Molecular mechanisms of cell death induced in glioblastoma by experimental and antineoplastic drugs: New and old drugs induce apoptosis in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most aggressive astrocytic tumors; it is resistant to most chemotherapeutic agents currently available and is associated with a poor patient survival. Thus, the development of new anticancer compounds is urgently required. Herein, we studied the molecular mechanisms of cell death induced by the experimental drugs resveratrol and MG132 or the antineoplastic drugs cisplatin and etoposide on a human GBM cell line (D54) and on primary cultured mouse astrocytes (PCMAs). Caspases, Bcl-2, inhibitors of apoptosis proteins (IAP) family members, and p53 were identified as potential molecular targets for these drugs. All drugs had a cytotoxic effect on D54 cells and PCMAs, with a similar inhibitory concentration (IC50) after 24 h. However, MG132 and cisplatin were more effective to induce apoptosis and autophagy than resveratrol and etoposide. Cell death by apoptosis involved the activation of caspases-3/7, -8, and -9, increased lysosomal permeability, LC3 lipidation, poly-(ADP-ribose) polymerase (PARP)-1 fragmentation, and a differential expression of genes related with apoptosis and autophagy like Mcl-1, Survivin, Noxa, LC3, and Beclin. In addition, apoptosis activation was partially dependent on p53 activation. Since experimental and antineoplastic drugs yielded similar results, further work is required to justify their use in clinical protocols.

Mutant p53 gain of function induces HER2 over-expression in cancer cells.

BACKGROUND: HER2 over-expression is related with a poor prognosis in patients with invasive breast cancer tumors. Clinical associations have reported that somatic mutations of p53 more frequently detected in cases of sporadic breast cancer of the HER2 subtypes, besides a high percentage of HER2-amplifying tumors carry germline mutations of p53. The mechanisms responsible for the acquisition of oncogenic functions of p53 mutant proteins (mtp53), known as Gain of Function (GOF), over HER2 expression have not been reported. The objective of this study was to evaluate a possible relationship between p53 mutants and HER2 regulation. METHODS: HER2 expression (transcription and protein), as well as HER2 protein stabilization have been evaluated after inducing or silencing of p53 mutants' expression in cell lines. Finally, we evaluated the interaction of the p53 mutants over the HER2 receptor promoter. RESULTS: Higher HER2 expression in cell lines harboring endogenous mtp53 compared with wt or null expression of p53 cell lines. Transfection of p53 mutants (R248Q and R273C) in cell lines increased the expression of HER2. Silencing of p53 mutants, decrease HER2 expression. The p53 mutants R248Q and R273C significantly increase the luciferase activity on the HER2 promoter, and both mutants also promote acetylation of H3 and H4 histones binding in it. CONCLUSIONS: These findings show for the first time that p53 mutants induce over-expression of HER2 at transcriptional level of the HER2 protein. Our results could have clinical implications in breast cancer and other types of cancer where HER2 is over-expressed and used as a therapy target.

Resveratrol decreases Rad51 expression and sensitizes cisplatin­resistant MCF­7 breast cancer cells.

Resveratrol (RES), a polyphenol compound with anti­proliferative properties, has been previously evaluated for its beneficial effects against a variety of tumour cells. The current study elucidated the means by which RES enhances the anti­proliferative effects of cisplatin (CIS) on MCF­7 cells, focusing on the inhibitory effects on DNA repair of double­strand breaks (DSBs). Chemoresistant MCF­7 cells (MCF­7R) were generated by continuous exposure to low concentrations of CIS (10 µM CIS­IC40) during 5 passages, with the IC50 value increasing ~3­fold. Using an MTT assay, we estimated the changes in IC50 for CIS in MCF­7, T47­D, MDA­MB­231 and MCF­7R cells in the presence of RES. The relative transcript level of Nbs­1, Mre­11 and Rad­50 genes was assessed using RT­qPCR analysis. Rad51 and H2AX [pSer139] protein expression was determined by western blot analysis. RES at 50 and 100 µM significantly enhanced the anti­proliferative effects of CIS in both MCF­7 and MCF­7R cells, decreasing the IC50 values for CIS to one­tenth and one­sixth, respectively. A total of 100 µM RES decreased the relative transcript levels of homologous recombination (HR) initiation complex components and the Rad51 protein level in MCF­7 and MCF­7R cells. After 48 h of CIS DNA damage, the levels of Rad51 protein increased, but this effect was inhibited by 100 µM RES. RES also maintained serine 139 phosphorylation of histone H2AX, suggesting that RES prevents the repair of DSBs. It was observed that RES exerts an antagonistic effect over CIS on the activation of Rad51 and sustained phosphorylation of H2AX. The results suggest that RES in combination with DNA damage­based therapy has potential as a strategy to overcome resistance and provide much safer and more effective treatment for breast cancer.

Nicotinamide sensitizes human breast cancer cells to the cytotoxic effects of radiation and cisplatin.

