scholarly journals Restricting Glutamine Uptake Enhances NSCLC Sensitivity to Third-Generation EGFR-TKI Almonertinib

2021 ◽  
Vol 12 ◽  
Author(s):  
Yaming Liu ◽  
Xianming Ge ◽  
Jinlong Pang ◽  
Yuhan Zhang ◽  
Hao Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Colony Formation ◽  
A549 Cells ◽  
Glutamine Metabolism ◽  
Wild Type ◽  
Secondary Resistance ◽  
Submicroscopic Structure ◽  
Related Proteins ◽  
Egfr Tkis ◽  
Glutamine Uptake

The emergence of secondary resistance is the main failure cause of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) as a targeted therapy for non-small cell lung cancer (NSCLC). EGFR mutations of NSCLC cells can markedly increase glutamine transporter (SLC1A5) expression, thereby increasing glutamine metabolism. Glutamine metabolites can activate EGFR downstream signals, including mTOR, ERK1/2, STAT3, etc., which is an important cause for the decreased sensitivity of NSCLC to EGFR-TKIs. CCK8 and Annexin V/PI assays were conducted to detect the effects of Almonertinib and/or V9302 on the proliferation and apoptosis of NSCLC cells. Proteomics was used to determine the effect of Almonertinib on energy metabolism-related proteins in NSCLC. siRNA transfection was performed to study the effect of SLC1A5 down-regulation on cell proliferation. In addition, the effects of drugs on colony formation capacity were determined by colony formation assay. Immunofluorescence and Western blot were utilized to detect the apoptosis- and autophagy-related proteins expression. DAPI staining was utilized to detect the effect of drugs on the nucleus. Transmission electron microscope was used to observe the changes of submicroscopic structure such as autophagosomes and nucleus of cells. mCherry-GFP-LC3B tandem fluorescent protein was to used to detect the level of autophagy flux. Tumor-bearing nude mouse model was utilized to detect the effect of V9302 on the anti-tumor effect of Almonertinib in vivo. As a result, Almonertinib suppressed H1975 and A549 cell proliferation depended on its dosage and treatment duration, and it also induced apoptosis. A549 cells with wild-type EGFR had lower sensitivity to Almonertinib. The expression of SLC1A5 was up-regulated by stimulating with low concentration of Almonertinib in NSCLC cells. SLC1A5 was highly expressed in A549 cells with wild-type EGFR. Glutamine deletion or SLC1A5 inhibition/silencing inhibited the proliferation of NSCLC cells, and decreased cellular glutamine uptake. The combination of SLC1A5 inhibitor V9302 and Almonertinib had a synergistic inhibitory effect on the proliferation of NSCLC. V9302 enhanced the effect of Almonertinib in apoptosis-inducing in NSCLC cells. The combination of V9302 and Almonertinib might induce apoptosis by inhibiting autophagy.

scholarly journals Role of Protein Kinase G in Growth and Glutamine Metabolism of Mycobacterium bovis BCG

2005 ◽  
Vol 187 (16) ◽  
pp. 5852-5856 ◽  
Author(s):  
Liem Nguyen ◽  
Anne Walburger ◽  
Edith Houben ◽  
Anil Koul ◽  
Stefan Muller ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mycobacterium Bovis ◽  
Glutamine Metabolism ◽  
Protein Kinase G ◽  
In Vitro Growth ◽  
Wild Type ◽  
Putative Operon ◽  
Glutamine Uptake

ABSTRACT The survival of pathogenic mycobacteria in macrophages requires the eukaryotic enzyme-like serine/threonine protein kinase G. This kinase with unknown specificity is secreted into the cytosol of infected macrophages and inhibits phagosome-lysosome fusion. The pknG gene is the terminal gene in a putative operon containing glnH, encoding a protein potentially involved in glutamine uptake. Here, we report that the deletion of pknG did not affect either glutamine uptake or intracellular glutamine concentrations. In vitro growth of Mycobacterium bovis BCG lacking pknG was identical to that of the wild type. We conclude that in M. bovis BCG, glutamine metabolism is not regulated by protein kinase G.

