Cytotoxic and toxicogenomic effects of silibinin in bladder cancer cells with different TP53 status.

Silibinin is a natural phenol found in the seeds of the milk thistle plant. Recent data have shown its effectiveness for preventing/treating bladder tumours. Therefore, in this study we investigated the cytotoxic and toxicogenetic activity of silibinin in bladder cancer cells with different TP53 statuses. Two bladder urothelial carcinoma cell lines were used: RT4 (wild-type TP53 gene) and T24 (mutated TP53 gene). Cell proliferation, clonogenic survival, apoptosis rates, genotoxicity and relative expression profile of FRAP/mTOR, FGFR3, AKT2 and DNMT1 genes and of miR100 and miR203 were evaluated. Silibinin promoted decreased proliferation and increased late apoptosis in TP53 mutated cells. Increased early apoptosis rates, primary DNA damage, and decrease of cell colonies in the clonogenic survival assay were detected in both RT4 and T24 cell lines. Down-regulation of FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 expression occurred in RT4 cells. Modulation of miR203 was observed in both cell lines. In conclusion, despite the reduction of clone formation in both cell lines, the toxicogenomic effect of silibinin on FRAP/mTOR, AKT2, FGFR3, DNMT1 and miR100 was dependent on the TP53 status. Taken together, the data confirmed the role of silibinin as an antiproliferative compound, whose mechanism of action was related to the TP53 status.

Immunohistochemical expression of DNA methyltransferases 1, 3a, and 3b in actinic cheilitis and lip squamous cell carcinomas.

BACKGROUND: Epigenetic modifications, including DNA methylation of tumor suppressor genes carried out by DNA methyltransferases (DNMTs), are important events in carcinogenesis. Although there are studies concerning to its expression in several cancer types, DNMTs expression pattern is not known in photoinduced lip carcinogenesis. The aim of this study was to investigate the immunoexpression of DNMTs 1, 3a, and 3b in lip precancerous lesion (actinic cheilitis) and cancer. METHODS: Thirty cases of actinic cheilitis (AC), thirty cases of lip squamous cell carcinoma (LSCC), and twenty cases of non-neoplastic tissue (NNT) were selected for immunohistochemical investigation of DNMTs 1, 3a, and 3b. RESULTS: Nuclear DNMT 1 immunoreactivity was significantly higher in the LSCC group (68.6%) compared with NNT (47%), and nuclear DNMT 3b was higher in LSCC (70.9%) than in NNT (37.9%) and in AC (44%). Only DNMT 3a showed both higher nuclear and cytoplasmic expression in AC (35.9% and 35.5%, respectively) than in NNT (4.4% and 16.1%, respectively) and LSCC (8.8% and 13.2%, respectively) (P < 0.05). CONCLUSIONS: The results suggested that DNMT 3a could play a key role in the methylation process of initial steps of UV carcinogenesis present in AC while DNMT 3b could be responsible for de novo methylation in already established lip cancer.

Deregulation of DNMT1, DNMT3B and miR-29s in Burkitt lymphoma suggests novel contribution for disease pathogenesis.

Methylation of CpG islands in promoter gene regions is frequently observed in lymphomas. DNA methylation is established by DNA methyltransferases (DNMTs). DNMT1 maintains methylation patterns, while DNMT3A and DNMT3B are critical for de novo DNA methylation. Little is known about the expression of DNMTs in lymphomas. DNMT3A and 3B genes can be regulated post-transcriptionally by miR-29 family. Here, we demonstrated for the first time the overexpression of DNMT1 and DNMT3B in Burkitt lymphoma (BL) tumor samples (69% and 86%, respectively). Specifically, the treatment of two BL cell lines with the DNMT inhibitor 5-aza-dC decreased DNMT1 and DNMT3B protein levels and inhibited cell growth. Additionally, miR-29a, miR-29b and miR-29c levels were significantly decreased in the BL tumor samples. Besides, the ectopic expression of miR-29a, miR-29b and miR-29c reduced the DNMT3B expression and miR-29a and miR-29b lead to increase of p16(INK4a) mRNA expression. Altogether, our data suggest that deregulation of DNMT1, DNMT3B and miR29 may be involved in BL pathogenesis.

Role of heme oxygenase-1 in demethylating effects on SKM-1 cells induced by decitabine.

