Development of assays to support identification and characterization of modulators of DExH-box helicase DHX9.

DHX9 is a DExH-box RNA helicase that utilizes hydrolysis of all four nucleotide triphosphates (NTPs) to power cycles of 3' to 5' directional movement to resolve and/or unwind double stranded RNA, DNA, and RNA/DNA hybrids, R-loops, triplex-DNA and G-quadraplexes. DHX9 activity is important for both viral amplification and maintaining genomic stability in cancer cells; therefore, it is a therapeutic target of interest for drug discovery efforts. Biochemical assays measuring ATP hydrolysis and oligonucleotide unwinding for DHX9 have been developed and characterized, and these assays can support high-throughput compound screening efforts under balanced conditions. Assay development efforts revealed DHX9 can use double stranded RNA with 18-mer poly(U) 3' overhangs and as well as significantly shorter overhangs at the 5' or 3' end as substrates. The enzymatic assays are augmented by a robust SPR assay for compound validation. A mechanism-derived inhibitor, GTPγS, was characterized as part of the validation of these assays and a crystal structure of GDP bound to cat DHX9 has been solved. In addition to enabling drug discovery efforts for DHX9, these assays may be extrapolated to other RNA helicases providing a valuable toolkit for this important target class.

Development and validation of a prediction model for postoperative intensive care unit admission in patients with non-cardiac surgery.

BACKGROUND: Accurately forecasting patients admitted to the intensive care units (ICUs) after surgery may improve clinical outcomes and guide the allocation of expensive and limited ICU resources. However, studies on predicting postoperative ICU admission in non-cardiac surgery have been limited. OBJECTIVE: To develop and validate a prediction model combining pre- and intraoperative variables to predict ICU admission after non-cardiac surgery. METHODS: This study is based on data from the Vital Signs DataBase (VitalDB) database. Predictors were selected using the least absolute shrinkage and selection operator regression method and logistic regression to develop a nomogram and an online web calculator. The model was internally verified by 1000-Bootstrap resampling. Performance of model was evaluated using area under the receiver operating characteristic curve (AUC), calibration curve and Brier score. The Youden's index was used to find the optimal nomogram's probability threshold. Clinical utility was assessed by decision curve analysis. RESULTS: This study included 5216 non-cardiac surgery patients; of these, 812 (15.6%) required postoperative ICU admission. Potential predictors included age, ASA classification, surgical department, emergency surgery, preoperative albumin level, preoperative urea nitrogen level, intraoperative crystalloid, intraoperative transfusion, intraoperative catheterization, and surgical time. A nomogram was constructed with an AUC of 0.917 (95% CI: 0.907-0.926) and a Brier score of 0.077. The Bootstrap-adjusted AUC was 0.914; the adjusted Brier score was 0.078. The calibration curve showed good agreement between predicted and actual probabilities; and the decision curve indicated clinical usefulness. Finally, we established an online web calculator for clinical application (https://xuzhikun.shinyapps.io/postopICUadmission1/). CONCLUSION: We developed and internally validated an easy-to-use nomogram for predicting ICU admission after non-cardiac surgery.

Identification of 2-Sulfonyl/Sulfonamide Pyrimidines as Covalent Inhibitors of WRN Using a Multiplexed High-Throughput Screening Assay.

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC50 = 22 nM, KD = 40 nM, KI = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC50 ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.

Targeted therapy for intractable cancer on the basis of molecular profiles: An open-label, phase II basket trial (Long March Pathway).

