Large Lung Cancer Cell Line Research Articles

The black cumin seeds oil with Curcuma xanthorrhiza extract preparation increases IFN-γ expression

Introduction: According to empirical evidence, Curcuma xanthorrhiza (CX) and black cumin (BC) have been utilized extensively in Indonesia. The primary components of Curcuma xanthorrhiza and black cumin (CXBC) formulations are BC oil and CX extract. This study aimed to assess the influence of CXBC active compounds (polyphenols, flavonoids, and thymoquinone) on IFN-g expression in HTB-183 cells. This study aimed to determine the levels of vitamins and minerals in CXBC preparation and the potential for immunomodulatory activities. Methods: We engaged in experimental laboratory research. Observing the expression of interferon-gamma (IFN-γ) was used to evaluate the immunomodulatory activities of the HTB-183 human large lung cancer cell line. Results: The results demonstrated that the CXBC supplement included vitamins (A, C, and E) and minerals (potassium, calcium). CXBC preparations boosted HTB-183 cell IFN-γ expression. Conclusion: The study's findings indicate that the CXBC formulation contains a high concentration of vitamins and minerals. CXBC formulations augment IFN-γ expression.

Combination of black cumin seeds with Curcuma xanthorrhiza extract as an antioxidant and immune-modulator agent in the COVID-19 pandemic era

The COVID-19 pandemic demands changes in using of medicinal plants. Empirically, the medicinal plants of Curcuma xanthorrhiza (CX) and black cumin (BC) have been used massively in the era of the COVID-19 pandemic. Curcuma xanthorrhiza and black cumin (CXBC) preparations have been developed with the main ingredients of BC oil and CX extract. The purpose of the study was to determine the antioxidant and immune-modulatory activities of CXBC preparations active substances (polyphenols, flavonoids, thymoquinone). We conducted experimental laboratory research. The immunomodulatory activity test was carried out on human large lung cancer cell line (HTB-183 cells) by observing the expression of tumor necrosis factor alfa (TNF-α) and interleukine 10 (IL-10). The results showed that the CXBC preparation contained 4% thymoquinone, 25.87 mg/ml polyphenols, and 41.86 mg/dl flavonoids. CXBC preparations contain vitamins (A, C, and E) and minerals (potassium, calcium). The antioxidant activity of the CXBC preparation was included in the strong category with IC50=54.87 ppm. CXBC preparations increased TNF-a expression and decreased IL-10 expression in HTB-183 cells. Based on the study results, it can be concluded that the CXBC preparation contains 4% thymoquinone, 25.87 mg/ml polyphenol, 41.86 mg/dl flavonoid, and a high level of vitamin and minerals. CXBC preparations have potent antioxidant activity, increase TNF-α and decrease IL-10 expression.

Nm23-H1 was involved in regulation of KAI1 expression in high-metastatic lung cancer cells L9981

The tetraspanin KAI1/CD82 was identified as a tumor metastasis suppressor that down-regulated in malignant progression of lung cancer. However, the underlying mechanism of anti-metastasis role of KAI1 in lung cancer is hardly known. In this paper, we sought to study the function and regulatory mechanism of KAI1 in high metastasis lung cancer cell line. KAI1 expression was detected in high/low metastatic large lung cancer cell line L9981/NL9980 by quantitative real-time polymerase chain reaction (qRT-PCR). The tumor suppressor function of KAI1 was determined by wound healing assay after over-expression or knockdown of KAI1 in L9981 or NL9980 cells. Invasion assay was performed to detect the invasion ability of L9981 by transfection of KAI1. The effect of tumor suppressor p53 on KAI1 expression was measured by western blot and luciferase assay. Then the regulation of KAI1 due to over-expression of metastasis suppressor nm23-H1 was monitored by qRT-PCR, western blot and reporter gene assay. The progression of L9981 cells after p53 and nm23-H1 expression was detected by invasion assay. Also, methylation status of KAI1 promoter in NL9980 and L9981 cells were examined by bisulfite sequencing and methylation-specific PCR. We found that KAI1 is down-regulated in high metastatic L9981 cells compare with NL9980 cells. The migration and invasion of L9981 cells were remarkably suppressed in vitro by KAI1 transfection. The migration ability of NL9980 was enhanced by inhibition of KAI1. Furthermore, KAI1 expression was induced after over-expression of p53 or nm23-H1, while cell invasion was inhibited in L9981 cells. The results of reporter analysis indicated that KAI1 promoter region between -922 to -846 could response to nm23-H1. In addition, we discovered only slight methylation of KAI1 promoter, which showed that loss expression of KAI1 in L9981 cells may not due to promoter methylation. The results suggested that nm23-H1 was involved in the KAI1-regulated inhibition of metastasis in lung cancer cells. More insights into the relationship between KAI1 and other metastasis suppressors will pave the way for the elucidation of anti-metastasis mechanism in lung cancer.

Germline polymorphisms and survival of lung adenocarcinoma patients: a genome-wide study in two European patient series.

