad, suppressed Bcl2 phosphorylation followed by PARP-1 deactivation and caspase 3 and 7 activation. 7Me-IEITC treatment arrested the progression of KLE cells in S-phase and caused CDC25 and cyclin-D1 downregulation. Pre-treatment with ascorbic acid abrogated 7Me-IEITC induced apoptosis in ECC-1 and KLE cells, suggesting that 7Me-IEITC mediated cytotoxicity is primarily through ROS production.

Conclusion

7Me-IEITC demonstrated promising cytotoxic effects in endometrial cancer cell line model.     

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination

Objectives

Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines.

Methods

Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed.

Results

Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively.

Conclusions

Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.     

Metformin impairs the growth of liver kinase B1-intact cervical cancer cells

Objective

Metformin is one of the most widely used drugs for the treatment of type 2 diabetes. Recent investigations demonstrated that application of metformin reduces cancer risk. The present study aimed to determine the role of liver kinase B1 (LKB1) in the response of cervical cancer cells to metformin.

Methods

LKB1 expression and the integrity of LKB1-AMPK signaling were determined with immunoblot in 6 cervical cancer cell lines. Cellular sensitivity to metformin was analyzed with MTT assay.

Results

Metformin inhibited growth of cervical cancer cells, C33A, Me180, and CaSki, but was less effective against HeLa, HT-3, and MS751 cells. Analyzing the expression status and the integrity of LKB1-AMPK-mTOR signaling, we found that cervical cancer cells sensitive to metformin were LKB1 intact and exerted an integral AMPK-mTOR signaling response after the treatment. Ectopic expression of LKB1 with stable transduction system or inducible expression construct in endogenous LKB1 deficient cells improved the activation of AMPK, promoted the inhibition of mTOR, and prompted the sensitivity of cells to metformin. In contrast, knock-down of LKB1 compromised cellular response to metformin. Our further investigation demonstrated that metformin could induce both apoptosis and autophagy in cervical cancer cells when LKB1 is expressed.

Conclusions

Metformin is a potential drug for the treatment of cervical cancers, in particular to those with intact LKB1 expression. Administration of cell metabolism agonists may enhance LKB1 tumor suppression, inhibit cell growth, and reduce tumor cell viability via the activation of LKB1-AMPK signaling.     

GRP78 induced by estrogen plays a role in the chemosensitivity of endometrial cancer

Objective

Molecular chaperone 78 kDa glucose-regulated protein (GRP78) is a residential protein in the endoplasmic reticulum (ER) that is induced by an unfolded-protein response triggered under many kinds of stress against a cell. GRP78 is also known to act as an anti-apoptotic factor by protecting ER-stress-induced cell death. In this study, we examined the significance of GRP78 expression in endometrial cancer.

Methods

Tissue samples obtained from patients with a diagnosis of enodometrial cancer were subjected to immunohistochemistry and RT-PCR to determine protein and mRNA expression levels of GRP78 and estrogen receptor α. We used Western blot and RT-PCR to examine whether estrogen induced GRP78 expression in cancer cell lines. Western blots and MTT assays of GRP78 siRNA transfected Ishikawa and HHUA cells were used to demonstrate whether GRP78 is involved in chemoresistence.

Results

GRP78 was highly expressed in well and moderately differentiated endometrial carcinoma. Estrogen induced GRP78 expression, which was correlated with cell viability and resistance to paclitaxel and cisplatin. Western blot analysis indicated that active caspase-3 and the 85-kDa protein poly (ADP-ribose) polymerase (PARP) were increased by incubation with either paclitaxel or cisplatin, suggesting that the apoptotic pathway was involved in cancer-drug-induced cell death.

Conclusions

These results may open up a novel therapeutic strategy for endometrial cancer: namely, the targeting of GRP78 to sensitize the tumor cell to chemotherapy.     

Simvastatin,an HMG-CoA reductase inhibitor,exhibits anti-metastatic and anti-tumorigenic effects in endometrial cancer

Objective

Our goal was to evaluate the effects of simvastatin on endometrial cancer cell lines and primary cultures of endometrial cancer cells.

