External magnetic field have significant effects on diversity of magnetotactic bacteria in sediments from Yangtze River, Chagan Lake and Zhalong Wetland in China.

Magnetotactic bacteria (MTB) can rapidly relocate to optimal habitats by magnetotaxis, and play an important role in iron biogeochemical cycling. This study aimed to evaluate the contribution of the external magnetostatic field to the diversity of MTB in freshwater sediments from Yangtze River (Changjiang River, CJ), Chagan Lake (CGH) and Zhalong Wetland (ZL). The magnetic field intensity was tightly associated with the community richness of MTB in CJ, whereas it was closely related to the diversity of MTB in CGH and ZL (p < 0.05), elucidating a significant variation in the community composition of MTB. Magnetic exposure time appeared more significant correlation with community richness than diversity for MTB in CJ and CGH (p < 0.05), while an opposite relationship existed in ZL (p < 0.01). Herbaspirillum (93.81-96.48 %) dominated in the sediments of these surfacewatesr regardless of waterbody types, while it shifted to Magnetospirillum in ZL under 100 Gs magnetic field. The network connectivity and stability of MTB deteriorate with the increase of magnetic field intensity. Functional analysis showed that the Two-component system and ABC transporter system of MTB obviously responded to magnetic field intensity and exposure time. Our findings will pave the way to understanding the response mechanism of MTB community in freshwater sediments to the external magnetostatic field.

Scutellaria baicalensis enhances 5-fluorouracil-based chemotherapy via inhibition of proliferative signaling pathways.

Fluoropyridine-based chemotherapy remains the most widely used treatment for colorectal cancer (CRC). In this study, we investigated the mechanism by which the natural product Scutellaria baicalensis (Huang Qin; HQ) and one of its main components baicalin enhanced 5-fluorouracil (5-FU) antitumor activity against CRC. Cell proliferation assays, cell cycle analysis, reverse-phase protein array (RPPA) analysis, immunoblot analysis, and qRT-PCR were performed to investigate the mechanism(s) of action of HQ and its active components on growth of CRC cells. HQ exhibited in vitro antiproliferative activity against drug resistant human CRC cells, against human and mouse CRC cells with different genetic backgrounds and normal human colon epithelial cells. In vivo animal models were used to document the antitumor activity of HQ and baicalin. The mechanism of growth inhibitory activity of HQ is due to inhibition of proliferative signaling pathways including the CDK-RB pathway. In addition, HQ enhanced the antitumor effects of 5-FU and capecitabine in vivo. Furthermore, we identified baicalin as an active component of HQ. The combination of baicalin and 5-FU demonstrated synergistic activity against 5-FU-resistant RKO-R10 cells. The combination significantly inhibited in vivo tumor growth greater than each treatment alone. RPPA results showed that the signaling pathway alterations in CRC cells were similar following HQ and baicalin treatment. Together, these results indicate that HQ and its component baicalin enhance the effect of 5-fluorouracil-based chemotherapy via inhibition of CDK-RB pathway. These findings may provide the rational basis for developing agents that can overcome the development of cellular drug resistance. Video Abstract.

Potential and whole-genome sequence-based mechanism of elongated-prismatic magnetite magnetosome formation in Acidithiobacillus ferrooxidans BYM.

A magnetosome-producing bacterium Acidithiobacillus ferrooxidans BYM (At. ferrooxidans BYM) was isolated and magnetically screened. The magnetosome yield from 0.5896 to 13.1291 mg/g was achieved under different aeration rates, ferrous sulfate, ammonium sulfate, and gluconic acid concentrations at 30 â„ƒ. TEM observed 6-9 magnetosomes in size of 20-80 nm irregularly dispersed in a cell. STEM-EDXS and HRTEM-FFT implied that the elongated-prismatic magnetite magnetosomes with {110} crystal faces grown along the [111] direction. Whole-genome sequencing and annotation of BYM showed that 3.2 Mb chromosome and 47.11 kb plasmid coexisted, and 322 genes associated with iron metabolism were discovered. Ten genes shared high similarity with magnetosome genes were predicted, providing sufficient evidence for the magnetosome-producing potential of BYM. Accordingly, we first proposed a hypothetic model of magnetosome formation including vesicle formation, iron uptake and mineralization, and magnetite crystal maturation in At. ferrooxidans. These indicated that At. ferrooxidans BYM would be used as a commercial magnetosome-producing microorganism.

Biomineralization and biotechnological applications of bacterial magnetosomes.

