Noor Azmi Mat Adenan

Cancer-Associated Fibroblasts Promote Proliferation of Endometrial Cancer Cells

Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.

Hypoxia enhances the viability, growth and chondrogenic potential of cryopreserved human adipose-derived stem cells

Cryopreservation is the only existing method of storage of human adipose-derived stem cells (ASCs) for clinical use. However, cryopreservation has been shown to be detrimental to ASCs, particularly in term of cell viability. To restore the viability of cryopreserved ASCs, it is proposed to culture the cells in a hypoxic condition. To this end, we aim to investigate the effect of hypoxia on the cryopreserved human ASCs in terms of not only cell viability, but also their growth and stemness properties, which have not been explored yet. In this study, human ASCs were cultured under four different conditions: fresh (non-cryopreserved) cells cultured in 1) normoxia (21% O2) and 2) hypoxia (2% O2) and cryopreserved cells cultured in 3) normoxia and 4) hypoxia. ASCs at passage 3 were subjected to assessment of viability, proliferation, differentiation, and expression of stemness markers and hypoxia-inducible factor-1 alpha (HIF-1α). We found that hypoxia enhances the viability and the proliferation rate of cryopreserved ASCs. Further, hypoxia upregulates HIF-1α in cryopreserved ASCs, which in turn activates chondrogenic genes to promote chondrogenic differentiation. In conclusion, hypoxic-preconditioned cryopreserved ASCs could be an ideal cell source for cartilage repair and regeneration.

Comparative study on Toxoplasma infection between Malaysian and Myanmar pregnant women

BackgroundToxoplasma gondii, an obligate intracellular protozoan parasite, causes a disease called toxoplasmosis which can sometimes be acquired congenitally by a newborn from an infected mother. This study aimed to determine the seroprevalence of Toxoplasma infection and its associated risks among 219 and 215 pregnant women from Malaysia and Myanmar, respectively.MethodsAnti-Toxoplasma IgG and IgM antibodies were screened by using standard commercial ELISA kits. The socio-demographic, obstetrics and risk factors associated with Toxoplasma infection data were compared between the two countries.ResultsThe overall prevalence of Toxoplasma infection in Malaysian pregnant women (42.47%; 95% CI = 36.11-49.09) was significantly higher (p < 0.05) than Myanmar pregnant women (30.70%; 95% CI = 27.92-37.16). By univariate analysis, this study identified that age group, education, parity, awareness on toxoplasmosis and consumption of undercooked meat were significantly associated (p < 0.05) with Toxoplasma seropositive Malaysian pregnant women but none of these factors associated with Toxoplasma seropositive Myanmar pregnant women. In comparison using univariate analysis between the two countries, it was found that Toxoplasma seropositive Malaysian pregnant women was associated with aged 30 years and above, secondary or lower-secondary level of education, the third trimester of pregnancy, having one child or more, lacking awareness of toxoplasmosis, absence of bad obstetrics history, having no history of close contact with cats or soil, living on a farm and also consumption of undercooked meat, unpasterized milk or untreated water. Avidity measurement was used to confirm the stages of Toxoplasma infection in pregnant women who were positive for both IgG and IgM antibodies and found all were infected in the past.ConclusionFrom our study, Toxoplasma screening and its risk measurement in pregnant women is firmly recommended for monitoring purposes and assisting proper management, including diagnosis and treatment during antenatal period. Also, it is necessary to initiate preventive measures for Toxoplasma infection among reproductive-age women in general and seronegative pregnant women in particular. Avidity measurement should be incorporated in Toxoplasma routine screening, especially with the availability of a single serum sample to assist in the diagnosis.

Acupressure as adjuvant treatment for the inpatient management of nausea and vomiting in early pregnancy: A double-blind randomized controlled trial

To evaluate the efficacy of acupressure at the Neiguan point (Pericardium [P]6) as adjuvant treatment during inpatient management of severe nausea and vomiting in pregnancy.

