Invasion Of A549 Cells Research Articles

CircCCT3 Enhances Invasion and Epithelial-Mesenchymal Transition (EMT) of Non-Small-Cell Lung Cancer (NSCLC) via the miR-107/Wnt/FGF7 Axis

Background CircRNAs play a role in a variety of biological processes, including tumorigenesis. circCCT3 has been shown to regulate cancer initiation and progression. Unfortunately, whether circCCT3 is involved in non-small-cell lung cancer (NSCLC) metastasis remains unclear. Methods Our study utilized RT-qPCR to examine gene expression levels. A transwell assay was used to measure invasion ability of cells. Starbase software and TargetScan software were used to predict target genes. Results circCCT3 knockdown attenuated invasion and epithelial-mesenchymal transition (EMT) of A549 and Calu-1 cells. miR-107 mimics could rescue circCCT3-induced invasion and EMT. Next, miR-107 mimics and circCCT3 knockdown suppressed Wnt3a and FGF7 expression. An miR-107 inhibitor promoted Wnt3a and FGF7 expressions. Finally, FGF7 greatly restored miR-107-inhibited invasion and EMT of A549 cells. Conclusion Here, we reveal a molecular mechanism circCCT3/miR-107/Wnt/FGF7 responsible for NSCLC metastasis.

Glycitin exerts anticancer effect on human lung cancer cells through induction of apoptosis, cell cycle arrest, and inhibition of PI3K/AKT signal pathway

Purpose: To determine the effect of glycitin on PI3K/AKT signaling, migration, invasion, apoptosis, and cell cycle in A549 lung cancer cells.Methods: 3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays were used to determine the proliferation and colony generation potency of A549 cells, respectively, after treatment with glycitin (0 - 120 μM). Apoptosis in A549 cells was measured using DAPI and Annexin V/PI-FITC assays. Cell cycle arrest was assessed byflow cytometry, while the effect of glycitin on migration and invasion of A549 cells was determined by Transwell assay. The effect of glycitin on expressions of proteins associated with PI3K/AKT signaling in A549 cells was measured using western blotting.Results: Glycitin significantly inhibited the proliferation and colony generation potential of A549 cells (p < 0.05). The antiproliferative effects of glycitin on A549 cells were mediated through stimulation of apoptosis and cell cycle arrest at G0/G1-phase. The compound also distorted normal cellular morphology by causing membrane damage and nuclear fragmentation. The proportion of cells in the G0/G1-phase increased after glycitin treatment, when compared to the other two phases, demonstrating cell cycle arrest (p < 0.05). Glycitin suppressed the migration and invasion of A549 cells. However, Western blotting results showed that glycitin down-regulated the expressions of PI3K/AKT signaling proteins in A549 cells (p < 0.05).Conclusion: Glycitin produced significant anticancer effect on A549 cells via enhanced apoptosis, induction of cell cycle arrest, and inhibition of PI3K/AKT signalling. Moreover, it suppressed the migration and invasion of A549 cells. Therefore, glycitin is a potential lead molecule for the development of a therapeutic agent for invasive lung cancer.

Circ_CSPP1 Regulates the Development of Non-small Cell Lung Cancer via the miR-486-3p/BRD9 Axis.

In this study, we explored the role of circ_CSPP1 in non-small cell lung cancer (NSCLC) using NSCLC cell lines (A549 and H1299) and human bronchial epithelioid cells (16HBE). The differential expression of circ_CSPP1, miR-486-3p and BRD9 in NSCLC by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot in A549 cells, H1299 cells, 16HBE cells, NSCLC tissues and healthy lung tissues. Dual-luciferase reporter assay was conducted to verify the interaction between circ_CSPP1 and miR-486-3p or miR-486-3p and BRD9. The effect of circ_CSPP1/miR-486-3p/BRD9 axis on NSCLC cell proliferation was evaluated using cell counting kit-8 assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine assay. Additionally, transwell assays were performed to evaluate the effect of circ_CSPP1/miR-486-3p/BRD9 axis on A549 and H1299 cell migration and invasion. The effect of circ_CSPP1 on tumor tumorigenesis of A549 cells in vivo was determined by xenograft tumor model and immunohistochemistry assay. Circ_CSPP1 and BRD9 expression were upregulated, while miR-486-3p expression was downregulated in tumor tissues of NSCCL patients and A549 and H1299 cells. Circ_CSPP1 specifically bound miR-486-3p, and miR-486-3p could target BRD9. Circ_CSPP1 upregulation promoted proliferation, invasion and migration of NSCLC cells, circ_CSPP1 knockdown or miR-486-3p upregulation had the opposite effects. Circ_CSPP1 knockdown-induced effects were reverted by miR-486-3p inhibition. Similarly, the effects of miR-486-3p upregulation on NSCLC cell proliferation, invasion and migration were reversed by BRD9 overexpression. In addition, circ_CSPP1 silencing inhibited tumor growth in nude mice. Circ_CSPP1 promoted A549 and H1299 cell malignancy by competitively inhibiting BRD9 and binding to miR-486-3p.