Magnetosomes intracellularly biomineralized by Magnetotactic bacteria (MTB) are membrane-enveloped nanoparticles of the magnetic minerals magnetite (Fe3O4) or greigite (Fe3S4). MTB thrive in oxic-anoxic interface and exhibit magnetotaxis due to the presence of magnetosomes. Because of the unique characteristic and bionavigation inspiration of magnetosomes, MTB has been a subject of study focused on by biologists, medical pharmacologists, geologists, and physicists since the discovery. We herein first briefly review the features of MTB and magnetosomes. The recent insights into the process and mechanism for magnetosome biomineralization including iron uptake, magnetosome membrane invagination, iron mineralization and magnetosome chain assembly are summarized in detail. Additionally, the current research progress in biotechnological applications of magnetosomes is also elucidated, such as drug delivery, MRI image contrast, magnetic hyperthermia, wastewater treatment, and cell separation. This review would expand our understanding of biomineralization and biotechnological applications of bacterial magnetosomes.

Overcoming acquired resistance to third-generation EGFR inhibitors by targeting activation of intrinsic apoptotic pathway through Mcl-1 inhibition, Bax activation, or both.

Treatment of EGFR-mutant non-small cell lung cancer (NSCLC) with mutation-selective third-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs) such as osimertinib has achieved remarkable success in the clinic. However, the immediate challenge is the emergence of acquired resistance, limiting the long-term remission of patients. This study suggests a novel strategy to overcome acquired resistance to osimertinib and other third-generation EGFR-TKIs through directly targeting the intrinsic apoptotic pathway. We found that osimertinib, when combined with Mcl-1 inhibition or Bax activation, synergistically decreased the survival of different osimertinib-resistant cell lines, enhanced the induction of intrinsic apoptosis, and inhibited the growth of osimertinib-resistant tumor in vivo. Interestingly, the triple-combination of osimertinib with Mcl-1 inhibition and Bax activation exhibited the most potent activity in decreasing the survival and inducing apoptosis of osimertinib-resistant cells and in suppressing the growth of osimertinib-resistant tumors. These effects were associated with increased activation of the intrinsic apoptotic pathway evidenced by augmented mitochondrial cytochrome C and Smac release. Hence, this study convincingly demonstrates a novel strategy for overcoming acquired resistance to osimertinib and other 3rd generation EGFR-TKIs by targeting activation of the intrinsic apoptotic pathway through Mcl-1 inhibition, Bax activation or both, warranting further clinical validation of this strategy.

Preparation, characterization, and in vitro release kinetics of doxorubicin-loaded magnetosomes.

The doxorubicin (DOX) was successfully coupled to the magnetosomes from Acidithiobacillus ferrooxidans (At. ferrooxidans) by genipin bridging. The parameters (magnetosome concentration, DOX concentration, genipin concentration-, and cross-link time) expected for temperature significantly influenced the coupling rate. Bacterial magnetosome-doxorubicin complexes (BMDCs) were characterized by transmission electron microscope (TEM), particle size analyzer and Fourier transform infrared spectroscopy. Results indicated that BMDCs exhibited a mean particle size of 83.98 mm and displayed a negative charge. The chemical reaction occurring between CO and NH group and the physical adsorption predominated by electrostatic interaction were found to involve in coupling. BMDCs can release 40% of DOX in simulated gastrointestinal conditions within 38 h. Kinetic models including Higuchi, Korsmeyer-Peppas, Zero order, First order, Hixon-Crowell, Baker-Lonsdale, and Weibull and Gompertz were utilized to explore the release mechanism of DOX from BMDCs. All models were found to fit well (r2 ≥ 0.8144) with the release data and the Gompertz was the best fit model (r2 = 0.9742), implying that the complex mechanisms involving Fickian and Gompertz diffusion contributed to the release. These findings suggested that magnetosomes from At. ferrooxidans have great potential applications in biomedical and clinical fields as the carrier of target drug delivery systems in the future.

[In vivo study on the effects of intercellular adhesion operon of Staphylococcus epidermidis on the inflammation associated with bacteria-fungal mixed biofilm].

