EGFR Oncogenic Mutations in NSCLC Impair Macrophage Phagocytosis and Mediate Innate Immune Evasion Through Up-Regulation of CD47.

INTRODUCTION: EGFR-mutated NSCLC is characterized by an immunosuppressive microenvironment that confers limited clinical effectiveness to anti-PD-1 or PD-L1 antibodies. Despite the discouraging outcomes of immunotherapy, novel immune checkpoints are constantly emerging, among which the specific vulnerability for therapeutic intervention in the context of EGFR-mutated NSCLC remains unresolved. METHODS: Data sets of patient- and cell line-levels were used for screening and mutual validation of association between EGFR mutation and a panel of immune checkpoint-related genes. Regulatory mechanism was elucidated through in vitro manipulation of EGFR signaling pathway and evaluated by immunoblot analysis, quantitative polymerase chain reaction, flow cytometry, immunofluorescence staining, and chromatin immunoprecipitation. In vivo investigation of different therapeutic strategies were conducted using both immunocompetent and immunodeficient mouse models. RESULTS: Among all screened immune checkpoints, CD47 emerged as the candidate most relevant to EGFR activation. Mechanistically, EGFR mutation constitutively activated downstream ERK and AKT pathways to respectively up-regulate the transcriptional factors c-Myc and NF-κB, both of which structurally bound to the promotor region of CD47 and actively transcribed this "don't eat me" signal. Impaired macrophage phagocytosis was observed on introduction of EGFR-sensitizing mutations in NSCLC cell line models, whereas CD47 blockade restored the phagocytic capacity and augmented tumor cell killing in both in vitro and in vivo models. Remarkably, the combination of anti-CD47 antibody with EGFR tyrosine kinase inhibitor revealed an additive antitumor activity compared with monotherapy of either antitumor agent in both immunocompetent and adaptive immunity-deficient mouse models. CONCLUSIONS: EGFR-sensitizing mutation facilitates NSCLC's escape from innate immune attack through up-regulating CD47. Combination therapy incorporating CD47 blockade holds substantial promise for clinical translation in developing more effective therapeutic approaches against EGFR-mutant NSCLC.

DLGAP5 promotes lung adenocarcinoma growth via upregulating PLK1 and serves as a therapeutic target.

BACKGROUND: Human discs large-associated protein 5 (DLGAP5) is reported to play a pivotal role in regulating the cell cycle and implicate in tumorigenesis and progression of various cancers. Our current research endeavored to explore the prognostic value, immune implication, biological function and targeting strategy of DLGAP5 in LUAD through approaches including bioinformatics, network pharmacology analysis and experimental study. METHODS: Multiple databases, including TCGA, GEO, CPTAC and Human Protein Atlas, were utilized to explore the expression and clinical significance of DLGAP5 in LUAD. The genetic alterations of DLGAP5 were assessed through cBioPortal and COSMIC databases. The relationship between DLGAP5 expression and genetic abnormalities of driver genes in LUAD was analyzed through TIMER2.0 database. CancerSEA database was utilized to explore the function of DLGAP5 in 14 different states in LUAD at single-cell resolution. GDSC database was utilized to analyze the impact of DLGAP5 on IC50 of frequently-used anti-LUAD drugs. CIBERSORT method and TIMER2.0 database was utilized to explore the relationship between DLGAP5 and tumor immune infiltration. Network pharmacology was applied to screen potential DLGAP5 inhibitor. In vitro and in vivo experiments were utilized to evaluate biological function and downstream targets of DLGAP5, and the effect of screened DLGAP5 inhibitor on LUAD growth. RESULTS: High DLGAP5 expression was commonly observed in LUAD and associated with mutation of major driver genes, poor prognosis, high IC50 values of frequently-used anti-LUAD drugs, increasing immune infiltration and elevated immune checkpoint blockade-related genes in LUAD. PLK1 was revealed as a potential DLGAP5 downstream target in LUAD. DLGAP5 overexpression or knockdown significantly promoted or inhibited LUAD cell proliferation and PLK1 expression. PLK1 overexpression well rescued DLGAP5 knockdown-induced cell proliferation inhibition, or vice versa. Furthermore, by virtual screening of an investigational drug library from the DrugBank database, AT9283 was screened and identified as a novel DLGAP5 inhibitor. AT9283 effectively suppressed growth of LUAD cells both in vitro and in vivo. DLGAP5 overexpression significantly reversed AT9283-induced proliferation inhibition. Moreover, AT9283 significantly suppressed DLGAP5 and PLK1 expression, while DLGAP5 overexpression significantly reversed AT9283-induced PLK1 suppression. CONCLUSION: Our research has demonstrated that DLGAP5 is upregulated in LUAD and exhibits a strong correlation with unfavorable prognosis. Furthermore, DLGAP5 assumes a significant function in the regulation of tumor immunity and treatment outcome of immune checkpoint inhibitors. Of note, we found that DLGAP5 promotes cell proliferation of LUAD via upregulating PLK1. Targeting DLGAP5 by AT9283, our newly identified DLGAP5 inhibitor, suppresses LUAD growth. DLGAP5 may become a promising prognostic biomarker and therapeutic target for patients with LUAD.