SEC61G participates in endoplasmic reticulum stress by interacting with CREB3 to promote the malignant progression of lung adenocarcinoma.

As the most common type of lung cancer, lung adenocarcinoma (LUAD) poses a great threat to human health worldwide and severely compromises the quality of life of the patients. The present study aimed to explore the potential pathogenesis of LUAD. Reverse transcription-quantitative PCR and western blotting were applied to measure the expression levels of SEC61 translocon subunit γ (SEC61G) and cyclic AMP-responsive element-binding protein 3 (CREB3). Western blotting was also used to determine the expression of endoplasmic reticulum (ER) stress-, apoptosis- and migration-related proteins. Cell Counting Kit-8, colony formation, TUNEL, wound healing and Transwell assays were used, respectively, to determine the viability, proliferation, apoptosis, migration and invasion of LUAD A549 cells. The association between SEC61G and CREB3 was verified by co-immunoprecipitation assay. The results revealed that SEC61G was upregulated in A549 cells and its downregulation could activate ER stress. It was also found that silencing SEC61G inhibited the malignant development of LUAD through ER stress. In addition, SEC61G was verified to participate in ER stress in LUAD via CREB3 and silencing SEC61G exerted inhibitory effects on the malignant progression of LUAD by regulating CREB3. In summary, SEC61G participated in ER stress and its knockdown exerted inhibitory effects on A549 cells via regulating CREB3, which suggests that SEC61G may be a potential therapy for patients with LUAD.

Metformin suppresses lung adenocarcinoma by downregulating long non-coding RNA (lncRNA) AFAP1-AS1 and secreted phosphoprotein 1 (SPP1) while upregulating miR-3163

ABSTRACT AFAP1-AS1 plays a pro-tumor role in lung cancer. However, no investigation has focused on whether it is involved in the anticancer activity of metformin (Met) in the treatment of lung adenocarcinoma (LUAD). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of long non-coding (lnc)RNA AFAP1-AS1, the microRNA (miR)-3163, and secreted phosphoprotein 1 (SPP1) in LUAD tissues, or of A549 and H3122 cells. Cell Counting Kit-8, wound scratch, and cell invasion assays were performed to evaluate the effect of the overexpression of lncRNA AFAP1-AS1, miR-3163, and SPP1 on the malignant behaviors of A549 and H3122 cells. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway-related proteins were detected by Western blot analysis. Dual luciferase reporter or RIP assays were used to determine the interplay between AFAP1-AS1 and miR-3163, or of miR-3163 and SPP1. Met inhibits the malignant characteristics of A549 and H3122 cells in vitro. GEPIA database analysis showed that AFAP1-AS1 is a highly expressed lncRNA in LUAD tissues, which was validated by RT-qPCR. Overexpression of AFAP1-AS1 suppressed the met-mediated anti-tumor activity in A549 and H3122 cells, while AFAP1-AS1 silencing promoted it. Met inhibited AFAP1-AS1 expression, which resulted in reduced proliferation, migration, and invasion in A549 and H3122 cells. This led to AFAP1-AS1-mediated suppression of miR-3163 and, subsequently, the upregulation of SPP1. Met exerts its antitumor activities by regulating the AFAP1-AS1/miR-3163/SPP1/PI3K/Akt/mTOR axis. Our findings deepen our understanding of mechanisms underlying anti-tumor effect of Met in LUAD.

Cepharanthine Regulates Autophagy via Activating the p38 Signaling Pathway in Lung Adenocarcinoma Cells.