OBJECTIVE: To study the effect of intercellular adhesion (ica) operon of Staphylococcus epidermidis on the inflammation associated with mixed biofilm of Staphylococcus epidermidis and Candida albicans on endotracheal tube material in rabbits. METHODS: The standard strains of Staphylococcus epidermidis RP62A (ica operon positive, positive group) and ATCC12228 (ica operon negative, negative group) were taken to prepare a bacterial solution with a concentration of 1×10 6 CFU/mL, respectively. Then, the two bacterial solutions were mixed with the standard strain of Candida albicans ATCC10231 of the same concentration to prepare a mixed culture solution at a ratio of 1∶1, respectively. The mixed culture solution was incubated with endotracheal tube material for 24 hours. The formation of mixed biofilm on the surface of the material was observed by scanning electron microscope. Thirty New Zealand rabbits, aged 4-6 months, were divided into two groups ( n=15), and the endotracheal tube materials of the positive group and the negative group that were incubated for 24 hours were implanted beside the trachea. The body mass of rabbits in the two groups was measured before operation and at 1, 3, and 7 days after operation. At 1, 3, and 7 days after operation, the levels of interleukin 1ß (IL-1ß), IL-6, tumor necrosis factor α (TNF-α), and monocytechemotactic protein 1 (MCP-1) were detected by using an ELISA test kit. At 7 days after operation, the formation of mixed biofilm on the surface of the endotracheal tube materials was observed by scanning electron microscope, the inflammation and infiltration of tissues around the materials were observed by HE staining, and the bacterial infections in heart, lung, liver, and kidney were observed by plate colony counting method. RESULTS: Scanning electron microscope observation showed that the mixed biofilm structure was obvious in the positive group after 24 hours in vitro incubation, but no mixed biofilm formation was observed in the negative group. In vivo studies showed that there was no significant difference in body mass between the two groups before operation and at 1, 3, and 7 days after operation ( P>0.05). Compared with the negative group, the levels of MCP-1 and IL-1ß at 1 day, and the levels of IL-1ß, MCP-1, IL-6, and TNF-α at 3 and 7 days in the positive group all increased, with significant differences ( P<0.05). Scanning electron microscope observation showed that a large amount of Staphylococcus epidermis and mixed biofilm structure were observed in the positive group, and a very small amount of bacteria was observed in the negative group with no mixed biofilm structure. HE staining of surrounding tissue showed inflammatory cell infiltration in both groups, and neutrophils and lymphocytes were more in the positive group than in the negative group. There was no significant difference in the number of bacterial infections in heart and liver between the two groups ( P>0.05). The number of bacterial infections in lung and kidney in the positive group was higher than that in negative group ( P<0.05). CONCLUSION: In the mixed infection of Staphylococcus epidermidis and Candida albicans, the ica operon may strengthen the structure of the biofilm and the spread of the biofilm in vivo, leading to increased inflammatory factors, and the bacteria are difficult to remove and persist.

Discovery of Small Molecule Bak Activator for Lung Cancer Therapy.

Rationale: Bak is a major proapoptotic Bcl2 family member and a required molecule for apoptotic cell death. High levels of endogenous Bak were observed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Increased Bak expression was correlated with poor prognosis of NSCLC patients, suggesting that Bak protein is an attractive target for lung cancer therapy. The BH3 domain functions as death domain and is required for Bak to initiate apoptotic cell death. Thus, the BH3 domain is attractive target for discovery of Bak agonist. Methods: The BH3 death domain binding pocket (aa75-88) of Bak was chosen as a docking site for screening of small molecule Bak activators using the UCSF DOCK 6.1 program suite and the NCI chemical library (300,000 small molecules) database. The top 500 compounds determined to have the highest affinity for the BH3 domain were obtained from the NCI and tested for cytotoxicity for further screening. We identified a small molecule Bak activator BKA-073 as the lead compound. The binding affinity of BKA-073 with Bak protein was analyzed by isothermal titration calorimetry (ITC) assay. BKA-073-mediated Bak activation via oligomerization was analyzed by a cross-linking with Bis (maleimido) hexane (BMH). Sensitivity of BKA-073 to lung cancer cells in vitro was evaluated by dynamic BH3 profiling (DBP) and apoptotic cell death assay. The potency of BKA-073 alone or in combination with radiotherapy or Bcl2 inhibitor was evaluated in animal models. Results: We found that BKA-073 binds Bak at BH3 domain with high affinity and selectivity. BKA-073/Bak binding promotes Bak oligomerization and mitochondrial priming that activates its proapoptotic function. BKA-073 potently suppresses tumor growth without significant normal tissue toxicity in small cell lung cancer (SCLC) and NSCLC xenografts, patient-derived xenografts, and genetically engineered mouse models of mutant KRAS-driven cancer. Bak accumulates in radioresistant lung cancer cells and BKA-073 reverses radioresistance. Combination of BKA-073 with Bcl-2 inhibitor venetoclax exhibits strong synergy against lung cancer in vivo. Conclusions: Development of small molecule Bak activator may provide a new class of anticancer agents to treat lung cancer.

