sincalide and Lung-Neoplasms

Small cell lung cancer (SCLC) with high c-Myc expression is prone to relapse and metastasis, leading to extremely low survival rate. Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor Abemaciclib plays a key role in the treatment of tumors, but the effects and mechanisms on SCLC remain unclear. This study was to analyze the effect and molecular mechanism of Abemaciclib in inhibiting proliferation, migration and invasion of SCLC with high c-Myc expression, with a view to expanding a new direction for reducing the recurrence and metastasis.. Proteins interacting with CDK4/6 were predicted using the STRING database. The expressions of CDK4/6 and c-Myc in 31 cases of SCLC cancer tissues and paired adjacent normal tissues were analyzed by immunohistochemistry. The effects of Abemaciclib on the proliferation, invasion and migration of SCLC were detected by CCK-8, colony formation assay, Transwell and migration assay. Western blot was used to detect the expressions of CDK4/6 and related transcription factors. Flow cytometry was used to analyze the effects of Abemaciclib on the cell cycle and checkpoint of SCLC.. The expression of CDK4/6 was associated with c-Myc by STRING protein interaction network. c-Myc can directly modalize achaete-scute complex homolog 1 (ASCL1), neuronal differentiation 1 (NEUROD1) and Yes-associated protein 1 (YAP1). Moreover, CDK4 and c-Myc regulate the expression of programmed cell death ligand 1 (PD-L1). Immunohistochemistry showed that the expressions of CDK4/6 and c-Myc in cancer tissues were higher than those in adjacent tissues(P<0.0001). CCK-8, colony formation assay, Transwell and migration assay verified that Abemaciclib could effectively inhibit the proliferation, invasion and migration of SBC-2 and H446OE(P<0.0001). Western blot analysis further showed that Abemaciclib not only inhibited CDK4 (P<0.05) and CDK6 (P<0.05), but also affected c-Myc (P<0.05), ASCL1 (P<0.05), NEUROD1 (P<0.05) and YAP1 (P<0.05), which are related to SCLC invasion and metastasis. Flow cytometry showed that Abemaciclib not only inhibited the cell cycle progression of SCLC cells (P<0.0001), but also significantly increased PD-L1 expression on SBC-2 (P<0.01) and H446OE (P<0.001).. Abemaciclib significantly inhibits the proliferation, invasion, migration and cell cycle progression of SCLC by inhibiting the expressions of CDK4/6, c-Myc, ASCL1, YAP1 and NEUROD1. Abemaciclib can also increase the expression of PD-L1 in SCLC.. 【中文题目：Abemaciclib抑制c-Myc高表达小细胞肺癌
增殖、侵袭和迁移的生物学功能的研究】 【中文摘要：背景与目的 c-Myc高表达小细胞肺癌（small cell lung cancer, SCLC）因易复发转移导致患者生存率极低，细胞周期依赖性激酶4和6（cyclin-dependent kinases 4 and 6, CDK4/6）抑制剂Abemaciclib在多种肿瘤的治疗中均起到关键作用，但其对SCLC的作用及机制仍不明晰。本研究旨在分析Abemaciclib抑制c-Myc高表达的SCLC增殖、侵袭和迁移的作用和分子机制，以期为减少患者的复发和转移拓展新的方向。方法 通过STRING数据库分析了CDK4/6和c-Myc蛋白相互作用网络，免疫组化实验分析了31例SCLC癌组织和配对的相邻正常组织中CDK4/6及c-Myc的表达，CCK-8实验和平板克隆实验检测Abemaciclib对SCLC增殖的影响，Transwell及划痕实验检测Abemaciclib对SCLC的侵袭和迁移能力的影响，Western blot检测Abemaciclib对CDK4/6及相关转录因子的影响，流式细胞术分析了Abemaciclib对SCLC的细胞周期和免疫检查点的调节作用。结果 通过STRING蛋白相互作用网络分析发现CDK4/6的表达均与c-Myc相关，c-Myc可以直接调节ASCL1（achaete-scute complex homolog 1）、NEUROD1（neuronal differentiation 1）和Yes相关蛋白1（Yes-associated protein 1, YAP1）的表达，CDK4和c-Myc均调控程序性死亡配体1（programmed cell death ligand 1, PD-L1）的表达。通过免疫组化实验发现，CDK4/6及c-Myc在SCLC癌组织中的表达高于癌旁组织（P<0.0001）。CCK-8实验、平板克隆实验、Transwell实验及划痕实验验证了Abemaciclib可以有效地抑制SBC-2和H446OE的增殖、侵袭和迁移（P<0.0001）。Western blot进一步分析了Abemaciclib在抑制SBC-2和H446OE细胞内CDK4（P<0.05）和CDK6（P<0.05）的同时，也影响了c-Myc（P<0.05）、ASCL1（P<0.05）、NEUROD1（P<0.05）和YAP1（P<0.05）等与SCLC浸润和转移相关的蛋白。流式细胞术发现Abemaciclib不仅抑制SCLC的细胞周期进程（P<0.0001），还可以提高SBC-2（P<0.01）和H446OE（P<0.001）细胞表面PD-L1的表达。结论 Abemaciclib明显抑制SCLC的增殖、侵袭、迁移和细胞周期进程，其通过抑制CDK4/6下调c-Myc、ASCL1、YAP1和NEUROD1蛋白表达的同时，也提高了PD-L1的表达。
】 【中文关键词：肺肿瘤；细胞周期依赖性激酶4/6；c-Myc】.

Topics: Adaptor Proteins, Signal Transducing; B7-H1 Antigen; Cell Proliferation; Humans; Lung Neoplasms; Neoplasm Recurrence, Local; Sincalide; Small Cell Lung Carcinoma; Transcription Factors

Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutations in non-small cell lung cancer (NSCLC). To prolong the survival of the patients, EGFR tyrosine kinase inhibitors (TKIs) resistance in NSCLC is a major challenge that needs to be addressed urgently, and this study focuses on investigating the mechanism of cigarette smoke (CS) induced Gefitinib resistance in NSCLC.. PC-9 and A549 cells were cultured in vitro and treated with 1 µmol/L Gefitinib for 4 h and 10% cigarette smoke extract (CSE) for 48 h. Western blot was used to detect Sirtuin 3 (Sirt3) and superoxide dismutase 2 (SOD2) protein expressions; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS); CCK-8 kit was used to detect cell activity, and EdU was used to detect cell proliferation ability. Sirt3 overexpression plasmid (OV-Sirt3) was transfected in PC-9 and A549 cells and treated with 1 µmol/L Gefitinib for 4 h and 10% CSE for 48 h after N-acetylcysteine (NAC) action. The expressions of Sirt3 and SOD2 were detected by Western blot; the ROS level in the cells was detected by DCFH-DA probe, and the cell activity was detected by CCK-8.. CSE induced an increase in the 50% inhibitory concentration (IC50) of both PC-9 and A549 cells to Gefitinib (P<0.01) and enhanced the proliferation of PC-9 and A549 cells, suggesting that CS induced Gefitinib resistance in NSCLC. ROS was involved in CSE-induced Gefitinib resistance (P<0.05). CSE induced low expressions of Sirt3 and SOD2 (P<0.01), and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients (P<0.05). OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance (P<0.05) and significantly reduced ROS production. NAC reversed CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05).. The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.. 【中文题目：烟草烟雾通过ROS/Sirt3/SOD2通路
诱导NSCLC细胞吉非替尼耐药】 【中文摘要：背景与目的 表皮生长因子受体（epidermal growth factor receptor, EGFR）基因突变是非小细胞肺癌（non-small cell lung cancer, NSCLC）最常见的驱动基因突变。为延长患者生存时间，NSCLC EGFR酪氨酸激酶抑制剂（tyrosine kinase inhibitors, TKIs）耐药是目前急需解决的重大难题。本研究主要探究烟草烟雾（cigarette smoke, CS）诱导NSCLC发生吉非替尼耐药的机制。方法 体外培养PC-9、A549细胞，分别经1 µmol/L吉非替尼处理4 h、10%CS萃取物（CS extract, CSE）处理48 h。Western blot检测沉默蛋白3（Sirtuin 3, Sirt3）、超氧化物歧化酶2（superoxide dismutase 2, SOD2）蛋白表达，使用DCFH-DA探针检测细胞内活性氧（reactive oxygen species, ROS）水平，CCK-8试剂盒检测细胞活性，EdU检测细胞增殖能力。Sirt3过表达质粒（OV-Sirt3）转染于PC-9和A549细胞中、N-乙酰半胱氨酸乙酯（N-acetylcysteine, NAC）作用于细胞后分别经1 µmol/L吉非替尼处理4 h和10%CSE处理48 h。Western blot检测Sirt3、SOD2蛋白表达，DCFH-DA探针检测细胞中的ROS水平，CCK-8检测细胞活性。结果 CSE均可促使PC-9、A549细胞对吉非替尼的半数抑制浓度（50% inhibitory concentration, IC50）提高（P<0.01），并且增强PC-9和A549细胞的增殖能力，提示CS可诱导NSCLC吉非替尼耐药；ROS参与CSE诱导的吉非替尼耐药（P<0.05）；CSE诱导Sirt3、SOD2低表达（P<0.01），且Sirt3/SOD2与肺癌患者不良预后有关（P<0.05）；OV-Sirt3的PC-9、A549细胞可逆转CSE诱导的吉非替尼耐药（P<0.05）且显著降低ROS生成；NAC可逆转CSE诱导的PC-9、A549细胞吉非替尼耐药（P<0.05）。结论 ROS/Sirt3/SOD2通路参与了CS诱导的NSCLC吉非替尼耐药。
】 【中文关键词：肺肿瘤；烟草烟雾；ROS；Sirt3/SOD2；吉非替尼耐药】.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cigarette Smoking; Drug Resistance, Neoplasm; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Reactive Oxygen Species; Sincalide; Sirtuin 3

Tumor necrosis factor receptor-associated factor 3 (TRAF3) has specific regulatory effects on a wide range of diseases, including tumors. However, the effect and mechanism of TRAF3 on lung adenocarcinoma (LUAD) are still unknown. The aim of the present study was to make clear the role and potential mechanism of TRAF3 in LUAD.. TIMER2.0 database and western blot were applied to detect the expression of TRAF3 in lung adenocarcinoma tissue. Kaplan-Meier Plotter database was utilized to explore the effect of TRAF3 on the clinical prognosis of lung adenocarcinoma patients. Specific siRNA was used to inhibit the expression of TRAF3 in LUAD cells (A549 and H1299). CCK-8 and EdU assays were performed for assessing LUAD cells proliferation. Wound healing assay and transwell assay were performed for determining cells migration. CCK-8 assay was used to assess the response of the LUAD cells to paclitaxel. TIMER2.0 bioinformatics and western blot were employed to detect the effects of TRAF3 on pyroptosis in LUAD.. TRAF3 was highly expressed in lung adenocarcinoma tissues and cell lines. Patients with TRAF3 hyperexpression had a good prognosis compared to those with lower expression. TRAF3 inhibition notably induced proliferation and migration of LUAD cells. Inhibition of TRAF3 also weakened the sensitivity of LUAD cells to paclitaxel. Moreover, bioinformatics results showed that TRAF3 was positively correlated with the expression of pyroptosis-related genes in LUAD. Western blot assays showed that TRAF3 inhibition visibly decreased the expression of apoptosis-associated speck-like protein (ASC), cleaved caspase-1 and matured- IL-1β.. Inhibition of TRAF3 promotes the proliferation and migration of LUAD cells, and reduces the sensitivity of LUAD cells to paclitaxel. The effects of TRAF3 on LUAD cells were mediated in part by caspase-1-dependent pyroptosis.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Caspases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Paclitaxel; Pyroptosis; Sincalide; TNF Receptor-Associated Factor 3

MiR-19b-3p has been reported in several types of human cancer. Nevertheless, the expression profile and biological functions of miR-19b-3p remain unclear in non-small cell lung cancer (NSCLC). The expression level of miR-19b-3p was evaluated in NSCLC tissues and cell lines using qRT-PCR. Survival analysis was performed using Kaplan-Meier curves, while the prognostic significance of miR-19b-3p was analyzed using Cox regression analysis in 80 NSCLC patients. The effects of miR-19b-3p on cell proliferation and invasion capacities were analyzed using CCK-8, crystal violet, and transwell assays. Target genes of miR-19b-3p were assessed using luciferase reporter assay, qRT-PCR, Western blot and rescue experiments. MiR-19b-3p was found to be upregulated in human NSCLC tissues and cell lines. The expression of miR-19b-3p was observed to be closely associated with TNM stage and metastasis. High expression of miR-19b-3p was found to be capable of predicting poor clinical prognosis in NSCLC patients. Whilst overexpression of miR-19b-3p was demonstrated to promote the proliferation and invasion of NSCLC cells, knockdown of miR-19b-3p showed an opposite inhibitory effect. Bioinformatics analysis and luciferase reporter assays confirmed that HOXA9 is a direct target of miR-19b-3p. Functional assays demonstrated that NSCLC cell proliferation and invasion were promoted by miR-19b-3p via negative regulation of HOXA9. Finally, overexpression of HOXA9 was shown to partially reverse the tumor promoting effect of miR-19b-3p. This study indicates that miR-19b-3p is a crucial prognostic biomarker of NSCLC, and that targeting of the miR-19b-3p/HOXA9 axis may be a promising strategy in NSCLC therapy.