Methods

Cell proliferation in the ECC-1 and Ishikawa endometrial cancer cell lines and primary cultures of endometrial cancer cells was assessed by MTT assay. Apoptosis and cell cycle were detected by Annexin V assay and propidium iodide staining, respectively. Reactive oxygen species and cell adhesion were assessed using ELISA assays. Invasion was analyzed using a transwell invasion assay. Mitochondrial DNA damage was confirmed using qPCR. The effects of simvastatin on the AKT/mTOR and MAPK pathways were determined by Western blotting.

Results

Simvastatin inhibited cell proliferation in a dose-dependent manner in both endometrial cancer cell lines and 5/8 primary cultures of endometrial cancer cells. Simvastatin treatment resulted in G1 cell cycle arrest, a reduction in the enzymatic activity of HMG-CoA, induction of apoptosis as well as DNA damage and cellular stress. Treatment with simvastatin resulted in inhibition of the MAPK pathway and exhibited differential effects on the AKT/mTOR pathway in the ECC-1 and Ishikawa cells. Minimal change in AKT phosphorylation was seen in both cell lines. An increase in phosphorylated S6 was seen in ECC-1 and a decrease was seen in Ishikawa. Treatment with simvastatin reduced cell adhesion and invasion (p < 0.01) in both cell lines.

Conclusion

Simvastatin had significant anti-proliferative and anti-metastatic effects in endometrial cancer cells, possibly through modulation of the MAPK and AKT/mTOR pathways, suggesting that statins may be a promising treatment strategy for endometrial cancer.     

Tetrathiomolybdate induces doxorubicin sensitivity in resistant tumor cell lines

Objective

Doxorubicin is a potent anti-cancer agent with efficacy against a broad range of tumors, including endometrial cancer. Doxorubicin produces reactive oxygen species (ROS) resulting in cytotoxicity. Tetrathiomolybdate (TM), a copper-chelating agent, is known to target a cellular antioxidant enzyme copper/zinc-superoxide dismutase. This study tests the hypothesis that TM can modulate antioxidants in tumor cells and render doxorubicin resistant tumor cells sensitive to doxorubicin.

Methods

The anti-cancer activities of doxorubicin and TM, as single agents and in combination, were assessed. Flow cytometric and immunoblot analysis were conducted to investigate the induction of apoptosis and changes in apoptotic signaling pathways.

Results

Doxorubicin-induced growth inhibition was observed in each endometrial cancer cell line (ECC-1, AN3CA, and KLE) tested with cell specificity. ECC-1 and KLE cells were found to have increased resistance to doxorubicin than AN3CA cells. Moreover, doxorubicin mediated apoptosis was greater in the AN3CA cell line than ECC-1 and KLE. The combination of doxorubicin with a sub-cytotoxic level of TM was significantly more effective at inducing apoptosis in doxorubicin resistant cell lines.

Conclusion

Our results highlight the therapeutic potential of TM to sensitize tumor cells to doxorubicin for endometrial cancer treatment.     

Metformin targets ovarian cancer stem cells in vitro and in vivo

Purpose

Studies in non-gynecologic tumors indicate that metformin inhibits growth of cancer stem cells (CSC). Diabetic patients with ovarian cancer who are taking metformin have better outcomes than those not taking metformin. The purpose of this study was to directly address the impact of metformin on ovarian CSC.

Methods

The impact of metformin on ovarian cancer cell line growth and viability was assessed with trypan blue staining. Aldehyde dehydrogenase (ALDH) expressing CSC were quantified using FACS®. Tumor sphere assays were performed to determine the impact of metformin on cell line and primary human ovarian tumor CSC growth in vitro. In vivo therapeutic efficacy and the anti-CSC effects of metformin were confirmed using both tumor cell lines and ALDH(+) CSC tumor xenografts.

Results

Metformin significantly restricted the growth of ovarian cancer cell lines in vitro. This effect was additive with cisplatin. FACS analysis confirmed that metformin reduced ALDH(+) ovarian CSC. Consistent with this, metformin also inhibited the formation of CSC tumor spheres from both cell lines and patient tumors. In vivo, metformin significantly increased the ability of cisplatin to restrict whole tumor cell line xenografts. In addition, metformin significantly restricted the growth of ALDH(+) CSC xenografts. This was associated with a decrease in ALDH(+) CSC, cellular proliferation, and angiogenesis.