Impact of elemental iron on human spermatozoa and mouse embryonic development in a defined synthetic culture medium

There is a paucity of studies on effect of iron in embryo culture procedures. This study aims to ascertain the optimal, tolerance and toxic levels of iron in a protein-free embryo culture medium (PFM) to determine the effect of iron on embryonic development. The application of PFM in assisted reproductive technologies (ART) is intended to eliminate disease transmission and improve ART treatment outcome. The optimal, tolerance and toxic levels of iron on human spermatozoa and mouse embryos were determined by challenging them with different levels of iron (ferric iron; Fe+3). Human normozoospermic semen samples (n=24) and days 1-4 Quakenbush Special (Qs) mouse embryos (n=1160) were incubated in PFM supplemented with different concentrations of Fe+3 over different periods of time. 2.0μg/mL (35.8μM) of Fe+3 was the optimal level of Fe+3 for human spermatozoa with a tolerance range of 0.5-2μg/mL; whereas a level of 0.11μg/mL (2μM) of Fe+3 was the optimum for day 2 embryos. Levels of ferric iron at 0.11 to 2.8μg/mL appear to enhance spermatozoa motility, preserve its DNA integrity and possibly increase percentage of blastocysts developed but levels of ferric iron >16μg/mL is hazardous for both spermatozoa and embryos. In spite of beneficial effects of iron it is premature to recommend its supplementation in embryo culture media because of the known deleterious nature of iron and the paucity of toxicological data. Toxicological studies must be performed following which it can be decided whether it is safe to consider iron as a supplement in human embryo and spermatozoa culture media.

Embryo culture conditions are significantly improved during uninterrupted incubation: A randomized controlled trial

A parallel group superiority prospective randomised controlled trial was devised to compare the culture characteristics of human pre-implantation stage embryos during uninterrupted culture in a time lapse incubator (TLI) versus the conventional model of interrupted culture in a standard incubator (SI) under low oxygen tension using a single step medium. 221 patients aged 35-and-under, 124 patients aged between 36 and 39 and 86 patients aged 40-and-over years were randomised and cultured either in a SI or in a TLI. Patients in the three age groups were distributed between the TLI and SI in a 1:1 ratio. The development of embryos on days 2, 3 and 5, and the clinical pregnancy and implantation rates were recorded. The fertilisation rate, development of day 2 and clinical pregnancy rates were similar in both treatments but the 8-cell development rate in all age groups combined (p = 0.016), blastocyst development rate (p = 0.0022) and the implantation rate (p = 0.0022) was significantly higher for the uninterrupted culture. These findings demonstrated significant differences between the two incubation groups. It also indicated less efficacious embryonic development with age in both treatments which appeared more pronounced in the conventional incubator. In conclusion uninterrupted culture is superior compared to the interrupted incubation culture system.

High throughput silencing identifies novel genes in endometrioid endometrial cancer

OBJECTIVE To validate the gene expression profile obtained from the previous microarray analysis and to further study the biological functions of these genes in endometrial cancer. From our previous study, we identified 621 differentially expressed genes in laser-captured microdissected endometrioid endometrial cancer as compared to normal endometrial cells. Among these genes, 146 were significantly up-regulated in endometrial cancer. MATERIALS AND METHODS A total of 20 genes were selected from the list of up-regulated genes for the validation assay. The qPCR confirmed that 19 out of the 20 genes were up-regulated in endometrial cancer compared with normal endometrium. RNA interference (RNAi) was used to knockdown the expression of the upregulated genes in ECC-1 and HEC-1A endometrial cancer cell lines and its effect on proliferation, migration and invasion were examined. RESULTS Knockdown of MIF, SOD2, HIF1A and SLC7A5 by RNAi significantly decreased the proliferation of ECC-1 cells (p < 0.05). Our results also showed that the knockdown of MIF, SOD2 and SLC7A5 by RNAi significantly decreased the proliferation and migration abilities of HEC-1A cells (p < 0.05). Moreover, the knockdown of SLC38A1 and HIF1A by RNAi resulted in a significant decrease in the proliferation of HEC1A cells (p < 0.05). CONCLUSION We have identified the biological roles of SLC38A1, MIF, SOD2, HIF1A and SLC7A5 in endometrial cancer, which opens up the possibility of using the RNAi silencing approach to design therapeutic strategies for treatment of endometrial cancer.