Topics: Biomarkers; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gentian Violet; Humans; Lung Neoplasms; MicroRNAs; Prognosis; Sincalide

To investigate the molecular mechanism by which miR-20a-5p regulates HOXB13 gene expression and inhibits lung cancer cell proliferation.. The expression levels of HOXB13 mRNA and protein in lung cancer A549 cells transfected with HOXB13 overexpression plasmid or HOXB13 siRNA were detected with real-time fluorescence quantitative PCR (qRT-PCR) and Western blotting. CCK-8 and EdU assays were used to examine the effect of modulation of HOXB13 expression on cell proliferation. We screened possible binding miRNAs of HOXB13 by bioinformatics analysis. In A549 cells transfected with miR-20a-5p mimic or miR-20a-5p inhibitor, the expression level of miR-20a-5p was detected by qRT-PCR and the protein expression of HOXB13 was determined with Western blotting. CCK-8 and EdU assays were used to assess the effect of miR-20a-5p overexpression on the proliferation of A549 cells. miR-20a-5p mimic and HOXB13 overexpression plasmids were co-transfected into A549 cells, and the changes in cell proliferation were evaluated with CCK-8 and EdU assays.. HOXB13 overexpression obviously promoted the proliferation of A549 cells (. miR-20a-5p inhibits proliferation of lung cancer cells by down-regulating the expression of HOXB13.

Topics: A549 Cells; Apoptosis; Cell Line, Tumor; Cell Proliferation; Homeodomain Proteins; Humans; Lung Neoplasms; MicroRNAs; Sincalide

To seek out the mechanism by which C1QTNF6 mediates lung adenocarcinoma (LUAD).. Differentially expressed mRNAs and miRNAs in LUAD were analyzed using bioinformatics. In LUAD cells, C1QTNF6 mRNA and miR-184 expression were evaluated with qRT-PCR, and C1QTNF6 protein level was assessed by western blot. Cellular behaviors were assessed by colony formation, CCK-8, Transwell, and wound healing methods. The binding ability of miR-184 to C1QTNF6 was observed by dual-luciferase assay.. High expression of C1QTNF6 in LUAD stimulated cancer cellular behaviors. MiR-184 was lowly expressed in LUAD and downregulated C1QTNF6 expression. MiR-184 restrained LUAD cell processes by targeting C1QTNF6.. MiR-184 repressed LUAD cell processes via mediating C1QTNF6. MiR-184 and C1QTNF6 are expected to be indicators for LUAD treatment.

Topics: Adenocarcinoma of Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Collagen; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; RNA, Messenger; Sincalide

Lung adenocarcinoma (LUAD) is a predominant malignancy, and its high mortality prompts us to incessantly probe the relevant targeted treatment. This work intended to study the molecular mechanism of ESPL1 in LUAD. Bioinformatics analysis was performed for pan-cancer and prognosis analysis as well as target gene prediction. Expression of ESPL1 mRNA and let-7c-5p was determined via qRT-PCR, and western blot was employed to detect protein level of ESPL1. Dual-luciferase reporter gene method verified the interaction between ESPL1 and let-7c-5p. Thereafter, CCK-8, wound healing, Transwell, and flow cytometry assays were utilized to investigate proliferation, migration, and apoptosis of LUAD cells. The results revealed that ESPL1 was upregulated in LUAD, which was associated with poor prognosis. Overexpressed ESPL1 promoted LUAD cells to invade, proliferate, and migrate. Furthermore, ESPL1 was a target gene of let-7c-5p. Let-7c-5p was downregulated in LUAD cells, and played a suppressive role in LUAD malignant development, while reversed by ESPL1. Taken together, it was posited that let-7c-5p/ESPL1 may be underlying therapeutic targets of LUAD.

Topics: Adenocarcinoma of Lung; Apoptosis; Cell Movement; Cell Proliferation; Humans; Lung Neoplasms; MicroRNAs; RNA, Messenger; Separase; Sincalide

Lung squamous cell carcinoma (LSCC) is a prevalent and progressive subtype of lung cancer. This study aimed to substantiate the regulatory effect of the PAK2/SOX2/DEK axis on the LSCC development. LSCC tissues (n = 83) and adjacent normal tissues were collected and SOX2 expression was determined by qRT-PCR and Western blotting. Correlation between SOX2 expression and the prognosis of LSCC patients was then explored utilizing Kaplan-Meier analysis. Co-immunoprecipitation and glutathione-S-transferase pull-down assays were conducted to validate the binding of SOX2 to DEK. Gain- and loss- of function assays were then performed on LSCC cells, with CCK-8 and Transwell assays applied to detect the malignant behaviors of cells. A mouse xenograft model of LSCC was further established for in vivo validation. The expression levels of SOX2, PAK2 and DEK were up-regulated in LSCC tissues and cells. SOX2 overexpression was correlated with poor prognosis of LSCC patients. Knockdown of SOX2 weakened the viability and the migratory and invasive potential of LSCC cells. Further, PAK2 directly interacted with SOX2. PAK2 overexpression accelerated the malignant phenotypes of LSCC cells through interplay with SOX2. Moreover, SOX2 activated the expression of DEK, and silencing DEK attenuated the malignant behaviors of LSCC cells. In conclusion, PAK2 could bind to the transcription factor SOX2 and thus activate the expression of DEK, thereby driving the malignant phenotypes of LSCC cells both in vivo and in vitro.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Chromosomal Proteins, Non-Histone; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Laryngeal Neoplasms; Lung; Lung Neoplasms; Mice; MicroRNAs; Oncogene Proteins; p21-Activated Kinases; Poly-ADP-Ribose Binding Proteins; Sincalide; SOXB1 Transcription Factors

Lung cancer (LC) remains a threatening health issue worldwide. Methyltransferase-like protein 3 (METTL3) is imperative in carcinogenesis via m6A modification of microRNAs (miRNAs). This study estimated the effect of METTL3 in LC by regulating m6A methylation-mediated pri-miR-663 processing.. miR-663 expression in 4 LC cell lines and normal HBE cells was determined using RT-qPCR. A549 and PC9 LC cells selected for in vitro studies were transfected with miR-663 mimics or inhibitor. Cell viability, migration, invasion, proliferation, and apoptosis were detected by CCK-8, Transwell, EdU, and flow cytometry assays. The downstream target genes and binding sites of miR-663 were predicted via Starbase database and validated by dual-luciferase assay. LC cells were delivered with oe-METTL3/sh-METTL3. Crosslinking between METTL3 and DGCR8 was verified by co-immunoprecipitation. Levels of m6A, miR-663, and pri-miR-663 were measured by m6A dot blot assay and RT-qPCR. m6A modification of pri-miR-663 was verified by Me-RIP assay. Finally, the effects of METTL3 in vivo were ascertained by tumor xenograft in nude mice.. miR-663 was upregulated in LC cells, and miR-663 overexpression promoted cell proliferation, migration, invasion, and inhibited apoptosis, but miR-663 knockdown exerted the opposite effects. miR-663 repressed SOCS6 expression. SOCS6 overexpression annulled the promotion of miR-663 on LC cell growth. METTL3 bound to DGCR8, and METTL3 silencing elevated the levels of pri-miR-663 and m6A methylation-modified pri-miR-663, and suppressed miR-663 maturation and miR-663 expression. METTL3 facilitated tumor growth in mice through the miR-663/SOCS6 axis.. METTL3 promotes LC progression by accelerating m6A methylation-mediated pri-miR-663 processing and repressing SOCS6.