Purpose: We evaluated he effects of molecular guided-targeted therapy for intractable cancer. Also, the epidemiology of druggable gene alterations in Chinese population was investigated. Materials and methods: The Long March Pathway (ClinicalTrials.gov identifier: NCT03239015) is a non-randomized, open-label, phase II trial consisting of several basket studies examining the molecular profiles of intractable cancers in the Chinese population. The trial aimed to 1) evaluate the efficacy of targeted therapy for intractable cancer and 2) identify the molecular epidemiology of the tier II gene alterations among Chinese pan-cancer patients. Results: In the first stage, molecular profiles of 520 intractable pan-cancer patients were identified, and 115 patients were identified to have tier II gene alterations. Then, 27 of these 115 patients received targeted therapy based on molecular profiles. The overall response rate (ORR) was 29.6% (8/27), and the disease control rate (DCR) was 44.4% (12/27). The median duration of response (DOR) was 4.80 months (95% CI, 3.33-27.2), and median progression-free survival (PFS) was 4.67 months (95% CI, 2.33-9.50). In the second stage, molecular epidemiology of 17,841 Chinese pan-cancer patients demonstrated that the frequency of tier II gene alterations across cancer types is 17.7%. Bladder cancer had the most tier-II alterations (26.1%), followed by breast cancer (22.4%), and non-small cell lung cancer (NSCLC; 20.2%). Conclusion: The Long March Pathway trial demonstrated a significant clinical benefit for intractable cancer from molecular-guided targeted therapy in the Chinese population. The frequency of tier II gene alterations across cancer types supports the feasibility of molecular-guided targeted therapy under basket trials.

Systematic review and meta-analysis of type B aortic dissection involving the left subclavian artery with a Castor stent graft.

Objective: Despite the rapid development of thoracic endovascular aortic repair (TEVAR), it is still a challenge to maintain the blood flow of the branch arteries above the aortic arch in Stanford type B aortic dissection involving the left subclavian artery (LSA). The Castor stent graft is an integrated, customized, single-branch stent that enables reconstruction of the LSA. The purpose of this systematic review and meta-analysis was to assess the efficacy of the Castor stent graft for type B aortic dissection. Materials and methods: An extensive electronic literature search (PROSPERO registration number: CRD42022322146) was undertaken to identify all articles published up to August 2022 that described thoracic aortic repair with branch stents in the treatment of type B aortic dissection involving the LSA. The quality of the included studies was analyzed using the MINORS criteria. The primary outcome measures were the technical success rate, early mortality rate, endoleak rate, and 1-year survival rate. The secondary outcome measures were the stroke rate, left upper extremity ischemia rate, and target vessel patency rate. Results: Eleven studies involving 415 patients were eligible for this meta-analysis. The LSA was successfully preserved in all procedures. The technical success rate was 97.5% (95% CI: 0.953-0.991); the intraoperative endoleak rate was 0.1% (95% CI: 0.000-0.012); the intraoperative LSA patency rate was 99.52%; the intraoperative LSA stent deformation and stenosis rate was 0.15% (95% CI: 0.000-0.051); the early type I endoleak rate was 1.6% (95% CI: 0.003-0.035); the 30-day mortality rate was 0.96%; the early reintervention rate was 0.9% (95% CI: 0.000-0.040); and the perioperative stroke rate was 0% (95% CI: 0.000-0.005). The 1-year survival rate was 99.7% (95% CI: 0.976-1.000). The half-year LSA patency rate was 99.3%, the 1-year LSA patency rate was 97.58%, and the 2-year LSA patency rate was 95.23%. During the follow-up period, the leakage rate was 0.3% (95% CI: 0.000-0.017), the incidence of left upper extremity ischemia rate was 0.5% (95% CI: 0.000-0.035), and the deformation and stenosis rate of the LSA stent was 2.2% (95% CI: 0.06-0.046). Conclusion: This meta-analysis shows that endovascular repair of type B aortic dissection using the Castor stent-graft may be technically feasible and effective. However, this conclusion needs to be interpreted with caution, as the quality of evidence for all outcomes is between low and very low. Systematic review registration: [https://www.crd.york.ac.uk/prospero/], identifier [CRD42022322146].

Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer.

Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both  ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy-resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). SUMMARY: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.

Methionine oxidation of CLK4 promotes the metabolic switch and redox homeostasis in esophageal carcinoma via inhibiting MITF selective autophagy.