MiR-381 enhances the sensitivity of non-small cell lung cancer to radiotherapy by targeting ROCK2 to regulate NF-κB signaling pathway

2020 ◽  
Author(s):  
Jiuning Huang ◽  
Jing Zeng ◽  
Shuping Zhang ◽  
Jundong Cai ◽  
Wulong Wang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Western Blot ◽  
Signaling Pathway ◽  
A549 Cells ◽  
Small Cell ◽  
Small Cell Lung ◽  
Tumor Tissues ◽  
Related Proteins ◽  
Radiotherapy Treatment

Abstract BackgroundWe investigated the effect of miR-381 on the sensitivity of non-small cell lung cancer (NSCLC) to radiotherapy, and examined its possible mechanism.MethodsNSCLC A549 cells and miR-381 overexpression and gene silencing cell lines were treated with radiotherapy. The cell proliferation was tested by CCK-8 assay and colony formation. Flow cytometry and TUNEL were used to detect the cell apoptosis. The expression of nuclear factor kappa B (NF-κB) signaling pathway related proteins were detected by western blot. NF-κB signaling pathway activator and inhibitor cell lines were further constructed and the above experiments were repeated. Double luciferase assay was used to verify the target of miR-381. Furthermore, a nude mouse xenograft model was constructed and treated with radiotherapy. The tumor volume and tumor weight were measured. The expression of PCNA protein in tumor tissues was observed by immunohistochemistry. The apoptosis related proteins in tumor tissues were detected by western blot.ResultsThe mRNA expression of miR-381 was increased after radiotherapy treatment. Radiotherapy treatment also can inhibit the proliferation and promote apoptosis of A549 cells. Compared with radiotherapy group, cell proliferation was significantly decreased and apoptosis was significantly increased in miR-381 overexpression group (p < 0.05). Moreover, ROCK2 is a target of miR-381, and overexpression of miR-381 can down-regulate the expression of ROCK2 protein. In nude mice, miR-381 mimic interference can reduce cell tumorigenicity and proliferation, and increase the apoptosis. However, the indicators above were contrary in miR-381 silencing group. Verification experiments further verify that NF-κB signaling pathway activator can reverse the role of miR-381.ConclusionMiR-381 overexpression could enhance the sensitivity of NSCLC to radiotherapy by targeting ROCK2 to inhibit NF-κB signaling pathway.

scholarly journals PTENInhibits Cell Proliferation, Promotes Cell Apoptosis, and Induces Cell Cycle Arrest via Downregulating the PI3K/AKT/hTERTPathway in Lung Adenocarcinoma A549 Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Xiao-Xiao Lu ◽  
Lan-Yu Cao ◽  
Xi Chen ◽  
Jian Xiao ◽  
Yong Zou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Lung Adenocarcinoma ◽  
Cell Cycle Arrest ◽  
Cell Apoptosis ◽  
A549 Cells ◽  
Akt Pathway ◽  
Wild Type ◽  
Cycle Arrest ◽  
Lung Adenocarcinoma A549

PTENplays an essential role in tumorigenesis and both its mutation and inactivation can influence proliferation, apoptosis, and cell cycle progression in tumor cells. However, the precise role ofPTENin lung cancer cells has not been well studied. To address this, we have generated lung adenocarcinoma A549 cells overexpressing wild-type or mutantPTENas well as A549 cells expressing a siRNA directed toward endogenousPTEN. Overexpression of wild-typePTENprofoundly inhibited cell proliferation, promoted cell apoptosis, caused cell cycle arrest at G1, downregulated p-AKT, and decreased expression of the telomerase proteinhTERT. In contrast, in cells expressing aPTENdirected siRNA, the opposite effects on cell proliferation, apoptosis, cell cycle arrest, p-AKT levels, andhTERTprotein expression were observed. A549 cells transfected with aPTENmutant lacking phosphatase activity (PTEN-C124A) or an empty vector (null) did not show any effect. Furthermore, using the PI3K/AKT pathway blocker LY294002, we confirmed that the PI3K/AKT pathway was involved in mediating these effects ofPTEN. Taken together, we have demonstrated thatPTENdownregulates the PI3K/AKT/hTERTpathway, thereby suppressing the growth of lung adenocarcinoma cells. Our study may provide evidence for a promising therapeutic target for the treatment of lung adenocarcinoma.