Topics: Animals; Humans; Lung Neoplasms; Methylation; Methyltransferases; Mice; Mice, Nude; MicroRNAs; RNA-Binding Proteins; Sincalide; Suppressor of Cytokine Signaling Proteins

Circular RNAs (circRNAs) are crucial for the pathogenesis of nonsmall lung cancer (NSCLC). Here, we set out to unravel the precise function of circRNA CD226 (circCD226) in NSCLC pathogenesis. The exosomes from serum specimens were observed by transmission electron microscopy. CircCD226, miR-1224-3p and high mobility group AT-hook 2 (HMGA2) were quantified by qRT-PCR, western blot and immunohistochemistry. Actinomycin D and Ribonuclease (RNase) R treatments and subcellular localization assay were used for circCD226 characterization. Cell viability, proliferation, migration, invasion and sphere formation abilities were gauged by CCK-8, EDU, wound-healing, transwell and sphere formation assays, respectively. Directed relationships among circCD226, miR-1224-3p and HMGA2 were examined by RNA pull-down, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The abundance of circCD226 was elevated in serum exosomes, tissues and cells of NSCLC. NSCLC serum exosomes enhanced NSCLC cell proliferation, migration, invasion and stemness. Loss of circCD226 impeded cell proliferation, migration, invasion and stemness in vitro , as well as tumor growth in vivo . Mechanistically, circCD226 sponged miR-1224-3p, and miR-1224-3p targeted HMGA2. CircCD226 involved the posttranscriptional regulation of HMGA2 through miR-1224-3p. Moreover, the miR-1224-3p/HMGA2 axis was identified as a functionally downstream effector of circCD226 in regulating NSCLC cell behaviors. Our study identifies circCD226 as a potential driver in NSCLC development depending on the regulation of miR-1224-3p/HMGA2 axis.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dactinomycin; Humans; Lung Neoplasms; MicroRNAs; Ribonucleases; RNA, Circular; Sincalide

STK11 is a frequently mutated tumor suppressor in lung adenocarcinoma (LUAD). STK11 mutations also lead to dramatic changes in the tumor microenvironment. Studies have shown that strengthening the killing effect of NK cells is vital for effective cancer treatment. Nonetheless, the mechanism of STK11 mutation in modulating the killing effect of NK cells on LUAD cells remains unclear. The expression of STK11, Ki67, and IFN-γ in tumor tissues was evaluated by immunohistochemistry. The contents of T cells, NK cells, and macrophages were analyzed by immunofluorescence assay. The expression of IL2, IL6, and IFN-γ was detected by ELISA. STK11 expression in LUAD cell line was evaluated by qRT-PCR. CCK-8 and colony formation assay were used to measure proliferative ability of LUAD cells and the viability of NK cells. Flow cytometry was utilized to analyze cell apoptosis and NK cell content. Transwell assay was utilized to measure the chemotactic capability of NK cells. In vivo experiments validated the effect of abnormal expression of STK11 on tumor growth. LUAD patients with STK11 mutation had a high tumor proliferative ability. Forced expression of STK11 could substantially constrain the proliferation of LUAD cells and induce cell apoptosis. In addition, STK11 deletion significantly reduced the infiltration level of NK cells and inhibited the viability and chemotactic ability of NK cells as well as their killing effect on LUAD cells. In vivo animal experiment results demonstrated that STK11 deletion significantly reduced NK cell infiltration and promoted LUAD tumor growth. This study revealed the mechanism of STK11 mutation regulating NK cytotoxicity and promoting tumor development, providing scientific basis for the exploration of STK11-related LUAD therapeutic targets and theoretical reference for developing new NK cell-based immunotherapy.

Topics: Adenocarcinoma of Lung; Animals; Cell Proliferation; Gene Expression Regulation, Neoplastic; Interleukin-2; Interleukin-6; Ki-67 Antigen; Killer Cells, Natural; Lung Neoplasms; Mutation; Sincalide; Tumor Microenvironment

Although our previous genome-wide association study (GWAS) has identified chromosome 2q33.1 as a susceptibility locus for non-small cell lung cancer (NSCLC), the causal variants remain unclear. The aims of this study were to identify the causal variants in 2q33.1 and to explore their biological functions in NSCLC.. CCK-8, colony formation, EdU incorporation, Transwell, and quantitative real-time polymerase chain reaction assays were applied to examine variant function. The tumor xenograft model was used to examine variant function. The missense variant rs3769823 (A > G), which caused the substitution of lysine with arginine at amino acid 14 in caspase-8 (caspase-8K14R), was identified as a potential causal candidate in 2q33.1. Compared with the wild type caspase-8 (caspase-8WT) group, the caspase-8K14R group had higher expression of caspase-8 and cleaved caspase-8. Caspase-8K14R inhibited the proliferation and metastasis of human lung cancer cell lines. These results suggested that rs3769823 is the causal variant in chromosome 2q33.1 and is involved in an apoptosis pathway, leading to a decreased risk of NSCLC.

