H1299 Cells Research Articles

Tetraspanin 3 promotes NSCLC cell proliferation via regulation of β1 integrin intracellular recycling

BackgroundThe involvement of tetraspanins in cancer development has been widely implicated. In this study, the function and molecular mechanisms of tetraspanin 3 (TSPAN3) in non-small cell lung cancer (NSCLC) cells were explored.MethodsTissue samples from patients diagnosed with NSCLC were analyzed by immunohistochemistry, western blotting, and real-time polymerase chain reaction (PCR) to indicate the involvement of TSPAN3 in cancer progression. In the meantime, we also performed exhaustive mechanistic studies using A549 and H460 cells in vitro through a variety of methods including western blotting, real-time PCR, immunofluorescent staining, coimmunoprecipitation, cell proliferation assay, and nocodazole (NZ) washout assay. Proper statistical analysis was implemented wherever necessary in this study.ResultsTSPAN3 was found to be highly expressed in lung cancer cells and tissues. Moreover, high levels of TSPAN3 positively correlated with poor differentiation, lymph node involvement, advanced pathological tumor-node-metastasis stage, and poor prognosis in patients with NSCLC. TSPAN3 showed potential to promote the proliferation of NSCLC cells in vitro and in vivo. Specifically, TSPAN3 was found to interact with β1 integrin via the LEL domain, thereby facilitating the sorting of β1 integrin into Rab11a endosomes and promoting β1 integrin recycling and upregulation.ConclusionsOur findings reveal TSPAN3 may represent a potentially valuable therapeutic target for NSCLC.

FBXO11 Mediates Ubiquitination of ZEB1 and Modulates Epithelial-to-Mesenchymal Transition in Lung Cancer Cells.

Epithelial-to-mesenchymal transition (EMT) affects the invasion and migration of cancer cells. Here, we show that FBXO11 recognizes and promotes ubiquitin-mediated degradation of ZEB1. There is a strong association between FBXO11 and ZEB1 in non-small cell lung cancer (NSLC) in a clinical database. FBXO11 interacts with ZEB1, a core inducer of EMT. FBXO11 leads to increased ubiquitination and proteasomal degradation of ZEB1. Depletion of endogenous FBXO11 causes ZEB1 protein accumulation and EMT in A549 and H1299 cells, while overexpression of FBXO11 reduces ZEB1 protein abundance and cellular invasiveness. Importantly, the depletion of ZEB1 suppresses the increased migration and invasion of A549 and H1299 cells promoted by the depletion of FBXO11. The same results are shown in xenograft tumors. High FBXO11 expression is associated with a favorable prognosis in NSLC. Collectively, our study demonstrates that FBXO11 modulates EMT by mediating the stability of ZEB1 in lung cancer cells.

Unveiling the antitumor synergy between pazopanib and metformin on lung cancer through suppressing p-Akt/ NF-κB/ STAT3/ PD-L1 signal pathway

Pazopanib, an inhibitor of the VEGF receptor tyrosine kinase, has demonstrated significant antitumor effects in lung cancer. However, its application as a standard treatment for this type of cancer is limited by its drug resistance and toxicity. Metformin has the potential to combat lung cancer by modifying the tumor’s immune microenvironment.In this study, we investigated the potential antitumor effects and the associated underlying molecular mechanisms of the combination of pazopanib and metformin in lung cancer. In vitro studies were conducted using the A549 and H460 lung cancer cell lines, whereas urethane-induced lung cancer-bearing mice were used for in vivo assessments. The urethane-induced mice received oral administration of pazopanib (50 mg/kg) and/or metformin (250 mg/kg) for a duration of 21 days.The results indicated that the MTT assay demonstrated a combined cytotoxic effect of the pazopanib/metformin combination in H460 and A549 cells, as evidenced by CI and DRI analyses. The observed increase in annexin V levels and the corresponding increase in Caspase-3 activity strongly suggest that this combination induced apoptosis.Furthermore, the pazopanib/metformin combination significantly inhibited the p-Akt/NF-κB/IL-6/STAT3, HIF1α/VEGF, and TLR2/TGF-β/PD-L1 pathways while also increasing CD8 expression in vivo. Immunohistochemical analysis revealed that these antitumor mechanisms were manifested by the suppression of the proliferation marker Ki67. In conclusion, these findings revealed that metformin augments the antitumor efficacy of pazopanib in lung cancer by simultaneously targeting proliferative, angiogenic, and immunogenic signaling pathways, metformin enhances the antitumor effectiveness of pazopanib in lung cancer, making it a promising therapeutic option for lung cancer.

