HK2 and LDHA upregulation mediate hexavalent chromium-induced carcinogenesis, cancer development and prognosis through miR-218 inhibition.

Hexavalent chromium [Cr(VI)] is one of the most common environmental contaminants due to its tremendous industrial applications, but its effects and mechanism remain to be investigated. Our previous studies showed that Cr(VI) exposure caused malignant transformation and tumorigenesis. This study showed that glycolytic proteins HK2 and LDHA levels were statistically significant changed in blood samples of Cr(VI)-exposed workers and in Cr-T cells compared to the control subjects and parental cells. HK2 and LDHA knockdown inhibited cell proliferation and angiogenesis, and higher HK2 and LDHA expression levels are associated with advanced stages and poor prognosis of lung cancer. We found that miR-218 levels were significantly decreased and miR-218 directly targeted HK2 and LDHA for inhibiting their expression. Overexpression of miR-218 inhibited glucose consumption and lactate production in Cr-T cells. Further study found that miR-218 inhibited tumor growth and angiogenesis by decreasing HK2 and LDHA expression in vivo. MiR-218 levels were negatively correlated with HK2 and LDHA expression levels and cancer development in human lung and other cancers. These results demonstrated that miR-218/HK2/LDHA pathway is vital for regulating Cr(VI)-induced carcinogenesis and human cancer development.

Elevated NOX4 promotes tumorigenesis and acquired EGFR-TKIs resistance via enhancing IL-8/PD-L1 signaling in NSCLC.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used for human non-small-cell lung cancer (NSCLC) treatment. However, acquired resistance to EGFR-TKIs is the major barrier of treatment success, and new resistance mechanism remains to be elucidated. In this study, we found that elevated NADPH oxidase 4 (NOX4) expression was associated with acquired EGFR-TKIs resistance. Gefitinib is the first-generation FDA-approved EGFR-TKI, and osimertinib is the third-generation FDA-approved EGFR-TKI. We demonstrated that NOX4 knockdown in the EGFR-TKI resistant cells enabled the cells to become sensitive to gefitinib and osimertinib treatment, while forced expression of NOX4 in the sensitive parental cells was sufficient to induce resistance to gefitinib and osimertinib in the cells. To elucidate the mechanism of NOX4 upregulation in increasing TKIs resistance, we found that knockdown of NOX4 significantly down-regulated the expression of transcription factor YY1. YY1 bound directly to the promoter region of IL-8 to transcriptionally activate IL-8 expression. Interestingly, knockdown of NOX4 and IL-8 decreased programmed death ligand 1 (PD-L1) expression, which provide new insight on TKIs resistance and immune escape. We found that patients with higher NOX4 and IL-8 expression levels showed a shorter survival time compared to those with lower NOX4 and IL-8 expression levels in response to the anti-PD-L1 therapy. Knockdown of NOX4, YY1 or IL-8 alone inhibited angiogenesis and tumor growth. Furthermore, the combination of NOX4 inhibitor GKT137831 and gefitinib had synergistic effect to inhibit cell proliferation and tumor growth and to increase cellular apoptosis. These findings demonstrated that NOX4 and YY1 were essential for mediating the acquired EGFR-TKIs resistance. IL-8 and PD-L1 are two downstream targets of NOX4 to regulate TKIs resistance and immunotherapy. These molecules may be used as potential new biomarkers and therapeutic targets for overcoming TKIs resistance in the future.

MKRN2 knockout causes male infertility through decreasing STAT1, SIX4, and TNC expression.

Makorin-2 (Mkrn2) is an evolutionarily conserved gene whose biological functions are not fully known. Although recent studies have shed insights on the potential causes of male infertility, its underlining mechanisms still remain to be elucidated. We developed a Mrkn2 knockout mice model to study this gene and found that deletion of Mkrn2 in mice led to male infertility. Interestingly, the expression level of signal transducer and activator of the transcription (STAT)1 was significantly decreased in MKRN2 knockout testis and MEF cells. Co-IP assay showed an interaction between MKRN2 and STAT1. Moreover, our results further indicated that MKRN2 regulated the expression level of SIX4 and tenascin C (TNC) via the EBF transcription factor 2 (EBF2) in mice. The results of our study will provide insights into a new mechanism of male infertility.