Topics: Apoptosis; Arginine; Carcinoma, Non-Small-Cell Lung; Caspase 8; Caspases; Genome-Wide Association Study; Humans; Ligands; Lung Neoplasms; Lysine; Sincalide; TNF-Related Apoptosis-Inducing Ligand

Recent literature have indicated that the malignancy of cancer cells is modulated by hsa_circ_0000423 (named circPPP1R12A) through the way of translating protein. Herein, we investigated the role and latent mechanisms of circPPP1R12A in Non-Small Cell Lung Cancer (NSCLC).. CircPPP1R12A expression was measured by qRT-PCR. The malignancy of NSCLC was determined by CCK-8, TUNEL assay, Wound healing, Transwell and Western blotting assays. The underlying mechanisms of circPPP1R12A were confirmed by Western blotting and qRT-PCR assays.. CircPPP1R12A expression in NSCLC tissues was higher than that of neighboring normal tissues. CircPPP1R12A showed an upregulated expression in NSCLC cells. Upregulation of circPPP1R12A could promote the cell viability of NSCLC cells and reduce the apoptosis of NSCLC cells, while it could not promote cell invasion and migration. The reduction of cell viability and apoptosis was discovered in NSCLC cells with the silencing of circPPP1R12A, but circPPP1R12A knockdown does not inhibit cell invasion and migration. There was something interesting that circPPP1R12A encoding protein circPPP1R12A-73aa was found in NSCLC cells. Mutations in circPPP1R12a-73AA might disrupt the function of circPPP1ra-73AA in A549 and H1299 cells. Next, we found that circPPP1R12A caused the increased growth of NSCLC cells by activating AKT signaling pathway.. In summary, our study proved that NSCLC cell proliferation was promoted by circPPP1R12A-73aa translated from circPPP1R12A through the AKT pathway, which could throw some light on the understanding of the mechanism of NSCLC.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Proto-Oncogene Proteins c-akt; Signal Transduction; Sincalide

This study aimed to identify the molecular mechanism underlying NEAT1 regulation of non-small-cell lung cancer (NSCLC) autophagy and apoptosis. The expression levels of NEAT1, miR-128-3p, and ADAM28 in NSCLC tissues and cells were examined using qRT-PCR. The relationships between NEAT1, miR-128-3p, and ADAM28 expression levels and prognosis of NSCLC patients were investigated using the Kaplan-Meier analysis method. The interactions between NEAT1, miR-128-3p, and ADAM28 were confirmed by dual-luciferase reporter assay or FISH assay. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry assays, respectively. Apoptosis and autophagy-related proteins Bax, cleaved caspase-3, cleaved caspase-9, Bcl-2, LC3, Beclin-1, and p62 were analyzed by western blotting. Finally, an in vivo NSCLC mouse xenograft model was established to confirm the in vitro data. We showed NEAT1 and ADAM28 expression levels were upregulated, while the miR-128-3p level was downregulated in NSCLC tissues and cells. NSCLC patients with high NEAT1 expression levels, low miR-128-3p levels, or high ADAM28 levels had significantly reduced overall survival times. Silencing of NEAT1 inhibited NSCLC cell autophagy and promoted apoptosis by sponging miR-128-3p. MiR-128-3p directly targeted ADAM28 and suppressed ADAM28 expression, which led to deactivation of the JAK2/STAT3 signaling pathway. Furthermore, ADAM28 overexpression attenuated miR-128-3p overexpression-induced apoptosis and autophagy inhibition in NSCLC by increasing the phosphorylation of JAK2 and STAT3. NEAT1 depletion or miR-128-3p overexpression inhibited autophagy and promoted apoptosis in vivo by suppressing ADAM28. In other word, silencing NEAT1 inhibited autophagy and then promoted NSCLC cell apoptosis by deactivating the JAK2/STAT3 signaling pathway through regulation of miR-128-3p/ADAM28 axis.

Topics: ADAM Proteins; Animals; Apoptosis; Autophagy; bcl-2-Associated X Protein; Beclin-1; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Mice; MicroRNAs; Proto-Oncogene Proteins c-bcl-2; RNA, Long Noncoding; Sincalide

Non-small cell lung carcinoma (NSCLC) is a subtype of lung cancer with unfavorable outcome. Autophagy, a mechanism responsible for cellular component degradation, has been recorded to play either a positive or negative regulatory role in apoptosis. Tectonin Beta-Propeller Repeat Containing 1 (TECPR1) is recognized relevant to autophagy. This study aimed to investigate the molecular mechanisms through which TECPR1 regulates NSCLC cell apoptosis.. Analysis of TECPR1 expression in the subcategories of NSCLC was conducted using GEPIA. Survival analysis for NSCLC patients was performed with Kaplan-Meier's plotter. The interaction between ATG5 and TECPR1 was predicted by STRING and validated through co-immunoprecipitation. NSCLC cells were transfected with short hairpin RNA against ATG5 and/or ATG5/TECPR1 overexpression plasmids, followed by viability and apoptosis assay using CCK-8 and flow cytometry. Expressions of TECPR1, ATG5, LC3-II/LC3-I, P62, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in NSCLC cells with or without transfection were assessed by qRT-PCR and/or Western blot.. TECPR1 was low-expressed in LUAD and LUSC samples as well as NSCLC cells. Higher TECPR1 expression was associated with better outcomes. TECPR1 overexpression and ATG5 overexpression both decreased viability, promoted apoptosis, upregulated Bax and LC3-II/LC3-I, and downregulated P62 and Bcl-2. TECPR1 could form a complex with ATG5 in NSCLC cells. ATG5 was upregulated by TECPR1 overexpression and could positively modulate TECPR1 expression. ATG5 knockdown induced effect oppositely to TECPR1 overexpression, and this effect reversed the TECPR1 overexpression-induced effect and vice versa.. TECPR1 induces NSCLC cell apoptosis via ATG5 upregulation-induced autophagy promotion.

Topics: Apoptosis; Autophagy; Autophagy-Related Protein 5; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Membrane Proteins; RNA, Small Interfering; Sincalide; Up-Regulation

Non-small cell lung cancer (NSCLC) results in high mortality and has gained increasing attention. C-Phycocyanin (C-PC) has been identified as a potential therapeutic inhibitor for NSCLC, but its underlying mechanism remains obscure. The gene expression of the long noncoding RNA neighbour of BRCAI RNA 2 (NBR2) in NSCLC cells was evaluated by quantitative reverse transcription-PCR. The cell capacity for proliferation and migration was examined by EdU and wound-healing assays. Furthermore, the viability and apoptosis of cells was measured with CCK-8 and annexin V/PI, respectively. Next, the protein level of activation of adenosine monophosphate- activated protein kinase and the rapamycin kinase (mTOR) signalling pathway-associated molecules was evaluated by western blotting. H292 cells were pre-treated with C-PC or transfected with plasmids encoding NBR2 or the shNBR2 plasmid, to over-express or knock down NBR2 expression, respectively. NBR2 expression was robustly down-regulated in NSCLC cell lines compared with a normal cell line (BEAS-2B). NBR2 over-expression inhibited migration and promoted apoptosis of H292 cells. Treatment of H292 cells with C-PC enhanced NBR2 levels in a dose- and time-dependent manner. Downregulation of NBR2 in H292 cells inhibited the activity of C-PC on cell proliferation, viability and clone formation. Further mechanistic investigation showed that the down-regulation of NBR2 abolished the modulatory effects of C-PC on the AMPK/mTOR signalling pathway. In conclusion, C-PC inhibits H292 cell growth by enhancing the NBR2/AMPK signalling pathway.