Cepharanthine (CEP) is an alkaloid extracted from Stephania cepharantha Hayata. This compound has been reported as a promising anti-tumor drug, although its potential molecular mechanism is not fully understood. Here, we studied the anti-tumor effect of CEP on human lung cancer cells and evaluated its molecular mechanism. The A549 cells were treated with CEP, the cell viability was measured by 3-(4, 5-dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide (MTT) assay, and formation of autophagosome was observed by acridine orange staining under a fluorescence microscope. The cell migration and invasion were determined by wound healing and transwell assay. The protein levels of autophagy-associated molecules, light chain 3 (LC3)p38 and phospho-p38 in A549 cells, were determined by western blot analysis. The results showed that CEP inhibited cell proliferation, migration and invasion in A549 cells. Moreover, we found that CEP resulted in significant increases in levels of the autophagy marker protein LC3 in A549 cells. The number of intracellular acid dye follicular bright red fluorescence in A549 cells was significantly increased after CEP treatment. At the molecular levels, CEP markedly increased the phosphorylation of p38 in A549 cells. The knockdown of p38 expression by siRNA-p38 impaired the autophagy-regulating effect of CEP. Our results indicated that CEPregulated autophagy was an anti-tumor effect and not a protective response to CEP. Taken together, these results demonstrated that CEP regulated autophagy by activating the p38 signaling pathway, which could be provided a potential application for preventing lung cancer.

TXNDC9 knockdown inhibits lung adenocarcinoma progression by targeting YWHAG.

Lung adenocarcinoma (LUAD) is the most common form of lung cancer and with the highest mortality rate. Therefore, the identification and development of effective methods for the treatment of LUAD is of great importance. The present study aimed to investigate the role of thioredoxin domain-containing protein 9 (TXNDC9) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein γ (YWHAG; also known as 14-3-3γ) in the progression of LUAD. The expression of TXNDC9 and its association with the survival of patients with LUAD was analyzed using Encyclopedia of RNA Interactomes. Reverse transcription-quantitative PCR and western blot analysis were used to detect TXNDC9 mRNA and protein expression levels, respectively, in in vitro studies. To investigate the role of TXNDC9 in the progression of LUAD, TXNDC9 was silenced using small interfering RNA transfection. Furthermore, the viability, proliferation, migration, invasiveness and apoptosis of TXNDC9-silenced A549 cells were detected using Cell Counting Kit (CCK)-8, colony formation, wound healing, Transwell and TUNEL assays, respectively. The association between TXNDC9 and YWHAG was analyzed using STRING and Gene Expression Profiling Interactive Analysis databases, as well as co-immunoprecipitation assays. Subsequently, YWHAG was overexpressed to similarly determine effects of YWHAG on viability, proliferation, migration, invasiveness and apoptosis of A549 cells. TXNDC9 expression was markedly upregulated in lung cancer cells, particularly A549 cells, and silencing of TXNDC9 expression suppressed the viability of the lung cancer cells. The results also revealed that TXNDC9 silencing exerted inhibitory effects on the viability, proliferation, migration and invasiveness of A549 cells, whereas the apoptotic rate was increased. Similar to TXNDC9, YWHAG expression was also upregulated in the A549 cells. Furthermore, TXNDC9 was demonstrated to bind to YWHAG and was positively associated with YWHAG. YWHAG overexpression reversed the inhibitory effects of TXNDC9 silencing on LUAD, as evidenced by increased viability, proliferation, migration and invasiveness, and decreased apoptosis, of A549 cells. The present study demonstrated that the knockdown of TXNDC9 exerted suppressive effects on LUAD, whereas YWHAG overexpression reversed the inhibitory effects of TXNDC9 silencing on LUAD. Therefore, TXNDC9 silencing may exert protective effects against LUAD by targeting YWHAG.

Human bone marrow mesenchymal stem cell-derived exosomes containing microRNA-425 promote migration, invasion and lung metastasis by down-regulating CPEB1.