MiR-6884-5p inhibits proliferation and epithelial-mesenchymal transition in non-small cell lung cancer cells

BackgroundNon-small cell lung cancer (NSCLC) is associated with a high mortality and morbidity rate. MicroRNAs participate in tumorigenesis, progression and metastasis of NSCLC. However, miR-6884-5p has not been previously studied. This study aimed to investigate the role of miR-6884-5p in NSCLC and explore its underlying mechanisms. MethodsWe used miR-6884-5p mimics and inhibitors to assess its effects in NSCLC. miR-6884-5p expression levels in NSCLC cell lines were quantified using qRT-PCR. Cell viability was determined using a cell-counting kit 8 assay. Western blot analysis was employed to measure apoptotic proteins. The impact of miR-6884-5p on cell proliferation was assessed via colony formation assay. Furthermore, Transwell assays were utilized to visualize and quantify the effects of miR-6884-5p on NSCLC migration and invasion. ResultsmiR-6884-5p mimic significantly inhibited NSCLC cell proliferation to 71.21 % and 72.26 % of control at 5 days of culture time in H460 and HC9 cells (both p < 0.01), respectively, while miR-6884-5p inhibitor significantly promoted cell proliferation to 119.66 % and 126.44 % of control at 5 days of culture time in H460 and HC9 cells (both p < 0.05), respectively. In addition, miR-6884-5p promoted apoptosis by reducing the anti-apoptotic protein B-cell lymphoma 2 (BCL2) protein and increasing apoptotic protein BCL2 associated X protein (all p < 0.01 at least). Moreover, miR-6884-5p effectively suppressed transforming growth factor β1-induced epithelial-mesenchymal transition, as evidenced by the restored expression of E-cadherin (p < 0.01), N-cadherin (p < 0.01) and Vimentin (p < 0.05), leading to the inhibition of migration and invasion in NSCLC cell lines. ConclusionsOur findings demonstrate that miR-6884-5p can inhibit NSCLC cell proliferation, migration, and invasion, suggesting its potential as a therapeutic target for NSCLC treatment.

Therapeutic effects of paeonol on non‑small cell lung cancer cells via regulation of the MAPK pathway.

The present study aimed to investigate the molecular mechanisms by which paeonol impedes DNA damage repair, induces apoptosis and inhibits cell viability via the mitogen-activated protein kinase (MAPK) pathway. Firstly, normal human bronchial epithelial cells (BEAS-2B) and non-small cell lung cancer cells (H1299) were employed in the study as cellular models. Following cultivation, the cells were divided into experimental and control groups, and were treated with different concentrations of paeonol. Subsequently, various techniques, including western blotting, Cell Counting Kit-8, colony formation, TUNEL and comet assays were conducted to evaluate the effects of paeonol on cell viability, colony-forming ability, apoptosis levels and DNA damage in H1299 cells. According to the experimental results, paeonol significantly reduced the viability and colony formation ability of H1299 cells, but substantially increased apoptosis and DNA damage. These effects were enhanced in response to higher concentrations of paeonol. Furthermore, western blot analysis revealed that paeonol treatment decreased the protein levels of B-cell lymphoma 2 and breast cancer susceptibility gene 1, while it increased the expression levels of cleaved-PARP, cleaved-caspase 3, γH2AX and P21. Additionally, the phosphorylated levels of extracellular signal-regulated kinase 1, c-Jun N-terminal kinase and P38 within the MAPK signaling pathway were diminished. Collectively, the present study demonstrated that paeonol may inhibit the metabolic activity and proliferative capability of H1299 cells, and that it could promote apoptosis and obstruct DNA damage repair by modulating the MAPK signaling pathway.

