Novel structured ADAM17 small-molecule inhibitor represses ADAM17/Notch pathway activation and the NSCLC cells' resistance to anti-tumour drugs.

Background and aims: The outcomes of current treatment for non-small cell lung cancer (NSCLC) are unsatisfactory and development of new and more efficacious therapeutic strategies are required. The Notch pathway, which is necessary for cell survival to avert apoptosis, induces the resistance of cancer cells to antitumour drugs. Notch pathway activation is controlled by the cleavage of Notch proteins/receptors mediated by A disintegrin and metalloproteinase 17 (ADAM17); therefore, ADAM17 is a reliable intervention target for anti-tumour therapy to overcome the drug resistance of cancer cells. This work aims to develop and elucidate the activation of Compound 2b, a novel-structured small-molecule inhibitor of ADAM17, which was designed and developed and its therapeutic efficacy in NSCLC was assessed via multi-assays. Methods and results: A lead compound for a potential inhibitor of ADAM17 was explored via pharmacophore modelling, molecular docking, and biochemical screening. It was augmented by substituting two important chemical groups [R1 and R2 of the quinoxaline-2,3-diamine (its chemical skeleton)]; subsequently, serial homologs of the lead compound were used to obtain anoptimized compound (2b) with high inhibitory activity compared with leading compound against ADAM17 to inhibit the cleavage of Notch proteins and the accumulation of the Notch intracellular domain in the nuclei of NSCLC cells. The inhibitory activity of compound 2b was demonstrated by quantitative polymerase chain reaction and Western blotting. The specificity of compound 2b on ADAM17 was confirmed via point-mutation. Compound 2b enhanced the activation of antitumor drugs on NSCLC cells, in cell lines and nude mice models, by targeting the ADAM17/Notch pathway. Conclusion: Compound 2b may be a promising strategy for NSCLC treatment.

Molecular characterization based on tumor microenvironment-related signatures for guiding immunotherapy and therapeutic resistance in lung adenocarcinoma.

Background: Although the role of tumor microenvironment in lung adenocarcinoma (LUAD) has been explored in a number of studies, the value of TME-related signatures in immunotherapy has not been comprehensively characterized. Materials and Methods: Consensus clustering was conducted to characterize TME-based molecular subtypes using transcription data of LUAD samples. The biological pathways and immune microenvironment were assessed by CIBERSORT, ESTIMATE, and gene set enrichment analysis. A TME-related risk model was established through the algorithms of least absolute shrinkage and selection operator (Lasso) and stepwise Akaike information criterion (stepAIC). Results: Four TME-based molecular subtypes including C1, C2, C3, and C4 were identified, and they showed distinct overall survival, genomic characteristics, DNA methylation pattern, immune microenvironment, and biological pathways. C1 had the worst prognosis and high tumor proliferation rate. C3 and C4 had higher enrichment of anti-tumor signatures compared to C1 and C2. C4 had evidently low enrichment of epithelial-mesenchymal transition (EMT) signature and tumor proliferation rate. C3 was predicted to be more sensitive to immunotherapy compared with other subtypes. C1 is more sensitive to chemotherapy drugs, including Docetaxel, Vinorelbine and Cisplatin, while C3 is more sensitive to Paclitaxel. A five-gene risk model was constructed, which showed a favorable performance in three independent datasets. Low-risk group showed a longer overall survival, more infiltrated immune cells, and higher response to immunotherapy than high-risk group. Conclusion: This study comprehensively characterized the molecular features of LUAD patients based on TME-related signatures, demonstrating the potential of TME-based signatures in exploring the mechanisms of LUAD development. The TME-related risk model was of clinical value to predict LUAD prognosis and guide immunotherapy.

The clinical and genomic distinctions of Class1/2/3 BRAF-mutant colorectal cancer and differential prognoses.

BRAF mutations are the oncogenic drivers in colorectal cancer and V600 mutations (Class1), which lead to RAS-independent active monomers, are the most common mutation types. BRAF non-V600 mutants can be further classified as RAS-independent active dimers (Class2) and RAS-dependent impaired kinase (Class3). We retrospectively reviewed the mutational profiles of 328 treatment-naïve colorectal tumors with BRAF mutations detected using capture-based hybrid next-generation sequencing targeting 400 + cancer-related genes. The clinical and genetic distinctions of patients harboring Class1/2/3 BRAF mutations were investigated, which revealed that tumors with Class1 BRAF mutations showed more unique genomic profiles than those with Class2/3 mutations. Also, by using an external dataset from cBioPortal, we demonstrated that patients with Class3 BRAF mutations had the best survival outcomes compared to the other two subgroups. These findings promoted the development of anti-BRAF strategies by distinguishing BRAF mutant subgroups.

PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies.

In spite of impressive success in treating hematologic malignancies, adoptive therapy with chimeric antigen receptor modified T cells (CAR T) has not yet been effective in solid tumors, where identification of suitable tumor-specific antigens remains a major obstacle for CAR T-cell therapy due to the "on target off tumor" toxicity. Protein tyrosine kinase 7 (PTK7) is a member of the Wnt-related pseudokinases and identified as a highly expressed antigen enriched in cancer stem cells (CSCs) from multiple solid tumors, including but not limited to triple-negative breast cancer, non-small-cell lung cancer, and ovarian cancer, suggesting it may serve as a promising tumor-specific target for CAR T-cell therapy. In this study, we constructed three different PTK7-specific CAR (PTK7-CAR1/2/3), each comprising a humanized PTK7-specific single-chain variable fragment (scFv), hinge and transmembrane (TM) regions of the human CD8α molecule, 4-1BB intracellular co-stimulatory domain (BB-ICD), and CD3ζ intracellular domain (CD3ζ-ICD) sequence, and then prepared the CAR T cells by lentivirus-mediated transduction of human activated T cells accordingly, and we sequentially evaluated their antigen-specific recognition and killing activity in vitro and in vivo. T cells transduced with all three PTK7-CAR candidates exhibited antigen-specific cytokine production and potent cytotoxicity against naturally expressing PTK7-positive tumor cells of multiple cancer types without mediating cytotoxicity of a panel of normal primary human cells; meanwhile, in vitro recursive cytotoxicity assays demonstrated that only PTK7-CAR2 modified T cells retained effective through multiple rounds of tumor challenge. Using in vivo xenograft models of lung cancers with different expression levels of PTK7, systemic delivery of PTK7-CAR2 modified T cells significantly prevented tumor growth and prolonged overall survival of mice. Altogether, our results support PTK7 as a therapeutic target suitable for CAR T-cell therapy that could be applied for lung cancers and many other solid cancers with PTK7 overexpression.

Radiosensitizing effect of gold nanoparticle loaded with small interfering RNA-SP1 on lung cancer: AuNPs-si-SP1 regulates GZMB for radiosensitivity.

Radioresistance is a major challenge that largely limits the efficacy of radiotherapy in lung cancer. Gold nanoparticles (AuNPs) are emerging as novel radiosensitizers for cancer patients. Therefore, this study was designed to explore the radiosensitizing effect and mechanism of AuNPs loaded with small interfering RNA (siRNA)-SP1 (AuNPs-si-SP1) on lung cancer. AuNPs-si-SP1 was prepared by the noncovalent binding between AuNPs and siRNA-SP1. The adsorption capacity of AuNPs to siRNA-SP1 was analyzed by gel electrophoresis. The cell uptake of AuNPs-si-SP1 was observed under a laser confocal microscopy. Silencing efficacy of AuNPs-si-SP1 was validated by RT-qPCR and Western blot analysis. Cell viability was determined by CCK-8 assay, radiosensitization by plate colony formation assay, cell apoptosis and cell cycle by flow cytometry, and DNA double strand breaks by immunofluorescence in the presence or absence of AuNPs-si-SP1 or GZMB. The downstream mechanism of SP1 was predicted by bioinformatics analysis, followed by verification by Western blot analysis. Subcutaneous tumorigenesis in nude mice was established to verify the radiosensitization of AuNPs-si-SP1 and GZMB in vivo. AuNPs-si-SP1 effectively absorbed SP1 siRNA and was highly internalized by A549 cells to reduce SP1 protein expression. AuNPs-si-SP1 or GZMB overexpression promoted cells to G2/M phase, DNA double strand breaks, and enhanced radiosensitivity. SP1 could repress GZMB expression in lung cancer cells. In vivo experiments manifested that AuNPs-si-SP1 could inhibit the growth of solid tumor in nude mice to achieve radiosensitization by inhibiting SP1 to upregulate GZMB. AuNPs-si-SP1 might increase the radiosensitivity of lung cancer by inhibiting SP1 to upregulate GZMB.

Novel small molecule inhibitors of the transcription factor ETS-1 and their antitumor activity against hepatocellular carcinoma.