ObjectiveBone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) could mediate the malignancy of tumor cells by transmitting targeted cargo. Therein, this study intends to explore the function of BMSC-Exos transmitting microRNA-425 (miR-425)/cytoplasmic polyadenylation binding protein 1 (CPEB1) in lung cancer growth.MethodsmiR-425 and CPEB1 levels in cancer tissues and cells were measured. BMSCs and their exosomes were collected and identified. After intervention with BMSC-Exos, miR-425 or CPEB1, invasion and migration of A549 and NCI-H1299 cells in vitro, and lung metastasis of A549 cells in vivo were observed. The relationship between miR-425 and CPEB1 was verified.ResultsmiR-425 was highly expressed while CPEB1 was lowly expressed in lung cancer tissues of patients. CPEB1 was the direct target of miR-425. Down-regulating miR-425 or up-regulating CPEB1 decreased cell invasion and migration ability of A549 and NCI-H1299 cells, as well as decreased the number of lung metastasis lesions in vivo. After co-culture with BMSC-Exos, A549 and NCI-H1299 cells showed promoted migration and invasion in vitro and A549 cells demonstrated increased lung metastasis in vivo. Down-regulated miR-425 or up-regulated CPEB1 reversed the promotion of BMSC-Exos on lung cancer cell invasion, migration and lung metastasis.ConclusionBMSC-Exos could deliver miR-425 to inhibit CPEB1 expression in lung cancer cells, thereby promoting the malignant biological properties of lung cancer cells and their metastasis in vivo.

LINC00342 induces metastasis of lung adenocarcinoma by targeting miR-15b/TPBG.

In this study, the function and regulation of long non-coding RNA (lncRNA) LINC00342 in lung adenocarcinoma were investigated. From The Cancer Genome Atlas (TCGA) datasets and Gene Expression Omnibus (GEO) datasets, LINC00342 was found to be up-regulated in lung adenocarcinoma. The high expression of LINC00342 was also validated in lung cancer cell lines. LINC00342 induced invasion and epithelial-mesenchymal transition (EMT) process of A549 cells. By analyzing GEO datasets, TPBG was confirmed positively correlated to LINC00342 and highly expressed in lung adenocarcinoma. In addition, TPBG induced invasion and EMT process of A549 cells. Through bioinformatics analysis and luciferase assay, miR-15b was validated as a direct target of both LINC00342 and TPBG. Ectopic miR-15 expression repressed LINC00342 and TPBG. Interestingly, LINC00342 overexpression inhibited miR-15b and induced TPBG, whereas ectopic TPBG unchanged LINC00342 and miR-15b levels. In conclusion, LINC00342 promotes metastasis of lung adenocarcinoma through inducing TPBG targeted by miR-15b.

Anti‐proliferative and metastasis‐inhibiting effect of carbon ions on non‐small cell lung adenocarcinoma A549 cells

Objective Carbon ions suppress the effect of glutathione peroxidase 4 (GPX4) on the proliferation and metastasis of lung adenocarcinoma A549 cells. Methods The clonogenic formation ability of A549 cells was calculated with different ray doses of radiation. Cell proliferation, cycle, and apoptosis of A549 cells after different ray irradiation were detected by cck8 and flow cytometry. The transwell assay was used to detect the cell migration and invasion after different rays of radiation to A549 cells. The GPX4 matrix metalloproteinase 2 and E-cadherin expression levels were assessed by immunofluorescence and western blot. Results At the dose of 4 Gy, A549 cell viability was 22.95 ± 5.00% and 55.52 ± 3.26% when treated with carbon ions and X-rays, respectively. The distribution of A549 cells arrested in the G2 phase by carbon ion irradiation was positively correlated with radiation dose, with a most significant increase at 4 Gy irradiation (t = 4.303, p < 0.05). The results of apoptosis after irradiation showed were no significant differences in apoptosis at 1 and 2 Gy of X-ray compared with 0 Gy, and 4 Gy irradiation significantly induced apoptosis in A549 cells (t = 30.520, p < 0.05). The apoptotic rate of carbon ion-irradiated cells increased in a dose-dependent manner and was the highest at 4 Gy (t = 17.356, p < 0.05). The transwell assays showed significantly decreased migration and invasion of both ray-irradiated cells compared with the 0 Gy control cells. The carbon ions of 2 and 4 Gy significantly inhibited A549 cell migration (t2Gy = 9.260, t4Gy = 19.052, both p < 0.05) and invasion compared with those in the control cells (t2Gy = 6.141, t4Gy = 12.700, both p < 0.05). Treatment with 2 and/or 4 Gy doses of carbon ion reduced GPX4 (t2Gy = 8.170, t4Gy = 13.811, both p < 0.05) and matrix metalloproteinase 2 (t2Gy = 207.620, t4Gy = 93.170, both p < 0.05) protein expression, but E-cadherin expression was increased (t2Gy = 62.811, t4Gy = 102.106, both p < 0.05). Conclusion Compared with X-ray irradiation, carbon ions have a higher relative biological effectiveness and can increase the proportion of A549 cells arrested in the G2/M stage to induce apoptosis, thereby inhibiting proliferation. At the same dose with X-ray, carbon ions may inhibit the migration and invasion of A549 cells by regulating the expression of GPX4, matrix metalloproteinase 2, and E-cadherin.

