Silibinin Induces Both Apoptosis and Necroptosis with Potential Anti-tumor Efficacy in Lung Cancer.

BACKGROUND: Lung cancer incidence is steadily on the rise, posing a growing threat to human health. The search for therapeutic drugs from natural active substance and elucidating their mechanism have been the focus of anti-tumor research. OBJECTIVE: In our work, Silibinin (SiL) was chosen as a possible substance that could inhibit lung cancer. and its effects on inducing tumor cell death have been studied. METHODS: CCK-8 analysis and morphological observation were used to assess the cytotoxic impacts of SiL on lung cancer cells in vitro. The alterations in mitochondrial membrane potential (MMP) and apoptosis rate of cells were detected by flow cytometry. The level of lactate dehydrogenase (LDH) release out of cells was measured. The expression changes of apoptosis or necroptosis-related proteins were detected using western blotting. Protein interactions among RIPK1, RIPK3 and MLKL were analyzed using the co-immunoprecipitation technique. In vivo, SiL was evaluated for its antitumor effects using LLC tumor-bearing mice with mouse lung cancer. RESULTS: With an increased dose of SiL, the proliferation ability of A549 cells was considerably inhibited, and the accompanying cell morphology changed. The results of flow cytometry showed that after SiL treatment, MMP levels decreased, and the proportion of cells undergoing apoptosis increased. The proteins associated with apoptosis were upregulated and activated. The amount of LDH released from the cells increased following SiL treatment, accompanied by augmented expression and phosphorylation levels of necroptosis-related proteins. The co-IP assay further confirmed necrosome formation induced by SiL. Furthermore, Necrosulfonamide (an MLKL inhibitor) increased the apoptotic rate of SiL-treated cells and aggravated the cytotoxic effect of SiL, indicating that necroptosis blockade could switch cell death to apoptosis and increase the inhibitory effect of SiL on A549 cells. In LLC-bearing mice, gastric administration of SiL significantly inhibited tumor growth. CONCLUSIONS: This study helped clarify the anti-tumor mechanism of SiL against lung cancer, elucidating its role in dual induction of apoptosis and necroptosis. In particular, necroptosis blockade could switch cell death to apoptosis and increase the inhibitory effect of SiL. Our work provided an experimental basis for the research on cell death induced by SiL and revealed its possible applications for improving the management of lung cancer.

Complete genome of Priestia filamentosa H146 isolated from tobacco leaves.

We report the complete genome of Priestia filamentosa H146 isolated from tobacco leaves. H146 contained a circular chromosome and five circular plasmids. A total of 4,669 genes were predicted, of which 4,372 genes were in the chromosome and other genes were located on plasmids. The genome sequence data provide an important basis for studying Priestia filamentosa.

Regulation of macrophage polarization by targeted metabolic reprogramming for the treatment of lupus nephritis.

Lupus nephritis (LN) is a severe and common manifestation of systemic lupus erythematosus (SLE) that is frequently identified with a poor prognosis. Macrophages play an important role in its pathogenesis. Different macrophage subtypes have different effects on lupus-affected kidneys. Based on their origin, macrophages can be divided into monocyte-derived macrophages (MoMacs) and tissue-resident macrophages (TrMacs). During nephritis, TrMacs develop a hybrid pro-inflammatory and anti-inflammatory functional phenotype, as they do not secrete arginase or nitric oxide (NO) when stimulated by cytokines. The infiltration of these mixed-phenotype macrophages is related to the continuous damage caused by immune complexes and exposure to circulating inflammatory mediators, which is an indication of the failure to resolve inflammation. On the other hand, MoMacs differentiate into M1 or M2 cells under cytokine stimulation. M1 macrophages are pro-inflammatory and secrete pro-inflammatory cytokines, while the M2 main phenotype is essentially anti-inflammatory and promotes tissue repair. Conversely, MoMacs undergo differentiation into M1 or M2 cells in response to cytokine stimulation. M1 macrophages are considered pro-inflammatory cells and secrete pro-inflammatory mediators, whereas the M2 main phenotype is primarily anti-inflammatory and promotes tissue repair. Moreover, based on cytokine expression, M2 macrophages can be further divided into M2a, M2b, and M2c phenotypes. M2a and M2c have anti-inflammatory effects and participate in tissue repair, while M2b cells have immunoregulatory and pro-inflammatory properties. Further, memory macrophages also have a role in the advancement of LN. Studies have demonstrated that the polarization of macrophages is controlled by multiple metabolic pathways, such as glycolysis, the pentose phosphate pathway, fatty acid oxidation, sphingolipid metabolism, the tricarboxylic acid cycle, and arginine metabolism. The changes in these metabolic pathways can be regulated by substances such as fish oil, polyenylphosphatidylcholine, taurine, fumaric acid, metformin, and salbutamol, which inhibit M1 polarization of macrophages and promote M2 polarization, thereby alleviating LN.