Nomogram based on circulating lymphocyte subsets for predicting radiation pneumonia in esophageal squamous cell carcinoma.

Purpose: Currently, the relationship between radiation pneumonia (RP) and circulating immune cell in patients with esophageal squamous cell carcinoma (ESCC) remains unclear. This study aimed to explore the relationship between RP and circulating lymphocyte subsets in patients with ESCC receiving chemoradiotherapy (CRT), and develop a nomogram model to predict RP. Since we should implement clinical intervention to ≥ grade 2 RP, a nomogram model for ≥ grade 2 RP was also established to provide an early warning. Patients and methods: This study retrospectively included 121 patients with ESCC receiving CRT from Guangxi Medical University Cancer Hospital from 2013 to 2021. Independent factors associated with occurrence of RP and ≥ grade 2 RP were identified by univariate and multivariate logistic regression analysis in the training cohort, and incorporated into nomograms. The predictive accuracy and discrimination of the model was assessed using Concordance Index (C-index), calibration curve and decision curve analysis (DCA). And each model was internally validated. Additionally, to verify the optimized predictive performance of the nomograms, the area under the ROC curve (AUC) of each nomogram was compared to that of single independent risk factors, lung V10 and lung V20, respectively. Moreover, each model was further evaluated for risk stratification to identify populations at high risk of RP and ≥ grade 2 RP. Results: Multivariate analysis suggested that TNM stage, post-RT percentage of CD8+ T cell, and lung V15 were independent predictive factors of RP. Besides, pre- and post-RT percentage of CD8+ T cell, and V15 were independent factors of ≥ grade 2 RP. The C-indexes of RP and ≥ grade 2 RP nomograms were 0.809 (95% CI: 0.715-0.903) and 0.787 (95% CI: 0.685-0.889) in the training cohort, respectively. And the C-indexes of RP and ≥ grade 2 RP nomograms were 0.718 (95% CI: 0.544-0.892) and 0.621 (95% CI: 0.404-0.837) in the validation cohort, respectively. The calibration curves showed that the predicted values of model agreed well with actual observations. Moreover, DCA results indicated the applicability and accuracy of the models to predict RP and ≥ grade 2 RP. After stratification, the incidence of the high-risk group was significantly higher than that of the low-risk group with respect to either RP or ≥ grade 2 RP. Conclusion: TNM stage, post-RT percentage of CD8+ T cell, and lung V15 were the independent predictors of RP toxicity. Pre- and post-RT percentage of CD8+ T cell, and lung V15 were the independent factors of ≥ grade 2 RP toxicity. The nomograms based on circulating lymphocyte subsets can robustly predict RP and ≥ grade 2 RP, guiding clinicians in risk stratification and early intervention.

Prediction of the Mechanism of Sodium Butyrate against Radiation-Induced Lung Injury in Non-Small Cell Lung Cancer Based on Network Pharmacology and Molecular Dynamic Simulations and Molecular Dynamic Simulations.

Background: Radiation-induced lung injury (RILI) is a severe side effect of radiotherapy for non-small cell lung cancer (NSCLC) ,and one of the major hindrances to improve the efficacy of radiotherapy. Previous studies have confirmed that sodium butyrate (NaB) has potential of anti-radiation toxicity. However, the mechanism of the protective effect of NaB against RILI has not yet been clarified. This study aimed to explore the underlying protective mechanisms of NaB against RILI in NSCLC through network pharmacology, molecular docking, molecular dynamic simulations and in vivo experiments. Methods: The predictive target genes of NaB were obtained from the PharmMapper database and the literature review. The involved genes of RILI and NSCLC were predicted using OMIM and GeneCards database. The intersectional genes of drug and disease were identified using the Venny tool and uploaded to the Cytoscape software to identify 5 core target genes of NaB associated with RILI. The correlations between the 5 core target genes and EGFR, PD-L1, immune infiltrates, chemokines and chemokine receptors were analyzed using TIMER 2.0, TIMER and TISIDB databases. We constructed the mechanism maps of the 3 key signaling pathways using the KEGG database based on the results of GO and KEGG analyses from Metascape database. The 5 core target genes and drug were docked using the AutoDock Vina tool and visualized using PyMOL software. GROMACS software was used to perform 100 ns molecular dynamics simulation. Irradiation-induced lung injury model in mice were established to assess the therapeutic effects of NaB. Results: A total of 51 intersectional genes involved in NaB against RILI in NSCLC were identified. The 5 core target genes were AKT1, TP53, NOTCH1, SIRT1, and PTEN. The expressions of the 5 core target genes were significantly associated with EGFR, PD-L1, immune infiltrates, chemokines and chemokine receptors, respectively. The results from GO analysis of the 51 intersectional genes revealed that the biological processes were focused on the regulation of smooth muscle cell proliferation, oxidative stress and cell death, while the three key KEGG pathways were enriched in PI3K-Akt signal pathway, p53 signal pathway, and FOXO signal pathway. The docking of NaB with the 5 core target genes showed affinity and stability, especially AKT1. In vivo experiments showed that NaB treatment significantly protected mice from RILI, with reduced lung histological damage. In addition, NaB treatment significantly inhibited the PI3K/Akt signaling pathway. Conclusions: NaB may protect patients from RILI in NSCLC through multiple target genes including AKT1, TP53, NOTCH1, SIRT1 and PTEN, with multiple signaling pathways involving, including PI3K-Akt pathway, p53 pathway, and FOXO pathways. Our findings effectively provide a feasible theoretical basis to further elucidate the mechanism of NaB in the treatment of RILI.