Corrigendum: Regulation of microrna-497-targeting AKT2 influences tumor growth and chemoresistance to cisplatin in lung cancer.

[This corrects the article DOI: 10.3389/fcell.2020.00840.].

Discovery of a New CDK4/6 and PI3K/AKT Multiple Kinase Inhibitor Aminoquinol for the Treatment of Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is a lethal malignancy lacking effective treatment. The Cyclin-dependent kinases 4/6 (CDK4/6) and PI3K/AKT signal pathways play pivotal roles in carcinogenesis and are promising therapeutic targets for HCC. Here we identified a new CDK4/6 and PI3K/AKT multi-kinase inhibitor for the treatment of HCC. Methods: Using a repurposing and ensemble docking methodology, we screened a library of worldwide approved drugs to identify candidate CDK4/6 inhibitors. By MTT, apoptosis, and flow cytometry analysis, we investigated the effects of candidate drug in reducing cell-viability,inducing apoptosis, and causing cell-cycle arrest. The drug combination and thermal proteomic profiling (TPP) method were used to investigate whether the candidate drug produced antagonistic effect. The in vivo anti-cancer effect was performed in BALB/C nude mice subcutaneously xenografted with Huh7 cells. Results: We demonstrated for the first time that the anti-plasmodium drug aminoquinol is a new CDK4/6 and PI3K/AKT inhibitor. Aminoquinol significantly decreased cell viability, induced apoptosis, increased the percentage of cells in G1 phase. Drug combination screening indicated that aminoquinol could produce antagonistic effect with the PI3K inhibitor LY294002. TPP analysis confirmed that aminoquinol significantly stabilized CDK4, CDK6, PI3K and AKT proteins. Finally, in vivo study in Huh7 cells xenografted nude mice demonstrated that aminoquinol exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil with the combination treatment showed the highest therapeutic effect. Conclusion: The present study indicates for the first time the discovery of a new CDK4/6 and PI3K/AKT multi-kinase inhibitor aminoquinol. It could be used alone or as a combination therapeutic strategy for the treatment of HCC.

Discovery of vanoxerine dihydrochloride as a CDK2/4/6 triple-inhibitor for the treatment of human hepatocellular carcinoma.

BACKGROUND: Cyclin-dependent kinases 2/4/6 (CDK2/4/6) play critical roles in cell cycle progression, and their deregulations are hallmarks of hepatocellular carcinoma (HCC). METHODS: We used the combination of computational and experimental approaches to discover a CDK2/4/6 triple-inhibitor from FDA approved small-molecule drugs for the treatment of HCC. RESULTS: We identified vanoxerine dihydrochloride as a new CDK2/4/6 inhibitor, and a strong cytotoxicdrugin human HCC QGY7703 and Huh7 cells (IC50: 3.79 µM for QGY7703and 4.04 µM for Huh7 cells). In QGY7703 and Huh7 cells, vanoxerine dihydrochloride treatment caused G1-arrest, induced apoptosis, and reduced the expressions of CDK2/4/6, cyclin D/E, retinoblastoma protein (Rb), as well as the phosphorylation of CDK2/4/6 and Rb. Drug combination study indicated that vanoxerine dihydrochloride and 5-Fu produced synergistic cytotoxicity in vitro in Huh7 cells. Finally, in vivo study in BALB/C nude mice subcutaneously xenografted with Huh7 cells, vanoxerine dihydrochloride (40 mg/kg, i.p.) injection for 21 days produced significant anti-tumor activity (p < 0.05), which was comparable to that achieved by 5-Fu (10 mg/kg, i.p.), with the combination treatment resulted in synergistic effect. Immunohistochemistry staining of the tumor tissues also revealed significantly reduced expressions of Rb and CDK2/4/6in vanoxerinedihydrochloride treatment group. CONCLUSIONS: The present study isthe first report identifying a new CDK2/4/6 triple inhibitor vanoxerine dihydrochloride, and demonstrated that this drug represents a novel therapeutic strategy for HCC treatment.