Targeting c-Myc to Overcome Acquired Resistance of EGFR Mutant NSCLC Cells to the Third-Generation EGFR Tyrosine Kinase Inhibitor, Osimertinib.

Osimertinib (AZD9291 or TAGRISSO) is a promising and approved third-generation EGFR tyrosine kinase inhibitor (TKI) for treating patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations or the resistant T790M mutation. However, the inevitable emergence of acquired resistance limits its long-term efficacy. A fuller understanding of the mechanism of action of osimertinib and its linkage to acquired resistance will enable the development of more efficacious therapeutic strategies. Consequently, we have identified a novel connection between osimertinib or other EGFR-TKIs and c-Myc. Osimertinib rapidly and sustainably decreased c-Myc levels primarily via enhancing protein degradation in EGFR-mutant (EGFRm) NSCLC cell lines and xenograft tumors. c-Myc levels were substantially elevated in different EGFRm NSCLC cell lines with acquired resistance to osimertinib in comparison with their corresponding parental cell lines and could not be reduced any further by osimertinib. Consistently, c-Myc levels were elevated in the majority of EGFRm NSCLC tissues relapsed from EGFR-TKI treatment compared with their corresponding untreated baseline c-Myc levels. Suppression of c-Myc through knockdown or pharmacologic targeting with BET inhibitors restored the response of resistant cell lines to osimertinib. These findings indicate that c-Myc modulation mediates the therapeutic efficacy of osimertinib and the development of osimertinib acquired resistance. Furthermore, they establish c-Myc as a potential therapeutic target and warrant clinical testing of BET inhibition as a potential strategy to overcome acquired resistance to osimertinib or other EGFR inhibitors. SIGNIFICANCE: This study demonstrates a critical role of c-Myc modulation in mediating therapeutic efficacy of osimertinib including osimertinib acquired resistance and suggests targeting c-Myc as a potential strategy to overcome osimertinib acquired resistance.

Plasma S100ß and neuron-specific enolase, but not neuroglobin, are associated with early cognitive dysfunction after total arch replacement surgery: A pilot study.

ABSTRACT: To investigate whether plasma concentrations of S100ß protein, neuron-specific enolase (NSE), and neuroglobin (NGB) correlate with early postoperative cognitive dysfunction (POCD) in patients undergoing total arch replacement.This prospective study analyzed 40 patients who underwent total arch replacement combined with stented elephant trunk implantation at our hospital between March 2017 and January 2019. Cognitive function was assessed using the Mini-mental State Examination (MMSE) preoperatively, on the day after extubation and on day 7 after surgery. Plasma levels of S100ß, NSE, and NGB POCD were assayed preoperatively and at 1, 6, and 24 hours after cardiopulmonary bypass. POCD was defined as a decrease of at least 1 unit in the MMSE score from before surgery until day 7, and patients were stratified into those who experienced POCD or not. The 2 groups were compared in clinicodemographic characteristics and plasma levels of the 3 proteins.Plasma levels of all 3 biomarkers increased significantly during and after cardiopulmonary bypass. Levels of S100ß and NSE, but not NGB, were significantly higher in the 15 patients who showed POCD than in the remainder who did not. For prediction of early POCD, S100ß showed an area under the receiver operating characteristic curve (AUC) of 0.71 (95% confidence interval [CI] 0.55-0.87), sensitivity of 48%, and specificity of 87%. The corresponding values for NSE were 0.77 (95%CI 0.60-0.94), 92%, and 67%. Together, S100ß and NSE showed an AUC of 0.81 (95%CI 0.66-0.96), sensitivity of 73%, and specificity of 80%. NGB did not significantly predict early POCD (AUC 0.62, 95%CI 0.43-0.80).Plasma S100ß protein and NSE, but not NGB, may help predict early POCD after total arch replacement.

Visible light regulates anthocyanin synthesis via malate dehydrogenases and the ethylene signaling pathway in plum (Prunus salicina L.).