Conclusions

Metformin can restrict the growth and proliferation of ovarian cancer stem cells in vitro and in vivo. This was true in cell lines and in primary human CSC isolates. These results provide a rationale for using metformin to treat ovarian cancer patients.     

Differential in vitro sensitivity to patupilone versus paclitaxel in uterine and ovarian carcinosarcoma cell lines is linked to tubulin-beta-III expression

Objective

To compare the in vitro sensitivity/resistance to patupilone versus paclitaxel in uterine and ovarian carcinosarcomas (CS).

Methods

Five primary carcinosarcoma cell lines, two from uterine and three of ovarian origin, were evaluated for growth rate and tested for their in vitro sensitivity/resistance to patupilone versus paclitaxel by MTS assays. To identify potential mechanisms underlying the differential sensitivity/resistance to patupilone, expression levels of β-tubulin III (TUBB3) were determined with quantitative-real-time-polymerase-chain-reaction (q-RT-PCR) in primary uterine and ovarian CS cell lines and in 26 uterine and 9 ovarian CS fresh-frozen-tissues.

Results

No appreciable difference in sensitivity to patupilone versus paclitaxel was noted in ovarian CS cell lines, or when uterine and ovarian CS cell lines were compared in their response to paclitaxel. In contrast, uterine CS cell lines were found to be significantly more sensitive to patupilone than to paclitaxel (P < 0.002) and demostrated lower IC_50s to patupilone (range 0.76-0.93 nM) when compared to ovarian CS (range 1.9-3.4 nM, p < 0.05). Higher levels of TUBB3 were detected in uterine CS cell lines and fresh frozen tissues when compared to ovarian CS (P < 0.05).

Conclusions

Uterine CS cell lines are significantly more sensitive than ovarian CS cell lines to patupilone versus paclitaxel. High expression of TUBB3 is associated with sensitivity to patupilone in primary CS cell lines and may act as a genetic marker to predict chemotherapy efficacy. Patupilone may represent a promising drug in the treatment of this subset of rare but highly aggressive gynecological tumors.     

Metformin is associated with improved survival in endometrial cancer

Objective

Preclinical evidence suggests that metformin exhibits anti-tumorigenic effects in endometrial cancer. We sought to investigate the association of metformin on endometrial cancer outcomes.

Methods

A multi-institutional IRB-approved retrospective cohort analysis was conducted comparing endometrial cancer patients with diabetes mellitus who used metformin (based on medication review at the time of diagnosis) to those who did not use metformin from 2005 to 2010. Metformin use on treatment related outcomes (TTR: time to recurrence; RFS: recurrence free survival; OS: overall survival) were evaluated using univariate and multivariate modeling.

Results

24% (363/1495) endometrial cancer patients were diabetic, of whom 54% used metformin. Metformin users were younger and heavier than non-users, though nearly all were postmenopausal and obese. 75% of both groups had endometrioid histology. Stage, grade, and adjuvant therapy distributions were similar. Metformin users had improved RFS and OS. Non-metformin users had 1.8 times worse RFS (95% CI: 1.1–2.9, p = 0.02) and 2.3 times worse OS (95% CI: 1.3–4.2, p = 0.005) after adjusting for age, stage, grade, histology and adjuvant treatment. Metformin use was not associated with TTR.

Conclusion

Metformin use was associated with improved RFS and OS but not TTR, most likely due to improving all-cause mortality. Its role in modifying cancer recurrence remains unclear. Prospective studies that capture metformin exposure prior to, during and post endometrial cancer treatment may help define the role of metformin upon cancer specific and overall health outcomes.     

Surgical staging for endometrial cancer in the elderly - is there a role for lymphadenectomy?

Objectives

We sought to evaluate the effect of systematic lymphadenectomy (LND) on endometrial cancer-specific survival in an elderly population.

