Silencing expression of the NANOG gene and changes in migration and metastasis of urinary bladder cancer cells.

INTRODUCTION: It has been proved that expression of the NANOG gene is observed not only in embryonic-derived malignancies, but also in breast cancer, ovarian cancer, cervix cancer and bladder cancer. NANOG overexpression is correlated with high activity of MMP-2 and MMP-9. The aim of the study was to evaluate the changes in the malignant phenotype of T24 bladder cancer cells with modulated expression of the NANOG gene. MATERIAL AND METHODS: Human urinary bladder cancer cells T24 (HTB-4) were cultivated under standard conditions. Transfection of the cells with silencing constructions was performed with the application of Lipofectamine 2000 (Invitrogen) reagent. Evaluation of changes in the expression level of individual genes was performed using qRTPCR. Changes in the protein level were evaluated using the Human ELISA Kit (Abcam). The invasion capability of transfected cells was tested using Matrigel Invasion Chambers (BD Biosciences). The changes in cell migration were assessed with a wound-healing assay. RESULTS: The qRTPCR evaluation showed that silencing the NANOG gene in T24 cells led to the decrease of mRNA for the MMP-2 gene to the level of 62.4% and the MMP-9 gene to the level of 76%. The cells with modulated expression of the NANOG gene migrated slower in the Matrigel invasion assay and in the wound-healing assay. The immunoenzymatic test showed a decrease in the protein level of MMP-9. CONCLUSIONS: The transcriptional activity of the NANOG gene might be connected with some aspects of bladder cancer cell metastasis in vitro and has an influence on MMP-2 and MMP-9 expression levels.

Synthesis and in vitro Evaluation of the Anticancer Potential of New Aminoalkanol Derivatives of Xanthone.

A series of 15 derivatives of xanthone were synthesized and evaluated for the anticancer activity. The structure of the tested compounds was diversified to establish structureactivity relationships. The following evaluations were carried out: cytotoxicity-proliferation tests, apoptosis detection, expression of apoptosis and proliferation-related genes, expression and activity of gelatinases A and B, wound migration assays, and cell adhesion to MatrigelTMcoated plates. Four compounds (7, 12, 13 and 15) displayed direct cytotoxicity at micromolar concentrations toward the studied cell lines. They also significantly affected the expression of proliferationapoptosis markers, and 13 demonstrated as strong influence as α-mangostin, that served as a natural standard in our study. These four compounds also decreased the expression and activity of gelatinases, and inhibited the migration-motility potential of cancer cells. The influence of compounds 7 and 12 on MMPs mRNA levels even exceeded the activity of α-mangostin and shRNA-mediated silencing; zymography revealed that 7, 13 and 15 were as equally active as α-mangostin, despite their higher IC50 values. The highest activity to inhibit motility and migration of cancer cells was demonstrated by 7, 12, 15, and by α-mangostin; and this was almost equal to shRNA-mediated silencing. Structural features predetermining compound activity were: substitution at position C4 instead of C2, and presence of a chlorine atom and allyl moiety. These results indicate that synthesis of aminoalkanol derivatives of xanthone may lead to successful establishment of new potential anticancer chemicals.

MMP-9 directed shRNAs as relevant inhibitors of matrix metalloproteinase 9 activity and signaling.