Exploration of the Active Component and Mechanisms of Shengyu Decoction Against Myelosuppression Using Network Pharmacology and in vitro Experimental Validation.

Background: Chemotherapy-induced myelosuppression (CIM) is a common adverse reaction with a high incidence rate that seriously affects human health. Shengyu Decoction (SYD) is often used to treat CIM. However, its pharmacodynamic basis and therapeutic mechanisms remain unclear. Purpose: This study aimed to clarify the active components and mechanisms of SYD in CIM. Methods: LC-QTOF/MS was used to identify the absorbable components of SYD. A series of network pharmacology methods have been applied to explore hub targets and potential mechanisms. Molecular docking was used to identify the binding ability of potential active ingredients and hub targets. Finally, in vitro experiments were performed to validate these findings. Results: In this study, 33 absorbable prototype components were identified using LC-QTOF/MS. A total of 62 possible targets of SYD in myelosuppression were identified. KEGG pathway enrichment analyses showed that some signaling pathways such as PI3K-Akt and HIF-1 may be the mechanisms by which it functions. Among them, we verified the PI3K-Akt pathway. 6 Hub proteins were screened by Protein-protein interaction (PPI) network analysis. Molecular docking results showed that four absorbable components in SYD showed good binding with six Hub targets. The effectiveness of the four predicted compounds and the mechanism were verified in vitro. It has also been shown that the active component could promote the proliferation of bone marrow stromal cells (BMSCs) and block apoptosis of BMSCs, which may be related to the PI3K-Akt pathway. This result is consistent with the network pharmacology approach and molecular docking predictions. Conclusion: Our results provided not only the candidate active component of SYD, but also a new insights into mechanism of SYD in the treatment of CIM.

Harnessing External Irradiation for Precise Activation of Metal-Based Agents in Cancer Therapy.

Cancer is a significant global health issue. Platinum-based chemotherapy drugs, including cisplatin, are crucial in clinical anti-cancer treatment. However, these drugs have limitations such as drug resistance, non-specific distribution, and irreversible toxic and side effects. In recent years, the development of metal-based agents has led to the discovery of other anti-cancer effects beyond chemotherapy. Precise spatiotemporal controlled external irradiation can activate metal-based agents at specific sites and play a different role from traditional chemotherapy. These strategies can not only enhance the anti-cancer efficiency, but also show fewer side effects and non-cross-drug resistance, which are ideal approaches to solve the problems caused by traditional platinum-based chemotherapy drugs. In this review, we focus on various metal-based agent-mediated cancer therapies that are activated by three types of external irradiation: near-infrared (NIR) light, ultrasound (US), and X-ray, and give some prospects. We hope that this review will promote the generation of new kinds of metal-based anti-cancer agents.

ß-Hydroxybutyrate Protects Against Cisplatin-Induced Renal Damage via Regulating Ferroptosis.

Cisplatin is a particularly potent antineoplastic drug. However, its usefulness is restricted due to the induction of nephrotoxicity. More recent research has indicated that ß-hydroxybutyrate (ß-HB) protects against acute or chronic organ damage as an efficient healing agent. Nonetheless, the therapeutic mechanisms of ß-HB in acute kidney damage caused by chemotherapeutic drugs remain unclear. Our study developed a model of cisplatin-induced acute kidney injury (AKI), which involved the administration of a ketogenic diet or ß-HB. We analyzed blood urea nitrogen (BUN) and creatinine (Cr) levels in serum, and used western blotting and immunohistochemical staining to assess ferroptosis and the calcium/calmodulin-dependent kinase kinase 2 (Camkk2)/AMPK pathway. The mitochondrial morphology and function were examined. Additionally, we conducted in vivo and in vitro experiments using selective Camkk2 inhibitor or activator to investigate the protective mechanism of ß-HB on cisplatin-induced AKI. Exogenous or endogenous ß-HB effectively alleviated cisplatin-induced abnormally elevated levels of BUN and Cr and renal tubular necrosis in vivo. Additionally, ß-HB reduced ferroptosis biomarkers and increased the levels of anti-ferroptosis biomarkers in the kidney. ß-HB also improved mitochondrial morphology and function. Moreover, ß-HB significantly attenuated cisplatin-induced cell ferroptosis and damage in vitro. Furthermore, western blotting and immunohistochemical staining indicated that ß-HB may prevent kidney injury by regulating the Camkk2-AMPK pathway. The use of the Camkk2 inhibitor or activator verified the involvement of Camkk2 in the renal protection by ß-HB. This study provided evidence of the protective effects of ß-HB against cisplatin-induced nephrotoxicity and identified inhibited ferroptosis and Camkk2 as potential molecular mechanisms.