Copper(II) and Zinc(II) complexes of new water-soluble Schiff base ligands and their antiproliferative properties towards mesothelioma cell line

In this work, three Schiff base ligands (HL1-HL3) containing a triphenyl phosphonium cation and their Cu(II) and Zn(II) complexes with the general formulae of [M(L)Cl2] were synthesized and their structures were characterized by spectroscopic and analytical methods. Crystal structure of complex [Zn(L1)Cl2] was determined. In the structure of the complex, each Zn(II) ion is four coordinated binding to phenolate oxygen and imine nitrogen atoms of the ligand L1 and two chloride atoms in approximately tetrahedral geometry. The equilibrium geometry of three Schiff base ligands (HL1-HL3) and their Cu(II) and Zn(II) complexes were calculated using the density functional theory (DFT/B3LYP) method with the 6–31++G(d,p) basis set for the C, H, Cl, N, O, P atoms and LANL2DZ for the I atom. Frontier molecular orbitals (HOMO, LUMO) analyses were conducted for the optimized geometries of the compounds, and chemical reactivity descriptors such as hardness, softness, electrophilicity, and electronegativity were examined. Additionally, the molecular electrostatic potential (MEP) of all compounds was modeled using DFT calculations. These calculations were thoroughly examined, and their implications were discussed. The ligands and their metal complexes were investigated for their DNA binding properties. The compounds showed comparable DNA binding properties to ethidium bromide (EB) and 5-fluorouracyl (5-Fu) with DNA binding constant (Kb) 1.5–6.25 × 105 M−1. The cytotoxic properties of the compounds towards HUVEC and H2452 (mesothelioma cell line) cells were investigated using an MTS assay. The IC50 values determined for HUVEC cells were found to range between 27.28 µg/mL ([Cu(L2)Cl2]) and 273.1 µg/mL ([Zn(L2)Cl2]), while in H2452 cells, they varied from 39.48 µg/mL ([Cu(L2)Cl2]) to 319.3 µg/mL ([Zn(L3)Cl2]). The compounds HL1, HL3, and [Zn(L3)Cl2] exhibited antioxidant activity at the highest concentration (750 µg/mL) only, while [Zn(L1)Cl2], HL2, [Zn(L2)Cl2], [Cu(L2)Cl2], and [Cu(L3)Cl2] showed antioxidant activity at 250 µg/mL.

PARD6A promotes lung adenocarcinoma cell proliferation and invasion through Serpina3.

Par6α encoded by PARD6A is a member of the PAR6 family and is reported to promote cancer initiation and progression. PARD6A is frequently upregulated in different types of cancers, but its regulatory role in lung cancer progression is yet to be established. In this study, we analyzed the PARD6A expression in biopsies from lung adenocarcinoma (LUAD) patients, and the survival probability using LUAD tissue microarray (TMA) and online datasets from TCGA and GEO. We conducted in vitro and in vivo assays to assess the role of PARD6A in regulating lung cancer progression, including proliferation, wound healing, transwell, RNA-seq, and subcutaneous tumor mice models. Our findings revealed that PARD6A is highly expressed in cancer tissues from LUAD patients and is associated with poor prognosis in LUAD patients. In vitro assays showed that PARD6A promoted cell proliferation, migration, and invasion. The transcriptome sequencing identified Serpina3 as one of the key downstream molecules of PARD6A. Ectopic expression of Serpina3 rescued impaired proliferation, migration, and invasion in PARD6A-knocking down H1299 cells, whereas silencing Serpina3 impeded enhanced proliferation, migration, and invasion in PARD6A-overexpressing H1975 cells. Our findings suggest that PARD6A promotes lung cancer progression by inducing Serpina3, which may be a promising therapeutic target.

The Proteolytic Activity of Neutrophil-Derived Serine Proteases Bound to the Cell Surface Arming Lung Epithelial Cells for Viral Defense.

