transfected with siNT control. However, this effect was abrogated in cells after BRCA1 silencing. Similar results were obtained using a second BRCA1 siRNA oligonucleotide. The effect of BRCA1 silencing on vinorelbine induced apoptosis was also assessed by measuring the percentage of subG1 apoptotic cell population. Bicalutamide Again, vinorelbineinduced apoptosis was increased in the three cell lines transfected with siNT. However, this effect was reduced following silencing of BRCA1. Again, similar results were obtained with siBRCA12. Selection for vinorelbine resistance induces down regulation of BRCA1 To further examine the association between BRCA1 expression and sensitivity to vinorelbine, we determined whether selection for resistance would result in down regulation of BRCA1 expression.
Both REN and MSTO 211H cell lines were exposed to increasing concentrations of vinorelbine leading to PARP Inhibitor the isolation of two isogenic cell lines with log fold resistance to vinorelbine compared to their parental counterparts. Resistance was associated with loss of BRCA1 expression and reduction in vinorelbineinduced apoptosis measured by PARP and caspase 9 cleavage.Vinorelbine induced significant increases in caspase 3 activity in transfected parental for resistance to vinorelbine as suggested by functional genetic studies. Accordingly, we proceeded to investigate the frequency of loss of BRCA1 expression in MPM tumours. We performed immunohistochemistrybased assessment of BRCA1 expression using a recently reported methodology.
By using a predefined threshold of less than 10% of BRCA1 expression for defining immunonegativity, we conducted a centralized pathological review of 144 archival mesothelioma specimens. Expression of BRCA1 was classified as immunonegative in 38.9% of Receptor Tyrosine Kinase Signaling mesotheliomas. We then assessed the potential correlation between BRCA1 protein expression and histology. The MPM samples in this study were representative of the three mesothelioma histological subtypes, including 36.1% epithelioid, 11% sarcomatoid, and 56.26% biphasic. The highest percentage of BRCA1 immunonegativity was observed in the sarcomatoid MPM tumours. However, these data did not achieve statistical significance. We can therefore conclude that BRCA1 immunodeficiency is not restricted by histological subtype. Discussion Our results strongly support a role for BRCA1 as an essential mediator of vinorelbine induced apoptosis in mesothelioma.
Given the potentially large genetic variation inherent in our panel of cell lines and the potential for multiple genes to regulate drug sensitivity, it was surprising to see that BRCA1 alone was so strongly correlated with vinorelbine induced toxicity. However, our data do not support gsk3 a causal link between BRCA1 expression and vinorelbine treatment, which may be related to epigenetic, transcriptional institutionalized or posttranslational effects of the drug. Moreover, BRCA1 targeted RNA interference induced resistance to vinorelbine. The mechanisms by which reduced BRCA1 expression results in resistance to spindle poisons, such as vinorelbine, have not been defined and are under investigation. However, it is suggested that these may be due to loss of BRCA1 mediated mitotic spindle checkpoint activation and apoptotic regulation.