In lung cancer, the survival of patients with the same clinical stage varies widely for unknown reasons. In this two-phase study, we examined the hypothesis that germline variations influence the survival of patients with lung adenocarcinoma. First, we analyzed existing genotype and clinical data from 289 UK-resident patients with lung adenocarcinoma, identifying 86 single nucleotide polymorphisms (SNPs) that associated with survival (p < 0.01). We then genotyped these candidate SNPs in a validation series of 748 patients from Italy that resulted genetically compatible with the UK series based on principal component analysis. In a Cox proportional hazard model adjusted for age, sex and clinical stage, four SNPs were confirmed on the basis of their having a hazard ratio (HR) indicating the same direction of effect in the two series and p < 0.05. The strongest association was provided by rs2107561, an intronic SNP of PTPRG, protein tyrosine phosphatase, receptor type, G; the C allele was associated with poorer survival in both patient series (pooled analysis loge HR = 0.31; 95% CI: 0.15-0.46, p = 8.5 × 10(-5) ). PTPRG mRNA levels in 43 samples of lung adenocarcinoma were 40% of those observed in noninvolved lung tissue from the same patients. PTPRG overexpression significantly inhibited the clonogenicity of A549 lung carcinoma cells and the anchorage-independent growth of the NCI-H460 large cell lung cancer line. These four germline variants represent promising candidates that, with further study, may help predict clinical outcome. In addition, the PTPRG locus may have a role in tumor progression.

EXPRESSION AND PURIFICATION OF HUMAN ARP1 PROTEIN AND RAPID PREPARATION OF POLYCLONAL ANTIBODY

Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

Methylation profile difference in human high-metastatic large cell lung cancer cell line L9981 and low-metastatic large cell lung cancer line NL9980

背景与目的肺癌是人类肿瘤中最容易发生侵袭转移的恶性肿瘤之一。影响肺癌患者预后及生存的最主要因素是远处转移。本文旨在分析具有相似遗传背景和不同转移潜能的人大细胞肺癌细胞株间基因甲基化的差异，为深入研究肿瘤转移机制提供充分的目标基因选择和发现肿瘤相关的新基因的机会。方法应用全基因组甲基化芯片杂交技术分析具有相同遗传背景和不同转移潜能的人大细胞肺癌细胞株L9981和NL9980之间差异甲基化基因，并运用生物信息学的方法分析这些差异基因。结果与NL9980相比，在L9981细胞株中有1 552个高甲基化DNA片断共涉及735个基因，其中656个已知基因及79个未知基因。低甲基化DNA片段1 787个，涉及809个基因，其中698个已知基因及111个未知基因。这些基因涉及细胞生物进程及其调节、基因表达、信号传导、细胞通讯、细胞运动、细胞粘附及血管生成等。结论抑癌基因及信号通路负向调节基因的高甲基化及癌基因、细胞粘附因子的低甲基化可能与L9981的侵袭性及克隆能力强有关。

Construction of the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene

It has been proven that nm23-H1 gene is an important metastaticsuppressed gene of lung cancer. In order to screen the differential expression genes related to nm23-H1 , we constructed the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene by suppression subtractive hybridization (SSH) in this study, which lay a solid foundation for further screening and cloning metastatic-related genes of nm23-H1. The forward and reverse suppression subtractive cDNA libraries were constructed in the human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene (L9981 and L9981-nm23-H1) by SSH method. The positive clones were preliminarily screened by bluewhite colony, and precisely identified by PCR. The suppression subtractive cDNA libraries were successfully constructed in the human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981). After the blue-white screening, about three hundred positive clones in the forward subtracted library and four hundred positive clones in the reverse subtracted library were obtained. Ramdom analysis of 96 clones in each library with colony PCR methods showed that 84 clones in the forward subtracted library and 83 clones in the reverse subtracted library contained (300-750) bp inserts. SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981) are successfully constructed by SSH and T/A cloning technology. The expression of nm23-H1 gene in the human large cell lung cancer cell lines may affect the differential expression of some metastatic-related genes.

Primary experimental results of methylseleninic acid on the proliferation inhibition and apoptotic induction in human high-metastatic large cell lung cancer cell line L9981

It has been proved that selenium has remarkable effects in the prevention of cancer and proliferation inhibition for breast cancer and prostate cancer. Up to now, little is known, however, if methylseleninic acid (MSA) has the anticancer effect on lung cancer or not. The objective of this study is to detect the effect of MSA on proliferation inhibition and apoptotic induction for human high-metastatic large cell lung cancer cell line L9981, and to explore the molecular mechanisms. The changes of proliferation, clone formation, apoptotic level and cell cycles were detected in L9981 by trypan blue staining, clone formation suppression test, and flow cytometry before and after treating with different concentration of MSA. The expression level of proliferative-related and apoptotic-related genes was also determined in L9981 by flow cytometry. (1)The proliferation ability of L9981 was remarkably inhibited at the concentration of 0.5μmol/L of MSA (P < 0.05), and the cells were arrested at G0/G1 phase after treating with the same concentration. (2)Apoptosis of L9981 was remarkably induced by MSA at the concentration of 2.5μmol/L (P < 0.05). (3)The clone formation ability of L9981 was significantly suppressed by MSA at the concentration of 5.0μmol/L (P < 0.05). (4)The expression levels of P53, P21, Fas, FasL and Bax were remarkably up-regulated after treatment with MSA. (1)MSA can significantly suppress the proliferation and clone formation ability of human high-metastatic large cell lung cancer cell line L9981, and also induce apoptosis of L9981. (2)The anticancer effects of MSA might be related to regulate the expression of cell cycle-related genes and apoptotic-related genes in the human high-metastatic large cell lung cancer line L9981.