Baicalein Inhibits the SMYD2/RPS7 Signaling Pathway to Inhibit the Occurrence and Metastasis of Lung Cancer.

Objective This study investigated the potential effects of Baicalein on proliferation, migration, and invasion of human lung cancer A549 and NCI-H1299 cells and its possible mechanisms. Methods The effects of Baicalein on proliferation and invasion of A549 and NCI-H1299 cells were detected by MTT assay, clonogenesis assay, and Transwell assay. A subcutaneous transplanted tumor model was used to evaluate the effects of SMYD2 and Baicalein on the proliferation of lung cancer. Baicalein inhibited in SMYD2/RPS7 signaling pathway in tumor cells was also detected by qRT-PCR. Results Baicalein significantly inhibited the growth of lung cancer cells. In addition, Baicalein significantly reduced the rate of A549 and NCI-H1299 cell invasion and clone formation in a dose-dependent manner. Animal experiments showed that both SMYD2 and Baicalein treatments could inhibit lung cancer tumor proliferation. Mechanism studies suggest that Baicalein inhibits the SMYD2/RPS7 signaling pathway. Conclusion These results indicated that Baicalein could inhibit the proliferation, migration, and invasion of LUNG cancer A549 and NCI-H1299 cells. Baicalein inhibits cell proliferation by downregulating the SMYD2/RPS7 signaling pathway.

The Role of RNA-Binding Protein HuR in Lung Cancer by RNA Sequencing Analysis.

Background: The overexpression of human antigen R (HuR) has been proven in various types of cancer and is associated with the poor survival lung cancer patients. HuR overexpression stabilizes the mRNA of tumor-promoting genes by binding with 3′-UTR AU-rich elements. However, the role of HuR in the proliferation of lung cancer is unclear. Methods: HuR expression was assessed using immunohistochemistry of tumor tissue samples from ten patients with lung cancer and ten patients with benign lung disease. Gene, protein, mRNA, and lncRNA changes in A549 HuR knockdown (KD) cells were assessed by single-cell RNA sequencing analysis. Furthermore, cell proliferation, migration, and invasion were determined by Cell Counting Kit-8 (CCK-8) assays and Transwell assays with or without Matrigel. The cell cycle was assessed by propidium iodide staining. The protein level, mRNA level and half-life of PLK1 were detected by western blotting and RT-qPCR. Results: In clinical patients, the expression of HuR was significantly higher in lung cancer patients than in patients with benign lung disease. RNA sequencing analysis of A549 HuR knockdown cells revealed that the main function of HuR was related to ribonucleoprotein complex biogenesis. HuR was found to regulate signaling pathways mainly related to the spliceosome, RNA transport and the cell cycle. HuR KD suppressed the proliferation, migration and invasion of A549 cells, indicating its promotive role in these processes. Conclusion: These results demonstrate that HuR plays an important role in the progression of lung cancer.

Trichosanthin inhibits cell growth and metastasis by promoting pyroptosis in non-small cell lung cancer.

BackgroundA number of studies have demonstrated that trichosanthin (TCS) can induce apoptosis in numerous types of tumor cell lines. However, whether TCS can induce pyroptosis has not yet been reported. This study aimed to investigate the role of TCS and its inhibitory effect on tumor growth by modulating pyroptosis in non-small cell lung cancer (NSCLC).MethodsEffects of different concentrations of TCS on the cell viability, proliferation, migration and invasion of NSCLC were detected by Cell Counting Kit-8 (CCK-8), colony formation, migration, and invasion assays. Immunofluorescence was used to detect the effect of TCS on the expression of pyroptosis marker protein gasdermin-D (GSDMD)-N in A549 cells. A tumor xenograft animal model was established by injecting A549 cells into nude mice.ResultsIn the present study, we found that TCS significantly inhibited the proliferation, migration, and invasion of A549 cells in a concentration-dependent manner. In addition, TCS at a high concentration (40 µg/mL) significantly promoted the expression of pyroptosis-related proteins [GSDMD-N, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and GSDMD], which showed an inhibitory effect on the pyroptosis of A549 cells. Additionally, we found that necrosulfonamide (NSA) significantly reversed the inhibitory effect of high concentrations of TCS on the pyroptosis of A549 cells. The in vivo experiments showed that TCS effectively reduced the tumor volume and inhibited the expression of Ki-67, whereas it increased the expression of GSDMD-N.ConclusionsTaken together, these results indicated that TCS could inhibit the progression of NSCLC by promoting pyroptosis. These findings provide further information on the possible underlying mechanism of TCS in the treatment of NSCLC.