BACKGROUND: Metabolic reprogramming and redox homeostasis contribute to esophageal squamous cell carcinoma (ESCC). CDC-like kinase 4 (CLK4) is a dual-specificity kinase that can phosphorylate substrates' tyrosine or serine/threonine residue. However, the role and mechanism of CLK4 in ESCC remain unknown. METHODS: CLK4 expression was analysed using publicly available datasets and confirmed in ESCC tissues and cell lines. The biological roles of CLK4 were studied with gain and loss-of-function experiments. Mass spectrometry was employed to examine the effects of CLK4 on metabolic profiling. In vitro kinase assay, co-immunoprecipitation, glutathione S-transferase pulldown, chromatin immunoprecipitation and luciferase reporter were used to elucidate the relationship among CLK4, microphthalmia-associated transcription factor (MITF), COP1 and ZRANB1. RESULTS: CLK4 down-regulation was observed in ESCC cell lines and clinical samples and associated with the methylation of its promoter. Low levels of CLK4 promoted ESCC development by affecting the purine synthesis pathway and nicotinamide adenine dinucleotide phosphate (NADPH)/nicotinamide adenine dinucleotide phosphate (NADP+ ) ratio. Interestingly, CLK4 inhibited ESCC development by blocking MITF-enhanced de novo purine synthesis and redox balance. Mechanistically, wild type CLK4 (WT-CLK4) but not kinase-dead CLK4-K189R mutant phosphorylated MITF at Y360. This modification promoted its interaction with E3 ligase COP1 and its K63-linked ubiquitination at K308/K372, leading to sequestosome 1 recognition and autophagic degradation. However, the deubiquitinase ZRANB1 rescued MITF ubiquitination and degradation. In turn, MITF bound to E- rather than M-boxes in CLK4 promoter and transcriptionally down-regulated its expression in ESCC. Clinically, the negative correlations were observed between CLK4, MITF, and purine metabolic markers, which predicts a poor clinical outcome of ESCC patients. Notably, CLK4 itself was a redox-sensitive kinase, and its methionine oxidation at M307 impaired kinase activity, enhanced mitochondria length and inhibited lipid peroxidation, contributing to ESCC. CONCLUSIONS: Our data highlight the potential role of CLK4 in modulating redox status and nucleotide metabolism, suggesting potential therapeutic targets in ESCC treatment.

Elevated Stratifin promotes cisplatin-based chemotherapy failure and poor prognosis in non-small cell lung cancer.

Drug resistance is a key factor in the treatment failure of clinical non-small cell lung cancer (NSCLC) patients after adjuvant chemotherapy. Here, our results provide the first evidence that eukaryotic translation initiation factor 2b subunit delta (EIF2B4)-Stratifin (SFN) fusion and increased SFN expression are associated with chemotherapy tolerance and activation of the phosphatidylinositol 3 kinase/v-akt murine thymoma viral oncogene (PI3K/Akt) signaling pathway in NSCLC patients, suggesting that SFN might have potential prognostic value as a tumor biomarker for the prognosis of patients with NSCLC.

Systematic analysis using a bioinformatics strategy identifies SFTA1P and LINC00519 as potential prognostic biomarkers for lung squamous cell carcinoma.

Lung cancer has high incidence and mortality rates, in which lung squamous cell carcinoma (LUSC) is a primary type of non-small cell lung carcinoma (NSCLC). The aim of our study was to discover long non-coding RNAs (lncRNAs) associated with diagnose and prognosis for LUSC. RNA sequencing data obtained from LUSC samples were extracted from The Cancer Genome Atlas database (TCGA). Two prognosis-associated lncRNAs (including SFTA1P and LINC00519) were selected from LUSC samples, and the expression levels were also verified to be associated abnormal in LUSC clinical samples. Our findings demonstrate that lncRNAs SFTA1P and LINC00519 exert important functions in human LUSC and may serve as new targets for LUSC diagnosis and therapy.

Discovery of Aminopyrazole Derivatives as Potent Inhibitors of Wild-Type and Gatekeeper Mutant FGFR2 and 3.

Fibroblast growth factor receptors (FGFR) 2 and 3 have been established as drivers of numerous types of cancer with multiple drugs approved or entering late stage clinical trials. A limitation of current inhibitors is vulnerability to gatekeeper resistance mutations. Using a combination of targeted high-throughput screening and structure-based drug design, we have developed a series of aminopyrazole based FGFR inhibitors that covalently target a cysteine residue on the P-loop of the kinase. The inhibitors show excellent activity against the wild-type and gatekeeper mutant versions of the enzymes. Further optimization using SAR analysis and structure-based drug design led to analogues with improved potency and drug metabolism and pharmacokinetics properties.

Discovery of 2,6-Dimethylpiperazines as Allosteric Inhibitors of CPS1.