The collaboration between cellular proteases and host cells is pivotal in mounting an effective innate immune defense. Of particular interest is the synergistic interaction between cathepsin G (CatG) and neutrophil elastase (NE), which are proteases secreted by activated neutrophils, and the human alveolar basal epithelial cell line (A549) and the human lung epithelial-like cell line (H1299), because of the potential implications for viral infection. Our study aimed to investigate the binding capacity of CatG and NE on the surface of A549 and H1299 cells through preincubation with purified CatG and NE; thereby, the proteolytic activity could be detected using activity-based probes. Both CatG and NE were capable of binding to the cell surface and exhibited proteolytic activity, leading to increased cell surface levels of MHC I molecules, which is crucial for displaying the endogenous antigenic repertoire. In addition, CatG cleaved the S2' site of the SARS-CoV-2 spike protein at two specific sites (815RS816 and 817FI818) as well as NE (813SK814 and 818IE819), which potentially leads to the destruction of the fusion peptide. Additionally, furin required the presence of Ca2+ ions for the distinct cleavage site necessary to generate the fusion peptide. Overall, the findings suggest that CatG and NE can fortify target cells against viral entry, underscoring the potential significance of cell surface proteases in protecting against viral invasion.

Novel DNA Repair Inhibitors Targeting XPG to Enhance Cisplatin Therapy in Non-Small Cell Lung Cancer: Insights from In Silico and Cell-Based Studies.

NSCLC is marked by low survival and resistance to platinum-based chemotherapy. The XPG endonuclease has emerged as a promising biomarker for predicting the prognosis of cisplatin-treated patients and its downregulation having been reported to increase cisplatin efficacy. This study presents an integrated strategy for identifying small molecule inhibitors of XPG to improve cisplatin therapy in NSCLC. A structure-based virtual screening approach was adopted, including a structural and physicochemical analysis of the protein, and a library of small molecules with reported inhibitory activities was retrieved. This analysis identified Lys84 as a crucial residue for XPG activity by targeting its interaction with DNA. After molecular docking and virtual screening calculations, 61 small molecules were selected as potential XPG inhibitors, acquired from the ChemBridge database and then validated in H1299 cells, a NSCLC cell line exhibiting the highest ERCC5 expression. The MTS assay was performed as a first screening approach to determine whether these potential inhibitors could enhance cisplatin-induced cytotoxicity. Overall, among the eight compounds identified as the most promising, three of them revealed to significantly increase the impact of cisplatin. The inherent cytotoxicity of these compounds was further investigated in a non-tumoral lung cell line (BEAS-2B cells), which resulted in the identification of two non-cytotoxic candidates to be used in combination with cisplatin in order to improve its efficacy in NSCLC therapy.

Association of Wild-Type TP53 with Downregulation of Lovastatin Sensitivity in Human Non-Small Cell Lung Cancer Cells.

Statins inhibit 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway, and reduce cholesterol synthesis. They also have been demonstrated to improve prognosis in patients with various cancers, suggesting a potential anti-cancer effect of statins. However, there is no consensus on the molecular targets of statins for their anti-cancer effects. Docetaxel (DOC) is a microtubule-stabilizing agent currently used as a chemotherapeutic drug in several cancers, including lung cancer. Interestingly, the anti-cancer effects of either drug that are related to abnormal or wild-type TP53 gene have been implied. Therefore, the drug sensitivity of DOC and lovastatin in human lung cancer cells was evaluated. We found that H1355 (mutant TP53-E285K), CL1 (mutant TP53-R248W), and H1299 (TP53-null) human non-small cell lung cancer cells were more sensitive to lovastatin than A549 and H460 cells expressing wild-type TP53. Conversely, A549 and H460 cells showed higher sensitivity to DOC than H1299 and CL1 cells, as demonstrated by the MTT assay. When endogenous TP53 activity was inhibited by pifithrin-α in A549 and H460 cells, lovastatin sensitivities significantly increased, and cancer cell viabilities markedly reduced. These results indicate that TP53 status is associated with the anti-cancer effect of statins in human lung cancer cells. Mutated or null TP53 status is correlated with higher statin sensitivity. Furthermore, DOC-resistant H1299 (H1299/D8) cells showed significant sensitivity to lovastatin treatment compared to DOC-resistant A549 (A549/D16) cells, indicating a potential application of statins/chemotherapy combination therapy to control wild-type and abnormal TP53-containing human lung tumors.

Gossypol Inhibits Metastasis of Lung Cell Carcinoma by Reversing Epithelial to Mesenchymal Transition and Suppressing Proteases Activity.