Poly(ADP-ribose) polymerase (PARP) inhibitors enhance the effect of DNA alkylating agents on BRCA1­ and BRCA2-deficient cell lines. The aim of this study was to analyze the effect of the PARP inhibitor nicotinamide (NAM) on breast cancer cells with different BRCA1 expression or function, such as BRCA1­deficient MDA-MB-436 cells, low expression BRCA1 MCF-7 cells, and the BRCA1 wild­type MDA-MB-231 cells, to demonstrate its effects as a chemo­ or radiosensitizing agent. PARP activity was analyzed in MDA-MB-436, MCF-7 and MDA-MB-231 breast cancer cells subjected or not to NAM. Inhibition of PARP by NAM in the presence of DNA damage was examined by Alexa Fluor 488 immunofluorescence. Crystal violet assays were used to test growth inhibition and the chemo­ and radiosensitization effects of NAM were investigated using clonogenic assays. Significant differences among data sets were determined using two-tailed ANOVA and Bonferroni tests. We demonstrated that NAM reduces PARP activity in vitro, and in cells subjected or not to DNA damage, it also reduces the viability of breast cancer cell lines and synergyzes the cytotoxicity of cisplatin in MDA-MB-436 and MCF-7 cells. Downregulation of PARP1 with siRNA led to modest growth inhibition, which was further increased by cisplatin. Nicotinamide also induced radiosensitization in MDA-MB-436 and MDA-MB-231 cells. In conclusion, NAM may be used as a chemo­ or radiosensitizing agent regardless of the BRCA1 status in breast cancer.

Association between ERCC1 and XPA expression and polymorphisms and the response to cisplatin in testicular germ cell tumours.

BACKGROUND: Cisplatin cures over 80% of testicular germ cell tumours (TGCTs), and nucleotide-excision repair (NER) modifies the sensitivity to cisplatin. We explored the association between NER proteins and their polymorphisms with cisplatin sensitivity (CPS) and overall survival (OS) of patients with non-seminomatous (ns)-TGCTs. METHODS: The expression of ERCC1 and XPA and the presence of γH2AX were evaluated in cancer cell lines and in fresh ns-TGCTs. The ERCC1 protein was also determined in ns-TGCTs. The differences between CPS and non-CPS cell lines and patients were analysed by Student's t- or χ(2)-tests. The differences in OS were analysed using the log-rank test, and the hazard ratios (HRs) were calculated using the Cox model. RESULTS: High ERCC1 expression was observed in the non-CPS cells, and both ERCC1 and γH2AX expressions were augmented after cisplatin treatment. Increased ERCC1 expression was also identified in non-CPS patients. Neither polymorphism was associated with either CPS or OS. The presence of ERCC1 was associated with non-CPS (P=0.05) and adjusted in the prognosis groups. The HR in ERCC1-negative and non-CPS patients was >14.43, and in ERCC1-positive and non-CPS patients the HR was >11.86 (P<0.001). CONCLUSIONS: High levels of ERCC1 were associated with non-CPS, suggesting that ERCC1 could be used as a potential indicator of the response to cisplatin and prognosis in ns-TGCTs.

Differential gene expression between skin and cervix induced by the E7 oncoprotein in a transgenic mouse model.

HPV16 E7 oncoprotein expression in K14E7 transgenic mice induces cervical cancer after 6 months of treatment with the co-carcinogen 17ß-estradiol. In untreated mice, E7 also induces skin tumors late in life albeit at low penetrance. These findings indicate that E7 alters cellular functions in cervix and skin so as to predispose these organs to tumorigenesis. Using microarrays, we determined the global genes expression profile in cervical and skin tissue of young adult K14E7 transgenic mice without estrogen treatment. In these tissues, the E7 oncoprotein altered the transcriptional pattern of genes involved in several biological processes including signal transduction, transport, metabolic process, cell adhesion, apoptosis, cell differentiation, immune response and inflammatory response. Among the E7-dysregulated genes were ones not previously known to be involved in cervical neoplasia including DMBT1, GLI1 and 17ßHSD2 in cervix, as well as MMP2, 12, 14, 19 and 27 in skin.

Gene expression profile of cervical and skin tissues from human papillomavirus type 16 E6 transgenic mice.

BACKGROUND: Although K14E6 transgenic mice develop spontaneous tumors of the skin epithelium, no spontaneous reproductive tract malignancies arise, unless the transgenic mice were treated chronically with 17beta-estradiol. These findings suggest that E6 performs critical functions in normal adult cervix and skin, highlighting the need to define E6-controlled transcriptional programs in these tissues. METHODS: We evaluated the expression profile of 14,000 genes in skin or cervix from young K14E6 transgenic mice compared with nontransgenic. To identify differentially expressed genes a linear model was implemented using R and the LIMMA package. Two criteria were used to select the set of relevant genes. First a set of genes with a Log-odds > or = 3 were selected. Then, a hierarchical search of genes was based on Log Fold Changes. RESULTS: Microarray analysis identified a total of 676 and 1154 genes that were significantly up and down-regulated, respectively, in skin from K14E6 transgenic mice. On the other hand, in the cervix from K14E6 transgenic mice we found that only 97 and 252 genes were significantly up and down-regulated, respectively. One of the most affected processes in the skin from K14E6 transgenic mice was the cell cycle. We also found that skin from transgenic mice showed down-regulation of pro-apoptotic genes and genes related to the immune response. In the cervix of K14E6 transgenic mice, we could not find affected any gene related to the cell cycle and apoptosis pathways but did observe alterations in the expression of immune response genes. Pathways such as angiogenesis, cell junction and epidermis development, also were altered in their gene expression profiles in both tissues. CONCLUSION: Expression of the HPV16 E6 oncoprotein in our model alters expression of genes that fell into several functional groups providing insights into pathways by which E6 deregulate cell cycle progression, apoptosis, the host resistance to infection and immune function, providing new opportunities for early diagnostic markers and therapeutic drug targets.