ß-HB protects against cisplatin-induced renal damage both in vivo and in vitro.Moreover, ß-HB is effective in attenuating cisplatin-induced lipid peroxidation and ferroptosis.The regulation of energy metabolism, as well as the treatment involving ß-HB, is associated with Camkk2.

Gut microbiota mediates the protective effects of ß-hydroxybutyrate against cisplatin-induced acute kidney injury.

The gut microbiota has been reported to be perturbed by chemotherapeutic agents and to modulate side effects. However, the critical role of ß-hydroxybutyrate (BHB) in the regulation of the gut microbiota and the pathogenesis of chemotherapeutic agents related nephrotoxicity remains unknown. We conducted a comparative analysis of the composition and function of gut microbiota in healthy, cisplatin-challenged, BHB-treated, and high-fat diet-treated mice using 16 S rDNA gene sequencing. To understand the crucial involvement of intestinal flora in BHB's regulation of cisplatin -induced nephrotoxicity, we administered antibiotics to deplete the gut microbiota and performed fecal microbiota transplantation (FMT) before cisplatin administration. 16 S rDNA gene sequencing analysis demonstrated that both endogenous and exogenous BHB restored gut microbiota dysbiosis and cisplatin-induced intestinal barrier disruption in mice. Additionally, our findings suggested that the LPS/TLR4/NF-κB pathway was responsible for triggering renal inflammation in the gut-kidney axis. Furthermore, the ablation of the gut microbiota ablation using antibiotics eliminated the renoprotective effects of BHB against cisplatin-induced acute kidney injury. FMT also confirmed that administration of BHB-treated gut microbiota provided protection against cisplatin-induced nephrotoxicity. This study elucidated the mechanism by which BHB affects the gut microbiota mediation of cisplatin-induced nephrotoxicity by inhibiting the inflammatory response, which may help develop novel therapeutic approaches that target the composition of the microbiota.

An overview of the regulatory role of annexin A1 in the tumor microenvironment and its prospective clinical application (Review).

Although annexin A1 (ANXA1), a 37 kDa phospholipid­binding anti­inflammatory protein expressed in various tissues and cell types, has been investigated extensively for its regulatory role in cancer biology, studies have mainly focused on its intracellular role. However, cancer cells and stromal cells expressing ANXA1 have the ability to transmit signals within the tumor microenvironment (TME) through autocrine, juxtacrine, or paracrine signaling. This bidirectional crosstalk between cancer cells and their environment is also crucial for cancer progression, contributing to uncontrolled tumor proliferation, invasion, metastasis and resistance to therapy. The present review explored the important role of ANXA1 in regulating the cell­specific crosstalk between various compartments of the TME and analyzed the guiding significance of the crosstalk effects in promotion or suppressing cancer progression in the development of cancer treatments. The literature shows that ANXA1 is critical for the regulation of the TME, indicating that ANXA1 signaling between cancer cells and the TME is a potential therapeutic target for the development of novel therapeutic approaches for impeding cancer development.

Population pharmacokinetic analyses of methotrexate in pediatric patients: a systematic review.