Carbamoyl phosphate synthetase 1 (CPS1) is a potential synthetic lethal target in LKB1-deficient nonsmall cell lung cancer, where its overexpression supports the production of pyrimidine synthesis. In other cancer types, CPS1 overexpression and activity may prevent the accumulation of toxic levels of intratumoral ammonia to support tumor growth. Herein we report the discovery of a novel series of potent and selective small-molecule inhibitors of CPS1. Piperazine 2 was initially identified as a promising CPS1 inhibitor through a high-throughput screening effort. Subsequent structure-activity relationship optimization and structure-based drug design led to the discovery of piperazine H3B-616 (25), a potent allosteric inhibitor of CPS1 (IC50 = 66 nM).

Small Molecule Inhibition of CPS1 Activity through an Allosteric Pocket.

Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.

The R882H DNMT3A hot spot mutation stabilizes the formation of large DNMT3A oligomers with low DNA methyltransferase activity.

DNMT3A (DNA methyltransferase 3A) is a de novo DNA methyltransferase responsible for establishing CpG methylation patterns within the genome. DNMT3A activity is essential for normal development, and its dysfunction has been linked to developmental disorders and cancer. DNMT3A is frequently mutated in myeloid malignancies with the majority of mutations occurring at Arg-882, where R882H mutations are most frequent. The R882H mutation causes a reduction in DNA methyltransferase activity and hypomethylation at differentially-methylated regions within the genome, ultimately preventing hematopoietic stem cell differentiation and leading to leukemogenesis. Although the means by which the R882H DNMT3A mutation reduces enzymatic activity has been the subject of several studies, the precise mechanism by which this occurs has been elusive. Herein, we demonstrate that in the context of the full-length DNMT3A protein, the R882H mutation stabilizes the formation of large oligomeric DNMT3A species to reduce the overall DNA methyltransferase activity of the mutant protein as well as the WT-R882H complex in a dominant-negative manner. This shift in the DNMT3A oligomeric equilibrium and the resulting reduced enzymatic activity can be partially rescued in the presence of oligomer-disrupting DNMT3L, as well as DNMT3A point mutations along the oligomer-forming interface of the catalytic domain. In addition to modulating the oligomeric state of DNMT3A, the R882H mutation also leads to a DNA-binding defect, which may further reduce enzymatic activity. These findings provide a mechanistic explanation for the observed loss of DNMT3A activity associated with the R882H hot spot mutation in cancer.

METTL3 and ALKBH5 oppositely regulate m6A modification of TFEB mRNA, which dictates the fate of hypoxia/reoxygenation-treated cardiomyocytes.

N6-methyladenosine (m6A) mRNA modifications play critical roles in various biological processes. However, no study addresses the role of m6A in macroautophagy/autophagy. Here, we show that m6A modifications are increased in H/R-treated cardiomyocytes and ischemia/reperfusion (I/R)-treated mice heart. We found that METTL3 (methyltransferase like 3) is the primary factor involved in aberrant m6A modification. Silencing METTL3 enhances autophagic flux and inhibits apoptosis in H/R-treated cardiomyocytes. However, overexpression of METTL3 or inhibition of the RNA demethylase ALKBH5 has an opposite effect, suggesting that METTL3 is a negative regulator of autophagy. Mechanistically, METTL3 methylates TFEB, a master regulator of lysosomal biogenesis and autophagy genes, at two m6A residues in the 3'-UTR, which promotes the association of the RNA-binding protein HNRNPD with TFEB pre-mRNA and subsequently decreases the expression levels of TFEB. Further experiments show that autophagic flux enhanced by METTL3 deficiency is TFEB dependent. In turn, TFEB regulates the expression levels of METTL3 and ALKBH5 in opposite directions: it induces ALKBH5 and inhibits METTL3. TFEB binds to the ALKBH5 promoter and activates its transcription. In contrast, inhibition of METTL3 by TFEB does not involve transcriptional repression but rather downregulation of mRNA stability, thereby establishing a negative feedback loop. Together, our work uncovers a critical link between METTL3-ALKBH5 and autophagy, providing insight into the functional importance of the reversible mRNA m6A methylation and its modulators in ischemic heart disease. Abbreviations: ACTB, actin beta; ALKBH5, alkB homolog 5, RNA demethylase; ANXA5, annexin A5; ATG, autophagy-related; BafA, bafilomycin A1; CASP3, caspase 3; ELAVL1, ELAV like RNA binding protein 1; FTO, FTO, alpha-ketoglutarate dependent dioxygenase; GFP, green fluorescent protein; GST, glutathione S-transferase; HNRNPD, heterogeneous nuclear ribonucleoprotein D; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LAD, left anterior descending; m6A, N6-methyladenosine; MEFs, mouse embryo fibroblasts; Mer, mutated estrogen receptor domains; METTL3, methyltransferase like 3; METTL14, methyltransferase like 14; mRFP, monomeric red fluorescent protein; MTORC1, mechanistic target of rapamycin kinase complex 1; NMVCs, neonatal mouse ventricular cardiomyocytes; PCNA, proliferating cell nuclear antigen; PE, phosphatidylethanolamine; PI, propidium iodide; PTMs, post-translational modifications; PVDF, polyvinylidenedifluoride; RIP, RNA-immunoprecipitation; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TFEB, transcription factor EB; TUBA: tublin alpha; WTAP, WT1 associated protein; YTHDF, YTH N6-methyladenosine RNA binding protein.