We evaluated the influence of heme oxygenase-1 (HO-1) gene inhibition in myelodysplastic syndrome (MDS) cell line SKM-1 on enhancement of the demethylating effects of decitabine on p15, and explored the possible mechanism. DNMT1 gene expression in SKM-1 cells was silenced by being transfected by a constructed siRNA with liposomes. The proliferation inhibition rates after drug treatment were detected by cell counting kit-8 assay. The apoptotic rates were detected by Annexin V/PI assay with flow cytometry. The expressions of p16, p15, TP73, CDH1, ESR1, and PDLIM4 mRNAs were detected by real-time PCR, and those of HO-1, DNMT1, DNMT3A, DNMT3B, HDAC, and p15 proteins were measured by western blot. The degree of methylation of the p15 gene was analyzed by using methylation-specific PCR (MSP). CCK-8 assay showed that after HO-1 gene expression was inhibited; the proliferation rate of SKM-1 cells treated by decitabine (70.91 ± 0.05%) was significantly higher than that of the control group (53.67 ± 0.05%). Flow cytometry showed that the apoptotic rate of SKM- 1 cells treated by decitabine in combination with HO-1 expression inhibition (44.25 ± 0.05%) exceeded that of the cells treated by this drug alone (37.70 ± 0.05%). MSP showed that inhibiting HO-1 expression significantly increased the degree of methylation of the p15 gene. As suggested by western blot, the degree of methylation of the p15 protein was changed after decitabine treatment when the expression of the HO-1 protein was changed, being associated with the affected DNMT1 expression. Inhibited HO-1 expression attenuated the hypermethylation of CDKN2B by suppressing DNMT1, which was conducive to treatment by cooperating with decitabine. In conclusion, the findings of this study provide valuable experimental evidence for targeted MDS therapy, and a theoretical basis for further studies.

Immunolocalization of DNMT1 and DNMT3a in salivary gland neoplasms.

OBJECTIVE: Salivary gland neoplasms pathogenesis has not been well established. DNA methylation occurs when methyl groups are added to cytosine nucleotides in specific areas of the gene by the enzyme DNA methyltransferase (DNMT). This chemical modification can alter gene expression without altering DNA sequence. While DNMT3a is mostly involved in de novo methylation, DNMT1 acts as a maintenance methyltransferase. We aimed to investigate the immunoexpression of DNMT3a and DNMT1 in minor salivary gland neoplasms, comparing it with normal tissue. MATERIAL: Forty-four formalin-fixed and paraffin-embedded samples of pleomorphic adenoma, adenoid cystic carcinoma, mucoepidermoid carcinoma and polymorphous low-grade adenocarcinoma were included in the study. The DNMT1 and DNMT3a proteins were identified by using a highly sensitive polymer-based system. RESULTS: Positive nuclear and cytoplasmic labeling for DNMT1 was observed in all samples, including the controls. Positive nuclear labeling for DNMT3a was found only in few neoplasms: 1 pleomorphic adenoma (9.0%), 2 adenoid cystic carcinoma (16.6%) and 1 mucoepidermoid (9.0%) cases. CONCLUSION: Our results were not able to demonstrate a clear correlation between DNMT1 and DNMT3a immunoexpression and salivary gland neoplasms development.

Changes in DNA methyltransferase induced by treatment with N-2-acetylaminofluorene.

We have compared the levels of DNA methyltransferases from rat liver and spleen in both sexes following a single injection of N-2-acetylaminofluorene (AAF). Enzyme extracts from treated animals were obtained at different intervals (2-34 days) after treatment. The extracts were assayed in the presence of chicken erythrocyte DNA and S-adenosyl-L-[Me-3H]methionine. A 55% increase in male rat-liver methyltransferase activity measured by Me-3H incorporation into DNA occurred on day 14. By contrast, female methyltransferase after a similar period revealed a 33% decrease in activity. Between days 21 and 34, there is a progressive return to normal methyltransferase levels. Spleen-derived enzyme studied between days 7 and 14, showed a decrease in methylating activity in both sexes. After replacing corn seed oil by ethanol as the vehicle for AAF injection, we observed a change in liver methyltransferase 48 h after injection. Quantification of radioactive eluates in m5C fractions together with the increase in the integrated area identified as m5C in HPLC chromatograms allowed positive identification of methylated products.