Methods

Data were obtained from the Surveillance, Epidemiology, and End Results (SEER) program from 1988 to 2006. Women who underwent primary hysterectomy for non-serous, non-clear cell endometrial carcinoma were included. Women were stratified by age (< 70, 70-79, and ≥ 80) and disease-specific survival (DSS) was analyzed via the Kaplan-Meier method and stratified by postoperative grade. Cohorts were compared using the log-rank test. In a simulated population, the disease-specific survival of women with pre-operative grade 1 endometrial carcinoma was calculated using a weighted average survival accounting for those upgraded at final pathology.

Results

Endometrial cancer was identified in 5759 women ≥ 80 years old. Disease specific survival at 5 years for the LND and no LND groups was 93.4% and 94.5% (p = 0.36) for grade 1, 84.4% and 85% (p = 0.97) for grade 2, and 65.9% and 60.9% (p = 0.002) for grade 3. In the simulated pre-operative grade 1 group, 5 year disease-specific survival (DSS) was 91% in the LND group and 92% in the no LND group.

Conclusion

In women older than 80, systematic lymphadenectomy is associated with improved DSS for high grade, but similar DSS for low grade endometrial cancer, consistent with what is seen with younger women. As there is no clear survival benefit to lymphadenectomy in elderly women presenting with low grade disease, the surgeon should carefully weigh the surgical risks and benefits in this patient population, which may be at higher risk for morbidity.     

Inhibin/activin betaE-subunit in uterine endometrioid adenocarcinoma and endometrial cancer cell lines: from immunohistochemistry to clinical testing?

Objective

Inhibins and activins are important regulators of the female reproductive system and have also been described to be involved in gynecologic cancer development. A new inhibin/activin subunit betaE has been identified, but only limited data on its expression in human uterine adenocarcinomas and endometrial cancer cell lines exist.

Methods

A series of 223 uterine endometrial adenocarcinomas were immunohistochemically analyzed with a specific antibody against the inhibin betaE-subunit. In addition, established endometrial cancer cell lines were analyzed for the synthesis of the inhibin betaE subunit by immunofluorescence and RT-PCR analysis.

Results

In this analysis, the inhibin betaE staining intensity was associated with histological grading along with a significant decrease in cases with ovarian invasion. However, inhibin-betaE did not affect patient survival nor did it constitute an independent prognostic parameter in endometrial adenocarcinoma patients. Additionally, the inhibin/activin betaE-subunit was found to be expressed in the human endometrial carcinoma cell lines HEC1a, HEC1b, Ishikawa, and RL95-2, although the level of expression was found to be highly differing among the cancer cell lines tested.

Conclusion

The isolated analysis of the inhibin betaE subunit might be of minor prognostic value in identifying high-risk patients. However, its differential expression in cancer of different histological differentiation and ovarian metastasis suggests a putative but yet undefined role in endometrial carcinogenesis. Whether this novel and not well studied inhibin subunit has a substantial function in the pathogenesis and malignant transformation of human endometrium is still under investigation.     

Anti-diabetic doses of metformin decrease proliferation markers in tumors of patients with endometrial cancer

Background

Metformin has been associated with reduced cancer risk. The mechanisms underlying this cancer protective effect remain unknown.

Methods

“Window of opportunity” study of metformin in women with operable endometrial cancer (EC). Eleven newly diagnosed, untreated, non-diabetic patients with EC received metformin 500 mg tid from diagnostic biopsy to surgery. Fasting plasma insulin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein 1 (IGFBP-1) and insulin-like growth factor binding protein 7 (IGFBP-7) measurements were taken before and after metformin treatment. Ki-67, pAMPK, and pS6 immunohistochemistry staining was performed on the endometrial cancer before and after metformin treatment and was compared to a control group of 10 women with EC who did not receive metformin.

Results

Metformin was administered for a mean of 36.6 days. None of the patients suffered side effects requiring withdrawal from the study. The study group comprised 8 patients with endometrioid EC, and 3 non-endometrioid EC, with a mean follow-up time of 57 months. Mean plasma insulin (p = 0.0005), IGF-1 (p = 0.001), and IGFBP-7 (p = 0.0098) were significantly reduced after metformin treatment. A clear reduction in ki-67 and pS6 expression was observed by both conventional light microscope analysis and digital image analysis with a significant mean reduction in percentage of cells staining for ki-67 (9.7%, P = 0.02) and pS6 (31%, P = 0.03). In the non-treated control group expression was similar between the biopsy and the surgical specimens.