Gossypol, a natural polyphenolic compound, possesses antivirus activity and induces cell death of different types of tumors. However, the efficacy of gossypol on lung carcinoma metastases and epithelial to mesenchymal transition remains unknown. The aim of the present work was to determine the cellular and molecular mechanism of the anti-cancer and anti-metastatic efficacies of gossypol on human lung carcinoma cells. Gossypol showed a marked suppression of the viability, motility, and invasion in H1299 and A549 cells. Zymography assay showed that gossypol was sufficient to suppress the activities of urokinase-type plasminogen activator and matrix metalloproteinase-2. Gossypol reversed TGF-β-induced epithelial to mesenchymal transition. Gossypol reduced vimentin, p-FAK, p-Src and p-paxillin. In vivo studies of gossypol were performed using subcutaneous inoculation and tail vein injection of A549 into immunodeficient BALB/c nude mice and severe combined immunodeficient mice.

SLC12A8 promotes the migration and invasion of non-small cell lung cancer (NSCLC) cells.

This study aims to investigate the impacts of SLC12A8 on the invasion, migration, and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) cells. GEPIA database was employed to examine SLC12A8 expression pattern in lung cancer cells. Subsequently, qRT-PCR and Western blot analyses were conducted to assess SLC12A8 expression in NSCLC tissues and cell lines. The overall prognosis of NSCLC patients was evaluated using Kaplan-Meier plot and univariate and multivariate COX regression curves. The knockdown of SLC12A8 was established using lentivirus-mediated shRNA in A549 and H1299 cells. Cell proliferation, invasion, migration, and apoptosis were evaluated using CCK-8 assay, transwell, and flow cytometry techniques, respectively. Western blot analysis was performed to measure the expression levels of EMT-related proteins (E-cadherin and vimentin). The expression level of SLC12A8 was found to be significantly higher in both NSCLC cell lines and tissues. High SLC12A8 expression was correlated with a poor prognosis in NSCLC patients. Knocking down SLC12A8 led to a significant decrease in proliferation, migration, and invasion abilities, while promoting apoptosis in NSCLC cells. Additionally, SLC12A8 knockdown resulted in decreased levels of N-cadherin and vimentin, along with increased E-cadherin expression. The results indicate that reducing SLC12A8 expression may suppress the malignant phenotype of NSCLC cells, as well as the EMT. SLC12A8 may serve as a target for the clinical management of NSCLC progression.

LRRC45 promotes lung cancer proliferation and progression by enhancing c-MYC, slug, MMP2, and MMP9 expression

BackgroundThe leucine-rich repeat-containing (LRRC) superfamily members are known for their significant roles in tumorigenesis and cellular proliferation. However, the specific regulatory role of LRRC45 in lung cancer remains unexplored. This study investigated the impact and underlying mechanisms of LRRC45 on the proliferative, migratory, and invasive capacities of lung adenocarcinoma (LUAD) cells, potentially identifying new targets for therapeutic intervention. Material and methodsThe importance of LRRC45 in lung cancer was analyzed using the online databases of UCSC Xena, TCGA, TISIDB, and UALCAN, whereas to detect target gene expression, we used the qRT-PCR, Western blot, and immunofluorescence confocal. The cell growth was monitored by colony formation assay and migration was examined by cell migration assay. Finally, a xenograft mouse tumor model using A549 ​cells was used to explore the in vivo effect of LRRC45 in lung cancer. ResultsInhibition of LRRC45 expression led to a notable decrease in proliferation, migration, and invasion of A549 and H1299 ​cells. LRRC45 silencing significantly reduced the tumor volume and improved the mice's survival. Additionally, inhibition of LRRC45 expression dramatically suppressed c-MYC, Slug, MMP2, and MMP9 expression. Overexpression of c-MYC and/or Slug in the LRRC45-deficient cells can partially or totally restore the LRRC45 deficiency-suppressed growth. Moreover, the overexpression of MMP2 and/or MMP9 could partially or totally restore LRRC45 deficiency-reduced cell metastasis. ConclusionsLRRC45 could promote the proliferative, migrative, and invasive capacities of lung cancer cells by increasing c-MYC, Slug, MMP2, and MMP9 expression, indicating the therapeutic implications and potential significance of these pathways in lung cancer.