Regulation of MicroRNA-497-Targeting AKT2 Influences Tumor Growth and Chemoresistance to Cisplatin in Lung Cancer.

BACKGROUND: MicroRNA-497 (miR-497) has been implicated in several cancers. Increasing studies demonstrate the role of AKT2 in cancers as an oncogene which is closely associated with tumor aggressiveness by enhancing cancer cell survival, migration and invasion However, miR-497/AKT2 axis in non-small cell lung cancer (NSCLC) remains unclear. METHODS: Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of miR-497 and its target gene. The function of miR-497 in lung cancer was investigated through in vitro and in vivo assays (cell proliferation assay, cell migration assay, colony formation assay, flow cytometry assay, immunoblotting and tumorigenesis assay). Luciferase reporter assay was conducted to confirm the target gene of miR-497. RESULTS: In this study, we found that miR-497 was significantly downregulated in tumor tissues and blood samples of lung cancer patients. To understand the potential mechanism of miR-497 in inhibiting tumor growth, we showed that miR-497 blocked the activation of AKT2 and regulated cell proliferation, cell migration, colony formation and increases chemosensitivity of H1299 cells to cisplatin by inhibiting AKT2. MiR-497 also inhibited tumor growth and suppressed expression of AKT2 at the protein and mRNA levels in mouse xenograft tumors. CONCLUSION: Taken together, our findings indicated that miR-497 suppresses the tumor growth by targeting AKT2, and the miR-497/AKT2 axis is a potential therapeutic target for NSCLC intervention.

Apigenin Inhibits IL-6 Transcription and Suppresses Esophageal Carcinogenesis.

Esophagus cancer is the seventh cause of cancer-related deaths globally. In this study, we analyzed interleukin 6 (IL-6) gene expression in human esophagus cancer patients and showed that IL-6 mRNA levels are significantly higher in tumor tissues and negatively correlated with overall survival, suggesting that IL-6 is a potential therapeutic target for esophagus cancer. We further demonstrated that apigenin, a nature flavone product of green plants, inhibited IL-6 transcription and gene expression in human esophagus cancer Eca-109 and Kyse-30 cells. Apigenin significantly and dose-dependently inhibited cell proliferation and promoted apoptosis while stimulating the cleaved PARP (poly ADP-ribose polymerase) (C-PARP) and caspase-8 expression. It suppressed VEGF (Vascular endothelial growth Factor) expression and tumor-induced angiogenesis. Pretreatment of cells with IL-6 could completely reverse apigenin-induced cellular changes. Finally, using a preclinical nude mice model subcutaneously xenografted with Eca-109 cells, we demonstrated the in vivo antitumor activity and mechanisms of apigenin. Taken together, this study revealed for the first time that apigenin is a new IL-6 transcription inhibitor and that inhibiting IL-6 transcription is one of the mechanisms by which apigenin exhibits its anticancer effects. The potential clinical applications of apigenin in treating esophagus cancer warrant further investigations.

Suppression of miR-143 contributes to overexpression of IL-6, HIF-1α and NF-κB p65 in Cr(VI)-induced human exposure and tumor growth.

