WITHDRAWN: Use of Tumor-Infiltrating Lymphocyte (TIL) Cell Therapy for Spindle Cell Carcinoma of the Breast: A Case Report

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HGFK1 Enhances the Anti-Tumor Effects of Angiogenesis Inhibitors via Inhibition of CD90+ CSCs in Hepatocellular Carcinoma.

The combination of anti-angiogenesis agents with immune-checkpoint inhibitors is a promising treatment for patients with advanced hepatocellular carcinoma (HCC); however, therapeutic resistance caused by cancer stem cells present in tumor microenvironments remains to be overcome. In this study, we report for the first time that the Kringle 1 domain of human hepatocyte growth-factor α chain (HGFK1), a previously described anti-angiogenesis peptide, repressed the sub-population of CD90+ cancer stem cells (CSCs) and promoted their differentiation and chemotherapy sensitivity mainly through downregulation of pre-Met protein expression and inhibition of Wnt/ß-catenin and Notch pathways. Furthermore, we showed that the i.p. injection of PH1 (a tumor-targeted and biodegradable co-polymer), medicated plasmids encoding Endostatin (pEndo), HGFK1 genes (pEndo), and a combination of 50% pEndo + 50% pHGFK1 all significantly suppressed tumor growth and prolonged the survival of the HCC-bearing mice. Importantly, the combined treatment produced a potent synergistic effect, with 25% of the mice showing the complete clearance of the tumor via a reduction in the microvessel density (MVD) and the number of CD90+ CSCs in the tumor tissues. These results suggest for the first time that HGFK1 inhibits the CSCs of HCC. Furthermore, the combination of two broad-spectrum anti-angiogenic factors, Endo and HGFK1, is the optimal strategy for the development of effective anti-HCC drugs.

Identification of bioactive compounds and potential mechanisms of scutellariae radix-coptidis rhizoma in the treatment of atherosclerosis by integrating network pharmacology and experimental validation.

OBJECTIVE: This study aims at investigating the potential targets and functional mechanisms of Scutellariae Radix-Coptidis Rhizoma (QLYD) against atherosclerosis (AS) through network pharmacology, molecular docking, bioinformatic analysis and experimental validation. METHODS: The compositions of QLYD were collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and literature, where the main active components of QLYD and corresponding targets were identified. The potential therapeutic targets of AS were excavated using the OMIM database, DrugBank database, DisGeNET database, CTD database and GEO datasets. The protein-protein interaction (PPI) network of common targets was constructed and visualized by Cytoscape 3.7.2 software. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis were performed to analyze the function of core targets in the PPI network. Molecular docking was carried out using AutoDockTools, AutoDock Vina, and PyMOL software to verify the correlation between the main components of QLYD and the core targets. Mouse AS model was established and the results of network pharmacology were verified by in vivo experiments. RESULTS: Totally 49 active components and 225 corresponding targets of QLYD were obtained, where 68 common targets were identified by intersecting with AS-related targets. Five hub genes including IL6, VEGFA, AKT1, TNF, and IL1B were screened from the PPI network. GO functional analysis reported that these targets had associations mainly with cellular response to oxidative stress, regulation of inflammatory response, epithelial cell apoptotic process, and blood coagulation. KEGG pathway analysis demonstrated that these targets were correlated to AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, IL-17 signaling pathway, MAPK signaling pathway, and NF-kappa B signaling pathway. Results of molecular docking indicated good binding affinity of QLYD to FOS, AKT1, and TNF. Animal experiments showed that QLYD could inhibit inflammation, improve blood lipid levels and reduce plaque area in AS mice to prevent and treat AS. CONCLUSION: QLYD may exert anti-inflammatory and anti-oxidative stress effects through multi-component, multi-target and multi-pathway to treat AS.

The long-term survival of the doublet regimen of concurrent chemoradiation therapy for locoregionally advanced nasopharyngeal carcinoma: a retrospective study.