A Novel 3-Benzyloxychromone From Celastrus orbiculatus Thunb. Exhibits Anticancer Effects on Non-Small Cell Lung Cancer Cells via GSK-3β-Dependent c-Jun/ATF2 Pathway.

Celastrus orbiculatus Thunb. is a vine used as a traditional Chinese medicinal herb. In this study, we focused on the anticancer cytotoxicity and underlying mechanism of previously unreported 3-oxygen-substituted isoflavone analogue (3-benzyloxychromone, 3-Boc) from the herb. Initially, we established cell line-derived xenograft mouse model using H1299 non-small cell lung cancer (NSCLC) cells and found that the ethanol crude extracts of the stem part of C. orbiculatus (200 mg/kg) could substantially suppress the growth of xenograft tumors in athymic nu/nu mice. We compared 3-Boc with three other flavonoid analogues isolated from the stem part of C. orbiculatus. Among these, 3-Boc showed the most potent cytotoxicity against H1299 and H1975 NSCLC cells. Colony formation, EdU incorporation and Annexin V-FITC/PI apoptosis assays demonstrated that 3-Boc induced growth inhibition primarily by inhibiting DNA replication and inducing apoptotic death of NSCLC cells. Structure-based target prediction and MD simulation suggested that 3-Boc potentially suppressed the activity of glycogen synthase kinase-3β (GSK-3β) by interacting with the ATP-binding site. Western blot analysis indicated that 3-Boc triggered the phosphorylation of Serine 21 of GSK-3α or Serine 9 of GSK-3β in a time- and dose-dependent manner. To investigate the dependency of GSK-3β, we established GSK-3β knockout in H1299 cells. Depletion of GSK-3β enhanced 3-Boc-induced cytotoxicity compared with wild-type counterparts through activated c-Jun/ATF2 signaling pathway. Altogether, our study highlights the anticancer potential of C. orbiculatus and the discovery of novel 3-oxygen-substituted chromone from the herb, which may have important implications for screening promising modulators of GSK-3β and related signaling pathways in the treatment of cancer.

Effects of 630nm laser on apoptosis, metastasis, invasion and epithelial-to-mesenchymal transition of human lung squamous cell carcinoma H520 cells mediated by hematoporphyrin derivatives.

Photodynamic therapy (PDT) has significant advantages in the treatment of malignant lung tumors. The research on the mechanism of PDT mediated by hematoporphyrin derivatives (HPD) and its cytotoxic effects on lung cancer cells has primarily focused on lung adenocarcinoma cells. However, the impact of HPD-PDT on lung squamous cell carcinoma has not been thoroughly studied. This study aimed to investigate the effects of 630nm laser on apoptosis, metastasis, invasion, and epithelial-mesenchymal transition (EMT) in human lung squamous cell carcinoma H520 cells mediated by HPD. H520 cells were divided into four groups: control group, photosensitizer group, irradiation group, and HPD-PDT group. Cell proliferation was assessed using CCK8 assay; cell apoptosis was detected by Hoechst33258 staining and flow cytometry; cell migration and invasion abilities were evaluated using wound-healing and invasion assays; and protein and mRNA expressions were analyzed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR)respectively. Results showed that HPD-PDT significantly inhibited cell proliferation, promoted apoptosis (P < 0.05), suppressed cell migration and invasion (P < 0.05), decreased Bcl-2 mRNA expression, and increased Bax and Caspase-9 mRNA expression(P < 0.05). Western blotting analysis indicated increased expression of Bax, Caspase-9, and E-cadherin, and decreased expression of Bcl-2, N-cadherin, and Vimentin (P < 0.05). In conclusion, 630nm laser mediated by HPD promoted cell apoptosis via upregulation of Bax and caspase-9, and downregulation of Bcl-2, and inhibited cell migration and invasion by regulating EMT in H520 cells.

Mitochondrial VDAC1 Silencing in Urethane-Induced Lung Cancer Inhibits Tumor Growth and Alters Cancer Oncogenic Properties.