scholarly journals C1QTNF6 regulates cell proliferation and apoptosis of NSCLC in vitro and in vivo

2020 ◽  
Author(s):  
Wei Zhang ◽  
Ganzhu Feng
Keyword(s):  
Cell Proliferation ◽  
Cancer Progression ◽  
Colony Formation ◽  
Cell Function ◽  
Flow Cytometric Analysis ◽  
A549 Cells ◽  
Migration And Invasion ◽  
Flow Cytometric

Objectives: Lung cancer has been reported as the leading cause of cancer-associated death in humans, and its incidence continues to increase in the world. A growing number of studies have shown that dysregulated genes are associated with the occurrence and poor prognosis of a variety of tumors, including NSCLC. C1q/tumor necrosis factor-related protein 6 (C1QTNF6), a member of the CTRP family, has been revealed to play a role in carcinogenesis and cancer progression. Nevertheless, the effects and mechanisms of C1QTNF6 in NSCLC remain unrevealed. Materials and methods: MTT and colony formation, flow cytometric and transwell assays were performed to explore the cell function. RT-PCR and western blot were used to analyze the mRNA and protein expression. Results: In this study, we found that C1QTNF6 significantly promoted the proliferation of SPCA1 and A549 cells by MTT and colony formation assays. In addition, downregulation of C1QTNF6 weakened the tumor growth in vivo. Besides, C1QTNF6 remarkably reduced apoptosis by flow cytometric analysis and TUNEL assay. Furthermore, the capability of migration and invasion was obviously enhanced when C1QTNF6 overexpression. Conclusion: Overall, our results demonstrated that inhibition of C1QTNF6 attenuated cell proliferation, migration, invasion and promoted apoptosis in vitro and in vivo of NSCLC. Based on the above results, our study provided us with a new and key perspective in understanding and treating NSCLC.

scholarly journals The Effects of 2,2',4'-Trihydroxychalcone on the Cell Proliferation, Metastasis, and Apoptosis in Human Lung Cancer Cell Line A549

2021 ◽  
Author(s):  
Jialin Sun ◽  
Zhanqi Cao ◽  
Shiwei Sun ◽  
Zhonghua Sun ◽  
Shuhong Sun ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Human Lung ◽  
Vasculogenic Mimicry ◽  
A549 Cells ◽  
Cancer Cell Line ◽  
Lung Cancer Cell Line ◽  
Human Lung Cancer ◽  
Lung Cancer Cell ◽  
Related Proteins

Abstract Objectives：Our study aimed to evaluate the antitumor effects of 2,2',4'-trihydroxychalcone (7a) on human lung cancer cell line A549. Methods：A549 cells were treated with different concentrations of 7a for different times. The cells without 7a were set as the negative control group. The cell proliferation, invasion, vasculogenic mimicry (VM) formation, heterogeneous adhesion, apoptosis were, respectively, measured by CCK-8, transwell invasion assay, vasculogenic mimicry assay, adhesion assay and flow cytometry. In addition, the expression of related proteins were examined via western blot or ELISA. Results：Our research found that 7a had a significant inhibitory effect on the survival rate of lung cancer A549 cells, while almost had no effect on human lung epithelial BEAS-2B cells and human venous endothelial cells (HUVECs). The migration rate, VM length, invasion and heterogeneous adhesion number of cells treated with 7a significantly decreased as the increase in concentration, while the apoptosis rate increased. Western blot analysis showed that 7a treatment significantly upregulated the expression of E-cadherin, cleaved PARP, Bax, caspase-3, and simultaneously downregulated the expression of MMP-2/9, Bcl-2, p-PI3K, p-Akt, p-MTOR, VEGF, E-selectin and N-cadherin. At the same time, ELISA results found that the pro-angiogenic factor VEGF level in culture media was reduced in the presence of 7a. Additionally, 7a could also reduce the nucleus NF-κB protein level, which would inhibit gene transcription of tumor activity-related proteins. Conclusion：7a might exert inhibitory effects on A549 cells via inhibiting cell proliferation, migration, VM formation, heterogeneous adhesion and inducing apoptosis through suppressing the PI3K/AKT/NF-κB signaling pathway, suggesting that 7a might have therapeutic potential for the treatment of lung cancer.