The transcription factor ETS-1 (E26 transformation specific sequence 1) is the key regulator for malignant tumor cell proliferation and invasion by mediating the transcription of the invasion/migration related factors, e.g. MMPs (matrix metalloproteinases). This work aims to identify the novel small molecule inhibitors of ETS-1 using a small molecule compound library and to study the inhibitors' antitumor activity against hepatocellular carcinoma (HCC). The luciferase reporter is used to examine the inhibition and activation of ETS-1's transcription factor activity in HCC cells, including a highly invasive HCC cell line, MHCC97-H, and five lines of patient-derived cells. The inhibition of the proliferation of HCC cells is examined using the MTT assay, while the invasion of HCC cells is examined using the transwell assay. The anti-tumor activity of the selected compound on HCC cells is also examined in a subcutaneous tumor model or intrahepatic tumor model in nude mice. The results show that for the first time, four compounds, EI1~EI-4, can inhibit the transcription factor activation of ETS-1 and the proliferation or invasion of HCC cells. Among the four compounds, EI-4 has the best activation. The results from this paper contribute to expanding our understanding of ETS-1 and provide alternative, the safer and more effective, HCC molecular therapy strategies.

[Mechanism and Research Progress of Microbiome in the Development of Lung Cancer].

Lung cancer is one of the most common malignant tumors with poor prognosis in China, and most patients are diagnosed as advanced patients. Studies have shown that the microecological characteristics of lung cancer patients are different from healthy people, and the microorganisms of the respiratory tract can affect the occurrence and development of lung cancer through various mechanisms. In recent years, the study of the correlation between microbiome and disease has become another research hotspot following the Human Genome Project. However, at present, there are relatively few studies on the characteristics of lung cancer and respiratory microbiome. Therefore, it is necessary to further explore the potential relationship between lung cancer and microbial flora. By studying the mechanism of action of respiratory microorganisms in the development of lung cancer, it is expected to provide a clearer scientific basis in the clinical diagnosis, treatment and prognosis assessment of lung cancer. This article reviews the current researches on microbial flora and lung cancer, and provides new ideas for clinical diagnosis and treatment of lung cancer.
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Electroacupuncture alleviates polycystic ovary syndrome-like symptoms through improving insulin resistance, mitochondrial dysfunction, and endoplasmic reticulum stress via enhancing autophagy in rats.

BACKGROUND: Electroacupuncture (EA), a treatment derived from traditional Chinese medicine, can effectively improve hyperandrogenism and insulin resistance in patients with polycystic ovary syndrome (PCOS), however, its underlying mechanisms remain obscure. This study aimed to investigate whether EA could mitigate PCOS-like symptoms in rats by regulating autophagy. METHODS: A rat model of PCOS-like symptoms was established by subcutaneous injection with dehydroepiandrosterone (DHEA), and then EA treatment at acupoints (ST29 and SP6) was carried out for 5 weeks. To inhibit autophagy in rats, intraperitoneal injection with 0.5 mg/kg 3-MA (an autophagy inhibitor) was performed at 30 min before each EA treatment. RESULTS: EA intervention alleviated PCOS-like symptoms in rats, which was partly counteracted by the combination with 3-MA. Moreover, DHEA-exposure-induced deficient autophagy in skeletal muscle was improved by EA treatment. EA-mediated improvements in insulin resistance, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in PCOS-like rats were counteracted by 3-MA pretreatment. Mechanically, EA attenuated autophagy deficiency-mediated insulin resistance in PCOS-like rats via inactivating mTOR/4E-BP1 signaling pathway. CONCLUSIONS: Taken together, our findings indicate that EA treatment ameliorates insulin resistance, mitochondrial dysfunction, and ER stress through enhancing autophagy in a PCOS-like rat model. Our study provides novel insight into the mechanisms underlying the treatment of EA in PCOS, which offers more theoretic foundation for its clinical application.

Ultra-high dose rate effect on circulating immune cells: A potential mechanism for FLASH effect?