OBJECTIVE: This study introduces innovative strategies, the doublet regimen of concurrent chemoradiotherapy, to ensure longer survival for locoregionally advanced nasopharyngeal carcinoma. METHODS: We retrospectively reviewed 104 locoregionally advanced nasopharyngeal carcinoma patients who underwent taxane combined platinum-based concurrent chemoradiotherapy in our center between January 2013 and December 2018. All statistical analyses were performed using the Kaplan-Meier method (SPSS 23.0). Different groups were compared with the Wilcoxon rank-sum test. RESULTS: Ultimately, 104 patients were selected for this study, including 18 and 86 who received either concurrent chemoradiation therapy alone or concurrent chemoradiation therapy plus adjuvant chemotherapy, respectively. The median follow-up time for progression free survival was 53.0 months (IQR 48.5-57.5). The 3-years progression-free survival (PFS), overall survival (OS), local-regional recurrence-free survival (LRRFS) and distant metastasis-free survival (DMFS) rates of the doublet regimen of concurrent chemotherapy for locoregionally advanced nasopharyngeal carcinoma were 85.9%, 96.0%, 96.0% and 90.8%, respectively. Additionally, we analyzed the subgroups and found that the 3-years PFS, OS, LRRFS and DMFS rates for stage III versus stage IVa were 97.8% versus 75.5% (P = 0.000), 100% versus 92.5% (P = 0.004), 100% versus 92.4% (P = 0.015) and 97.8% versus 82.8% (P = 0.002), respectively. During concurrent chemotherapy, acute chemotherapy adverse events of grade 3 or 4 was only 18.3%. Leukopenia was the most common acute chemotherapy adverse event (in 10 patients [9.6%]), followed by neutropenia (in 8 patients [7.6%]). CONCLUSION: The doublet regimen of taxane plus platinum concurrent chemoradiotherapy resulted in improved long-term survival of locoregionally advanced nasopharyngeal carcinoma patients, especially for local control rate and warrants further prospective evaluation.

PPP1R14D promotes the proliferation, migration and invasion of lung adenocarcinoma via the PKCα/BRAF/MEK/ERK signaling pathway.

Protein phosphatase 1 (PP1) inhibitors play a role in tumor progression through different mechanisms. Protein phosphatase 1 regulatory subunit 14D (PPP1R14D) is an inhibitor of PP1. However, the role of PPP1R14D in tumors and its mechanism of action are largely unknown. The purpose of the present study was to investigate the expression, function and mechanism of PPP1R14D in lung adenocarcinoma (LUAD). In the present study, GEPIA database analysis and immunohistochemistry demonstrated that PPP1R14D was highly expressed in LUAD tissues and that the expression of PPP1R14D in LUAD was negatively correlated with the age of patients and positively correlated with the 8th American Joint Committee on Cancer staging among patients. In addition, Kaplan­Meier Plotter database analysis showed that PPP1R14D expression was associated with lower survival rates in patients with LUAD. PPP1R14D knockdown significantly inhibited LUAD cell proliferation, migration and invasion and induced LUAD cell arrest at the G1 phase of the cell cycle. Mechanistic analyses revealed that PPP1R14D knockdown may inhibit cell proliferation, migration and invasion by inactivating PKCα/BRAF/MEK/ERK pathway signaling and its downstream key proteins c­Myc/Cyclin E1­CDK2 and MMP2/MMP9/Vimentin. Moreover, knockdown of PPP1R14D suppressed tumor growth in vivo. All these results showed that PPP1R14D plays an important role in LUAD tumorigenesis and may serve as a potential prognostic factor and therapeutic target in LUAD.

OLFM4 depletion sensitizes gallbladder cancer cells to cisplatin through the ARL6IP1/caspase-3 axis.