Hexavalent chromium [Cr(VI)] is a known occupational and environmental contaminant and carcinogen, but new mechanisms of Cr(VI)-induced carcinogenesis remain to be elucidated. In this study, we found that expression of miR-143 is decreased, whereas that of Interleukin 6 (IL-6) is increased in blood samples of Cr(VI)-exposing workers compared with corresponding unexposed workers. In addition, IL-6 was increased in human bronchial epithelial cells (BEAS-Cr) exposed to Cr(VI) compared with unexposed BEAS-2B cells. To further investigate the mechanisms by which Cr(VI) promotes these changes, we assessed the effects of miR-143 on gene expression and found that miR-143 suppressed expression of IL-6, HIF-1α and NF-κB p65, and that inhibiting miR-143 promoted expression of IL-6, HIF-1α and NF-κB p65. Interestingly, IL-6 regulated expression of HIF-1α, and HIF-1α transcriptionally regulated expression of IL-6. Experiments in animals showed that miR-143 inhibited tumor growth and angiogenesis by regulating IL-6/HIF-1α and downstream signaling pathways in vivo. These outcomes support the hypothesis that the miR-143/IL-6/HIF-1α pathway functions to regulate Cr(VI)-induced carcinogenesis.

CT-pathologic correlation in lung adenocarcinoma and squamous cell carcinoma.

Distinguishing lung adenocarcinoma from squamous cell carcinoma (SCC) is clinically important. Computed tomography (CT) scan is an economical, effective, noninvasive, commonly available, and quick diagnostic way for lung cancer. In this study, we aim to compare the CT characteristics in adenocarcinoma and SCC.Data from 275 cases (259 adenocarcinoma and 16 SCC) were retrospectively compared. CT characteristics, including lesion size and shape, single/multifocal lesions, location of the tumor, the margin of lobes, whether the lesion had deep lobulated margin, bronchial cut-off sign, signs of dilated bronchial arteries, signs of vascular bundle thickening, signs of short burrs, spinous processes, and pleural indentation, were compared in 148 cases (137 adenocarcinoma and 11 SCC).Patients with adenocarcinoma were more likely to be female (44.2% vs 25.0%, P = .017). Compared with SCC, adenocarcinomas were more likely to have deep lobulated margin (81.0% vs 54.5%, P = .038), less likely to have smooth lobes margin (2.7% vs 83.3%, P < .001), more likely to have vascular bundle thickening (37.2% vs 0, P = .016) and pleural indentation (59.9% vs 18.2%, P = .01), and marginally less likely to have dilated bronchial arteries (17.5% vs 45.5%, P = .064). No significant difference was observed regarding to characteristics, including tumor size, location of the tumor, signs of bronchial cut-off, dilated bronchial arteries, short burrs, or spinous processes.CT scan has the potential to help to distinguish lung adenocarcinoma and SCC in a fast and commonly available way. CT could be a rough but fast way to diagnosis, and may thus shorten the waiting time to treatment and allow more time for clinicians, patients, and their families to prepare for future treatment.

Magnetic resonance image manifestations of the atypical meningioma.

Through retrospective analysis of 13 cases of magnetic resonance image (MRI) manifestations of atypical meningiomas confirmed by operation and pathology in the First Affiliated Hospital of Xinxiang Medical University, the objective of this study was to evaluate the diagnostic value of MRI in order to improve the accuracy rate of preoperative diagnosis. In this retrospective analysis of MRI findings for atypical meningiomas in First Affiliated Hospital of Xinxiang Medical University from January to July in 2012, the location, morphology and tumor signals and other tumor imaging characteristics were covered. In 13 cases of atypical meningioma patients of this group, most tumors were located at typical sites (10/13), mainly the falx cerebri, parasagittal, convexity, saddle area. Only two cases were at atypical locations, 1 in the cerebellar hemisphere and 1 in a lateral ventricle. Most of the tumors showed T1 and T2 isointensity signals, and necrosis, calcification, and peritumoral edema were always featured. DWI showed isointensity in 11 cases (11/13), and hyperintensity in 2. Some 9 cases had dural tail signs, 12 had accurate positioning (12/13), and 2 were postoperative recurrences. MRI has high value in the diagnosis of atypical meningiomas, with important roles in early clinical diagnosis, treatment and prognosis evaluation.