Topics: Adenosine Monophosphate; AMP-Activated Protein Kinases; Annexin A5; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Lung Neoplasms; Phycocyanin; RNA, Long Noncoding; Sincalide; Sirolimus; TOR Serine-Threonine Kinases

Cancer-associated fibroblasts (CAFs) within the tumor microenvironment are key players in tumorigenesis and tumor development. Nevertheless, the regulatory mechanisms of CAFs on lung squamous cell carcinoma- (LUSC-) associated remain poorly elucidated.. The microarray dataset GSE22874, containing 30 specimens of primary culture of normal fibroblasts (NFs) and 8 specimens of cancer-associated fibroblasts (CAFs) samples derived from LUSC, was retrieved from the Gene Expression Omnibus (GEO) database and then calculated by using the R language (limma package) to identify differentially expressed genes (DEGs). CAF-conditioned medium (CAF-CM) was collected and used to culture LUSC cells, followed by assessment of cell proliferation, apoptosis, and oxidative stress levels by using CCK-8, annexin V-FITC/PI double staining and ELISA assays. Subsequently, COL10A1 was knocked down in CAFs to assess the role of COL10A1 in CAF regulation of LUSC behavior. Bioinformatics online analysis and MeRIP were applied to predict and test the m. Elevated expression of COL10A1 was found in LUSC-derived CAFs by GSE22874 dataset analysis. We discovered that COL10A1 and METTL3 was expressed in both LUSC cells and matched CAFs, while COL10A1 expression was prominently higher in CAFs than in LUSC cells. CAF-CM memorably encouraged LUSC cell proliferation and suppressed apoptosis-induced oxidative stress, which was reversed by interfering with COL10A1 expression in CAFs, suggesting that COL10A1 might be secreted by CAFs into the culture medium to exert its effects inside LUSC cells. Global m. COL10A1 secreted by CAFs could facilitate LUSC cell proliferation and repress apoptosis-induced oxidative stress, and the mechanism was due to elevated expression mediated by METTL3 promoting its mRNA m

Topics: Animals; Apoptosis; Cancer-Associated Fibroblasts; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Collagen Type X; Culture Media, Conditioned; Fibroblasts; Lung; Lung Neoplasms; Methylation; Methyltransferases; Mice; Mice, Nude; Oxidative Stress; RNA, Messenger; Sincalide

Circular RNA of vimentin (circ-VIM) is a predictor for poor prognosis of acute myeloid leukemia, but we had little information on its function in esophageal cancer (EC). Here we examined the effects of circ-VIM together with sevoflurane on immune escape and multiple oncogenic activities of EC.. Significant upregulation of circ-VIM and PD-L1 and downregulation of miR-124 were detected in EC tissues or cells. Circ-VIM sponged miR-124 and released its suppression on the downstream target PD-L1. Sevoflurane, independent of circ-VIM, also upregulated miR-124 to lower PD-L1 expression. By modulating miR-124/PD-L1 axis, silencing circ-VIM and applying sevoflurane both inhibited immune escape and multiple oncogenic activities of EC in vitro, and suppressed xenograft growth and lung metastases in vivo. The inactivation of Ras/ERK signaling pathway was involved in suppression of malignant phenotypes by silencing circ-VIM and sevoflurane treatment.. Silencing circ-VIM and applying sevoflurane, by separately regulating miR-124/PD-L1 axis, presented synergistic effects in inhibiting immune escape and multiple malignant phenotypes of EC cells.

Topics: Annexin A5; B7-H1 Antigen; Carcinogenesis; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Esophageal Neoplasms; Humans; Lung Neoplasms; MicroRNAs; RNA, Circular; Sevoflurane; Sincalide; Vimentin

Increasing evidence indicated that miR-2682-5p acted as a tumor suppressor in various cancers. The current study aimed to investigate the biological function of exosomal miR-2682-5p in non-small cell lung cancer (NSCLC).. The expression of miR-2682-5p in NSCLC tissues and adjacent non-tumor tissues, NSCLC cell lines and human embryonic lung fibroblast, as well as serum and serum exosomes of NSCLC patients and healthy donors was detected by RT-qPCR. The effects of miR-2682-5p on the viability, migration, and apoptosis of NSCLC cells were detected by CCK-8, Transwell, and flow cytometry assays. Dual-luciferase reporter gene and RNA immunoprecipitation assays were used to evalutate the relationship between miR-2682-5p and HDAC1.. Low expressed miR-2682-5p was found in tumor tissues, cell lines, serum, and serum exosomes of NSCLC patients. MiR-2682-5p overexpression suppressed NSCLC cell viability and migration and promoted apoptosis, while miR-2682-5p knockdown showed the opposite results. Furthermore, exosomes from healthy donor serum inhibited NSCLC cell viability and migration and promoted apoptosis. Dual-luciferase reporter gene and RNA immunoprecipitation assays verified that HDAC1 was a target of miR-2682-5p. HDAC1 overexpression abolished the effects of miR-2682-5p mimic on NSCLC cell viability, migration, and apoptosis. Chromatin immunoprecipitation assay indicated that HDAC1 bound to the promoter region of ADH1A. Upregulation of ADH1A counteracted the effects of HDAC1 overexpression on NSCLC cell viability, migration, and apoptosis.. Taken together, exosomal miR-2682-5p inhibited NSCLC cell viability and migration and promoted apoptosis by the HDAC1/ADH1A axis, and this result might provide a novel therapeutic target for NSCLC.

Topics: Alcohol Dehydrogenase; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Survival; Down-Regulation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase 1; Humans; Lung Neoplasms; MicroRNAs; Sincalide; Up-Regulation

Ultrasound-targeted microbubble destruction (UTMD) is a novel adjuvant tumor therapeutic method by enhancing exogenous gene transfection to target tissues. This study aims to investigate the role of microRNA-492 (miR-492) in non-small cell lung cancer (NSCLC) and further analyze the effects of UTMD-mediated miR-492 inhibitor on tumorigenesis.. The expression of miR-492 was detected by qRT-PCR. Co-transfection of microbubbles and miR-492 inhibitor with Lipofectamine 3000 was performed to achieve UTMD-mediated miR-492 inhibition in NSCLC cells. CCK-8 and Transwell assay were used to determine NSCLC cell proliferation, and the migration and invasion.. High expression of miR-492 was associated with poor prognosis in NSCLC patients. miR-492 inhibitor suppressed tumor cell proliferation, migration and invasion, and UTMD not only increased the transfection efficiency of miR-492 inhibitor, but also enhance the inhibitory effects on cell biological behaviors.. The results showed that the expression level of miR-492 was up-regulated in NSCLC tissue samples and cells. Silencing of miR-492 inhibited NSCLC cell proliferation, migration and invasion, and UTMD-mediated miR-492 inhibitor could promote more significant inhibition, which indicated that UTMD-mediated miR-492 inhibitor might provide a novel strategy for the treatment of NSCLC.KEY MESSAGESmiR-492 inhibitor inhibited cell proliferation, migration and invasion.UTMD-mediated miR-492 inhibitor can promote more significant inhibition.UTMD-mediated miR-492 inhibitor provide a new strategy for NSCLC.