Ophthalmic Disease Detection via Deep Learning With a Novel Mixture Loss Function.

With the popularization of computer-aided diagnosis (CAD) technologies, more and more deep learning methods are developed to facilitate the detection of ophthalmic diseases. In this article, the deep learning-based detections for some common eye diseases, including cataract, glaucoma, and age-related macular degeneration (AMD), are analyzed. Generally speaking, morphological change in retina reveals the presence of eye disease. Then, while using some existing deep learning methods to achieve this analysis task, the satisfactory performance may not be given, since fundus images usually suffer from the impact of data imbalance and outliers. It is, therefore, expected that with the exploration of effective and robust deep learning algorithms, the detection performance could be further improved. Here, we propose a deep learning model combined with a novel mixture loss function to automatically detect eye diseases, through the analysis of retinal fundus color images. Specifically, given the good generalization and robustness of focal loss and correntropy-induced loss functions in addressing complex dataset with class imbalance and outliers, we present a mixture of those two losses in deep neural network model to improve the recognition performance of classifier for biomedical data. The proposed model is evaluated on a real-life ophthalmic dataset. Meanwhile, the performance of deep learning model with our proposed loss function is compared with the baseline models, while adopting accuracy, sensitivity, specificity, Kappa, and area under the receiver operating characteristic curve (AUC) as the evaluation metrics. The experimental results verify the effectiveness and robustness of the proposed algorithm.

Resveratrol Mediates the Apoptosis of Triple Negative Breast Cancer Cells by Reducing POLD1 Expression.

Resveratrol (RSV) is known to possess anticancer properties in many types of cancers like breast cancer, in which POLD1 may serve as a potential target. However, the anticancer mechanism of RSV on triple negative breast cancer (TNBC) remains unclear. In the present study, the antitumor effects and mechanism of RSV on TNBC cells were analyzed by RNA sequencing (RNA-seq), which was then verified via cell counting kit-8 (CCK8), immunofluorescence, immunohistochemistry, Western Blot (WB), flow cytometry, and hematoxylin-eosin (HE) staining. According to the corresponding findings, the survival rate of MDA-MB-231 cells gradually decreased as RSV treatment concentration increased. The RNA-seq analysis results demonstrated that genes affected by RSV treatment were mainly involved in apoptosis and the p53 signaling pathway. Moreover, apoptosis of MDA-MB-231 cells induced by RSV was observed to be mainly mediated by POLD1. When treated with RSV, the expression levels of full length PARP1, PCNA, and BCL-2 were found to be significantly reduced, and the expression level of Cleaved-PARP1 as well as Cleaved-Caspase3 increased significantly. Additionally, the mRNA expression of POLD1 was significantly reduced after treatment with RSV, and the protein expression level was also inhibited by RSV in a concentration-dependent manner. The prediction of domain interaction suggested that RSV may bind to at least five functional domains of the POLD1 protein (6s1m, 6s1n, 6s1o, 6tny and 6tnz). Furthermore, after RSV treatment, the anti-apoptotic index (PCNA, BCL-2) of MDA-MB-231 cells was found to decrease while the apoptosis index (caspase3) increased. Moreover, the overexpression of POLD1 reduced the extent of apoptosis observed in MDA-MB-231 cells following RSV treatment. Moreover, animal experimental results showed that RSV had a significant inhibitory effect on the growth of live tumors, while POLD1 overexpression was shown to antagonize this inhibitory effect. Accordingly, this study's findings reveal that RSV may promote the apoptosis of TNBC cells by reducing the expression of POLD1 to activate the apoptotic pathway, which may serve as a potential therapy for the treatment of TNBC.

