ABSTRACT
PURPOSE: Although lorlatinib, the third generation of echinoderm microtubule protein 4-anaplastic lymphoma kinase (EML4-ALK) tyrosine kinase inhibitor (TKI), overcame the previous generation ALK-TKIs' drug resistance problems, but the mechanism of lorlatinib resistance remained unclear. Furthermore, optimal chemotherapy for lorlatinib-resistant non-small cell lung cancer (NSCLC) patients was still unknown. METHODS: A lorlatinib-resistant NSCLC cell line SNU-2535LR was generated by gradually increasing dose of lorlatinib to crizotinib-resistant cell line SNU-2535 in vitro. To study the resistance mechanism of SNU-2535LR cells, we applied CCK-8 assay to detect the sensitivity of crizotinib and the reverse effect of APR-246, a p53 activator, on lorlatinib-induced resistance and different chemotherapy drugs to SNU-2535LR cells. We also detected the expressions of EML4-ALK-related proteins of SNU-2535LR cells via western blot.Please confirm that author names have been identified correctly and are presented in the right order.Dear Editor: I have carefully confirmed that the author names have been identified correctly and are presented in right order.Thank you very much! Your sincerely BoXie RESULTS: The sensitivity of SNU-2535LR cells to lorlatinib was decreased significantly than that of SNU-2535 cells. EML4-ALK fusion was decreased both at protein level and DNA level in SNU-2535LR cells. More interesting, the crizotinib-resistant mutation ALK p.G1269A disappeared, while new TP53 mutation emerged in SNU-2535LR cells. APR-246 can reverse the lorlatinib resistance in SNU-2535LR cells, with a reversal index of 4.768. Compared with SNU-2535 cells, the sensitivity of SNU-2535LR cells to gemcitabine, docetaxel and paclitaxel was significantly increased (P < 0.05), but decreased to cisplatin (P < 0.05). CONCLUSION: This study demonstrated that the combination of p53 protein agonist and lorlatinib may provide a new therapeutic strategy for NSCLC patients with lorlatinib resistance and TP53 mutation. Furthermore, the results also provide guidance for selecting optimal chemo-regimens for NSCLC patients after ALK-TKIs failure.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Aminopyridines , Anaplastic Lymphoma Kinase/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Cell Line , Cisplatin/therapeutic use , Crizotinib/therapeutic use , Docetaxel/therapeutic use , Drug Resistance, Neoplasm/genetics , Humans , Lactams , Lactams, Macrocyclic/pharmacology , Lactams, Macrocyclic/therapeutic use , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Microtubule Proteins/genetics , Mutation , Paclitaxel/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyrazoles , Tumor Suppressor Protein p53/geneticsABSTRACT
It is laborious to diagnose the infections of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and Suid herpesvirus 1 (SuHV-1) because of the similar clinical symptoms in piglets. Staphylococcus aureus (S. aureus), Streptococcus suis (S. suis), Salmonella choleraesuis (S. choleraesuis, serotype: 6,7:c:1,5), and Escherichia coli (E. coli) are common secondary bacterial pathogens in viral infections. Furthermore, the mixed infection of these viral and bacterial pathogens is more and more common in practical swine breeding. Therefore, a TaqMan multiplex qPCR method for simultaneous detection and differentiation of their pathogen was established in this study by designing specific primers and probes for the E2 gene of CSFV, the ORF7 gene of PRRSV, the ORF1 gene of PCV2 and the gE gene of SuHV-1, the nuc gene of S. aureus, the ef-tu gene of S. suis, the ivnA gene of S. choleraesuis, and the 23S rRNA gene of E. coli, and its specificity, sensitivity, and reproducibility were subsequently tested. The results showed that TaqMan multiplex qPCR method showed a high specificity with no cross reaction between different viruses, and a good repeatability with its coefficient of variation lower than 5%. Besides, the sensitivity of this method was also at least 10 times higher compared with conventional PCR. Overall, this study provided a reliable multiplex TaqMan qPCR method for the diagnosis and differentiation of the mentioned pathogens in pigs, laying a certain technical basis for disease prevention and control.