Alterations in cellular metabolism are vital for cancer cell growth and motility. Here, we focused on metabolic reprogramming and changes in tumor hallmarks in lung cancer by silencing the expression of the mitochondrial gatekeeper VDAC1. To better mimic the clinical situation of lung cancer, we induced lung cancer in A/J mice using the carcinogen urethane and examined the effectiveness of si-m/hVDAC1-B encapsulated in PLGA-PEI nanoparticles. si-m/hVDAC1-B, given intravenously, induced metabolism reprogramming and inhibited tumor growth as monitored using MRI. Mice treated with non-targeted (NT) PLGA-PEI-si-NT showed many large size tumors in the lungs, while in PLGA-PEI-si-m/hVDAC-B-treated mice, lung tumor number and area were markedly decreased. Immunofluorescence staining showed decreased expression of VDAC1 and metabolism-related proteins and altered expression of cancer stem cell markers. Morphological analysis showed two types of tumors differing in their morphology; cell size and organization within the tumor. Based on specific markers, the two tumor types were identified as small cell (SCLC) and non-small cell (NSCLC) lung cancer. These two types of tumors were found only in control tumors, suggesting that PLGA-PEI-si-m/hVDAC1-B also targeted SCLC. Indeed, using a xenograft mouse model of human-derived SCLC H69 cells, si-m/hVDAC1-B inhibited tumor growth and reduced the expression of VDAC1 and energy- and metabolism-related enzymes, and of cancer stem cells in the established xenograft. Additionally, intravenous treatment of urethane-induced lung cancer mice with the VDAC1-based peptide, Retro-Tf-D-LP4, showed inhibition of tumor growth, and decreased expression levels of metabolism- and cancer stem cells-related proteins. Thus, silencing VDAC1 targeting both NSCLC and SCLC points to si-VDAC1 as a possible therapeutic tool to treat these lung cancer types. This is important as target NSCLC tumors undergo transformation to SCLC.

The Role of ADCY1 in Regulating the Sensitivity of Platinum-Based Chemotherapy in NSCLC.

Lung cancer has the highest fatality rate among malignant tumors in the world. Finding new biomarkers of drug resistance is of great importance in the prognosis of lung cancer patients. We found that the polymorphisms of Adenylate Cyclase 1 (ADCY1) are significantly associated with platinum-based chemotherapy resistance in lung cancer patients in our previous research. In this study, we wanted to identify the mechanism of ADCY1 affecting platinum resistance. We used an MTT assay to find if the expression of ADCY1 is associated with the sensitivity of cisplatin in A549, H1299, and A549-DDP cells. Then, we performed CCK-8 tests to detect the absorbance of these cells stimulated by ADCY1, which can discover the cell proliferation that is affected by ADCY1. We investigated cell apoptosis and cell cycles regulated by ADCY1 through the flow cytometry assay. RNA sequencing was used to find the downstream genes affected by ADCY1 which may be associated with drug resistance in lung cancer patients. ADCY1 has higher expression in lung cancer cells than in normal cells. ADCY1 can affect cisplatin resistance in lung cancer cells by regulating cell proliferation, cell apoptosis, and the cell cycle. It may control cell apoptosis by regulating the classical apoptosis biomarkers Bax and Bcl2. Our study showed that ADCY1 may be a new biomarker in the prognosis of lung cancer patients. Much work remains to be carried out to clarify the mechanism in this important emerging field.

PBAE-PEG based lipid nanoparticles for lung cell-specific gene delivery.

Delivery of modified mRNA encapsulated in lipid nanoparticles, exemplified by their successful use in COVID-19 vaccination, provides a framework for treating various genetic and acquired disorders. Herein, we developed PEGylated(PBAE-PEG) and non-PEGylated(PBAE) PBAE with lipids 4A3-SC8/DOPE/cholesterol/DOTAP to form lipid nanoparticles (LNPs) for mRNA delivery into different types of pulmonary cells in vivo. PBAE-PEG/LNP were highly active in transfecting HEK293T cells and air-liquid interfaced H441 cells in vitro. PBAE-PEG/LNP were used to express Cre-recombinase after administration to mice by intravenous injection, resulting in high transfection levels in pulmonary vascular endothelial cells. Intratracheal injection of both PBAE-PEG/LNP and PBAE/LNPs resulted in efficient and selective transfection of lung epithelial cells, identified by the expression of stabilized Cre-recombinase mRNA in club cells and alveolar type 2 cells. PBAE-PEG/LNP were most effective in transfecting alveolar type 2 cells after intratracheal injection, while PBAE/LNPs administered intratracheally were more effective in transfecting secretory airway cells. Cre-mediated recombination was specific to lung epithelial cells after intratracheal administration. Likewise, intravenous administration resulted in selective transfection of endothelial cells but not other pulmonary cell types, indicating their failure to cross the pulmonary endothelial-to-epithelial barrier. Moreover, 5-methoxyuridine modified mRNA was more efficient than unmodified mRNA in vivo but not in vitro.