Practices and Hindrances in Cancer Pain Management: Results of a National Multi-Cancer Center Survey Among Healthcare Professionals in China.

BACKGROUND: Inadequate control of cancer-related pain in China is an ongoing problem. This study investigated the practices of cancer pain (CP) management at major cancer centers in China and perceived hindrances and knowledge of CP management among health professionals. METHODS: From September to October 2019, a survey was conducted using electronic questionnaires via the internet to investigate the practices, and perceived hindrances and knowledge in managing CP among healthcare professionals from 7 provincial cancer centers in China. The questionnaire included demographic data, the professionals' practices among their own patients, their opinions regarding hindrances to CP management, and knowledge of CP management. RESULTS: We gathered validated responses from 411 anonymous healthcare professionals, with 82.2% (411/500) of response rate. Based on the analysis of these 411 questionnaires, the results demonstrated that CP was prevalent among patients with cancer, while moderate-to-severe pain took a great proportion. CP management was inadequate for a significant proportion of the patients with CP. Pain assessment, analgesic treatment, attention to adverse effects of analgesic, and multidisciplinary management were usually ineffectual in many cases. The duration of work experience did not significantly affect CP management. The respondents considered that both patients and healthcare professionals were responsible for the undermanagement of CP. Only 26 (6.3%) respondents were able to answer correctly all 10 of the professional questions regarding CP. CONCLUSION: CP is commonly undermanaged in China. Effective pain control requires the implementation of standards, and the sufficient attention and training of healthcare professionals.

Advanced-platelet-rich fibrin extract promotes adipogenic and osteogenic differentiation of human adipose-derived stem cells in a dose-dependent manner in vitro.

Advanced platelet-rich fibrin (A-PRF) is an autogenous biological material obtained from peripheral blood. A-PRF extract (A-PRFe) contains a high concentration of various cytokines that are increasingly appreciated for their roles in improving stem cell repairing function during tissue regeneration. However, the optimal A-PRFe concentration to stimulate stem cells is unknown. This study aimed to identify the optimal concentrations of A-PRFe to promote adipogenic and osteogenic differentiation of human adipose-derived stem cells (ASCs). We produced A-PRFe from A-PRF clots by centrifuging fresh peripheral blood samples and isolated and identified ASCs using surface CD markers and multilineage differentiation potential. Enzyme-linked immunosorbent assay (ELISA) showed the concentrations of several cytokines, including b-FGF, PDGF-BB, and others, increased gradually, peaked on day 7 and then decreased. Cell proliferation assays showed A-PRFe significantly stimulated ASC proliferation, and proliferation significantly increased at higher A-PRFe doses. The degree of adipogenic and osteogenic differentiation increased at higher A-PRFe concentrations in the culture medium, as determined by oil red O and alizarin red staining. Reverse transcription polymerase chain reaction (RT-PCR) showed that expression levels of genes related to adipogenic/osteogenic differentiation (PPARγ2, C/EBPα, FABP4, Adiponectin, and ALP, OPN, OCN, RUNX2), paracrine (HIF-1α, VEGF, IGF-2) and immunoregulation (HSP70, IL-8) function were higher in groups with a higher concentration of A-PRFe than in lower concentration groups. This study demonstrates that A-PRFe is ideal for use in ASC applications in regenerative medicine because it improves biological functions, including proliferation, adipogenic/osteogenic differentiation, and paracrine function in a dose-dependent manner.

Capsaicin Inhibits Proliferation and Induces Apoptosis in Breast Cancer by Down-Regulating FBI-1-Mediated NF-κB Pathway.