Topics: Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cell Transformation, Neoplastic; Female; Gene Expression; Humans; Lipids; Lung Neoplasms; Male; Microbubbles; MicroRNAs; Middle Aged; Real-Time Polymerase Chain Reaction; Sincalide; Transfection; Treatment Outcome; Ultrasonics; Ultrasonography

To examine the effects of Keratin 6A (KRT6A) protein on the proliferation, migration and invasion abilities of lung adenocarcinoma cells, and to analyse the relationship between the expression level of KRT6A protein and the survival prognosis of lung adenocarcinoma patients.. Western Blot was used to detect the expression of KRT6A protein in lung adenocarcinoma cell lines. CCK-8 experiment and colony formation assays were performed to detect the proliferation ability. Wound healing assay and transwell migration assay were conducted to detect the migration ability. Transwell invasion assay was conducted to detect the invasion ability. Immunohistochemistry was used to detect the expression of KRT6A protein in lung adenocarcinoma tissues.. We first found that the expression of KRT6A protein in lung adenocarcinoma cell lines was low. After overexpressed KRT6A protein in lung adenocarcinoma cells, we then found that KRT6A protein could not only inhibit the proliferation ability of lung adenocarcinoma cells but also inhibit them migration and invasion abilities. In addition, we also found that there had obvious difference in the expression of KRT6A protein in between patients. And through further analysis, we finally discovered that high expression of KRT6A protein was related to favourable prognosis in lung adenocarcinoma patients.. KRT6A protein inhibits the proliferation, migration and invasion abilities of lung adenocarcinoma cells, and high expression of KRT6A protein is a predictor of good prognosis in patients with lung adenocarcinoma.

Topics: Adenocarcinoma of Lung; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Keratin-6; Lung Neoplasms; Male; Neoplasm Invasiveness; Prognosis; Sincalide

Mutant EGFR Non-small cell lung cancer has benefit from gefitinib, but it has limited effect for wild-type EGFR tumors. Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicine, the plant Lithospermum erythrorhizon (zicao), not only can inhibit the tumor growth, but also overcome cancer drug resistance. Our aim is to investigate whether shikonin can enhance antitumor effect of gefitinib in EGFR wild-type lung cancer cells in vitro and in vivo.. CCK-8 was used to determine the proliferation of EGFR wild-type non-small cell lung cancer. Apoptosis and cell cycle were detected by flow cytometry. PKM2, STAT3, p-STAT3 and cyclinD1 were detected by Western blot. A549 tumor model was established to observe the antitumor effect of shikonin combination with gefitinib in vivo.. The results showed that combination of shikonin with gefitinib exhibited synergistic antitumor effect in vitro and in vivo. Its potential molecular mechanisms may be associated with inhibition of PKM2/STAT3/cyclinD1.. These results provide a promising therapeutic approach for the treatment of wild-type EGFR non-small cell lung cancer.

Topics: A549 Cells; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; Drug Synergism; ErbB Receptors; Gefitinib; Humans; Immunohistochemistry; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Naphthoquinones; Quinazolines; Signal Transduction; Sincalide; STAT3 Transcription Factor; Thyroid Hormone-Binding Proteins; Thyroid Hormones

The regional distributions and relative frequencies of some gastric endocrine cells of C57BL/6 mice were studied by immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin, somatostatin, gastrin, cholecystokinin (CCK)-8, glucagon and human pancreatic polypeptide (HPP) after subcutaneous implantation of murine lung carcinoma (3LL) cells.. The experimental animals were divided into two groups, one is non-implanted sham and the other is 3LL-implanted group. Samples were collected from the two regions of stomach (fundus and pylorus) at 28 d after implantation of 3LL cells (1 x 10(5) cell/mouse).. In this study, all the seven types of immunoreactive (IR) cells were identified except for HPP. Most of these IR cells in the gastric portion were generally spherical or spindle in shape (open-type cell) while cells showing round in shape (closed-type cell) were found occasionally. The regional distributions of gastric endocrine cells in the 3LL-implanted group were similar to those of non-implanted sham. However, significant decreases of some types of IR cells were detected in 3LL-implanted group compared to those of non-implanted sham. In addition, the IR cells showing degranulation were numerously detected in 3LL-implanted group. CGA-, serotonin- and somatostatin-IR cells in the fundus and pylorus regions, and gastrin-IR cells in the pylorus regions of 3LL-implanted groups significantly decreased compared to those of non-implanted sham. However, no changes on frequencies of CCK-8- and glucagon-IR cells were demonstrated between 3LL-implanted and non-implanted groups.. Endocrine cells are the anatomical units responsible for the production of gut hormones, and the change in their density would reflect a change in the capacity of producing these hormones. Implantation of tumor cell mass (3LL) induced severe quantitative changes of gastric endocrine cell density, and the abnormality in density of gastric endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.

Topics: Animals; Antibodies; Carcinoma, Lewis Lung; Chromogranin A; Chromogranins; Enteroendocrine Cells; Female; Gastric Fundus; Gastrins; Glucagon; Immunohistochemistry; Injections, Subcutaneous; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Pancreatic Polypeptide; Protein Precursors; Pylorus; Serotonin; Sincalide; Somatostatin