Comparative characteristics of spermatozoa harvested and cryopreserved in culture and cryoprotectant media with or without donor serum proteins

The objectives of this study is to evaluate the efficacy of protein-free media in the preparation, holding and crypreservation of spermatazoa for use in ART. Normozoospermic semen samples (N=71) were used to compare the effects of media on the survival and quality of spermatozoa when washed and cultured with different media with and without added proteins at 4°C, 15°C, 22°C and 37°C for 0, 4-7 and 24h. Survival and quality of spermatozoa were assessed after freeze-thaw with synthetic cryoprotectant with and without proteins. Ethics/IRB approval was obtained (Ref. 1073.52). Spermatozoa parameters were similar in all media after washing and culture for 24h. Post-thaw survival and quality of spermatozoa was not significantly different 24h after thawing of samples frozen in all cryoprotectant medium. In conclusion synthetic protein-free culture and cryoprotectant media are equal in efficacy to protein-containing media in culture and cryopreservation of spermatozoa . Use of these synthetic media are anticipated to significantly reduce the risk, potentially associated with conventional protein-containing media, of transmission of disease and possibly harmful undeclared proteins to the patient, baby and the healtcare worker. Synthetic media also ensure consistency of quality between batches of media.

Pregnancies generated in novel synthetic protein-free embryo culture medium

concentrations (Table 1).We also found that the percentage of embryos falling within the optimal ranges defined for cc2, t5 and s2 were different between the two groups as showed in Table 2. Based on these results we decided to perform a more detailed analysis in order to find the implications of O2 concentration for D3 embryo selection based on kinetic parameters.

Abstract 1078: Cancer-associated fibroblasts promote endometrial cancer cell proliferation in vitro and in vivo

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cancer-associated fibroblasts (CAFs) recently demonstrated tumor-promoting roles in various cancer types, yet their implication in endometrial cancer (EC) has not been fully explored. We isolated fibroblasts (CAFs) and its epithelial counterpart from human EC tissues using CD90- and CD326-antibodies conjugated magnetic beads, respectively. We collected CAFs secretion by concentrating spent supernatants from cells growing in media containing 2% fetal bovine serum. Both human EC cell lines and primary EC epithelial cells showed increased cell proliferation in a dose-dependent manner, following treatment with CAFs secretion. This was in contrast to the growth inhibitory effects shown with secretions from normal endometrial fibroblasts. EC cells also demonstrated increased cell motility and invasiveness in response to CAFs secretion. Using cytokine array and ELISA, we further identified several cytokines which were overexpressed in CAFs compared to normal endometrial fibroblasts: macrophage chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), growth regulated oncogene (GRO), regulated on activation, normal T cell expressed and secreted (RANTES) and vascular endothelial growth factor (VEGF). These cytokines may activate MAPK/Erk and/or PI3K/Akt pathways to promote EC cell proliferation, as shown by immunoblotting and ELISA. Indeed, CAFs-mediated EC proliferation was suppressed independently by specific inhibitors for PI3K/Akt (LY294002) and MAPK/Erk (U0126). To determine if CAFs promote EC tumor proliferation in vivo, we inoculated fluorescently labeled-epithelial cell and CAFs subcutaneously at the flank of nude mice in the ratio of 1:2 and 1:4. Tumor sizes were measured twice a week using calipers and monitored weekly using in vivo imaging. Inoculation of CAFs and normal fibroblasts alone did not induce any tumor growth; however, co-injection of EC with CAFs (1:4) showed approximately 3 times higher growth rate when compared to EC alone. More interestingly, there was no sign of tumor growth in mice inoculated with combination of EC and normal fibroblasts (1:4) at the end of experiment. Taken together, our data suggests that CAFs exert tumor-promoting effects both in vitro and in vivo partly via specific cytokines-mediated activation of MAPK/Erk and PI3K/Akt pathways. Delineating the roles of CAFs in EC tumor microenvironment may provide potential therapeutic targets for the treatment of EC. Citation Format: Ivy Chung, Kavita S. Subramaniam, Seng Tian Tham, Zahurin Mohamed, Noor Azmi Mat Adenan, Yin Ling Woo. Cancer-associated fibroblasts promote endometrial cancer cell proliferation in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2014-1078