Rotundic Acid Regulates the Effects of Let-7f-5p on Caco2 Cell Proliferation

2020 ◽  
Vol 20 ◽  
Author(s):  
Yuan Feng ◽  
Xinran Liu ◽  
Yueqing Han ◽  
Mantian Chen ◽  
Lin Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Colony Formation ◽  
Cancer Cell Line ◽  
Human Colon ◽  
Colon Cancer Cell Line ◽  
Inhibitory Effect ◽  
Human Colon Cancer ◽  
Caco2 Cell ◽  
Study Results ◽  
Rotundic Acid

Background & Objective: Nowadays, the interaction between natural products and microRNAs provides a promising field for exploring the chemo preventive agents for various cancers.As a member of microRNAs, the expression of let-7f-5p is universally down regulated in colorectal cancer (CRC). The present study aimed to uncover the function of let-7f-5p in the proliferation of human colon cancer cell line Caco2 and explored chemo preventive agents from natural resources that can prevent the development of CRC. Methods: Herein, Caco2 cells were transfected with let-7f-5p mimic and inhibitor to manipulate let-7f-5p levels, and the expression of let-7f-5p wasper formed by RT‑qPCR. Next, we determined how let-7f-5p regulates Caco2 cell proliferation by using MTT, wound-healing, cell cycle,and colony formation assays.Besides, to further understand the effect of let-7f-5p, we evaluated the protein level of AMER3 and SLC9A9 by using western blotting assays. Results: The results showed a suppressive function of let-7f-5p on Caco2 cell proliferation and then put forward a triterpenoid (rotundic acid, RA) which significant antagonized the effect of cell proliferation, restitution after wounding,and colony formation caused by let-7f-5p. Moreover, the western blot results further indicated that the inhibitory effect of RA might be due to its suppressive role in let-7f-5p-targeted AMER3 and SLC9A9 regulation. Conclusion: Our validation study results confirmed that let-7f-5p was a potent tumor suppressor gene of Caco2 cell proliferation,and RA showed as a regulator of the effect oflet-7f-5p on cell proliferation and then could be a potential chemo preventive agent for CRC treatment.

Identification of a new GLDC gene alternative splicing variant and its protumorigenic roles in lung cancer

2019 ◽  
Vol 15 (36) ◽  
pp. 4127-4139 ◽  
Author(s):  
Yingli Yuan ◽  
Luguo Sun ◽  
Xu Wang ◽  
Jingxian Chen ◽  
Mingnan Jia ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Colony Formation ◽  
Lactate Production ◽  
Cellular Transformation ◽  
Lung Cancer Cell Line ◽  
Stem Cell Marker ◽  
Brdu Incorporation ◽  
Clinical Samples

Aim: To clarify the regulatory roles of GLDCV1, the first identified truncated glycine decarboxylase (GLDC), on cancer stem cells and tumorigenesis. Materials & methods: RT-PCR or RT-qPCR, immunoblotting and immunohistochemical staining were applied to assess gene expression. MTT, BrdU incorporation and colony formation assays were used to examine cell proliferation capacity. Soft agar colony formation and in vivo transplantation were applied to evaluate cellular transformation and tumorigenesis. Results & conclusion: Expression of GLDCV1 or GLDC was enhanced in non-small-cell lung cancer cell line and clinical samples. GLDCV1 overexpression induced MRC5 cell proliferation, transformation and tumorigenesis. Additionally, GLDCV1 increased lactate production and cancer stem cell marker expression and activated ERK and P38 pathways. Our study gained deeper insight into GLDC oncogene.