Discovery of Selective Estrogen Receptor Covalent Antagonists for the Treatment of ERαWT and ERαMUT Breast Cancer.

Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Highly sensitive detection of multiple tumor markers for lung cancer using gold nanoparticle probes and microarrays.

Assay of multiple serum tumor markers such as carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen (CYFRA21-1), and neuron specific enolase (NSE), is important for the early diagnosis of lung cancer. Dickkopf-1 (DKK1), a novel serological and histochemical biomarker, was recently reported to be preferentially expressed in lung cancer. Four target proteins were sandwiched by capture antibodies attached to microarrays and detection antibodies carried on modified gold nanoparticles. Optical signals generated by the sandwich structures were amplified by gold deposition with HAuCl4 and H2O2, and were observable by microscopy or the naked eye. The four tumor markers were subsequently measured in 106 lung cancer patients and 42 healthy persons. The assay was capable of detecting multiple biomarkers in serum sample at concentration of <1 ng mL-1 in 1 h. Combined detection of the four tumor markers highly improved the sensitivity (to 87.74%) for diagnosis of lung cancer compared with sensitivity of single markers. A rapid, highly sensitive co-detection method for multiple biomarkers based on gold nanoparticles and microarrays was developed. In clinical use, it would be expected to improve the early diagnosis of lung cancer.

Kaempferol inhibits cell proliferation and glycolysis in esophagus squamous cell carcinoma via targeting EGFR signaling pathway.

Antitumor activity of kaempferol has been studied in various tumor types, but its potency in esophagus squamous cell carcinoma is rarely known. Here, we reported the activity of kaempferol against esophagus squamous cell carcinoma as well as its antitumor mechanisms. Results of cell proliferation and colony formation assay showed that kaempferol substantially inhibited tumor cell proliferation and clone formation in vitro. Flow cytometric analysis demonstrated that tumor cells were induced G0/G1 phase arrest after kaempferol treatment, and the expression of protein involved in cell cycle regulation was dramatically changed. Except the potency on cell proliferation, we also discovered that kaempferol had a significant inhibitory effect against tumor glycolysis. With the downregulation of hexokinase-2, glucose uptake and lactate production in tumor cells were dramatically declined. Mechanism studies revealed kaempferol had a direct effect on epidermal growth factor receptor (EGFR) activity, and along with the inhibition of EGFR, its downstream signaling pathways were also markedly suppressed. Further investigations found that exogenous overexpression of EGFR in tumor cells substantially attenuated glycolysis suppression induced by kaempferol, which implied that EGFR also played an important role in kaempferol-mediated glycolysis inhibition. Finally, the antitumor activity of kaempferol was validated in xenograft model and kaempferol prominently restrained tumor growth in vivo. Meanwhile, dramatic decrease of EGFR activity and hexokinase-2 expression were observed in kaempferol-treated tumor tissue, which confirmed these findings in vitro. Briefly, these studies suggested that kaempferol, or its analogues, may serve as effective candidates for esophagus squamous cell carcinoma management.