BACKGROUND AND OBJECTIVES: Methotrexate is widely utilized in the chemotherapy of malignant tumors and autoimmune diseases in the pediatric population, but dosing can be challenging. Several population pharmacokinetic models were developed to characterize factors influencing variability and improve individualization of dosing regimens. However, significant covariates included varied across studies. The primary objective of this review was to summarize and discuss population pharmacokinetic models of methotrexate and covariates that influence pharmacokinetic variability in pediatric patients. METHODS: Systematic searches were conducted in the PubMed and EMBASE databases from inception to 7 July 2023. Reporting Quality was evaluated based on a checklist with 31 items. The characteristics of studies and information for model construction and validation were extracted, summarized, and discussed. RESULTS: Eighteen studies (four prospective studies and fourteen retrospective studies with sample sizes of 14 to 772 patients and 2.7 to 93.1 samples per patient) were included in this study. Two-compartment models were the commonly used structural models for methotrexate, and the clearance range of methotrexate ranged from 2.32 to 19.03 L/h (median: 6.86 L/h). Body size and renal function were found to significantly affect the clearance of methotrexate for pediatric patients. There were limited reports on the role of other covariates, such as gene polymorphisms and co-medications, in the pharmacokinetic parameters of methotrexate pediatric patients. Internal and external evaluations were used to assess the performance of the population pharmacokinetic models. CONCLUSION: A more rigorous external evaluation needs to be performed before routine clinical use to select the appropriate PopPK model. Further research is necessary to incorporate larger cohorts or pool analyses in specific susceptible pediatric populations to improve the understanding of predicted exposure profiles and covariate identification.

Pharmacokinetics, metabolism, excretion and safety of iruplinalkib (WX-0593), a novel ALK inhibitor, in healthy subjects: a phase I human radiolabeled mass balance study.

BACKGROUND: Iruplinalkib is a novel anaplastic lymphoma kinase (ALK) inhibitor for the treatment of ALK-positive crizotinib-resistant NSCLC. RESEARCH DESIGN AND METHODS: A single oral dose of 120 mg/3.7 MBq [14C]iruplinalkib was administered to healthy subjects. Blood, urine and fecal samples were collected and analyzed for iruplinalkib and its metabolites. The safety of iruplinalkib was also assessed. RESULTS: Iruplinalkib was absorbed quickly and eliminated slowly from plasma, with a Tmax of 1.5 h and t1/2 of 28.6 h. About 88.85% of iruplinalkib was excreted at 312 h, including 20.23% in urine and 68.63% in feces. Seventeen metabolites of iruplinalkib were identified, and M3b (demethylation), M7 (cysteine conjugation), M11 (oxidative dehydrogenation and cysteine conjugation of M3b) and M12 (oxidative dehydrogenation and cysteine conjugation) were considered the prominent metabolites in humans. Iruplinalkib-related compounds were found to be covalently bound to proteins, accounting for 7.70% in plasma and 17.96% in feces, which suggested chemically reactive metabolites were formed. There were no serious adverse events observed in the study. CONCLUSIONS: Iruplinalkib was widely metabolized and excreted mainly through feces in humans. Unchanged iruplinalkib, cysteine conjugates and covalent protein binding products were the main drug-related compounds in circulation. Iruplinalkib was well tolerated at the study dose. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov (Identifier: Anonymized).

Research progress in nano-drug delivery systems based on the characteristics of the liver cancer microenvironment.

The liver cancer has microenvironmental features such as low pH, M2 tumor-associated macrophage enrichment, low oxygen, rich blood supply and susceptibility to hematotropic metastasis, high chemokine expression, enzyme overexpression, high redox level, and strong immunosuppression, which not only promotes the progression of the disease, but also seriously affects the clinical effectiveness of traditional therapeutic approaches. However, nanotechnology, due to its unique advantages of size effect and functionalized modifiability, can be utilized to develop various responsive nano-drug delivery system (NDDS) by using these characteristic signals of the liver cancer microenvironment as a source of stimulation, which in turn can realize the intelligent release of the drug under the specific microenvironment, and significantly increase the concentration of the drug at the target site. Therefore, researchers have designed a series of stimuli-responsive NDDS based on the characteristics of the liver cancer microenvironment, such as hypoxia, weak acidity, and abnormal expression of proteases, and they have been widely investigated for improving anti-tumor therapeutic efficacy and reducing the related side effects. This paper provides a review of the current application and progress of NDDS developed based on the response and regulation of the microenvironment in the treatment of liver cancer, compares the effects of the microenvironment and the NDDS, and provides a reference for building more advanced NDDS.

Metabolomic profiling reveals the mechanisms underlying the nephrotoxicity of methotrexate in children with acute lymphoblastic leukemia.

