Digital methylation-specific PCR: New applications for liquid biopsy.

Epigenetic analysis is a fundamental part of understanding pathophysiological processes with potential applications in diagnosis, prognosis, and assessment of disease susceptibility. Epigenetic changes have been widely studied in chronic obstructive pulmonary disease (COPD), but currently, there is no molecular marker used to improve the treatment of patients. Furthermore, this progressive disease is a risk factor for the development of more severe COVID-19. Methylation-specific polymerase chain reaction (MSP-PCR) plays an important role in the analysis of DNA methylation profiles, and it is one of the most widely used techniques. In this context, the combination of MSP-PCR with emerging PCR technologies, such as digital PCR (dPCR), results in more accurate analyses of the DNA methylation profile of the genes under study. In this study, we propose the application of the MSP-dPCR technique to evaluate the methylation profile of the ADAM33 gene from saliva samples and lung tissue biopsies of patients with COPD and COVID-19. MSP-dPCR generated a measurable prediction of gene methylation rate, with the potential application of this combined technology for diagnostic and prognostic purposes. It has also proven to be a powerful tool for liquid biopsy applications.

l-carvone decreases breast cancer cells adhesion, migration, and invasion by suppressing FAK activation.

Breast cancer is one of the most common types of cancer in the world and current therapeutic strategies present severe drawbacks. l-carvone (CRV), a monoterpene found in Mentha spicata (spearmint), has been reported to have potent anti-inflammatory activity. Here, we examined the role of CRV in breast cancer cell adhesion, migration and invasion in vitro and how this component could suppress the growth of Ehrlich carcinoma-bearing mice. In vivo, treatment with CRV significantly decreased tumor growth, increased tumor necrosis area, and reduced the expression of VEGF and HIF-1α in Ehrlich carcinoma-bearing mice. Furthermore, the anticancer efficacy of CRV was similar to currently used chemotherapy (Methotrexate), and the combination of CRV with MTX potentiated the chemotherapy effects. Further mechanistic investigation in vitro revealed that CRV modulates the interaction of breast cancer cells with the extracellular matrix (ECM) by disrupting focal adhesion, which was shown by scanning electron microscopy (SEM) and immunofluorescence. Moreover, CRV caused a decrease in ß1-integrin expression and inhibited focal adhesion kinase (FAK) activation. FAK is one of the most important downstream activators of several metastatic processes, including MMP-2 mediated invasion and HIF-1α/VEGF angiogenesis stimulus, both of which were found to be reduced in MDA-MB-231 cells exposed to CRV. Our results provide new insight about targeting ß1-integrin/FAK signaling pathway with CRV, which could be a new potential agent in the treatment of breast cancer.