BACKGROUND: As a natural compound extracted from a variety of hot peppers, capsaicin has drawn increasing attention to its anti-cancer effects against multiple human cancers including breast cancer. FBI-1 is a major proto-oncogene negatively regulating the transcription of many tumor suppressor genes, and plays a vital role in tumorigenesis and progression. However, whether FBI-1 is involved in capsaicin-induced breast cancer suppression has yet to be ascertained. This study aimed to investigate the effects of capsaicin on proliferation and apoptosis and its association with FBI-1 expression in breast cancer. METHODS: CCK-8 and morphological observation assay were employed to detect cell proliferation. Flow cytometry and TUNEL assay were conducted to detect cell apoptosis. RNA interference technique was used to overexpress or silence FBI-1 expression. qRT-PCR and/or Western blot analysis were applied to detect the protein expression of FBI-1, Ki-67, Bcl-2, Bax, cleaved-Caspase 3, Survivin and NF-κB p65. Xenograft model in nude mice was established to assess the in vivo effects. RESULTS: Capsaicin significantly inhibited proliferation and induced apoptosis in breast cancer in vitro and in vivo, along with decreased FBI-1, Ki-67, Bcl-2 and Survivin protein expression, increased Bax protein expression and activated Caspase 3. Furthermore, FBI-1 overexpression obviously attenuated the capsaicin-induced anti-proliferation and pro-apoptosis effect, accompanied with the above-mentioned proteins reversed, whereas FBI-1 silencing generated exactly the opposite response. In addition, as a target gene of FBI-1, NF-κB was inactivated by p65 nuclear translocation suppressed with capsaicin treatment, which was perceptibly weakened with FBI-1 overexpression or enhanced with FBI-1 silencing. CONCLUSION: This study reveals that FBI-1 is closely involved in capsaicin-induced anti-proliferation and pro-apoptosis of breast cancer. The underlying mechanism may be related to down-regulation of FBI-1-mediated NF-κB pathway. Targeting FBI-1 with capsaicin may be a promising therapeutic strategy in patients with breast cancer.

Synergistic Effect of MC-LR and C-Terminal Truncated HBx on HepG2 Cells and Their Effects on PP2A Mediated Downstream Target of MAPK Signaling Pathway.

C-terminally truncated hepatitis B virus (HBV) X (ctHBx) infection and exposure to microcystins-LR (MC-LR) can lead to human hepatitis and liver cancer, but the mechanism associated with their synergistically effects not been fully elucidated. The ctHBx (HBxΔ4 and HBxΔ32) lentivirus were constructed and transfected into the HepG2 cells. Then we investigated the function of MC-LR and ctHBx using the molecular biology approaches, including enzyme-linked immunosorbent assay, clone formation assay, scratch wound testing, transwell assays, carried out flow cytometry respectively to examine cell cycle and apoptosis in each group, and detected the related proteins of HBx, MEK/ERK/JNK/p38 in mitogen-activated protein kinase (MAPK) pathway and the downstream proteins such as cdc2, cdc25C, and p53 by western blotting. We found that the protein phosphorylase 2A (PP2A) enzyme activity in MC-LR and HBxΔ32/HBxΔ4 groups decreased more than in MC-LR and HBx group at the same time point and MC-LR concentration (P < 0.05). Meanwhile the proliferation, migration, invasion and colony formation capability of HepG2 cells were significantly enhanced in MC-LR and ctHBx groups (P < 0.05). In addition the proportion of S stage cells in the MC-LR-treated HBxΔ32/HBxΔ4 groups was significantly greater than that in the untreated groups (P < 0.05). Furthermore, the protein expression of MAPK signaling pathway including phospho-MEK1/2, ERKl/2, p38, and JNK were up-regulated by MC-LR and HBxΔ32, and the expression of cyclin-related proteins, including p53, cdc25C, and cdc2 were also activated (P < 0.05). Taken together, our findings revealed the essential significance of the MC-LR and ctHBx on the PP2A/MAPK/p53, cdc25C and cdc2 axis in the formation and development of HCC and identified MC-LR and ctHBx as potential causal cofactors of hepatocarcinogenesis.

Sodium Butyrate Combined with Docetaxel for the Treatment of Lung Adenocarcinoma A549 Cells by Targeting Gli1.

PURPOSE: This study is aimed to investigate the combined treating efficacy of sodium butyrate and docetaxel on proliferation and apoptosis of the lung adenocarcinoma A549 cell line based on Gli1 regulation in vitro and in vivo. MATERIALS AND METHODS: RNA interference method was used to overexpress Gli1 in A549 cells. Cells were treated with varying concentrations of sodium butyrate, docetaxel or both in combination. CCK-8, colony formation assay, Hoechst 33258 staining, flow cytometry and TUNEL assay were employed to detect proliferation, cell cycle and apoptosis. qRT-PCR and Western blot analysis were applied to detect the mRNA and protein expression of Gli1. In vivo tumorigenicity was detected by tumor transplantation in nude mice. Downstream protein levels of Gli1 were detected using Western blot assay. RESULTS: It was found that sodium butyrate or docetaxel alone, respectively, inhibited proliferation and promoted apoptosis of A549 cells in vitro and in vivo, while the combination of the two generated significantly higher responses, which were also effective in another lung adenocarcinoma cell line H1299. Furthermore, the combined therapy had an additive effect in suppressing Gli1 expression and regulating the expression of its downstream proteins that involve in proliferation, cell cycle and apoptosis of A549 cells in vitro and in vivo, including decreased protein expression of Ki-67, CDK1, CDK2, Cyclin D1, Bcl-2 and Survivin, and increased protein expression of Cyclin A, p21, Bax and cleaved-Caspase 3. On the other hand, Gli1 overexpression perceptibly reversed the above-mentioned additive effect in vitro and in vivo. CONCLUSION: This study demonstrates that the combined therapy of sodium butyrate and docetaxel additively inhibits proliferation and promotes apoptosis of A549 lung adenocarcinoma cells via suppressing Gli1 expression in vitro and in vivo. Targeting Gli1 by the combined therapy may provide new insights into the therapeutic management of patients with lung adenocarcinoma.