BACKGROUND: Methotrexate is widely recommended as a first-line treatment for the intensive systemic and consolidation phases of childhood acute lymphoblastic leukemia. However, methotrexate-induced nephrotoxicity is a severe adverse reaction, of which the mechanisms remain unclear. METHODS: An untargeted metabolomics analysis of serum from childhood acute lymphoblastic leukemia patients with delayed methotrexate excretion, with or without acute kidney injury, was performed to identify altered metabolites and metabolic pathways. An independent external validation cohort and in vitro HK-2 cell assays further verified the candidate metabolites, and explored the mechanisms underlying the nephrotoxicity of methotrexate. RESULTS: Four metabolites showed significant differences between normal excretion and delayed excretion, seven metabolites reflected the differences between groups with or without acute kidney injury, and six pathways were finally enriched. In particular, oxidized glutathione was confirmed as a candidate metabolite involved in the toxicity of methotrexate. We further explored the role of glutathione deprivation-induced ferroptosis on methotrexate cytotoxicity, and it was found that methotrexate overload significantly reduced cell viability, triggered reactive oxygen species and intracellular Fe2+ accumulation, and altered the expression of ferroptosis-related proteins in HK-2 cells. These methotrexate-induced changes were alleviated or reversed by the administration of a ferroptosis inhibitor, further suggesting that ferroptosis promoted methotrexate-induced cytotoxicity in HK-2 cells. CONCLUSIONS: Our findings revealed complex metabolomic profiles and provided novel insights into the mechanism by which ferroptosis contributes to the nephrotoxic effects of methotrexate.

Development of a UPLC-MS/MS method for simultaneous therapeutic drug monitoring of anti-hepatocellular carcinoma drugs and analgesics in human plasma.

In hepatocellular carcinoma treatment, sorafenib, oxaliplatin, 5-fluorouracil, capecitabine, lenvatinib, and donafenib are first-line drugs; regorafenib, apatinib, and cabozantinib are second-line drugs; and oxycodone, morphine, and fentanyl are commonly used analgesics. However, the high degree of inter- and intra-individual variability in the efficacy and toxicity of these drugs remains an urgent issue. Therapeutic drug monitoring (TDM) is the most reliable technical means for evaluating drug safety and efficacy. Therefore, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous TDM of three chemotherapy drugs (5-fluorouracil, oxaliplatin, and capecitabin), six targeted drugs (sorafenib, donafenib, apatinib, cabozantinib, regorafenib, and lenvatinib), and three analgesics (morphine, fentanyl, and oxycodone). We extracted 12 analytes and isotope internal standards (ISs) from plasma samples by magnetic solid phase extraction (mSPE) and separated them using a ZORBAX Eclipse Plus C18 column with water containing 0.1% formic acid and methanol containing 0.1% formic acid as the mobile phase. The analytical performance of our method in terms of sensitivity, linearity, specificity, carryover, precision, limit of quantification, matrix effect, accuracy, dilution integrity, extraction recovery, stability, and crosstalk of all the analytes under different conditions met all the criteria stipulated by the guidelines of the Chinese Pharmacopoeia and U.S. Food and Drug Administration. The response function was estimated at 10.0-10 000.0 ng/mL for sorafenib, donafenib, apatinib, cabozantinib, regorafenib, and lenvatinib, and 20.0-20 000.0 ng/mL for 5-fluorouracil, oxaliplatin, capecitabin, morphine, fentanyl, and oxycodone, with a correlation of > 0.9956 for all compounds. The precision and accuracy of all analytes were < 7.21% and 5.62%, respectively. Our study provides empirical support for a simple, reliable, specific, and suitable technique for clinical TDM and pharmacokinetics.

Iron metabolism-related indicators as predictors of the incidence of acute kidney injury after cardiac surgery: a meta-analysis.