Cholecystokinin (CCK)-A and CCK-B/gastrin receptors were evaluated with in vitro receptor autoradiography in 406 human tumors of various origins using a sulfated 125I-labeled CCK decapeptide analogue 125I-(D-Tyr-Gly, Nle28,3l)-CCK 26-33 and 125I-labeled Leu15-gastrin as radioligands. CCK-B/gastrin receptors were found frequently in medullary thyroid carcinomas (92%), in small cell lung cancers (57%), in astrocytomas (65%), and in stromal ovarian cancers (100%). They were found occasionally in gastroenteropancreatic tumors, breast, endometrial, and ovarian adenocarcinomas. They were either not expressed or rarely expressed in colorectal cancers, differentiated thyroid cancers, non-small cell lung cancers, meningiomas, neuroblastomas, schwannomas, glioblastomas, lymphomas, renal cell cancers, prostate carcinomas, and the remaining neuroendocrine tumors (i.e., pituitary adenomas, pheochromocytomas, paragangliomas, and parathyroid adenomas). CCK-A receptors were expressed rarely in tumors except in gastroenteropancreatic tumors (38%), meningiomas (30%), and some neuroblastomas (19%). The identified CCK-A and CCK-B receptors were specific and of high affinity in the subnanomolar range. The rank order of potency of various CCK analogues was: sulfated CCK-8 = L-364,718 >> nonsulfated CCK-8 = L-365,260 > or = gastrin for CCK-A receptors and sulfated CCK-8 > gastrin = nonsulfated CCK-8 > L-365,260 > L-364,718 for CCK-B receptors. CCK-B receptors could also be selectively and specifically labeled with a newly designed nonsulfated 125I-(D-Tyr-Gly, Nle28,31)-CCK 26-33. Gastrin mRNA measured by in situ hybridization was present in most CCK-B receptor-positive small cell lung cancers, breast tumors, and ovarian tumors, representing the molecular basis of a possible autocrine growth regulation of these tumors. Gastrin and CCK mRNAs were lacking in medullary thyroid cancers. Thus, these results may have pathogenic, diagnostic, differential diagnostic, and therapeutic implications.

Topics: Autoradiography; Breast Neoplasms; Carcinoma, Small Cell; Cholecystokinin; Female; Gastrins; Humans; Lung Neoplasms; Neoplasms; Neuroendocrine Tumors; Ovarian Neoplasms; Receptor, Cholecystokinin A; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Sincalide; Thyroid Neoplasms

Gastrin, cholecystokinin (CCK), and CCK-related peptides comprise a hormonal family characterized by an identical carboxy-terminal amino acid sequence, a domain critical for receptor binding. The addition of gastrin to small cell lung cancer (SCLC) cells causes a rapid and transient increase in the intracellular concentration of calcium ([Ca2+]i). Furthermore, gastrin acts as a direct growth factor through CCKB/gastrin receptors. We report here that the expression of the mRNA coding for CCKB/gastrin receptors correlates with the responsiveness of SCLC cells to gastrin in terms of Ca2+ mobilization and stimulation of clonal growth in semisolid medium. The GLC19 SCLC cell line had no detectable expression of CCKB/gastrin receptor mRNA. Accordingly, gastrin (1-100 nM) did not cause any measurable increase in [Ca2+]i. In contrast, the addition of cholecystokinin residues 26-33 (CCK-8) caused a rapid and transient increase in [Ca2+]i in this cell line. CCK-8 mobilized Ca2+ in a dose-dependent manner in the nanomolar range (half-maximal stimulatory concentration = 12 nM). Furthermore, the selective CCKA antagonist CAM-1481 inhibited the increase in [Ca2+]i induced by CCK-8 (half-maximal inhibitory concentration = 3 nM) in GLC19 but not in H510 cells. The selective CCKB/gastrin antagonist blocked the increase in [Ca2+]i induced by CCK-8 (half-maximal inhibitory concentration = 80 pM) in H510 but not in GLC19 cells. Thus, the effects of CCK-8 are mediated through CCKA receptors in GLC19 cells and via CCKB/gastrin receptors in H510 cells. CCK-8 markedly stimulated colony formation in GLC19 cells in a dose-dependent manner in the nanomolar range, whereas over the same concentration range, gastrin had no effect on clonal growth. CAM-1481 inhibited the CCK-stimulated colony formation in GLC19 but not in H510 cells. Our results show, for the first time, that CCKA receptors can mediate Ca2+ mobilization and growth in SCLC cells and that SCLC cells express two distinct functional CCK receptor subtypes.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Bradykinin; Calcium; Carcinoma, Small Cell; Clone Cells; DNA Primers; Dogs; Gastrins; Gene Expression; Humans; Kinetics; Lung Neoplasms; Molecular Sequence Data; Muridae; Polymerase Chain Reaction; Rats; Receptors, Cholecystokinin; RNA, Messenger; RNA, Neoplasm; Sequence Homology, Amino Acid; Sincalide; Tumor Cells, Cultured

Gastrin has been postulated to be a physiological growth factor, but compelling in vitro evidence of this has been difficult to obtain. In the present study we investigated whether small cell lung carcinoma cell lines could provide a useful model system to study the effects of gastrin on signal transduction and cell proliferation in vitro. We found that the addition of gastrin to small cell lung cancer cells loaded with the fluorescent Ca2+ indicator fura 2-tetraacetoxymethylester causes a rapid and transient increase in the intracellular concentration of Ca2+ ([Ca2+]i) followed by homologous desensitization. The [Ca2+]i response was especially prominent in the small cell lung carcinoma cell line H510. In this cell line, gastrin I, gastrin II, cholecystokinin residues 26-33 (CCK-8), and unsulfated CCK-8 increased [Ca2+]i in a concentration-dependent fashion with half-maximum effects at 7, 2.5, 3, and 5 nM, respectively. The Ca(2+)-mobilizing effects of gastrin and CCK-8 were prevented by proglumide, benzotript, and the specific gastrin/CCKB receptor antagonist L365260. Gastrin stimulated the clonal growth of H510 cells in semisolid (agarose-containing) medium, increasing both the number and the size of the colonies. Gastrin and CCK agonists were equally effective in promoting clonal growth. The broad-spectrum neuropeptide antagonists [D-Arg1,D-Phe5,D-Trp7,9,Leu11] substance P and [Arg6,D-Trp7,9,MePhe8] substance P (6-11) markedly inhibited gastrin-stimulated Ca2+ mobilization and clonal growth. These results show that gastrin acts as a direct growth factor through gastrin/CCKB receptors and demonstrate, for the first time, that these peptides can stimulate the proliferation of cells outside the gastrointestinal tract.

Topics: Benzamides; Calcium; Carcinoma, Small Cell; Cell Division; Gastrins; Humans; Lung Neoplasms; Proglumide; Sincalide; Tumor Cells, Cultured

The ability of cholecystokinin (CCK) to elevate intracellular Ca2+ levels in small cell lung cancer cells was investigated using the fluorescent Ca2+ indicator Fura 2. CCK-8 elevated the cytosolic Ca2+ levels in cell line NCI-H345 in a dose dependent manner. Nanomolar concentration of CCK-8 elevated cytosolic Ca2+ levels in the absence or presence of extracellular Ca2+. Potent CCK agonists such as gastrin-1 and nonsulfated CCK-8 but not inactive compounds such as CCK-27-32-NH2 elevated cytosolic Ca2+ levels. These data suggest that CCK receptors may regulate the release of Ca2+ from intracellular organelles in small cell lung cancer cells.

Topics: Bombesin; Calcium; Carcinoma, Small Cell; Cholecystokinin; Cytosol; Lung Neoplasms; Neurotensin; Receptors, Cell Surface; Sincalide; Structure-Activity Relationship