Light regulates anthocyanins synthesis in plants. Upon exposure to visible light, the inhibition of photosynthetic electron transfer significantly lowered the contents of anthocyanins and the expression levels of key genes involved in anthocyanins synthesis in plum fruit peel. Meanwhile, the expression levels of PsmMDH2 (encoding the malate dehydrogenase in mitochondria) and PschMDH (encoding the malate dehydrogenase in chloroplasts) decreased significantly. The contents of anthocyanins and the levels of the key genes involved in anthocyanin synthesis decreased significantly with the treatment of 1-MCP (an inhibitor of ethylene perception) but were enhanced by the exogenous application of ethylene. The ethylene treatment could also recover the anthocyanin synthesis capacity lowered by the photosynthetic electron transfer inhibition. Silencing PsmMDH2 and PschMDH significantly lowered the contents of anthocyanins in plum fruit. At low temperature, visible light irradiation induced anthocyanin accumulation in Arabidopsis leaves. However, the mmdh, chmdh, and etr1-1 mutants had significantly lower anthocyanins content and expressions of the key genes involved in anthocyanins synthesis compared to wild type. Overall, the present study demonstrates that both photosynthesis and respiration were involved in the regulation of anthocyanin synthesis in visible light. The visible light regulates anthocyanin synthesis by controlling the malate metabolism via MDHs and the ethylene signaling pathway.

Inflammatory Indicators and Hematological Indices in Contrast-Induced Nephropathy Among Patients Receiving Coronary Intervention: A Systematic Review and Meta-Analysis.

Strong inflammatory indicators such as C-reactive protein (CRP), high-sensitivity CRP (hsCRP), and hematological indices, including platelet to lymphocyte ratio (PLR), neutrophil to lymphocyte ratio (NLR), hematocrit (HCT), and red blood cell distribution width (RDW), may be related with contrast-induced nephropathy (CIN). Our meta-analysis aimed at exploring the relationship between these indicators and CIN incidence among patients undergoing coronary intervention. Clinical studies were retrieved from the electronic databases of PubMed, EMBASE, Google Scholar, Clinical Trials, and Science Direct from their inception to June 3, 2020. Meta-analysis was performed on pooled eligible studies. Finally, 26 studies involving 29 454 patients were included. Pooled analysis revealed that patients with higher CRP (odds ratio [OR] = 1.06, 95% CI: 1.01-1.12, P = .02), hsCRP (OR = 1.03, 95% CI: 1.01-1.06, P = .004), NLR (OR = 1.11, 95% CI: 1.01-1.20, P = .02), RDW (OR = 1.35, 95% CI: 1.19-1.53, P < .001), and lower HCT (OR = 0.94, 95% CI: 0.92-0.97, P = .003) all exhibited significantly higher CIN rates, but there was no significant association between PLR and CIN risk (OR = 1.12, 95% CI: 0.99-1.26, P = .07). Pre-angiography CRP/hsCRP and some hematological indices are associated with CIN.

Biochemical Characterization of Two Rhamnogalacturonan Lyases From Bacteroides ovatus ATCC 8483 With Preference for RG-I Substrates.

Rhamnogalacturonan lyase (RGL) cleaves backbone α-1,4 glycosidic bonds between L-rhamnose and D-galacturonic acid residues in type I rhamnogalacturonan (RG-I) by ß-elimination to generate RG oligosaccharides with various degrees of polymerization. Here, we cloned, expressed, purified and biochemically characterized two RGLs (Bo3128 and Bo4416) in the PL11 family from Bacteroides ovatus ATCC 8483. Bo3128 and Bo4416 displayed maximal activity at pH 9.5 and pH 6.5, respectively. Whereas the activity of Bo3128 could be increased 1.5 fold in the presence of 5 mM Ca2+, Bo4416 required divalent metal ions to show any enzymatic activity. Both of RGLs showed a substrate preference for RG-I compared to other pectin domains. Bo4416 and Bo3128 primarily yielded unsaturated RG oligosaccharides, with Bo3128 also producing them with short side chains, with yields of 32.4 and 62.4%, respectively. Characterization of both RGLs contribute to the preparation of rhamnogalacturonan oligosaccharides, as well as for the analysis of the fine structure of RG-I pectins.

The prognostic relevance of p53 and Ki-67 to chemotherapy sensitivity and prognosis in triple-negative breast cancer.