BACKGROUND: Some studies have found that ferroptosis plays an important role in the incidence of acute kidney injury (AKI) after cardiac surgery. However, whether iron metabolism-related indicators can be used as predictors of the incidence of AKI after cardiac surgery remains unclear. OBJECTIVES: We aimed to systematically evaluate whether iron metabolism-related indicators can be used as predictors of the incidence of AKI after cardiac surgery via meta-analysis.Search methods: The PubMed, Embase, Web of Science, and Cochrane Library databases were searched from January 1971 to February 2023 to identify prospective observational and retrospective observational studies examining iron metabolism-related indicators and the incidence of AKI after cardiac surgery among adults.Data Extraction and Synthesis: The following data were extracted by two independent authors (ZLM and YXY): date of publication, first author, country, age, sex, number of included patients, iron metabolism-related indicators, outcomes of patients, patient types, study types, sample, and specimen sampling time. The level of agreement between authors was determined using Cohen's κ value. The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of studies. Statistical heterogeneity across the studies was measured by the I2 statistic. The standardized mean difference (SMD) and 95% confidence interval (CI) were used as effect size measures. Meta-analysis was performed using Stata 15. RESULTS: After applying the inclusion and exclusion criteria, 9 articles on iron metabolism-related indicators and the incidence of AKI after cardiac surgery were included in this study. Meta-analysis revealed that after cardiac surgery, baseline serum ferritin (µg/L) (I2 = 43%, fixed effects model, SMD = -0.3, 95% CI:-0.54 to -0.07, p = 0.010), preoperative and 6-hour postoperative fractional excretion (FE) of hepcidin (%) (I2 = 0.0%, fixed effects model, SMD = -0.41, 95% CI: -0.79 to -0.02, p = 0.038; I2 = 27.0%, fixed effects model, SMD = -0.49, 95% CI: -0.88 to -0.11, p = 0.012), 24-hour postoperative urinary hepcidin (µg/L) (I2 = 0.0%, fixed effects model, SMD = -0.60, 95% CI: -0.82 to -0.37, p < 0.001) and urine hepcidin/urine creatinine ratio (µg/mmoL) (I2 = 0.0%, fixed effects model, SMD = -0.65, 95% CI: -0.86 to -0.43, p < 0.001) were significantly lower in patients who developed to AKI than in those who did not. CONCLUSION: After cardiac surgery, patients with lower baseline serum ferritin levels (µg/L), lower preoperative and 6-hour postoperative FE of hepcidin (%), lower 24-hour postoperative hepcidin/urine creatinine ratios (µg/mmol) and lower 24-hour postoperative urinary hepcidin levels (µg/L) are more likely to develop AKI. Therefore, these parameters have the potential to be predictors for AKI after cardiac surgery in the future. In addition, there is a need for relevant clinical research of larger scale and with multiple centers to further test these parameters and prove our conclusion.Trial Registration: PROSPERO identifier: CRD42022369380.

Integrated Analyses of the Expression and Prognostic Value of EPHB6 in Cervical Cancer and Its Correlation with Immune Infiltrates.

Among women, cervical cancer (CC) ranks as the third most frequent form of carcinoma and the fourth greatest cancer-related cause of deaths. There is increasing evidence that points to the dysregulation of EPH receptor B6 (EPHB6) in various cancers. On the other hand, neither the expression nor the function of EPHB6 in CC has been researched. In the first part of this investigation, we analyzed the data from the TCGA and discovered that the level of EPHB6 was much lower in CC tissues than in normal cervical tissues. ROC assays revealed that high EPHB6 expression had an AUC value of 0.835 for CC. The survival study revealed that both the overall and disease-specific survivals in this condition were considerably lower among patients who had a low EPHB6 level compared to those who had a high EPHB6 level. It is important to note that the multivariate COX regression analysis indicated that the expression of EPHB6 was an independent predictive factor. In addition to this, the C-indexes and calibration plots of a nomogram derived from multivariate assays revealed an accurate prediction performance among patients with CC. Immune infiltration analysis indicated that the expression of EPHB6 was positively associated with the levels of Tcm, TReg, B cells, T cells, iDC, T helper cells, cytotoxic cells, and DC, while negatively associated with NK CD56bright cells and neutrophils. In summary, the downregulation of EPHB6 was strongly linked to a more aggressive clinical development of CC, suggesting its potential utility as a diagnostic and therapeutic target in CC.

Development and validation of an UPLC-MS/MS method for simultaneous determination of fifteen targeted anti-cancer drugs in human plasma and its application in therapeutic drug monitoring.