BACKGROUND: Gallbladder cancer (GBC) is a highly lethal malignancy that carries an extremely poor prognosis due to its chemoresistant nature. Cisplatin (CDDP) is a first-line chemotherapeutic for GBC; however, patients experienced no benefit when treated with CDDP alone. The underlying mechanisms of CDDP resistance in GBC remain largely unknown. METHODS: Agilent mRNA microarray analysis was performed between paired GBC and paracarcinoma to explore differentially expressed genes that might underlie drug resistance. Gene Set Enrichment Analysis (GSEA) was employed to identify key genes mediating CDDP resistance in GBC, and immunohistochemistry was performed to validate protein expression and test correlations with clinicopathological features. In vitro and in vivo functional assays were performed to investigate the proteins' roles in CDDP resistance. RESULTS: Olfactomedin 4 (OLFM4) was differentially expressed between GBC and paracarcinoma and had the highest rank metric score in the GSEA. OLFM4 expression was increasingly upregulated from chronic cholecystitis to GBC in clinical tissue samples, and OLFM4 depletion decreased GBC cell proliferation and invasion. Interestingly, downregulation of OLFM4 reduced ARL6IP1 (antiapoptotic factor) expression and sensitized GBC cells to CDDP both in vitro and in vivo. The evidence indicated that CDDP could significantly increase Bax and Bad expression and activate caspase-3 cascade in OLFM4-depleted GBC cells through ARL6IP1. Clinically, lower OLFM4 expression was associated with good prognosis of GBC patients. CONCLUSIONS: Our results suggest that OLFM4 is an essential gene that contributes to GBC chemoresistance and could serve as a prognostic biomarker for GBC. Importantly, OLFM4 could be a potential chemotherapeutic target.

Knockdown CYP2S1 inhibits lung cancer cells proliferation and migration.

BACKGROUND: The incidence of lung cancer in Yunnan area ranks firstly in the world and underlying molecular mechanisms of lung cancer in Yunnan region are still unclear. We screened a novel potential oncogene CYP2S1 used mRNA microassay and bioinformation database. The function of CYP2S1 in lung cancer has not been reported. OBJECTIVE: To investigate the functions of CYP2S1 in lung cancer. METHODS: Immunohistochemistry and Real-time PCR were used to verify the expression of CYP2S1. Colony formation and Transwell assays were used to determine cell proliferation, invasion and migration. Xenograft assays were used to detected cell growth in vivo. RESULTS: CYP2S1 is significantly up-regulated in lung cancer tissues and cells. Knockdown CYP2S1 in lung cancer cells resulted in decrease cell proliferation, invasion and migration in vitro. Animal experiments showed downregulation of CYP2S1 inhibited lung cancer cell growth in vivo. GSEA analysis suggested that CYP2S1 played functions by regulating E2F targets and G2M checkpoint pathway which involved in cell cycle. Kaplan-Meier analysis indicated that patients with high CYP2S1 had markedly shorter event overall survival (OS) time. CONCLUSIONS: Our data demonstrate that CYP2S1 exerts tumor suppressor function in lung cancer. The high expression of CYP2S1 is an unfavorable prognostic marker for patient survival.

How to Improve the Well-Being of Youths: An Exploratory Study of the Relationships Among Coping Style, Emotion Regulation, and Subjective Well-Being Using the Random Forest Classification and Structural Equation Modeling.

The relationship between coping styles and subjective well-being (SWB) has recently received considerable empirical and theoretical attention in the scientific literature. However, the mechanisms underlying this relationship have primarily remained unclear. The present research aimed to determine whether emotion regulation mediated the relationship between coping styles and subjective well-being (SWB). Our hypothesis is based on the integration of theoretical models among 1,247 Chinese college students. The SWB questionnaire, Ways of Coping Questionnaire, and Emotion Regulation Questionnaire were used to correlate SWB, emotion regulation strategies, and coping styles, respectively. The random forest method was applied to predict life satisfaction and estimate the average variable importance to life satisfaction. The results indicated that positive coping can indirectly influence life satisfaction via cognitive reappraisal and indirectly influence expression suppression via positive affect. Negative coping can indirectly influence negative affect via expression suppression. Besides, negative coping was positively associated with both expression suppression and negative affect. Cognitive reappraisal was found to be positively associated with positive affect. The findings indicated that coping style is essential for the SWB of college students. These findings provide insight into how coping styles impact SWB and have implications for developing and assessing emotion regulation-based interventions.

Identification of a 4-lncRNA signature predicting prognosis of patients with non-small cell lung cancer: a multicenter study in China.