Overexpression of dual-specificity phosphatases 4 and 13 attenuates transforming growth factor β1-induced migration and drug resistance in A549 cells in vitro

Transforming growth factor-beta (TGFβ) proteins induce an epithelial-mesenchymal transition (EMT) programme that is associated with increased invasive and drug-resistant phenotype of carcinoma cells. In addition to the canonical pathway involving SMAD proteins, the mitogen-activated kinase (MAPK) pathway via extracellular signal-regulated kinases ½ (ERK1/2) is also involved in promoting and maintaining a mesenchymal phenotype by tumor cells following TGFβ signal activation. As dual-specificity phosphatases (DUSPs) regulate ERK1/2 activity by dephosphorylation, we aimed to examine DUSPs’ expression upon TGFβ stimulation and whether DUSPs play a role in the EMT and related phenotypes promoted by TGFβ1 in A549 cells. We found that TGFβ1 stimulation led to marked changes in several DUSP proteins, including significant decreases in DUSP4 and DUSP13 expressions. We then showed that the ectopic co-expression of DUSP4/13 suppresses TGFβ1-induced ERK1/2 phosphorylation and protein levels of the EMT transcription factors Snail and Slug proteins. We then demonstrated that DUSP4/13 co-expression partially inhibited TGFβ1-promoted migration, invasion, and chemoresistance in A549 cells. Collectively, this report provides data for the involvement of DUSP4/13 in malignant phenotypes regulated by TGFβ1 in A549 cells.

Hypoxic Microenvironment-Induced Reduction in PTEN-L Secretion Promotes Non-Small Cell Lung Cancer Metastasis through PI3K/AKT Pathway.

Objective Lung cancer is the leading cause of cancer-related deaths worldwide. The aim of this study was to investigate the effects of hypoxic microenvironment on PTEN-L secretion and the effects of PTEN-L on the metastasis of non-small cell lung cancer (NSCLC) and the potential mechanisms. Methods The expression levels of PTEN-L in NSCLC tissues, cells, and cell culture media were detected. The transfection of PTEN-L overexpression construct or HIF-1α-siRNAs was conducted to manipulate the expression of PTEN-L or HIF-1α. NSCLC cells were introduced into 200 μM CoCl2 medium for 72 hours under 37°C to simulate hypoxia. The proliferation and apoptosis of the A549 cells were determined by the Cell Counting Kit-8 assay and Annexin V-FITC/PI-stained flow cytometry assay, respectively. Wound healing assay and transwell invasion assay were used to measure the migration and invasion of A549 cells. The protein expression of PTEN, PTEN-L, PI3K/AKT pathway-related proteins, and HIF-1α was detected by Western blot. Results PTEN and PTEN-L are downregulated in lung cancer tissues and cells. The protein expression of PTEN-L in the culture medium of lung cancer cell lines is decreased. The hypoxic microenvironment inhibits PTEN-L secretion. The low level of PTEN-L promotes cell proliferation, migration, and invasion, as well as inhibits apoptosis of A549 cells. The overexpression of PTEN-L attenuated the activation of the PI3K/AKT pathway by the hypoxic microenvironment. The knockdown of HIF-1α upregulates PTEN-L secretion under hypoxia. Conclusions The hypoxic microenvironment inhibits PTEN-L secretion and thus activates PI3K/AKT pathway to induce proliferation, migration, and invasion promotion, and apoptosis inhibition in NSCLC cells.