Influence of surface conductivity on sensitivity of acoustic wave gas sensors based on multilayered structures.

The influences of the surface conductivity on the velocity of an acoustic wave (AW) in a multilayered material are studied theoretically with the transfer matrix method and the conductivity sensitivity of the AW sensor is presented. It is found that the velocity of the AW increases with decreasing surface conductivity and vice versa. The result is used to explain the abnormal response of AW sensors, in which the central frequencies of AW sensors increase after they sorb the detected gases. Meanwhile, the conductivity sensitivity is found to be related to the dielectric constants of the multilayered material and the electromechanical coupling coefficient of the sensor. Finally, the sensitivities of AW sensors based on multilayered structures are optimized by considering the influences of the surface conductivities of the sensors with different initial conductivities and thicknesses of the sensitive layers.

Experimental study of Love-wave immunosensors based on ZnO/LiTaO3 structures.

Experimental study of Love-mode immunosensors based on structures of ZnO/36 degrees YX-LiTaO3 is presented, in which the ZnO films with c-axis (002) orientation have been successfully grown on the 36 degrees YX-LiTaO3 substrates by RF magnetron sputtering technique. Then the Love-mode immunosensors based on the ZnO/36 degrees YX-LiTaO3 structures and monitoring antibody-antigen immunoreactions in aqueous solutions in real time are fabricated. The experimental results show that the optimal thickness of ZnO layers is about 1.20 microm in the structures deposited on 36 degrees YX-LiTaO3 substrates, which is much less than that of SiO2 overlayers about 6 microm. The antibody-antigen immunoreaction experiments also show that the frequency shifts of the sensors with 1.33 microm ZnO films are proportional to the concentration of antigen in solution as the concentration range less than 100 microg/ml.

[Preparation of cyclosporine A loaded mPEG-PLGA copolymer micelles and study its pharmacokinetics in rats].

To prepare cyclosporine A (CyA) loaded block copolymer micelles and observe its release behaviors in vitro and pharmacokinetics in rats, methoxylpoly (ethylene glycol)-poly (D, L-lactide-co-glycolide) (mPEG-PLGA) was synthesized by ring-opening copolymerization of lactide and glycolide in the presence of methoxylpoly (ethylene glycol) (mPEG) as initiator. The structure of the mPEG-PLGA copolymer was confirmed with 1H NMR and FT-IR. The cyclosporine A loaded micelles (CyA-PM) were prepared by solvent evaporation method and their morphology was observed by the transmission electron microscope (TEM). The mean size and size distribution were determined by dynamic light scattering (DLS). The release behaviors in vitro and pharmacokinetics in rats were investigated by HPLC method using cyclosporine A injection commercial agent, sandimmune, as the reference. The obtained CyA-PM showed spherical shape with the core-shell structure, the mean particle sizes are in the range of 136.1-141.9 nm. The drug loading amount and entrapment efficiency were increased and the particle size became smaller with decreasing the ratio of acetone to water. With the increasing of the amount of cyclosporine A fed the drug loading increased, entrapment efficiency decreased and the particle size had no change. CyA-PM showed significant sustained release behave in vitro compared with sandimmune and only 9.7% of encapsulated cyclosporine A was released after 12 hours, the release characteristics was well fitted with Higuchi equation (r = 0.999). The Pharmacokinetics study at equal administration dosage (5 mg x kg(-1)) in rats showed the half-life (t1/2) of CyA-PM extended and the area under concentration-time curve (AUC) increased compared to sandimmune. The results also showed that cyclosporine A concentration-time data were all in accord with two compartment model. Cyclosporine A loaded mPEG-PLGA micelles showed obviously solubility enhancement, sustained release and overcome the side effect and toxicity of sandimmune resulted from solubiling agent-polyoxyethylene castor oil (Cremophor EL) and might be developed as a novel dosage form of cyclosporine A.