INTRODUCTION: The main function of matrix metalloproteinases is the degradation of extracellular matrix components, which is related to changes in the proliferation of cells, their differentiation, motility, and death. MMPs play an important role in physiological processes such as embryogenesis, angiogenesis and tissue remodeling. The increase of MMPs activity is also observed in pathological conditions including tumorigenesis where MMP-2 (gelatinase A) and MMP-9 (gelatinase B) show the ability to degrade the basement membrane of vessels and they are involved in metastasis. The aim of our study was to verify the changes of MMP-9 enzymatic activity and the mobility of cells after inhibition of MMP-9 gene expression. MATERIAL AND METHODS: The oligonucleotide shRNA insert had been designed to silence MMP-9 gene expression and was cloned into the pSUPER.neo expression vector. The construct was introduced into the HeLa (CCL-2) cervical cancer cells by lipotransfection. Simultaneously in control cells MMP-9 were inhibited by doxycycline. Changes in activity of MMP-9 were analyzed by gelatin zymography and wound-healing assay. RESULTS/CONCLUSIONS: Gelatin zymography allowed us to confirm that activity of MMP-9 in cells transfected by shRNA-MMP-9 and treated by doxycycline were similar and significantly lower in comparison with control cells. Phenotypic tests of migration in vitro confirm statistically significant (P<0.05) changes in cell migration - control cells healed 3 to 5 times faster in comparison with transfected or doxycycline treated cells. Our studies show the significant role of MMP-9 in mobility and invasiveness of tumor cells, thus indicating a potential target point of interest for gene therapy.

[Silencing of the STAT3 gene expression activity and cancer cells metastatic potential at in vitro studies]. / Wyciszanie aktywnosci ekspresyjnej genu STAT3, a potencjal metastatyczny komórek nowotworowych w badaniach in vitro.

INTRODUCTION: The STAT proteins are the mediators in the signal transduction in between extracellular environment and nucleus. Based on its own activity STATs regulate expression of genes involved in normal and pathological cellular processes. Constitutive STAT3 activation, the results of different cytokines inductions, has been shown in many primary human cancers. STAT3, as an oncogenic protein, plays an important role in the regulation of autonomous properties of cancer cells. MATERIAL AND METHODS: In this study the effectiveness of the STAT3 gene expression activity silencing with RNA interference method was assessed. pSUPER.neo shRNA coding expression vector: shRNA-STAT3 and control vectors: shRNA-SCR, and pGFP were used. Effects of silencing of the examined gene was described as the phenotype changes of modulated HeLa (CCL-2) cancer cell line. To characterize modulated cancer cells phenotype changes two methods were applied: Wound Healing Assay and the stimulation to the apoptosis with anisomycin. RESULTS: According to control cells, the silencing of the STAT3 gene expression activity reduced the mobility of modulated cells as well after 24 as after 48 hours after modulation. Also, after anisomycin stimulation the increasing in apoptotic modulated cell death was seen. CONCLUSIONS: The inhibition of the activity of the STAT3 gene decreases HeLa cell migration, moreover the blocked STAT3 ability to the antyapoptotic gene expression activation leads to the increased susceptibility to apoptotic cell death.

The efficiency of silencing expression of the gene coding STAT3 transcriptional factor and susceptibility of bladder cancer cells to apoptosis.

AIM OF THE STUDY: Abnormalities in signaling as well as altered gene expression have been identified in numerous diseases, including cancer. The biological functions of signal transducer and activator of transcription 3 (STAT3) are very broad. It is thought that STAT3 can also contribute to oncogenesis. RNA interference (RNAi) is one of the most efficient tools for silencing gene expression within cells. The main goal of the study was to verify the effectiveness of STAT3 gene silencing and its influence on cell proliferation and activation of apoptosis in bladder cancer cells. MATERIAL AND METHODS: The study was conducted on cellular material, which was the stable human bladder cancer cell line T24. The synthesis of shRNA (short hairpin RNA) interfering with the STAT3 gene was based on pSUPER. neo expression vector. The gene expression at the mRNA level was determined by the real-time PCR method. The influence of STAT3 gene silencing on apoptosis induced in cells with modulated STAT3 expression was evaluated using parallel quantification of mono- and oligonucleosomal DNA degradation of genomic DNA. RESULTS: In transfected T24 cells, the STAT3 mRNA expression decreased to the level of 68.3% compared to the scrambled (SCR) control. Silencing the STAT3 gene induced changes in the phenotype of T24 cells. Statistically significant differences in cell proliferation (p = 0.0318) and apoptosis induction (p = 0.0376) were observed. CONCLUSIONS: Application of the designed shRNA for the STAT3 gene contributed to a decrease of expression of the examined gene. It also decreased the proliferation and increased the susceptibility to apoptosis in T24 bladder cancer cells.