MicroRNA-145 enhances lung cancer cell progression after exposure to lyophilized fertile hydatid cyst fluid of Echinococcus granulosus sensu stricto

There is increasing evidence that the secretory/excretory antigens of the larval stage of Echinococcus granulosus can induce both anticancer and oncogenic effects between parasite-derived metabolites and various cancer cells. The dual role of miR-145 as either a tumor suppressor or oncogene has already been reported in cancer. However, the mechanism by which miR-145 induces apoptosis in lung cancer cells treated with hydatid cyst fluid (HCF) remains unclear. The fertile HCF was obtained from sheep, purified and lyophilized. H1299 human lung cancer cells were then cultured into two groups: HCF-treated H1299 lung cancer cells and untreated H1299 cancer cells as control cells. Cell viability was assessed using MTT assay to evaluate the effects of HCF on the H1299 cells. Caspase-3 activity was assessed by fluorometric assay. In addition, mRNA expression levels of VGEF, vimentin, caspase-3, miRNA-145, Bax and Bcl-2 genes were quantified by real-time PCR. A scratch test was also performed to assess the effects of HCF on cell migration. The MTT assay revealed that the growth of H1299 cells increased when treated with 60 μg/mL of fertile HCF for 24 h. The fold change of caspase-3, miRNA-145, Bax/Bcl-2 ratio and caspase-3 activity was lower in HCF-treated H1299 cells compared to the control cell. The fold change in VGEF and vimentin gene expression was higher in the HCF-treated H1299 cells than in the control cell. The scratch test results showed that H1299 cell mobility increased 24 and 48 h after exposure to HCF. Our results suggest that the downregulation of miR-145 in HCF-treated H1299 cells may play a role as a possible oncogenic regulator of lung cancer growth. To confirm this assumption, further studies are required to evaluate the microRNA profile and effective oncogenes in vivo.

Overexpression of microRNA-611 inhibits TGF-β-induced epithelial-mesenchymal transition and migration in lung cancer cells through MAPKAP1

Metastasis is a major cause of death in patients with lung cancer (LC). microRNA-611 (miR-611), a miRNA, has been little studied in cancer. Here, we aimed to further elucidate the roles of miR-611 in epithelial-mesenchymal transition (EMT) and migration induced by transforming growth factor-β (TGF-β) in LC cells and the possible underlying mechanisms. miR-611 and MAPKAP1 expression was first identified in LC tissues from metastatic and nonmetastatic patients, and their expression was associated with overall survival. Gain- and loss-of-function experiments were performed to verify the impacts of miR-611 and MAPKAP1 on pAKT expression, EMT, and migration in LC cells treated with TGF-β. The interaction between miR-611 and MAPKAP1 was also determined with a luciferase reporter assay. In our study, miR-611 was expressed at low levels, and MAPKAP1 was highly expressed in LC tissues, which was associated with metastasis and short overall survival. Functionally, miR-611 inhibition or MAPKAP1 overexpression accelerated EMT and migration and upregulated pAKT in TGF-β-treated A549 and H1299 cells; miR-611 overexpression or MAPKAP1 silencing exerted the opposite effects as miR-611 inhibition or MAPKAP1 overexpression. Mechanistically, miR-611 could target and downregulate MAPKAP1. MAPKAP1 expression was also negatively correlated with miR-611 expression in LC tissues. In addition, miR-611 overexpression reduced the EMT and migration of TGF-β-treated A549 and H1299 cells by targeting MAPKAP1. In conclusion, miR-611 overexpression attenuated EMT and migration by targeting MAPKAP1 in TGF-β-induced LC cells, indicating that miR-611 is a biological target for LC treatment.