BACKGROUND: This study aimed to explore the prognostic function of p53 and Ki-67 protein expression in chemotherapy sensitivity and prognosis in triple-negative breast cancer (TNBC). METHODS: Patients who were confirmed with TNBC in Wenzhou Geriatric Hospital and Wenzhou Hospital of Traditional Chinese Medicine (including the Oncology Department, Tumor Surgery Department, and Gynecology Department) between January 2006 and February 2018 were included in this study. The expression of p53 and Ki-67 detected by immunohistochemistry, the rate of recurrence, and the objective curative effect evaluation at the end of the first-line rescue treatment were recorded for all patients. RESULTS: The patients were followed up to August 2020, and the median follow-up time was 9 years and 4 months. A total of 285 patients with TNBC were enrolled in the study. The patients ranged in age from 19 to 76 years old, with an average age of 53 years. The overall recurrence rate among the patients was 31.58%. The majority of cases (68.07%) were pathological stage I. The overall positive expression rates of Ki-67 and p53 were 53.33% and 56.84%, respectively. In the TNBC recurrence group, the positive rates of p53 and Ki-67 were 71.11% and 82.22%, respectively, which were significantly higher than those in the non-recurrence group. The positive rates of p53 and Ki-67 in the chemosensitive group were 96.05% and 92.11%, respectively, which were significantly higher than those in the non-chemosensitive group. Among all the TNBC patients, 128 patients had positive expression of both p53 and Ki-67, and 101 patients had negative expression of both p53 and Ki-67. The chemosensitivity rate of TNBC patients with positive expression of both Ki-67 and p53 was 98.53%, and that of TNBC patients with negative expression of both Ki-67 and p53 was 0.00%. The difference was statistically significant. The recurrence rate in TNBC patients with positive expression of both Ki-67 and p53 was 53.13%, and that in patients with negative expression of both Ki-67 and p53 was 6.93%. The difference was statistically significant. CONCLUSIONS: The expression of p53 and Ki-67 had prognostic relevance to chemotherapy sensitivity and prognosis in TNBC patients.

YAP1 Expression in SCLC Defines a Distinct Subtype With T-cell-Inflamed Phenotype.

INTRODUCTION: The clinical and biological significance of the newly described SCLC subtypes, SCLC-A, SCLC-N, SCLC-Y, and SCLC-P, defined by the dominant expression of transcription factors ASCL1, NeuroD1, YAP1, and POU2F3, respectively, remain to be established. METHODS: We generated new RNA sequencing expression data from a discovery set of 59 archival tumor samples of neuroendocrine tumors and new protein expression data by immunohistochemistry in 99 SCLC cases. We validated the findings from this discovery set in two independent validation sets consisting of RNA sequencing data generated from 51 SCLC cell lines and 81 primary human SCLC samples. RESULTS: We successfully classified 71.8% of SCLC and 18.5% of carcinoid cases in our discovery set into one of the four SCLC subtypes. Gene set enrichment analysis for differentially expressed genes between the SCLC survival outliers (top and bottom deciles) matched for clinically relevant prognostic factors revealed substantial up-regulation of interferon-γ response genes in long-term survivors. The SCLC-Y subtype was associated with high expression of interferon-γ response genes, highest weighted score on a validated 18-gene T-cell-inflamed gene expression profile score, and high expression of HLA and T-cell receptor genes. YAP1 protein expression was more prevalent and more intensely expressed in limited-stage versus extensive-stage SCLC (30.6% versus 8.5%; p = 0.0058) indicating good prognosis for the SCLC-Y subtype. We replicated the inflamed phenotype of SCLC-Y in the two independent validation data sets from the SCLC cell lines and tumor samples. CONCLUSIONS: SCLC subtyping using transcriptional signaling holds clinical relevance with the inflamed phenotype associated with the SCLC-Y subset.

Inhibition of ACK1 delays and overcomes acquired resistance of EGFR mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib.