scholarly journals LncRNA PCAT1 Interacts with DKC1 to Regulate Proliferation, Invasion and Apoptosis in NSCLC Cells via the VEGF/AKT/Bcl2/Caspase9 Pathway

2021 ◽  
Vol 30 ◽  
pp. 096368972098607
Author(s):  
Shi-Yuan Liu ◽  
Zhi-Yu Zhao ◽  
Zhe Qiao ◽  
Shao-Min Li ◽  
Wei-Ning Zhang
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Growth Factor ◽  
Cell Apoptosis ◽  
Rna Binding ◽  
A549 Cells ◽  
Nonsmall Cell Lung Cancer ◽  
Nsclc Cell ◽  
Loss Of Function ◽  
Proliferation And Invasion

Long noncoding RNAs (lncRNAs) are increasingly recognized as indispensable components of the regulatory network in the progression of various cancers, including nonsmall cell lung cancer (NSCLC). The lncRNA prostate cancer associated transcript 1 (PCAT1) has been involved in tumorigenesis of multiple malignant solid tumors, but it is largely unknown that what is the role of lncRNA-PCAT1 and how it functions in the progression of lung cancer. Herein, we observed that lncRNA PCAT1 expression was upregulated in both human NSCLC tissues and cell lines, which was determined by qualitative polymerase chain reaction analysis. Then, gain-and loss-of-function manipulations were performed in A549 cells by transfection with a specific short interfering RNA against PCAT1 or a pcDNA-PCAT1 expression vector. The results showed that PCAT1 not only promoted NSCLC cell proliferation and invasion but also inhibited cell apoptosis. Bioinformatics and expression correlation analyses revealed that there was a potential interaction between PCAT1 and the dyskerin pseudouridine synthase 1 (DKC1) protein, an RNA-binding protein. Then, RNA pull-down assays with biotinylated probes and transcripts both confirmed that PCAT1 directly bounds with DKC1 that could also promote NSCLC cell proliferation and invasion and inhibit cell apoptosis. Moreover, the effects of PCAT1 and DKC1 on NSCLC functions are synergistic. Furthermore, PCAT1 and DKC1 activated the vascular endothelial growth factor (VEGF)/protein kinase B (AKT)/Bcl-2/caspase9 pathway in NSCLC cells, and inhibition of epidermal growth factor receptor, AKT, or Bcl-2 could eliminate the effect of PCAT1/DKC1 co-overexpression on NSCLC cell behaviors. In conclusion, lncRNA PCAT1 interacts with DKC1 to regulate proliferation, invasion, and apoptosis in NSCLC cells via the VEGF/AKT/Bcl-2/caspase9 pathway.

scholarly journals CHN1 promotes epithelial–mesenchymal transition via the Akt/GSK-3β/Snail pathway in cervical carcinoma

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Haoqi Zhao ◽  
Lan Wang ◽  
Shufang Wang ◽  
Xihua Chen ◽  
Min Liang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Lymph Node ◽  
Signaling Pathway ◽  
Cervical Carcinoma ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Xenograft Tumor ◽  
Mesenchymal Transition ◽  
Gsk 3Β ◽  
Related Proteins