In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously detect 15 targeted anti-cancer drugs: aletinib, afatinib, apatinib, icotinib, dasatinib, erlotinib, gefitinib, crizotinib, lapatinib, regorafenib, ceritinib, sorafenib, vemurafenib, imatinib, and N-desmethyl imatinib. Plasma samples were processed using a new magnetic solid phase extraction technique to extract each drug. The 15 analytes and four isotope internal standards were separated using an Agilent Eclipse XDB-C18 column (50.0 × 2.1 mm, 1.7 µm) with water containing 0.1% formic acid and acetonitrile as the mobile phase. The method verification included specificity, calibration curves, carryover, accuracy, crosstalk, precision, stability, recovery, dilution integrity, and matrix effects. The results showed that the developed UPLC-MS/MS method met the requirements of the U.S. Food and Drug Administration guidelines for methodological validation and could be used to monitor plasma concentrations. The response function was established for concentration range of 2.5-2500.0 ng/mL for aletinib, afatinib, apatinib, icotinib, dasatinib, crizotinib, regorafenib, vemurafenib, and N-desmethyl imatinib and 10.0-10,000.0 ng/mL for erlotinib, ceritinib, imatinib, sorafenib, gefitinib, and lapatinib, with a coeffificient of correlation of > 0.9977 for all the compounds. The precision and accuracy of all the analytes were < 6.88% and 5.29%, respectively. The percentage recovery and matrix effect of all the analytes were 91.3-103% and 93.8-102% for three QC concentrations levels. The recovery and matrix effect for all the ISs ranged from 93.7% to 98.8% and 94.6-101%. Meanwhile, we also found that the plasma concentrations of these targeted anti-cancer drugs showed large individual differences, which is not conducive to the treatment of tumors. Therefore, therapeutic drug monitoring (TDM) of these 15 targeted anti-cancer drugs is necessary, and this method could be used for TDM and exploration of pharmacokinetics of the aforementioned 15 targeted anti-cancer drugs.

Wnt signaling pathway-derived score for predicting therapeutic resistance and tumor microenvironment in lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer. Due to tumor heterogeneity, understanding the pathological mechanism of tumor progression helps to improve the diagnosis process and clinical treatment strategies of LUAD patients. Methods: The transcriptome pattern, mutant expression and complete clinical information were obtained from the cancer genome atlas (TCGA) database and microarray data from gene expression omnibus (GEO) database. Firstly, we used single sample Gene Set Enrichment Analysis (ssGSEA) to estimate the activation of Wnt signaling pathway in each sample. Consensus clustering algorithm was used to classify LUAD samples into different subgroups according to the transcription patterns of 152 Wnt signaling pathway related genes. Then, ESTIMATE, ssGSEA and Gene Set Variation Analysis (GSVA) algorithms were used to assess the biological pathways and immunocytes infiltration between different subtypes. LASSO-COX algorithm was conducted to construct prognostic model. Kaplan-Meier and multivariate Cox analysis were performed to evaluate the predictive performance of risk model. Gene features were further confirmed using external datasets. Finally, we conducted vitro assay for validating hub gene (LEF1). Results: Based on the transcription patterns of 152 Wnt signaling pathway related genes, four different subtypes of LUAD patients were screened out by consensus clustering algorithm. Subsequently, it was found that patients with cluster A and B had massive immunocytes infiltration, and the survival rate of patients with cluster B was better than that of other subgroups. According to the coefficients in the LASSO- Cox model and the transcriptome patterns of these 18 genes, the risk score was constructed for each sample. The degree of malignancy of LUAD patients with high-risk subgroup was remarkable higher than that of patients with low-risk subgroup (p < 0.001). Subsequently, five top prognostic genes (AXIN1, CTNNB1, LEF1, FZD2, FZD4.) were screened, and their expression values were different between cancer and normal tissues. FZD2 and LEF1 were negatively related to ImmunoScore, and AXIN1 was negatively related to ImmunoScore. The significant correlation between LUAD patient risk score and overall survival (OS) was verified in external datasets. In the A549 cell line, knockdown of LEF1 can reduce the invasive and proliferation ability of LUAD cells. Conclusion: A innovative 18 genes predictive feature based on transcriptome pattern was found in patients with lung adenocarcinoma. These investigations further promote the insight of the prognosis of lung adenocarcinoma and may contribute to disease management at risk stratification.

Ultra-performance liquid chromatography-tandem mass spectrometry for simultaneous determination of 12 anti-tumor drugs in human plasma and its application in therapeutic drug monitoring.