Conclusions

This pilot trial presents biological evidence consistent with anti-proliferative effects of metformin in women with EC in the clinical setting.     

Induction of apoptosis by metformin in epithelial ovarian cancer: involvement of the Bcl-2 family proteins

Objective

The aims of the study were to evaluate the ability of metformin to induce apoptosis in epithelial ovarian cancer cell lines and to identify the pathways involved in this effect.

Methods

After treatment with metformin and/or cisplatin, OVCAR-3 and OVCAR-4 cellular apoptosis was assessed by flow cytometry and caspase 3/7 activity. Cell cycle analysis was also performed by flow cytometry as well. Modulation of protein expression of the Bcl-2 family after treatment with metformin and/or cisplatin was determined by Western blotting.

Results

Metformin induced apoptosis in OVCAR-3 and OVCAR-4 cell lines in an AMPK-independent manner and provoked a cell cycle arrest in the S and G2/M phase. Moreover, we established that metformin can induce apoptosis in OVCAR-3 and OVCAR-4 cells by activating caspases 3/7, down-regulating Bcl-2 and Bcl-xL expression, and up-regulating Bax and Bad expression. The induction of apoptosis by metformin was also enhanced by cisplatin and combination of these drugs did not modulate the expression of Bcl-2 family proteins in OVCAR-3 cell line, whereas the effect was enhanced in OVCAR-4 cell line.

Conclusion

Bcl-xL and Bcl-2 targeted strategies were suggested to constitute an effective therapeutic tool for the treatment of chemoresistant ovarian carcinoma, in conjunction with conventional chemotherapy. These data are relevant to ongoing translational research efforts and clinical trials exploring a possible protective effect of metformin against ovarian cancer, including Bcl-2 inhibition.     

Clinical, endocrine and metabolic effects of metformin vs N-acetyl-cysteine in women with polycystic ovary syndrome

Objective

To evaluate the clinical, endocrine and metabolic effects of metformin and N-acetyl-cysteine (NAC) in patients with polycystic ovary syndrome (PCOS).

Study design

In this prospective trial, 100 women with PCOS were randomly divided to receive metformin (500 mg p.o. three times daily) or NAC (600 mg p.o. three times daily) for 24 weeks. Hyperandrogenism, lipid profiles, hirsutism scores, menstrual irregularity, insulin sensitivity and tumour necrosis factor-α (TNF-α) levels were measured at baseline and after the treatment period.

Results

Both treatments resulted in a significant decrease in body mass index, hirsutism score, fasting insulin, HOMA index, free testosterone and menstrual irregularity compared with baseline values, and both treatments had equal efficacy. NAC led to a significant decrease in both total cholesterol and low-density lipoprotein levels, whereas metformin only led to a decrease in total cholesterol level. Although TNF-α levels increased following treatment for both groups, the difference from baseline was not significant.

Conclusions

Metformin and NAC appear to have comparable effects on hyperandrogenism, hyperinsulinaemia and menstrual irregularity in women with PCOS. The effects of metformin and NAC on insulin sensitivity are not associated with TNF-α.     

The BCL2 antagonist of cell death pathway influences endometrial cancer cell sensitivity to cisplatin

Objective

To identify pathways that influence endometrial cancer (EC) cell sensitivity to cisplatin and to characterize the BCL2 antagonist of cell death (BAD) pathway as a therapeutic target to increase cisplatin sensitivity.

Methods

Eight EC cell lines (Ishikawa, MFE296, RL 95-2, AN3CA, KLE, MFE280, MFE319, HEC-1-A) were subjected to Affymetrix Human U133A GeneChip expression analysis of approximately 22,000 probe sets. In parallel, endometrial cell line sensitivity to cisplatin was quantified by MTS assay, and IC₅₀ values were calculated. Pearson's correlation test was used to identify genes associated with response to cisplatin. Genes associated with cisplatin responsiveness were subjected to pathway analysis. The BAD pathway was identified and subjected to targeted modulation, and the effect on cisplatin sensitivity was evaluated.