BACKGROUND: Previous findings have indicated that the tumor, nodes, and metastases (TNM) staging system is not sufficient to accurately predict survival outcomes in patients with non-small lung carcinoma (NSCLC). Thus, this study aims to identify a long non-coding RNA (lncRNA) signature for predicting survival in patients with NSCLC and to provide additional prognostic information to TNM staging system. METHODS: Patients with NSCLC were recruited from a hospital and divided into a discovery cohort (n = 194) and validation cohort (n = 172), and detected using a custom lncRNA microarray. Another 73 NSCLC cases obtained from a different hospital (an independent validation cohort) were examined with qRT-PCR. Differentially expressed lncRNAs were determined with the Significance Analysis of Microarrays program, from which lncRNAs associated with survival were identified using Cox regression in the discovery cohort. These prognostic lncRNAs were employed to construct a prognostic signature with a risk-score method. Then, the utility of the prognostic signature was confirmed using the validation cohort and the independent cohort. RESULTS: In the discovery cohort, we identified 305 lncRNAs that were differentially expressed between the NSCLC tissues and matched, adjacent normal lung tissues, of which 15 are associated with survival; a 4-lncRNA prognostic signature was identified from the 15 survival lncRNAs, which was significantly correlated with survivals of NSCLC patients. This signature was further validated in the validation cohort and independent validation cohort. Moreover, multivariate Cox analysis demonstrates that the 4-lncRNA signature is an independent survival predictor. Then we established a new risk-score model by combining 4-lncRNA signature and TNM staging stage. The receiver operating characteristics (ROC) curve indicates that the prognostic value of the combined model is significantly higher than that of the TNM stage alone, in all the cohorts. CONCLUSIONS: In this study, we identified a 4-lncRNA signature that may be a powerful prognosis biomarker and can provide additional survival information to the TNM staging system.

Increased expression of the RNA-binding motif protein 47 predicts poor prognosis in non-small-cell lung cancer.

Lung cancer is the leading cause of cancer-associated mortality worldwide. In China, in particular, lung cancer mortality has markedly increased and is likely to continue to rise. RNA-binding proteins are pivotal to the development and progression of a variety of cancer types, including non-small cell lung cancer (NSCLC). RNA-binding motif protein 47 (RBM47) has been found to act as a tumor suppressor in breast cancer and NSCLC. However, to the best of our knowledge, RBM47 expression in NSCLC tissues has yet to be investigated. Analysis via the online database, Gene Expression Omnibus, revealed that RBM47 was upregulated in NSCLC and associated with pathological type, suggesting that RBM47 may play different roles in lung adenocarcinoma and lung squamous cell carcinoma. In the present study, the expression of RBM47 was examined by immunohistochemistry in 175 pairs of tumor and adjacent non-cancerous tissues resected from patients with NSCLC. The results indicated that the expression of RBM47 was significantly increased in NSCLC samples compared with that in the matched non-cancerous samples. Furthermore, RBM47 expression was higher in Xuanwei compared with that in non-Xuanwei NSCLC, suggesting that RBM47 is a more sensitive biomarker in Xuanwei NSCLC, and that it may serve as a candidate therapeutic target. In addition, RBM47 expression was associated with the pathological type, however not with the age, sex, lymph node metastasis, pT stage or pathological Tumor-Node-Metastasis stage of the patients. The increased expression level of RBM47 may indicate a worse overall survival rate for patients with NSCLC. In addition, multivariate survival analysis showed that the Xuanwei area is associated with poor prognosis for patients with NSCLC. In conclusion, the present study revealed that the upregulation of RBM47 accelerated the malignant progression of NSCLC, indicating that RBM47 may be a potential biomarker for NSCLC progression and a therapeutic target for NSCLC.

FAM136A immunoreactivity is associated with nodal involvement and survival in lung adenocarcinoma in a Chinese case series.

Lung cancer patients with lymph node metastasis usually had short overall survival and occurred distant metastases at the early stage. However, some of these people did have more prolonged survival. The underlying reason is still unclear. In this study, we found a novel molecule, family with sequence similarity 136, member A gene (FAM136A). First, we performed immunohistochemistry for FAM136A in 177 lung carcinoma tissues. Second, we carried out in vitro studies by using A549 and PC-9. We detected FAM136A immunoreactivity in 79 out of 177 (44.6%) lung carcinoma tissues, and the FAM136A status was significantly associated with tumor T stage, lymph node metastasis, and the Tumor-Node-Metastasis (TNM) staging system in these cases. Importantly, it was significantly associated with the overall survival of the patients with lymph node metastasis, especially FAM136A positive patients, who had worse outcomes. Subsequent in vitro experiments revealed that the proliferation activity and migration property decreased both A549 and PC-9 lung carcinoma cells transfected with siRNA-FAM136A, and apoptosis reduced. Meanwhile, the expression of CDK4 and CDK6 decreased. FAM136A status would be a potent, worse prognostic factor in lung cancer patients with lymph node metastasis. It would play a vital role in the proliferation, apoptosis, and migration properties of A549 and PC-9. In the future, We will focus on the uncovered signal mechanism between FAM136A and lung cancer.