Scutellarin potentiates vancomycin against lethal pneumonia caused by methicillin-resistant Staphylococcus aureus through dual inhibition of sortase A and caseinolytic peptidase P

The strategy of targeting virulence factor has received great attention as it barely develops bacterial resistance. Sortase A (SrtA) and caseinolytic peptidase P (ClpP), as important virulence factors, are considered to be ideal pharmacological targets for methicillin-resistant Staphylococcus aureus (MRSA) infection. Through screening hundreds of compounds, we found scutellarin, a natural flavonoid, markedly inhibited SrtA and ClpP activities of MRSA strain USA300 with an IC50 of 53.64 μg/mL and 107.00 μg/mL, respectively. Subsequently, we observed that scutellarin could inhibit the SrtA-related virulence of MRSA. To demonstrate whether scutellarin directly binding to SrtA, fluorescence quenching assay and molecular docking were performed and the results indicated that scutellarin directly bonded to SrtA molecule with a KA value of 7.58 × 104 L/mol. In addition to direct SrtA inhibition, scutellarin could also inhibit hemolytic activity of S. aureus by inhibiting the expression of Hla in a SrtA-independent manner. Further assays confirmed that scutellarin inhibited hemolysis by inhibiting ClpP. The combination of scutellarin and vancomycin showed enhancing inhibition of USA300 in vitro and in vivo, evidenced by decreased MIC from 3 μg/mL to 0.5 μg/mL and increased survival and improvement of lung pathology in pneumonia mice. Taken together, these results suggest that scutellarin exhibited di-inhibitory effects on SrtA and ClpP of USA300. The di-inhibition of virulence factors by scutellarin combined with vancomycin to prevent MRSA invasion of A549 cells and pneumonia in mice, indicating that scutellarin is expected to be a potential adjuvant against MRSA in the future.

Effects of artesunate on the malignant biological behaviors of non-small cell lung cancer in human and its mechanism

ABSTRACT We aimed to assess the effects of artesunate (ART) on the proliferation, migration, invasion and apoptosis of the non-small cell lung cancer cells A549 and H1299. The effects of ART and carboplatin (CBP) alone or in combination on the viability of A549 and H1299 cells were evaluated by MTT assay. The effects of 30 μg/ml ART on cell invasion, migration and apoptosis were evaluated by Transwell assay, scratch assay and flow cytometry, respectively. The protein expressions of human antigen R (HuR) and MMP-9 after treatment with 30 μg/ml ART for 48 h were detected by Western blotting. After 48 h of treatment, 9 μg/ml ART in combination with 7 μg/ml CBP exerted a mild synergistic effect on cell viability. The migration rates of cells treated with 30 μg/ml ART and number of invasive cells were significantly lower, and the apoptosis rates were higher than those of the DMSO-treated group. HuR and MMP-9 expressions in cells treated with 30 μg/ml ART for 48 h were significantly lower than those of the DMSO-treated group. ART suppresses the proliferation, migration and invasion of A549 and H1299 cells and induces their apoptosis, probably being associated with decreased expressions of HuR and MMP-9 proteins.

The mechanism of dioscin preventing lung cancer based on network pharmacology and experimental validation

Ethnopharmacological relevanceDioscorea nipponica Makino as a Chinese folk medicine has been used for the treatment of chronic bronchitis, cough, and asthma. Several studies have established the antimetastatic potential of Dioscorea nipponica Makino extract. Dioscin is a major bioactive compound in Dioscorea nipponica Makino and has anti-tumor property in lung cancer cell lines. However, the preventive effect of dioscin against lung cancer and its key mechanism haven't been identified yet. Aim of studyTo identify the prevention effect of dioscin on lung cancer and explore its key mechanism based on network pharmacology and experimental validation. MethodsThe potential targets of dioscin were obtained from the HERB database. The therapeutic targets of lung cancer were acquired from the GeneCards database. Protein-protein interaction network (PPI) was constructed in the STRING 11.0 database. The David database was used for enrichment analysis. Molecular Docking was finished by the AutoDock Vina. NSCLC cell lines and mouse lung cancer model were used to confirm the prevention effect of dioscin on lung cancer and its key mechanism. Results76 potential targets of dioscin were identified to be involved in lung cancer treatment, which refer to 512 biological processes, 47 molecular functions, 77 cellular components and 107 signal pathways. The molecular docking suggested that dioscin might bind to AKT1, Caspase3, TP53, C-JUN and IL-6. The DARTS indicated that dioscin could bind to AKT1. In vitro, dioscin could decrease proliferation, invasion and migration in A549 and PC-9 cells with the significant reduction in the expression of p-AKT, MMP2, and PCNA. In vivo, dioscin could reduce lung nodules, lung injury, and mortality in mouse lung cancer model with reducing the expression of p-AKT, MMP2, PCNA and increasing the expression of active-caspase3. ConclusionDioscin could prevent lung cancer and its key target is AKT1 kinase, a center protein of PI3K/AKT signaling pathway.