Results

Pearson's correlation analysis identified 1443 genes associated with cisplatin resistance (P < 0.05), which included representation of the BAD-apoptosis pathway. Small interfering RNA (siRNA) knockdown of BAD pathway protein phosphatase PP2C expression was associated with increased phosphorylated BAD (serine-155) levels and a parallel increase in cisplatin resistance in Ishikawa (P = 0.004) and HEC-1-A (P = 0.02) cell lines. In contrast, siRNA knockdown of protein kinase A expression increased cisplatin sensitivity in the Ishikawa (P = 0.02) cell line.

Conclusion

The BAD pathway influences EC cell sensitivity to cisplatin, likely via modulation of the phosphorylation status of the BAD protein. The BAD pathway represents an appealing therapeutic target to increase EC cell sensitivity to cisplatin.     

Effects of metformin on mammalian target of rapamycin in a mouse model of endometrial hyperplasia

Objective

The effects of metformin on S6K1, which is a crucial effector of mTOR signaling, and on endometrium were studied in a mouse model of endometrial hyperplasia induced by unopposed estradiol or tamoxifen.

Study design

Forty-eight oophorectomized Balb/c mice were randomly assigned to receive saline, tamoxifen citrate (4 mg/kg), 17-beta estradiol hemihydrate (4 mg/kg), metformin (50 mg/kg), tamoxifen citrate (4 mg/kg) with metformin (50 mg/kg), or estradiol (4 mg/kg) with metformin (50 mg/kg) for 3 days. Histological markers of uterotrophy, including luminal epithelial cell height and density of endometrial glands were quantified for each slide. Immunohistochemical expression of PCNA and S6K1 was evaluated. H-score was used for S6K1 expression. Statistical analysis was performed using Student's t-test for comparison of two continous variables and one-way ANOVA for comparison of multiple variables.

Results

Mice treated either with tamoxifen or estradiol had significantly increased density of endometrial glands and epithelial heights compared to vehicle-only or metformin-only group (p < 0.001). Addition of metformin to tamoxifen or estradiol treated mice significantly decreased the density of endometrial glands and epithelial cell heights (p < 0.05). Addition of metformin to tamoxifen significantly decreased the H-score of S6K1 (p < 0.05) and the immunohistochemical expression of PCNA (p < 0.05) in uterine lining epithelium, glandular and stromal cells. Addition of metformin to estradiol significantly decreased the H-score of S6K1 (p < 0.05) and the immunohistochemical expression of PCNA (p < 0.05) in uterine lining epithelium, glandular and stromal cells.

Conclusion

Metformin seems to have possible antiproliferative effects on the endometrium of estradiol or tamoxifen treated mice via inhibiting the mTOR mediated S6K1 activation.     

External validation of a nomogram predicting overall survival of patients diagnosed with endometrial cancer

Objectives

Nomograms are predictive models that provide the overall probability of a specific outcome. Nomograms have shown better individual discrimination than currently used staging systems in numerous tumor entities. Recently, a nomogram for predicting overall survival (OS) in women with endometrial cancer was introduced by Memorial Sloan-Kettering Cancer Center (MSKCC). The aim of this study was to test the validity of the MSKCC endometrial cancer nomogram using an independent, external patient cohort.

Methods

The MSKCC nomogram is based on five readily available clinical characteristics. A multi-institutional endometrial cancer database was used to test the nomogram's validity. All consecutive patients treated for endometrial cancer between December 1995 and May 2011 and who had all nomogram variables documented were identified for analysis.

Results

Seven hundred sixty-five eligible patients were identified and used for external validation analysis. In the Austrian patient cohort, median OS was 134 months, and 3-year and 5-year OS rates were 83.8% (95% CI, 80.6-86.5%) and 77.2% (95% CI, 43.5-80.5%), respectively. The nomogram concordance index was 0.71 (SE = 0.017; 95% CI, 0.68-0.74). The correspondence between the actual OS and the nomogram predictions suggests a good calibration of the nomogram in the validation cohort.

Conclusion

The MSKCC endometrial cancer nomogram was externally validated and was shown to be generalizable to a new and independent patient population. The nomogram provides a more individualized and accurate estimation of OS for patients diagnosed with endometrial cancer following primary therapy. The nomogram can be used for counseling patients more accurately and for better stratifying patients for clinical trials.