CD19 and CD30 CAR T-Cell Immunotherapy for High-Risk Classical Hodgkin's Lymphoma.

BACKGROUND: In clinical applications of CAR T-cell therapy, life-threatening adverse events including cytokine release syndrome and neurotoxicity can lead to treatment failure. Outcomes of patients treated with anti-CD30 CAR T- cell have been disappointing in relapsing/refractory (r/r) classical Hodgkin's Lymphoma (cHL). METHODS: In order to understand the applicable population of multiple CAR T-cell therapy, we examined the expression of CD19, CD20, and CD30 by immunohistochemistry (IHC) in 38 paraffin-embedded specimens of cHL. In the past two years, we found only one patient with cHL who is eligible for combined anti-CD19 and CD30 CAR T-cell treatment. This patient's baseline characteristics were prone to severe adverse events. We treated this patient with low doses and multiple infusions of anti-CD19 and CD30 CAR T-cell. RESULTS: The positive expression of CD19+ + CD30+ in Reed-Sternberg (RS) cells is approximately 5.2% (2/38). The patient we treated with combined anti-CD19 and CD30 CAR T-cell did not experience severe adverse events related to CAR T-cell therapy and received long term progression-free survival (PFS). CONCLUSION: For high risk r/r cHL patients, low doses of CAR T-cell used over different days at different times might be safe and effective. More clinical trials are warranted for CD19 and CD30 CAR T-cell combination therapy.

Knockdown of microsomal glutathione S-transferase 1 inhibits lung adenocarcinoma cell proliferation and induces apoptosis.

BACKGROUND: Lung cancer has been the most common cancer worldwide. Microsomal glutathione S-transferase 1 (MGST1) has been reported to play vital roles in oxidative stress, tumor occurrence and drug resistance. However, the biological function and molecular mechanism of MGST1 in lung adenocarcinoma (LUAD) has not yet been elucidated. METHODS: The expression of MGST1 in LUAD tissues and cell lines was evaluated by immunohistochemistry and western blotting, respectively. MGST1 was knocked down by shRNA lentivirus. Cell proliferation was evaluated by MTS, colony formation and EdU assays. Apoptosis was detected by flow cytometry. The potential molecules involved in cell proliferation and apoptosis were examined by western blotting. Finally, the effect of MGST1 on tumor growth in vivo was evaluated in a nude mouse xenograft model. RESULTS: TCGA database analysis and immunohistochemistry demonstrated that MGST1 was highly expressed in LUAD tissues. MGST1 expression in LUAD was correlated with AJCC stage and poor overall survival of patients. MGST1 knockdown significantly inhibited LUAD cell proliferation and induced apoptosis. Mechanistic analyses revealed that MGST1 knockdown might inhibit cell proliferation by inactivating the AKT/GSK-3ß pathway signaling and promote cell apoptosis by regulating the mitochondrial apoptosis pathway related proteins. Moreover, knockdown of MGST1 suppressed tumor growth in vivo. CONCLUSIONS: MGST1 plays an important role in LUAD tumorigenesis and might serve as a potential prognostic factor and therapeutic target in LUAD.

HDGF and PRKCA upregulation is associated with a poor prognosis in patients with lung adenocarcinoma.

Lung adenocarcinoma is the most common histologic subtype of lung cancer. The aim of the present study was to assess the expression of hepatoma-derived growth factor (HDGF) and protein kinase Cα (PRKCA) in lung adenocarcinoma (LADC), and to determine the association between the combined expression of these two proteins and clinicopathological characteristics of patients with LADC. The expression of HDGF and PRKCA mRNA was assessed by GEO database analysis, and HDGF and PRKCA protein levels were examined by immunohistochemistry using a tissue microarray. High HDGF and PRKCA expression was observed in LADC tissue compared to normal samples, and increased HDGF and PRKCA expression was associated with AJCC clinical stage, tumor classification, node classification, and lymph node metastasis. GEO database analysis revealed no significant differences between HDGF mRNA and PRKCA mRNA in LADC tissue. However, high PRKCA protein expression was associated with high HDGF protein expression, and patients with high HDGF and PRKCA expression exhibited poorer overall survival rates than patients with low expression levels of the two proteins. The results of the present study suggest that upregulation of both HDGF and PRKCA may be an unfavourable factor for lung adenocarcinoma progression.