OBJECTIVES: The emergence of acquired resistance to the third generation EGFR inhibitor, osimertinib (AZD9291 or TAGRISSO™), is an unavoidable huge clinical challenge. The involvement of ACK1, a non-receptor tyrosine kinase with an oncogenic function, in regulating cell response to osimertinib has not been investigated and thus is the focus of this study. MATERIAL AND METHODS: Drug effects on cell growth were evaluated by measuring cell numbers and colony formation. Apoptosis was monitored with flow cytometry for annexin V-positive cells and Western blotting for protein cleavage. Intracellular protein and mRNA alterations were detected with Western blotting and qRT-PCR, respectively. Drug effects on delaying osimertinib acquired resistance were determined using colony formation in vitro and xenografts in nude mice in vivo, respectively. Cell senescence was assayed by ß-galactosidase staining. RESULTS: Inhibition of ACK1 with the novel ACK1 inhibitor, (R)-9b synergized with osimertinib in inhibiting the growth of EGFR mutant NSCLC cell lines. Similar results were also generated with ACK1 gene knockdown. The combination of osimertinib and (R)-9b enhanced induction of apoptosis. In both in vitro and in vivo long-term resistance delay assays, the combination of (R)-9b and osimertinib clearly delayed the emergence of osimertinib-resistance. Further, the (R)-9b and osimertinib combination was also effective in inhibiting the growth of EGFR mutant NSCLC cell lines with acquired resistance to osimertinib, which possess elevated levels of ACK1, and the growth of osimertinib-resistant tumors in vivo. In some resistant cell lines, the combinations induced senescence in addition to induction of apoptosis. CONCLUSIONS: These novel findings suggest that ACK1 inhibition might be a potential and innovative strategy for delaying and overcoming osimertinb acquired resistance.

MT2A Promotes Oxaliplatin Resistance in Colorectal Cancer Cells.

We aimed to understand the molecular mechanism underlying the incidence of Oxaliplatin resistance in colorectal cancer. The Oxaliplatin-resistant (OR) HT29 colorectal cell line was established by long-term exposure to Oxaliplatin. Cell viability and proliferation were determined by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide and direct counting assays, respectively. Transcript level of metallothionein 2A (MT2A) was measured by real-time polymerase chain reaction. Protein levels of MT2A, BRCA1-associated RING domain 1 (BARD1), BRCA1, and ß-actin were quantified by immunoblotting. Direct interaction between MT2A with BARD1 and BRCA1 was analyzed by co-immunoprecipitation. Colocalization between of MT2A and BARD1 was determined by immunofluorescence. MT2A was upregulated in OR cells at both transcript and protein levels. Knockdown of MT2A in HT29 OR cells improved sensitivity to Oxaliplatin, while ectopic overexpression of MT2A conferred HT29 cells relative resistance to Oxaliplatin. We further demonstrated that MT2A interacted with and positively regulated BARD1/BRCA1 in colorectal cancer cells. BARD1 overexpression partially restored the compromised Oxaliplatin resistance elicited by MT2A deficiency in terms of both cell proliferation and viability. Our data highlighted the critical contributions of MT2A-BARD1/BRCA1 in Oxaliplatin resistance in colorectal cancer cells.

MEK or ERK inhibition effectively abrogates emergence of acquired osimertinib resistance in the treatment of epidermal growth factor receptor-mutant lung cancers.

BACKGROUND: The majority of patients with non-small cell lung cancer (NSCLC) harboring activating epidermal growth factor receptor (EGFR) mutations respond well to osimertinib (AZD9291), a third-generation, mutation-selective EGFR inhibitor. The current study focuses on determining whether targeting MEK/ERK signaling prevents or delays the development of acquired resistance to osimertinib. METHODS: Drug effects on cell survival were determined by measuring cell number alterations. Apoptosis was assessed with flow cytometry for the detection of annexin V-positive cells and with Western blotting for protein cleavage. Alterations of proteins in cells were detected with Western blotting. Drug effects on delaying the emergence of osimertinib resistance were evaluated with colony formation in vitro and xenografts in nude mice in vivo. RESULTS: Osimertinib combined with an MEK or ERK inhibitor synergistically decreased cell survival with enhanced induction of apoptosis in EGFR-mutant NSCLC cells but not in EGFR wild-type NSCLC cells. These combinations were also very effective in killing cell clones with primary intrinsic resistance to osimertinib. Continuous and intermittent pharmacologic inhibition of MEK/ERK signaling delayed the emergence of osimertinib resistance both in vitro and in vivo. CONCLUSIONS: These results provide strong preclinical evidence in support of targeting MEK/ERK signaling as a strategy for delaying or preventing acquired resistance to osimertinib in the clinic to improve the long-term therapeutic efficacy of osimertinib. From a clinical standpoint, the data support the evaluation of an intermittent treatment schedule of osimertinib in combination with an MEK or ERK inhibitor in patients with EGFR-mutated NSCLC.

Detection of plasma T790M mutation after the first generation EGFR-TKI resistance of non-small cell lung cancer in the real world.