Abstract Background Metastasis and invasion are crucial in determining the mortality of cervical carcinoma (CC) patients. The epithelial–mesenchymal transition (EMT) is now a universal explanation for the mechanisms of tumor metastasis. Α-chimeric protein (α-chimaerin, CHN1) plays an important role in the regulation of signal transduction and development. However, the molecular regulatory relationships between CHN1 and CC progression in relation to EMT have not yet been identified. Methods The expression of CHN1 in CC tissues, adjacent tissues, and lymph node metastases from CC patients was detected by immunohistochemistry. Upregulation and knockdown of CHN1 were achieved by transfection of CC cells. The effect of CHN1 on cell proliferation was determined by CCK-8 and plate clone formation assays. Changes in migration and invasion capabilities were evaluated using scratch migration and transwell invasion assays. The effect of CHN1 overexpression and interference on xenograft tumor growth was determined by tumor weight and pathological analyses. The expression of EMT-related mRNAs was measured by qRT-PCR in transfected CC cells. EMT-related proteins and Akt/GSK-3β/Snail signaling pathway-related proteins were also evaluated by western blotting. Results CHN1 was overexpressed in CC tissues and was associated with lymph node metastasis and low survival in CC patients. Overexpression of CHN1 promoted cell proliferation, migration, and invasion in CC cells. In contrast, silencing of CHN1 inhibited these phenomena. Overexpression of CHN1 promoted tumor formation in an in vivo xenograft tumor mouse model, with increased tumor volumes and weights. In addition, CHN1 induced the expression of EMT-related transcription factors, accompanied by the decreased expression of epithelial markers and increased expression of mesenchymal markers. The Akt/GSK-3β/Snail signaling pathway was activated by overexpression of CHN1 in vitro, and activation of this pathway was inhibited by the signaling pathway inhibitor LY294002. Conclusion These results suggest that CHN1 promotes the development and progression of cervical carcinoma via the Akt/GSK-3β/Snail pathway by inducing EMT.

scholarly journals Ovalitenone Inhibits the Migration of Lung Cancer Cells via the Suppression of AKT/mTOR and Epithelial-to-Mesenchymal Transition

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 638
Author(s):  
Kittipong Sanookpan ◽  
Nongyao Nonpanya ◽  
Boonchoo Sritularak ◽  
Pithi Chanvorachote
Keyword(s):  
Lung Cancer ◽  
Cell Migration ◽  
Cancer Cells ◽  
Colony Formation ◽  
Cancer Metastasis ◽  
Epithelial To Mesenchymal Transition ◽  
A549 Cells ◽  
Lung Cancer Cells ◽  
Migration And Invasion ◽  
Mesenchymal Transition

Cancer metastasis is the major cause of about 90% of cancer deaths. As epithelial-to-mesenchymal transition (EMT) is known for potentiating metastasis, this study aimed to elucidate the effect of ovalitenone on the suppression of EMT and metastasis-related behaviors, including cell movement and growth under detached conditions, and cancer stem cells (CSCs), of lung cancer cells. Methods: Cell viability and cell proliferation were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazo-liumbromide (MTT) and colony formation assays. Cell migration and invasion were analyzed using a wound-healing assay and Boyden chamber assay, respectively. Anchorage-independent cell growth was determined. Cell protrusions (filopodia) were detected by phalloidin-rhodamine staining. Cancer stem cell phenotypes were assessed by spheroid formation. The proteins involved in cell migration and EMT were evaluated by Western blot analysis and immunofluorescence staining. Results: Ovalitenone was used at concentrations of 0–200 μM. While it caused no cytotoxic effects on lung cancer H460 and A549 cells, ovalitenone significantly suppressed anchorage-independent growth, CSC-like phenotypes, colony formation, and the ability of the cancer to migrate and invade cells. The anti-migration activity was confirmed by the reduction of filopodia in the cells treated with ovalitenone. Interestingly, we found that ovalitenone could significantly decrease the levels of N-cadherin, snail, and slug, while it increased E-cadherin, indicating EMT suppression. Additionally, the regulatory signaling of focal adhesion kinase (FAK), ATP-dependent tyrosine kinase (AKT), the mammalian target of rapamycin (mTOR), and cell division cycle 42 (Cdc42) was suppressed by ovalitenone. Conclusions: The results suggest that ovalitenone suppresses EMT via suppression of the AKT/mTOR signaling pathway. In addition, ovalitenone exhibited potential for the suppression of CSC phenotypes. These data reveal the anti-metastasis potential of the compound and support the development of ovalitenone treatment for lung cancer therapy.

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