PURPOSE: "FLASH" radiotherapy (RT) is a potential paradigm-changing RT technology with marked tumor killing and normal tissue sparing. However, the mechanism of the FLASH effect is not well understood. We hypothesize that the ultra-high dose rate FLASH-RT significantly reduces the killing of circulating immune cells which may partially contribute to the reported FLASH effect. METHODS: This computation study directly models the effect of radiation dose rate on the killing of circulating immune cells. The model considers an irradiated volume that takes up A% of cardiac output and contains B% of total blood. The irradiated blood volume and dose were calculated for various A%, B%, blood circulation time, and irradiation time (which depends on the dose rate). The linear-quadratic model was used to calculate the extent of killing of circulating immune cells at ultra-high vs. conventional dose rates. RESULTS: A strong sparing effect on circulating blood cells by FLASH-RT was noticed; i.e., killing of circulating immune cells reduced from 90% to 100% at conventional dose rates to 5-10% at ultra-high dose rates. The threshold FLASH dose rate was determined to be ~40 Gy/s for mice in an average situation (A% = 50%), consistent with the reported FLASH dose rate in animal studies, and it was approximately one order of magnitude lower for humans than for mice. The magnitude of this sparing effect increased with the dose/fraction, reached a plateau at 30-50 Gy/fraction, and almost completely vanished at 2 Gy/fraction. CONCLUSION: We have calculated a strong sparing effect on circulating immune cells by FLASH-RT, which may contribute to the reported FLASH effects in animal studies.

Effect of autophagy on the resveratrol-induced apoptosis of ovarian cancer SKOV3 cells.

OBJECTIVE: This study aims to evaluate the relationship between apoptosis and autophagy induced by resveratrol (Res) in SKOV3 human ovarian cancer cell lines. METHODS: The experiment was divided into four groups: normal control group, Res group, Res combined with autophagy inhibitor 3-methyladenine (Res+3-MA) group, and Z-VAD-FMK group. SKOV3 cells were cultured and treated with Res and 3-MA for 24 hours. The processing concentration of Res was screened out by the Cell Counting Kit-8 (CCK8) assay. The cell survival rate was measured by the CCK8 assay. The expression of bule-associated protein light chain 3 beta 2 (LC3-II) and Beclin-1 was detected using Western blot analysis. The percentages of apoptotic and autophagic cells were analyzed using flow cytometry. RESULTS: The cell survival rate significantly decreased as Res concentration increased, and the differences were statistically significant (P < 0.05). The processing concentration of Res was 25 µmol/L. After treatment with Res for 24 hours, the expression levels of autophagy-related protein LC3 and Beclin-l were significantly higher than in the other groups. Furthermore, the expression of LC3 and Beclin-l significantly declined in the Res+3-MA group compared with the Res group. However, the percentage of autophagic cells significantly decreased from 37.0% ± 4.24% to 6.1% ± 0.28%, and the percentage of apoptotic cells significantly increased from 24% ± 4.55% to 67.0% ± 4.3%; and the differences were statistically significant ( P < 0.05). CONCLUSION: Res can induce autophagy to inhibit apoptosis in tumor SKOV3 cells, and inhibition of Res+3-MA could not only enhance the effects of chemotherapy but also prevent normal cells from tumorigenesis.

MicroRNA-144 functions as a tumor suppressor in gastric cancer by targeting cyclooxygenase-2.

Gastric cancer (GC) poses a serious public health threat and the 5-year survival rate of patients with GC is low. MicroRNAs (miRNAs/miRs) may serve oncogenic or tumor suppressor functions during tumorigenesis by regulating cell proliferation, apoptosis, migration and invasion and it has been demonstrated that they may be dysregulated in various types of cancer. The present study demonstrated that miR-144 and GATA4 were downregulated in GC tissues and cell lines and suggested that this may be due to hypermethylation. Additionally, miR-144 and GATA4 had synergistic effects on GC cells by repressing cell proliferation and inducing cell cycle arrest and apoptosis. The results of bioinformatics and a luciferase reporter assay indicated that cyclooxygenase-2 (COX-2) is a direct target of miR-144 and that miR-144 negatively regulated the expression of COX-2, which inhibits the viability of GC cells. GATA4 also induced a similar effect on COX-2. Taken together, the results of the present study may improve understanding of the underlying mechanism of miR-144 and GATA4 in GC.

Slug Is Associated With Tumor Metastasis and Angiogenesis in Ovarian Cancer.