Effect of Solanum lyratum Polysaccharide on Malignant Behaviors of Lung Cancer Cells by Regulating the Circ_UHRF1/miR-513b-5p Axis.

This study aimed to investigate the effect of Solanum lyratum polysaccharide on the malignant behavior of lung cancer cells and its possible mechanism. For this purpose, lung cancer A549 cells were cultured in vitro and treated with different doses (0.4, 0.8, 1.2 mg/mL) of Solanum lyratum polysaccharide for 24 h. Then cell proliferation was detected by the CCK-8 method and clone formation test. Transwell test was used to detect cell migration and invasion, and flow cytometry was used to detect cell apoptosis. The protein expressions of Bax and Bcl-2 in cells were detected by Western blotting, and the protein expressions of circ_UHRF1 and miR-513b-5p were detected by the qRT-PCR method. Pearson correlation was used to analyze the correlation between circ_UHRF1 and miR-513b-5p expressions in lung cancer tissues. Results showed that compared with the control group, the proliferation inhibition rate and apoptosis rate of A549 cells that intervened with the Solanum lyratum polysaccharide and expression of Bax protein in the cells were all increased (P<0.05), but the number of clones, migration and invasion and the protein expression of Bcl-2 were all decreased (P<0.05), and were dose-dependent. The expression of circ_UHRF1 in A549 cells that intervened with the S. lyratum polysaccharide was decreased (P<0.05), but the expression of miR-513b-5p was increased (P<0.05). The expression of circ_UHRF1 in lung cancer tissues was higher than that of adjacent tissues (P<0.05), and the expression of miR-513b-5p was lower than that of adjacent tissues (P<0.05). The expressions of circ_UHRF1 and miR-513b-5p in lung cancer tissues were negatively correlated (r=-0.861, P<0.05). Circ_UHRF1 could target miR-513b-5p, and the expression of miR-513b-5p in A549 cells knocking down circ_UHRF1 was increased. After knocking down circ_UHRF1, the proliferation inhibition rate and apoptosis rate of A549 cells and protein expression of Bax in the cells were all increased (P<0.05), but the number of clones, migration and invasion and the protein expression of Bcl-2 were all decreased (P<0.05). Up-regulation of circ_UHRF1 reduced the effects of S. lyratum polysaccharide on the proliferation, migration, invasion and apoptosis of A549 cells. In general, S. lyratum polysaccharide could inhibit the proliferation, migration and invasion of lung cancer A549 cells, and induce cell apoptosis. Its mechanism may be related to the regulation of the circ_UHRF1/miR-513b-5p axis.

Polyacetylenes from Codonopsis lanceolata Root Induced Apoptosis of Human Lung Adenocarcinoma Cells and Improved Lung Dysbiosis.

Codonopsis lanceolata is a perennial smelly herbaceous plant and widely employed for the treatment of various lung cancer and inflammation. However, the anticancer substances in C. lanceolata and their underlying mechanisms had not been well clarified. In this study, six compounds were obtained from the water extracts of C. lanceolata polyacetylenes (CLP) and then identified as syringin, codonopilodiynoside A, lobetyol, isolariciresinol, lobetyolin, and atractylenolide III. Treatment with CLP remarkably suppressed the cell proliferation, colony formation, migration, and invasion of A549 cells. Synergistic effects of lobetyolin and lobetyol were equivalent to the antiproliferative activities of CLP, while other compounds did not have any inhibition on the viabilities of A549 cells. CLP also reduced the expression of Ras, PI3K, p-AKT, Bcl-2, cyclin D1, and CDK4 but increased the expression of Bax, GSK-3β, clv-caspase-3, and clv-caspase-9, which could be reversed by the PI3K activator 740YP. Furthermore, CLP retarded the growths of tumor and lung pathogenic bacteria in mice. It demonstrated that lobetyolin and lobetyol were the main antitumor compounds in C. lanceolata. CLP induced cell apoptosis of lung cancer cells via inactivation of the Ras/PI3K/AKT pathway and ameliorated lung dysbiosis, suggesting the therapeutic potentials for treating human lung cancer.