No abstract

Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency ≥0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25× (25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.

Human osteosarcoma is a common primary malignancy of the bone in children and adolescents. It has been reported that curcumin is able to induce apoptosis in osteosarcoma MG63 cells through the mitochondrial pathway. However, whether curcumin is able to induce autophagy and the interaction between apoptosis and autophagy in osteosarcoma cells has yet to be fully elucidated. In the current study, it was determined that curcumin was able to significantly induce apoptosis, and lead to autophagy in MG63 cells. Notably, inhibition of apoptosis enhanced curcumin-induced autophagy due to upregulation of the c-Jun N-terminal kinase (JNK) signaling pathway. This finding was confirmed by the use of JNK-specific inhibitor, SP600125. Furthermore, our data showed that curcumin-induced apoptosis was increased when autophagy was completely inhibited by 3-methyladenine in MG63 cells. These results suggest that autophagy may have an important role in resistance to apoptosis when MG63 cells are incubated with curcumin. Thus, these results provide important insights into the interaction between apoptosis and autophagy in osteosarcoma cells and clinical treatment strategies using curcumin.