BACKGROUND: The epidermal growth factor receptor (EGFR) gene has been identified as the driving gene of non-small cell lung cancer (NSCLC), and EGFR-tyrosine kinase inhibitor (TKI) has shown efficacy, but acquired resistance is inevitable. It has been confirmed that the secondary EGFR Thr790Met (T790M) mutation accounts for about 50% of the mechanisms of acquired resistance to EGFR-TKI. The third-generation of EGFR-TKI has significantly efficacy in advanced T790M-positive NSCLC patients. Therefore, it is necessary to detect the status of T790M in patients with acquired resistance after first generation EGFR-TKI. The objective of this study was to investigate the positive rate of plasma test T790M mutation and its relationship with different clinical characteristics, and the frequency of T790M mutation in advanced EGFR-mutant NSCLC patients with acquired resistance after firstline EGFR-TKI treatment. METHODS: Patients from a single clinical center (Taizhou hospital) were recruited prospectively from September 2017 to June 2018. The eligibility criteria of the trial included the following: (I) aged 18 years or older, histologically confirmed NSCLC stage IIIB/st and EGFR mutation positive; (II) progressive disease (PD) after first generation EGFR-TKI by RECIST v1.1, with PFS>3 months; (III) no third generation TKI treatment. All patients signed informed consent, had 10 mL of blood drawn, and were evaluated for the presence of T790M gene by amplification refractory mutation system (ARMS). The study was approved by the Ethics Committee of Taizhou Hospital (ethical batch number: 201637). RESULTS: A total of 189 patients were included in the analysis. The overall T790M mutation rate of plasma detection was 36.51% (69/189). The positive rate of T790M mutation after the failure of first generation EGFR-TKI treatment was not correlated with the patient's age, sex, and the type of first generation TKI drugs. However, it was related to the mutation type of EGFR in baseline and the mode of progression according to reports by Wu et al. The frequency of T790M mutation among patients with initial exon 19 deletion mutation, exon 21 L858R point mutation, and other mutations were 45.45%, 26.19% and 33.33%, respectively. The mutation rate of T790M in 19del mutant patients was higher than that of L858R mutation and other mutations (P=0.026). The frequency of T790M mutation in local progression patients was 50% after the first generation TKI was resistant to drug treatment: in gradual progression it was 26.92%, and in dramatic progression it was 38.10%. The frequency of T790M mutation of patients with local progression was significantly higher (P=0.031). CONCLUSIONS: The patients with EGFR mutations after the first generation of EGFR-TKI-acquired resistance of NSCLC were evaluated for their plasma EGFR mutation status, and the overall T790M mutation rate of was 36.51%. The frequency of T790M mutation with initial mutation of 19 del was higher than that of L858R mutation and other mutations, and local progression was higher than that in patients with gradual progression and dramatic progression.

BRD4 Levels Determine the Response of Human Lung Cancer Cells to BET Degraders That Potently Induce Apoptosis through Suppression of Mcl-1.

Lung cancer consists of approximately 80% non-small cell lung cancer (NSCLC) and 20% small cell lung cancer (SCLC) and remains the leading cause of cancer-related deaths worldwide despite advances in early diagnosis, targeted therapy, and immunotherapy. Thus, novel therapies are still urgently needed. Bromodomain and extraterminal (BET) proteins, primarily comprised of BRD2, BRD3, and BRD4 proteins, function as epigenetic readers and master transcription coactivators and are now recognized cancer therapeutic targets. BET degraders such as ZBC260 and dBET represent a novel class of BET inhibitors that act by inducing BET degradation. The current study demonstrates the therapeutic efficacies of BET degraders, particularly ZBC260, against lung cancer, as well as understanding the underlying mechanisms and identifying molecular markers that determine cell sensitivity to BET degraders. A panel of NSCLC cell lines possessed similar response patterns to ZBC260 and dBET but different responses to BET inhibitor JQ-1. BRD levels, particularly BRD4, correlated positively with high sensitivity to BET degraders but not to JQ-1. BET degraders potently induced apoptosis in sensitive NSCLC cells and were accompanied by reduction of Mcl-1 and c-FLIP levels, which are critical for mediating induction of apoptosis and enhancement of TRAIL-induced apoptosis. Accordingly, ZBC260 exerted more potent activity than JQ-1 in vivo against the growth of NSCLC xenografts and patient-derived xenografts. These findings warrant future clinical validation of the efficacy of BET degraders in NSCLC, particularly those with high levels of BRD proteins, especially BRD4. SIGNIFICANCE: The current study demonstrates the potential of novel BET degraders in the treatment of lung cancer and warrants clinical validation of BET degraders in lung cancer with high levels of BRD4.