Knockdown of STIM1 expression inhibits non-small-cell lung cancer cell proliferation in vitro and in nude mouse xenografts.

Stromal interaction molecule 1 (STIM1) is a calcium-sensing protein localized in the membrane of the endoplasmic reticulum. The expression of STIM1 has been shown to be closely associated with cell proliferation. The aim of the present study was to investigate the role of STIM1 in the regulation of cancer progression and its clinical relevance. The data demonstrated that the expression of the STIM1 was significantly higher in non-small-cell lung cancer (NSCLC) tissues than in benign lesions and was associated with advanced NSCLC T stage. Knockdown of STIM1 expression in NSCLC cell lines A549 and SK-MES-1 significantly inhibited cell proliferation and induces A549 and SK-MES-1 cell arrest at the G2/M and S phases of the cell cycle. Western blotting showed that the expression of cyclin-dependent kinase (CDK) 1 and CDK2 were reduced while knockdown of STIM1 expression. Furthermore, knockdown of STIM1 in NSCLC cells significantly reduced the levels of xenograft tumor growth in nude mice. These data indicate that aberrant expression of the STIM1 protein may contribute to NSCLC progression. Future studies should focus on targeting STIM1 as a novel strategy for NSCLC therapy.

Curcumin inhibits cell proliferation and migration in NSCLC through a synergistic effect on the TLR4/MyD88 and EGFR pathways.

Despite the increasing number of available therapeutic methods, the prognosis of non­small cell lung cancer (NSCLC) remains poor. Furthermore, side effects are an important limiting factor in the treatment of NSCLC. Therefore, developing an efficacious, safe, affordable and easily accessible chemotherapeutic agent is necessary for NSCLC treatment. As a natural chemical produced by Zingiberaceae plants, curcumin exerts distinct antitumor effects on several tumor types. In the present study, curcumin was observed to inhibit not only cell proliferation and cell cycle transition, but also cell migration in NSCLC, as determined by a series of experiments (such as MTS assay, colony formation assay, flow cytometric analysis, Transwell migration assay and western blotting). Mechanistically, curcumin induced G2/M phase arrest by controlling cell cycle­ and epithelial­mesenchymal transition (EMT)­related checkpoints. Furthermore, curcumin significantly inhibited the expression of Toll­like receptor 4 (TLR4)/MyD88 and EGFR in a dose­ and time­dependent manner. Conversely, EGF reversed the inhibitory action of curcumin on TLR4/MyD88. In clinical specimens, TLR4 and MyD88 were highly expressed in NSCLC tissues, and a significant positive association was observed between TLR4 and MyD88 expression. These data suggested that curcumin may control the EGFR and TLR4/MyD88 pathways to synergistically downregulate downstream cell cycle­ and EMT­related regulators, in order to block cell proliferation and metastasis in NSCLC. These findings provide evidence for the clinical application of curcumin.

Prognostic value of tumor-infiltrating lymphocytes in melanoma: a systematic review and meta-analysis.

Tumor-infiltrating lymphocytes (TILs) are associated with prognosis in various tumors. However, it remains controversial whether the presence of TILs is related to an improved prognosis in melanoma. This meta-analysis confirmed the favorable prognostic role of the CD3+, CD4+, CD8+, FOXP3+, and CD20+ TILs in the overall survival of melanoma patients and found an association between the TILs present and improved overall survival. Additionally, subgroup analysis demonstrated that brisk TILs were obviously associated with OS, RFS and DSS/MSS. Thus, TILs may be a predictive biomarker in melanoma. This analysis will provide more insight into the study of TILs and predictive biomarker.

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters.