The effectiveness and safety of anti-tumor drugs are clinically important issues, and their therapeutic drug monitoring (TDM) is recommended. This study aimed to develop an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous TDM and exploration of clinical pharmacokinetics of anti-tumor drugs, including cyclophosphamide, ifosfamide, cisplatin, methotrexate, pemetrexed disodium, capecitabine, 5-fluorouracil, gemcitabine, doxorubicin, fulvestrant, tamoxifen, and irinotecan. After magnetic solid-phase extraction of plasma samples, the isotope internal standards and 12 anti-tumor drugs were separated using a ZORBAX Eclipse Plus C18 column (50.0 × 2.1 mm, 1.7 µm) with water containing 0.1% formic acid and acetonitrile as the mobile phase in a total run time of 5.0 min. The developed UPLC-MS/MS method was validated based on the Chinese Pharmacopoeia and the US Food and Drug Administration guidelines for bioanalytical method validation, including assessment of specificity, calibration curves, carryover, accuracy, crosstalk, precision, stability, recovery, dilution integrity, incurred sample reanalysis, and matrix effect. The results showed that a simple, fast, reliable, and specific UPLC-MS/MS method was developed and validated, and all the performance characteristics of the method met the requirements. The response function was established for concentration range of 0.10-25.00 µg/mL for gemcitabine, cyclophosphamide, ifosfamide, methotrexate, pemetrexed disodium, capecitabine, 5-fluorouracil, and cisplatin, and 0.05-12.50 µg/mL for doxorubicin, fulvestrant, tamoxifen, and irinotecan, with a coefficient of correlation of>0.9984 for all the compounds. The precision and accuracy of all the analytes were<6.5% and 5.9%, respectively. Hence, it could be used for TDM and exploration of pharmacokinetics of the aforementioned 12 anti-tumor drugs.

Study on the effect and mechanism of NFKBIA on cervical cancer progress in vitro and in vivo.

AIM: NFKBIA is frequently encountered. However, its expression and relevance of the proliferation, invasion, and migration in human cervical cancer (CC) remain unclear. The role and novel mechanism of NFKBIA in CC progression were investigated in this study. METHODS: We analyzed the expression of NFKBIA in CC and adjacent normal tissues and explored the proliferation, migration, and invasion of HeLa cells by treating with either wild-type NFKBIA plasmid or NFKBIA siRNA. Effect of NFKBIA on the epithelial-mesenchymal transition (EMT) and the ß-catenin-mediated transcription of target genes were evaluated subsequently. RESULTS: NFKBIA expression was lower in CC tissues than that of adjacent tissues. An obvious dysregulation of NFKBIA overexpression was revealed in CC cell proliferation, invasion, and migration, which differed from the effect of knockdown NFKBIA. NFKBIA overexpression facilitated the expression of both phosphorylated ß-catenin and E-cadherin protein. It inhibited the expression of vimentin, TWIST, as well as downstream targets of ß-catenin including c-MYC, TCF-4 and MMP14. Conversely, NFKBIA silencing elevated the expression of c-MYC, TCF-4, and MMP14, and promoted the EMT in HeLa cells. Both endogenous and exogenous NFKBIA interacted with ß-catenin. Moreover, ß-catenin overexpression stemmed effects of NFKBIA on the proliferation, migration, and invasion of HeLa cells. By overexpressing NFKBIA in vivo, the volume and size of tumors were notably decreased, while no obvious alteration was found in mice body weight. CONCLUSION: By inhibiting ß-catenin-mediated transcription, NFKBIA functioning as a tumor suppressor might be introduced as a novel anti-metastatic agent for the treatment of targeted CC.

A single nucleotide polymorphism in an R2R3 MYB transcription factor gene triggers the male sterility in soybean ms6 (Ames1).

KEY MESSAGE: Identification and functional analysis of the male sterile gene MS6 in Glycine max. Soybean (Glycine max (L.) Merr.) is an important crop providing vegetable oil and protein. The male sterility-based hybrid breeding is a promising method for improving soybean yield to meet the globally growing demand. In this research, we identified a soybean genic male sterile locus, MS6, by combining the bulked segregant analysis sequencing method and the map-based cloning technology. MS6, highly expressed in anther, encodes an R2R3 MYB transcription factor (GmTDF1-1) that is homologous to Tapetal Development and Function 1, a key factor for anther development in Arabidopsis and rice. In male sterile ms6 (Ames1), the mutant allele contains a missense mutation, leading to the 76th leucine substituted by histidine in the DNA binding domain of GmTDF1-1. The expression of soybean MS6 under the control of the AtTDF1 promoter could rescue the male sterility of attdf1 but ms6 could not. Additionally, ms6 overexpression in wild-type Arabidopsis did not affect anther development. These results evidence that GmTDF1-1 is a functional TDF1 homolog and L76H disrupts its function. Notably, GmTDF1-1 shows 92% sequence identity with another soybean protein termed as GmTDF1-2, whose active expression also restored the fertility of attdf1. However, GmTDF1-2 is constitutively expressed at a very low level in soybean, and therefore, not able to compensate for the MS6 deficiency. Analysis of the TDF1-involved anther development regulatory pathway showed that expressions of the genes downstream of TDF1 are significantly suppressed in ms6, unveiling that GmTDF1-1 is a core transcription factor regulating soybean anther development.