ZBTB7A promotes migration, invasion and metastasis of human breast cancer cells through NF-κB-induced epithelial-mesenchymal transition in vitro and in vivo.

It has been generally confirmed that zinc finger and BTB domain containing 7A (ZBTB7A) plays an important role in the occurrence and progression of malignant tumours, but the promotion or inhibition effect is related to tumour type. The mechanism between ZBTB7A and breast cancer is not well understood, so further research is needed. In this study, we first investigated the expression of ZBTB7A in tissue samples of clinical breast cancer patients, MDA-MB-231, MCF-7 and MCF-10A cells. Second, we overexpressed the ZBTB7A in MCF-7 cells and silenced the ZBTB7A in MDA-MB-231 cells using lentivirus transfection technology, respectively, and verified the effect of ZBTB7A on migration and invasion of breast cancer cell lines through in vitro cell function experiments, such as wound-healing assay, migration and invasion assay, quantitative real time reverse transcriptase (qRT-PCR) and western blot. Then, the correlation between the above influences, epithelial-mesenchymal transition (EMT) and NF-κB was analysed. Finally, in vivo tumour transplantation model in nude mice was established to verified the effect of ZBTB7A on metastasis of breast cancer MDA-MB-231 cells. In conclusion, ZBTB7A is highly expressed in cancer tissue, breast cancer cell line MDA-MB-231 and MCF-7. Meanwhile, the high expression of ZBTB7A may promote cell migration, invasion and tumour metastasis, which may be related to EMT events by regulating NF-κB.

Triptolide Suppresses Growth of Breast Cancer by Targeting HMGB1 in Vitro and in Vivo.

Triptolide has been indicated potent anti-cancer effect involving multiple molecular targets and signaling pathways. High-mobility group box 1 (HMGB1) is a highly conserved DNA-binding protein taking part in breast cancer development. The therapeutic effect of triptolide on HMGB1 has not been reported. Thus, our study aims to clarify the role of HMGB1 in triptolide-induced anti-growth effect on breast cancer in vitro and in vivo. We demonstrated that triptolide significantly suppressed growth of breast cancer cells by inhibition of cell viability, clonogenic ability. Further studies evidenced that triptolide treatment not only inhibited HMGB1 mRNA expression, but also decreased supernatant level of HMGB1 in vitro. In line with these observations, exogenous recombinant HMGB1 (rHMGB1) promoted cell proliferation of breast cancer, and triptolide reversed the rHMGB1-promoted proliferative effect. As well, triptolide enhanced the anti-proliferative activity of ethyl pyruvate (EP) (HMGB1 inhibitor). Furthermore, downstream correlation factors (Toll-like receptor 4 (TLR4) and phosphorylated-nuclear factor-kappaB (NF-κB) p65) of HMGB1 were significantly decreased in vitro after triptolide treatment. Consistantly, we confirmed that tumor growth was significantly inhibited after triptolide treatment in vivo. Meanwhile, immunohistochemical analyses showed that triptolide treatment significantly decreased the level of cytoplasmic HMGB1 and TLR4 expression, whereas the expression of NF-κB p65 was relatively higher in cytoplasm, and conversely lower in nucleus as compared to the control group. Collectively, these results demonstrate that triptolide suppresses the growth of breast cancer cells via reduction of HMGB1 expression in vitro and in vivo, which may provide new insights into the treament of breast cancer.

A Novel Animal Model of Induced Breast Precancerous Lesion in Tree Shrew.

Chinese tree shrew, an animal exhibited closer evolutionary relationship with humans compared to rodents, is getting increasingly attentions as an appealing experimental animal model for human diseases. However, a high-efficiency and stable method to establish tree shrew breast precancerous lesions model has not been clearly elucidated. Thus, the current study aimed to explore the way of establishing breast precancerous model in tree shrew and investigate the pathologic characteristics of induced breast precancerous lesions. The results indicated that 7,12-dimethylbenz(a)anthracene (DMBA) could induce breast lesions in tree shrews. However, comparing to DMBA alone, an addition of medroxyprogesterone acetate (MPA) to DMBA critically increased the rate of induced breast lesion in tree shrews. Half of induced breast lesions were intraductal papilloma and the others were atypical ductal hyperplasia. Induced lesions showed positive expression of estrogen receptor α (ERα), progesterone receptor (PR) and cytokeratin 5/6 (CK5/6), but negative expression of human epidermal growth factor receptor-2 (Her-2). The expression of B cell lymphoma-extra large (Bcl-xl) was significantly higher and the expression of B cell lymphoma 2 associated X protein (Bax) was significantly lower in the precancerous lesions (atypical ductal hyperplasia) compared to benign tumor (intraductal papilloma). These results suggest that DMBA is able to induce breast lesions in tree shrews. Combination of DMBA and MPA may be more effective to establish breast precancerous lesion tree shrew models. Tree shrew might be a promising animal model for studying the tumorogenesis of breast cancer.