Expression of MicroRNA-325-3p and its potential functions by targeting HMGB1 in non-small cell lung cancer.

MicroRNAs (miRNAs) can function as tumor suppressors and might provide an efficient strategy for annihilating cancer. Nevertheless, the potential role of miR-325-3p in NSCLC is still unknown. Here, we showed that miR-325-3p was decreased and HMGB1 was increased in 107 NSCLC patients. MiR-325-3p inhibition promoted cell invasion and proliferation, while miR-325-3p upregulation inhibited cell invasion and proliferation by using transwell and CCK8 assays. Using a bioinformatics method, we further showed that HMGB1 might be a direct target of miR-325-3p and is negatively regulated by miR-325-3p. Down-regulation of miR-325-3p predicts poor prognosis for NSCLC patients. These findings implied that miR-325-3p regulates cell invasion and proliferation via targeting HMGB1 and may be a potential prognostic marker for NSCLC.

Incidence and risk factors for acute lung injury after open thoracotomy for thoracic diseases.

BACKGROUND: Acute lung injury (ALI) is a major cause of morbidity and mortality after open thoracotomy. The purpose of the study was to identify the incidence and risk factors for ALI so as to prevent its occurrence and improve surgical results. METHODS: A prospective controlled study was carried out in 364 patients undergone open thoracotomy. Fifty-eight high risk elderly patients and 56 young patients as matched controls were prospectively entered into the study. The two groups were compared to identify the possible risk factors for ALI. RESULTS: ALI occurred exclusively in elderly patients, accounted for 2.7% of the whole series (10/364) and 7.9% of elderly patients (10/127). The mortality for patients with ALI was 30%, significantly higher than those without (1.0%, P=0.001). Upon univariate analysis, increased age, obesity, chronic obstructive pulmonary disease (COPD), poor spirometry, and positive fluid balance on postoperative day 1 were associated with increased risk of developing ALI. Upon multivariate analysis, only poor spirometry and excessive positive fluid balance on postoperative day 1 were revealed as independent risk factors for ALI. CONCLUSIONS: ALI after open thoracotomy has a high mortality. COPD and excessive positive fluid balance on the first postoperative day are significant predictors, suggesting stringent patient selection and timely conservative fluid management may be helpful in reducing this extremely devastating complication.

Tumor-associated macrophages provide a suitable microenvironment for non-small lung cancer invasion and progression.

OBJECTIVE: It remains largely unknown whether tumor-associated macrophages (TAMs) are involved in invasion and metastasis of human lung cancer. The aim of our study was to obtain an accurate overview of the broad range of changes occurring in monocytes that develop into TAMs, and the roles of TAMs during the progression of non-small cell lung cancer. METHODS: TAM was isolated from 98 primary lung cancer tissues by short term cultivation in serum-free medium. The mRNA expression levels of 9 genes, including EGF, Cathepsin K, Cathepsin S, COX-2, MMP-9, PDGF, uPA, VEGFA, HGF, were evaluated by real-time PCR in 98 NSCLC. The relationships between those gene expression levels and clinicopathological features were investigated. The effects of conditioned medium from TAMs on the invasive properties of different lung cancer cell lines were measured using Transwell chambers. RESULTS: We successfully achieved up to 95% purity of TAM, derived from 98 primary lung cancer tissues. TAM expressed high levels of Cathepsin K, COX-2, MMP-9, PDGF-B, uPA, VEGFA, and HGF. Phenotypic expression on TAMs, like MMP9, was shown to be correlated with disease progression by analyzing lung cancer tissues. Conditioned medium from TAM significantly increased cell migration and invasion in SPC-A1 cells, H460 cells and A549 cells. Anti-uPA and anti-MMP-9, but not anti-VEGF monoclonal antibodies, can inhibit TAM-induced invasion. The increase of invasiveness in the lung cancer cell lines was also correlated with their gelatinase activity, through MMP9. CONCLUSIONS: Short-term culture in serum free medium is an effective way to isolate TAM in NSCLC. The results of this study also demonstrated that those up-regulated genes in TAMs contributed to suitable microenvironments for lung cancer invasion and metastasis. These findings may be useful in developing novel therapeutic strategies to prevent lung cancer progression.