MATERIALS AND METHODS: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting.
RESULTS: The IC50 for imatinib on K562 was 362 nM compared to 3,952 nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down- regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562.
CONCLUSIONS: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.
MATERIALS AND METHODS: ER+ MCF7 and ER- MDA-MB-231 cell lines were subjected to two-dimensional electrophoresis (2-DE) and spots of interest were identified by matrix-assisted laser desorption/ionization time of- flight/time- of-flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis after downregulation of RhoGDIα using short interfering RNA (siRNA) and upregulated using GFP-tagged ORF clone of RhoGDIα.
RESULTS: The results showed a total of 35 proteins that were either up- or down-regulated in these cells. Here we identifed 9 and 15 proteins differentially expressed with silencing of RhoGDIα in MCF-7 and the MDA-MB-231 cells, respectively. In addition, 10 proteins were differentially expressed in the upregulation of RhoGDIα in MCF7, while only one protein was identified in the upregulation of RhoGDIα in MDA-MB-231. Based on the biological functions of these proteins, the results revealed that proteins involved in cell migration are more strongly altered with RhoGDI-α activity. Although several of these proteins have been previously indicated in tumorigenesis and invasiveness of breast cancer cells, some ohave not been previously reported to be involved in breast cancer migration. Hence, these proteins may serve as useful candidate biomarkers for tumorigenesis and invasiveness of breast cancer cells.
CONCLUSIONS: Future studies are needed to determine the mechanisms by which these proteins regulate cell migration. The combination of RhoGDIα with other potential biomarkers may be a more promising approach in the inhibition of breast cancer cell migration.
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.
MAIN METHODS: The expression of AR, 5α-reductase type1 (5αR1) and 5α-reductase type2 (5αR2) were examined in the bladder cancer cell line T24 and surgical pathology specimens. We also evaluated the status of androgen related cell proliferation and migration using the potent, non-aromatizable androgen agonist 5α-dihydrotestosterone (DHT).
KEY FINDINGS: DHT treatment significantly increased AR mRNA expression level, but not those of 5αR1 and 5αR2 in T24 cells. DHT also suppressed cellular migration with weaker and opposite effects on cell proliferation. A significant inverse correlation was detected between pT stage and AR, 5αR1 and 5αR2 immunoreactivity.
SIGNIFICANCE: Inverse correlations detected between tumor grade and AR/androgen metabolizing enzyme also suggested that the loss of AR and androgen-producing enzymes could be associated with tumor progression. Effects of DHT on cells also suggest that androgens may regulate cellular behavior.