[Effects of FBI-1 silencing on proliferation and apoptosis of triple-negative breast cancer cell line MDA-MB-231].

This work aimed to observe the effects of short hairpin RNA (shRNA)-silenced FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) on proliferation and apoptosis of triple-negative breast cancer cell line MDA-MB-231. qRT-PCR and Western blot analysis were applied to detect the mRNA and/or protein expression of FBI-1, Bcl-2, Bax, cleaved-Caspase 3 and Survivin. RNA interference method was used to silence FBI-1 expression in MDA-MB-231 cells. CCK-8 and colony formation assay were employed to detect the cell proliferation. Flow cytometry was employed for examining cell apoptosis. In vivo tumorigenicity of MDA-MB-231 cells was detected by tumor transplantation in nude mice. The results showed that the mRNA and protein expressions of FBI-1 were higher in MDA-MB-231 cells compared with those in normal human mammary epithelial cells MCF-10A. FBI-1 gene silencing inhibited proliferation and induced apoptosis of MDA-MB-231 cells in vitro, together with decreased Bcl-2 and Survivin protein expression, increased Bax protein expression and activated Caspase 3. Moreover, FBI-1 gene silencing inhibited the tumorigenesis of MDA-MB-231 cells in vivo. These results suggest that silencing of FBI-1 gene inhibits proliferation, induces apoptosis and suppresses the tumorigenesis of MDA-MB-231 cells.

Influencing factors and health risk assessment of microcystins in the Yongjiang river (China) by Monte Carlo simulation.

The Yongjiang river is a large, shallow, hyper-trophic, freshwater river in Guangxi, China. To investigate the presence of microcystin-RR, microcystin-LR, and microcystin-YR (MC-RR, MC-LR, and MC-YR) in the Yongjiang river and describe their correlation with environmental factors, as well as, assess health risk using Monte Carlo simulation, 90 water samples were collected at three sample points from March to December 2017. Results showed that during the monitoring period, total concentrations of MC-RR (TMC-RR), MC-YR (TMC-YR), and MC-LR (TMC-LR) varied from 0.0224 to 0.3783 µg/L, 0.0329 to 0.1433 µg/L, and 0.0341 to 0.2663 µg/L, respectively. Total phosphorus (TP) content appeared to be related to TMC-LR and the total concentrations of microcystins (TMCs), while pH and total nitrogen (TN)/TP ratio appeared to be related to TMC-RR and TMC-YR, respectively. Using the professional health risk assessment software @Risk7.5, the risks of dietary intake of microcystins (MCs), including the carcinogenic risk and non-carcinogenic risk, were evaluated. It was found that the carcinogenic risk of MC-RR from drinking water was higher than MC-LR and MC-YR, and the presence of MCs would lead to high potential health risks, especially in children. The carcinogenic risk of MC-RR to children was >1 × 10-4, the maximum allowance level recommended by the US Environmental Protection Agency; as for adults, it was >5 × 10-5, the maximum allowance level recommended by the International Commission on Radiological Protection. The non-carcinogenic hazard index (HI) of MC-RR, MC-YR, and MC-LR increased successively, indicating that MC-LR was more hazardous to human health than MC-YR and MC-RR, but its HI was <1. This suggests that MCs pose less risk to health. However, it is necessary to strengthen the protection and monitoring of drinking water source for effective control of water pollution and safeguarding of human health.

Hesperidin ameliorates lipopolysaccharide-induced acute lung injury in mice by inhibiting HMGB1 release.