BACKGROUND: Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800. RESULTS: Six recombinant strains harboring different plasmids with a single promoter (PP43, PHpaII, PBcaprE, PgsiB, PyxiE or PluxS) were constructed, and the analysis of the fibrinolytic activity showed that PP43 and PHpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by PP43 and PHpaII reached 140.5 ± 3.9 FU/ml and 110.8 ± 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive PP43 or PHpaII increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of PP43 or PHpaII could effectively enhance NK production. Eight triple-promoters with PP43 and PHpaII in different orders were constructed, and the highest yield of NK finally reached 264.2 ± 7.0 FU/ml, which was mediated by the promoter PHpaII-PHpaII-PP43. The scale-up production of NK that was promoted by PHpaII-PHpaII-PP43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL. CONCLUSIONS: Our studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by PHpaII-PHpaII-PP43, which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes.

SHIP1 inhibits cell growth, migration, and invasion in non­small cell lung cancer through the PI3K/AKT pathway.

Src homology 2­containing inositol­5'­phosphatase 1 (SHIP1) serves a vital role in the occurrence and development of hematological tumors, but there is limited knowledge regarding the role of SHIP1 in various solid tumors, including lung cancer. In the present study, the aim was to investigate the expression and functional mechanisms of SHIP1 in non­small cell lung cancer (NSCLC). The Gene Expression Omnibus database demonstrated that SHIP1 had low expression in NSCLC. Further studies using fresh tissues and cell lines also confirmed this observation. Biological function analyses revealed that SHIP1 overexpression notably suppressed cell growth, migration and invasion in vitro and in vivo in NSCLC. Mechanistic analyses indicated that SHIP1 inactivated the phosphoinositide 3­kinase (PI3K)/AKT pathway to suppress signals associated with the cell cycle and epithelial­mesenchymal transition. In clinical specimens, reduced SHIP1 is an unfavorable factor and is negatively associated with the T classification, N classification and clinical stage. Furthermore, patients with low SHIP1 levels exhibited reduced survival rate, compared with patients with high levels of the protein. Notably, the promoter of the SHIP1 gene lacks CpG islands, and the suppression of SHIP1 expression is not associated with epidermal growth factor receptor or Kirsten rat sarcoma mutations. Thus, the present study demonstrated that SHIP1 inhibits cell growth, migration and invasion in NSCLC through the PI3K/AKT pathway. Additionally, reduced SHIP1 expression may be an unfavorable factor for NSCLC.

Anticancer effect of the traditional Chinese medicine herb Maytenus compound via the EGFR/PI3K/AKT/GSK3ß pathway.

BACKGROUND: Cancer is a leading cause of death worldwide; folk anticancer medicinal plants have applied for cancer treatment. The Maytenus compound tablet as traditional Chinese compound medicine has been approved for alleviating hyperplasia of mammary glands, whether it can inhibit cancer still unknown. The study was to evaluate the anticancer activity of the Maytenus compound tablet. METHODS: MTS assay evaluated the anti-proliferation effect of the Maytenus compound on H226, A2058, 786O and HeLa cancer cells and two normal epithelial cell lines, namely, 16HBE and Hecate. Nude mouse xenograft tumor model using H226 and HeLa cells examined the drug's anticancer effect in vivo. Western blot assay studied the possible mechanism. RESULTS: The Maytenus compound indicated obvious ability to against proliferation in four strains of cancer cells, particularly against H226 cells by an IC50 of 85.47±10.06 µg/mL and against HeLa cells by an IC50 of 128.74±17.46 µg/mL. However, it had a low cytotoxicity in human normal epithelial cell lines 16HBE with an IC50 of 4,555.86±25.21 µg/mL and Hecate with an IC50 of 833.56±181.88 µg/mL. The Maytenus compound at the 2.45 g/kg oral dosages inhibited the proliferation of H226 cells and HeLa cells in nude mouse with inhibitory rates of 36.06% and 26.45%, respectively, and no organ toxicity. The Maytenus compound could significantly downregulate the expression of pEGFR, pPI3K, pAKT, pGSK3ß, ß-catenin, and c-MYC and upregulate the protein expression of GSK3ß. CONCLUSIONS: The Maytenus compound has significant anticancer activities against human cancer H226 and HeLa cells both in vitro and in vivo, highlighting it may be an anticancer medicine.