ABSTRACT
Objective:To investigate the effects of autophagy-mediated crizotinib resistance on cancer stem-like cell subsets in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALK + ALCL). Methods:The preliminary research of our group divided ALK + ALCL Karpas299 cell line into two subgroups: reporter unresponsive (RU) and reporter responsive (RR) cells through the implantation of Sox2 reporter genes, among which the RR cells had the characteristics of stem cells. Fluorescent labeled LC3 overexpressing RR and RU cells (RR-LC3 and RU-LC3) were constructed by lentiviral transfection technique, and the transfection efficiency was verified by using Western blotting and flow cytometry. RU-LC3 and RR-LC3 were treated with crizotinib at different concentrations (0, 250, 500, 1 000 nmol/L). The RED and GEN signals were detected by using double-signal flow cytometry to observe autophagy flux (RED represents the red signal B695 of the next generation of far-red fluorescent protein TagFP635 mKate; GEN represents the green signal from pH-sensitive GFP variant pHluorin B530), and the RED to GEV ratio represents autophagy flux. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect autophagy related genes ULK1, WIPI1 and LC3B mRNA expression levels in cells. The effects of different concentrations of crizotinib (250, 500, 1 000 nmol/L) combined with chloroquine (5, 10 μmol/L) on the cell survival were detected by using MTS assay. Results:RU-LC3 and RR-LC3 cells with overexpression of LC3 were successfully constructed. After induction of 250, 500 and 1 000 nmol/L crizotinib, the RED to GEN ratio in RU-LC3 cells was 1.135±0.017, 1.453±0.017 and 1.755±0.021, respectively; the RED to GEN ratio in RR-LC3 cells was 1.193±0.018, 2.116±0.013 and 3.307±0.189, respectively; the RED to GEN ratio in RU-LC3 cells and RR-LC3 cells showed a dose-dependent manner. The RED to GEN ratio in RR-LC3 cells was higher than that in RU-LC3 cells when treated with same concentrations of crizotinib, and the differences were statistically significant (all P < 0.01). The autophagy flux of RR-LC3 cells was larger than that of RU-LC3 cells. When treated without crizotinib, mRNA relative expression levels of ULK1, WIPI1 and LC3B in RR cells were higher than those in RU cells (1.69±0.05 vs.1.01±0.02, t = -1.62, P < 0.01; 1.24±0.04 vs. 1.03±0.05, t = -2.11, P < 0.01; 1.70±0.22 vs. 1.02±0.05, t = -1.74, P = 0.033). In the absence of chloroquine, the half-inhibitory concentration ( IC50) of crizotinib in RR cells was higher than that of RU cells (950 nmol/L vs. 709 nmol/L). After treated with chloroquine, IC50 of RU cells did not change, while IC50 of RR cells was decreased with the increase of chloroquine concentration. Conclusions:Compared with RU cells, autophagy reaction of cancer stem-like RR cells is more rapid and intense, which is considered to be one of the important reasons for their resistance to crizotinib.
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Objective:To compare the clinical effects of the three criteria for postoperative pulmonary complications (PPCs).Methods:The clinical data of patients underwent thoracoscopic lung resection between January 2021 and July 2021 in our hospital were retrospectively analyzed.PPCs were assessed using the Melbourne Group Scale (MGS), European Perioperative Clinical Outcome (EPCO) and Standardized Endpoints for Perioperative Medicine (StEP) criteria.The patients were divided into PPC group and non-PPC group according to the above criteria.The diagnostic rates of PPCs of the three criteria were recorded.Cohen′s weighted kappa coefficient was used to evaluate the agreement between the three criteria.Logistic regression method was used to analyze the association between PPCs diagnosed by different criteria and risk of adverse prognostic events developed.Results:A total of 397 patients who underwent thoracoscopic lung surgery were included in this study.The rate of PPCs diagnosed by MGS criterion was significantly lower than those by EPCO and StEP criteria ( P<0.001), and the rate of PPCs diagnosed by EPCO criterion was significantly higher than those by StEP criterion ( P<0.001). The diagnostic agreement between EPCO criterion and StEP criterion was good ( κ=0.624, P<0.001), while the diagnostic agreement between EPCO criterion, StEP criterion and MGS criterion was poor ( κ=0.101, P<0.001; κ=0.210, P<0.001). Univariate and multivariate logistic regression analysis showed that PPCs diagnosed by EPCO and StEP criteria increased the risk of adverse prognostic events developed ( P<0.001). Conclusions:The EPCO and StEP criteria are superior to MGS criterion with regard to the diagnostic and prognostic value for pulmonary complications following thoracoscopic lung resection, and the EPCO criterion had a higher sensitivity.
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Although myopathy has obvious heterogeneity in clinic,the exact mechanism of myopathy is not fully understood,and there is no effective biomarker,and there are one or some protein changes in the basic pathogenesis of myopathy.Proteomics is a discipline that explores the composition,expression and modification of proteins at a holistic level and is widely used in various fields of life sciences.The study of differential proteomics provides more scientific basis for explaining the pathogenesis of diseases,searching for new diagnostic markers and potential therapeutic targets.The application of proteomics in the study of muscular diseases deserves more attention from neurologists.
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Objective To study the clinical,pathological and genetic features of myofibrillar myopathy caused by BAG3 gene mutation.Methods The clinical features and pathological findings of a patient with myofibrillar myopathy were analyzed.Genomic DNA of the patient was extracted from peripheral blood and the next generation sequencing was performed to explore the mutation of genes about myopathies.Results The patient presented with nine-year-old onset myopathy characterized by progressive difficulty for squatting,rigid spine and muscle atrophy in the limbs symmetrically.Peripheral neurogenic damages were found on electromyography.On muscle biopsy,myogenic and neurogenic damages with rimmed vacuoles appeared,and the deposited materials were positive for sarcoglycan,dystrophin-R and dystrophin-C.There was a reported heterozygous mutation in the exons of the BAG3 gene (c.626C ＞ T).Conclusion There is no specificity of clinical manifestation in myofibrillar myopathy,and the diagnosis of this disease mainly depends on muscle biopsy and genetic screening.