Hesperidin (HDN), a flavanone glycoside, possesses anti-inflammatory properties and has been suggested to be able to modulate the lipopolysaccharide (LPS)-induced acute lung injury (ALI). High-mobility group box 1 (HMGB1) serves as an inflammatory cytokine when released extracellularly and is involved in the pathogenesis of diverse inflammatory disorders. The current study aimed to investigate the involvement of HMGB1 in HDN-induced immunoregulation of ALI. ALI in male BALB/c mice was induced by intranasal administration of LPS (0.5mg/kg). HDN (500mg/kg) was administered intragastrically 10days prior to LPS exposure. HDN significantly protected animals from LPS-induced ALI as evidenced by decreased elevation of the lung wet to dry weight ratio, total cells, neutrophils, macrophages, and myeloperoxidase (MPO) activity, associated with reduced lung histological damage. In the meantime, HDN pretreatment markedly inhibited the production of pro-inflammatory cytokines and chemokine, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Furthermore, HDN pretreatment dramatically inhibited the infiltration of macrophages and suppressed the expression and release of HMGB1 in vivo and in vitro. In addition, intranasal application of exogenous HMGB1 could result in lung injury which was also alleviated by HDN administration. These results suggest that HDN pretreatment protects mice from LPS-induced ALI via inhibiting the production of TNF-α and IL-6. Moreover, we found that HDN could inhibit the expression and release of HMGB1 via suppressing the infiltration of macrophages and production of MCP-1.

[Sodium butyrate inhibits HMGB1 expression and release and attenuates concanavalin A-induced acute liver injury in mice].

The purpose of the present study is to explore the protective effects of sodium butyrate (SB) pretreatment on concanavalin A (Con A)-induced acute liver injury in mice. The model animals were first administered intraperitoneally with SB. Half an hour later, acute liver injury mouse model was established by caudal vein injection with Con A (15 mg/kg). Then, levels of serous alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using standard clinical method by an automated chemistry analyzer, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were measured by ELISA, and pathological changes in hepatic tissue were observed by using HE staining and light microscopy. The expression and release of high-mobility group box 1 (HMGB1) were assessed by using reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry and ELISA. The results showed that the pretreatment of SB significantly protected Con A-treated mice from liver injury as evidenced by the decrease of serum ALT, AST (P < 0.01) and reduction of hepatic tissues necrosis. SB also decreased levels of serous TNF-α and IFN-γ (P < 0.01). Furthermore, the expression and release of HMGB1 were markedly inhibited by SB pretreatment (P < 0.05 or P < 0.01). These results suggest that the attenuating effect of SB on Con A-induced acute liver injury may be due to its role of reducing the TNF-α and IFN-γ production, and inhibiting HMGB1 expression and release.

Protective effects of hesperidin on concanavalin A-induced hepatic injury in mice.

Hesperidin (HDN) is a citrus bioflavonoid, which widely exists in many plants. Previous researches have proved that HDN has several functions such as anti-oxidant, anti-tumor, anti-inflammatory, immune regulation and so on. In the present study, we explored the protective effects of HDN on concanavalin A (Con A)-induced hepatic injury. Acute hepatic injury model was established successfully by intravenous administration of Con A (15 mg/kg) in male C57BL/6 mice, and HDN was pretreated for 10 days before Con A challenge. It was found that the hepatic injury was notably improved in HDN pretreated mice. Furthermore, hepatic oxidative stress and the production of proinflammatory cytokines including TNF-α and IFN-γ were decreased by HDN pretreatment. More importantly, compared with Con A-treated mice, the expression and releasing of HMGB1 and T-cell activation were markedly reduced in HDN pretreated mice. Thus, these results suggest that HDN protects mice from Con A-induced hepatic injury by suppressing hepatocyte oxidative stress, producing cytokines, expressing and releasing HMGB1 and activating T cells.

High-mobility group box 1 exacerbates CCl4-induced acute liver injury in mice.

High-mobility group box 1 (HMGB1) is a nuclear factor that can also serve as an imflammatory mediator once released into extracellular milieu. Therefore, HMGB1 has been recognized to play a pivotal role in inflammatory diseases such as sepsis, acute lung injury, ischemia reperfusion injury and type 1 diabetes. Nevertheless, its impact on carbon tetrachloride (CCl4)-induced hepatic injury is yet to be elucidated. In the present report, we demonstrated evidence indicating that high levels of HMGB1 were not only present in the necrotic area of liver but also in the serum after CCl4 challenge. In line with these observations, administration of exogenous recombinant HMGB1 exacerbated CCl4-induced hepatic injury, while HMGB1 blocking antibody provided protection for mice against CCl4-induced acute liver injury as evidenced by the decrease of serum transaminase and reduction of hepatic tissues necrosis. Mechanistic studies revealed that blockade of HMGB1 attenuated CCl4-induced MDA accumulation along with improved SOD and GSH activity. Treatment of mice with HMGB1 neutralizing antibody also significantly inhibited the production of proinflammatory mediators TNF-α and IL-6 along with attenuated HMGB1 expression and its extracellular release. Together, our data suggest an essential role for HMGB1 in CCl4-induced acute liver injury, while HMGB1 neutralizing antibody could be served as an effective regimen for preventing CCl4-induced acute liver injury.