Ovarian cancer is the most lethal gynecologic malignancy among women and usually initiated by the malignant transformation of epithelial cells. The progression of ovarian cancer involves a cascade of events, including tumor cell epithelial-mesenchymal transition (EMT), invasion, migration, and angiogenesis. Slug plays vital roles in the development of motile and invasive manner of cancer cells via EMT progression. The present work is devoted to investigate the effect of slug on the invasion and angiogenesis in ovarian cancer. The findings reveal that tumors with high expression of slug (44 of 60) represent higher tumor grade, lymph node metastasis, and worse prognosis than those with low expression (16 of 60; P < .05). We also identified a significant correlation between the slug and the microvessel density (MVD). Results of transwell migration assay showed that decreased slug induced by short hairpin RNA contributed to the repressed invasion and migration of SKOV3 cells. Additionally, the migration and tube formation capacity of human umbilical vein endothelial cells were markedly decreased in SKOV3-sh-conditioned medium compared to SKOV3 and SKOV3-NC. Furthermore, xenograft mouse models (SKOV3/SKOV3-sh cells injection into BALB/c nude mice) were developed to validate the effects of slug. The data confirmed that inhibited expression of slug extensively decreased the growth of tumor and MVD in vivo. Moreover, knockdown of slug can significantly reduce tumor angiogenesis of SKOV3 cells via ccn1/vascular endothelial growth factor. Thus, our present study demonstrates that slug is closely associated with tumor metastasis and angiogenesis in ovarian cancer.

RhNRG-1ß Protects the Myocardium against Irradiation-Induced Damage via the ErbB2-ERK-SIRT1 Signaling Pathway.

Radiation-induced heart disease (RIHD), which is a serious side effect of the radiotherapy applied for various tumors due to the inevitable irradiation of the heart, cannot be treated effectively using current clinical therapies. Here, we demonstrated that rhNRG-1ß, an epidermal growth factor (EGF)-like protein, protects myocardium tissue against irradiation-induced damage and preserves cardiac function. rhNRG-1ß effectively ameliorated irradiation-induced myocardial nuclear damage in both cultured adult rat-derived cardiomyocytes and rat myocardium tissue via NRG/ErbB2 signaling. By activating ErbB2, rhNRG-1ß maintained mitochondrial integrity, ATP production, respiratory chain function and the Krebs cycle status in irradiated cardiomyocytes. Moreover, the protection of irradiated cardiomyocytes and myocardium tissue by rhNRG-1ß was at least partly mediated by the activation of the ErbB2-ERK-SIRT1 signaling pathway. Long-term observations further showed that rhNRG-1ß administered in the peri-irradiation period exerts continuous protective effects on cardiac pump function, the myocardial energy metabolism, cardiomyocyte volume and interstitial fibrosis in the rats receiving radiation via NRG/ErbB2 signaling. Our findings indicate that rhNRG-1ß can protect the myocardium against irradiation-induced damage and preserve cardiac function via the ErbB2-ERK-SIRT1 signaling pathway.

Anticancer activity and molecular mechanism of resveratrol-bovine serum albumin nanoparticles on subcutaneously implanted human primary ovarian carcinoma cells in nude mice.

This study investigates the antitumor effects and functional mechanism of resveratrol-bovine serum albumin nanoparticles (RES-BSANP) on human primary ovarian carcinoma cells in nude mice. An implanted tumor model was established by injecting a suspension of the human primary ovarian cancer cell SKOV(3) into the subcutaneous tissue of nude mice. The tumor-bearing mice (n = 32) were randomly divided into 8 groups, which received intraperitoneal injections of normal saline (0.9%, 0.5 mL), BSA (1.5 mg/kg, 0.5 mL), or RES-BSANP or RES (200, 100, and 50 mg/kg, 0.5 mL), respectively, once a week for 4 weeks. The in vivo antitumor efficacy was evaluated by measurement of tumor volume, whereas morphological alterations were observed by transmission electron microscope (atomic force microscopy); TUNEL assays and immunoblotting for apoptotic and cell proliferation proteins were carried out to elucidate the possible mechanism. RES-BSANP was found to exhibit certain highly desirable characteristics such as innocuity, better dispersity, and water solubility; it affected the in vivo tissue/organ distribution of RES in a remarkable manner. The administration of RES-BSANP significantly retarded the growth of carcinomas in nude mice from the third week onwards, and the inhibition rate was markedly higher than in mice treated with RES (52.43% vs. 46.34%, p < 0.05), without causing weight loss (p > 0.05). Simultaneously, apoptotic and necrotic morphological characteristics were observed with electron microscopy in the tumor tissues of treated mice. TUNEL staining revealed that the tumors from RES-BSANP-treated mice exhibited a similar apoptotic index as RES control tumors. Western blot analysis of the protein expression profiles revealed that part of the mechanism may be mediated by triggering the release of cytochrome c from the intermembrane space and upregulating the expression of caspase-9 and caspase-3, suggesting that the mitochondrial apoptotic pathway was being activated.