Effects of transfection of nm23-H1 gene on β-catenin expression in human high-metastatic large cell lung cancer cell line L9981

To explore the effects of transfection of nm23-H1 gene on expression of β-catenin and phospho-β-catenin in human high-metastatic large cell lung cancer line L9981, and to provide evidence to elucidate the molecular mechanism of nm23-H1 mediated tumor metastatic suppression. To determine whether nm23-H1 contributes to cytoplasm and nuclear β-catenin and phospho-β-catenin expression, the expression level of β-catenin and phospho-β-catenin in cytoplasm and nucleus was detected in human high-metastatic large cell lung carcinoma cell lines including primary cell line L9981 with nm23-H1 gene deletion, L9981-nm23-H1 transfected with nm23-H1 gene, and L9981-pLXSN transfected with vector by Western blot. (1)β-catenin expression in L9981-nm23-H1 cytoplasm (IOD) (3 649±118) was significantly higher than that in L9981 (1 401±31) and L9981-pLXSN (1 350±55) cell lines (P < 0.001);(2)There was no statistical diffe-rence of the β-catenin expression in nucleus among L9981-nm23-H1 (2 945±68), L9981 (2 604±23) and L9981-pLXSN (2 652±53) cell lines (P > 0.05);(3)Phospho-β-cetenin expression of cytoplasm in L9981-nm23-H1 cell line (3 123±102) was significantly lower than that in L9981 (4 362±131) and L9981-pLXSN ( 4 500 ±117) cell lines (P < 0.001);(4)Phospho-β-catenin expression of nucleus in L9981-nm23-H1 (5 136±112) was significantly higher than that in L9981 (2 666±116) and L9981-pLXSN (2 661±66) cell lines (P < 0.001);(5)There was no statistical difference of β-catenin or phospho-β-catenin expression in cytoplasm and nucleus between L9981 and L9981-pLXSN cell lines (P > 0.05). (1)nm23-H1 gene transfection can remarkably upregulates the expression of cytoplasm β-catenin in human high-metastatic large cell lung cancer cell line L9981, but do not induce the nucleus accumulation of β-catenin; (2)Transfection of nm23-H1 gene can significantly upregulate the expression of phospho-β-catenin in nucleus and remarkably downregulate the expression of phospho-β-catenin in cytoplasm of L9981; (3)Regulation of the expression of the key modecule, β-catenin, in Wnt signal pathway might be the important melecular mechanisms which nm23-H1 gene controls "Lung Cancer Metastatic Suppresive Cascade" and reverves cancer metastasis in L9981.

Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines.

This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.

Chemosensitisation and drug accumulation effects of cyclosporin A, PSC-833 and verapamil in human MDR large cell lung cancer cells expressing a 190k membrane protein distinct from P-glycoprotein

The doxorubicin-selected multidrug resistant (MDR) human large cell lung cancer line COR-L23/R, lacks P-glycoprotein but shows a drug accumulation deficit. It does however overexpress a 190k membrane protein which shares an epitope with, but is otherwise distinct from, P-glycoprotein. The resistant cells show only a small sensitisation to vincristine and daunorubicin on treatment with cyclosporin A and its more potent analogue, PSC-833 despite an increase in drug accumulation. Verapamil, another effective resistance modifier in P-glycoprotein MDR cells, is slightly more effective. Fluorescent daunorubicin distributes in the cytoplasm and nucleus of sensitive parent COR-L23 cells but is confined to cytoplasmic perinuclear vesicles in resistant cells. Addition of cyclosporin A or PSC-833 slightly increases cytoplasmic fluorescence whereas verapamil also increases nuclear fluorescence. Resistance in this non-P-glycoprotein MDR line, COR-L23/R where these resistance modifiers have little effect may be associated with expression of the 190k protein.

Monoclonal antibodies that distinguish non-small cell from small cell lung cancer.

Two murine IgG2Ak monoclonal antibodies (703D4, 704A 1) were produced and characterized after immunization with a human large cell lung cancer line (NCI-H 157). These antibodies detect different epitopes on 31 kilodalton [35S]methionine incorporating protein(s). Radiobinding and immunohistochemical studies show these antibodies bind to most (11/13) human non-small cell lung cancer (adenocarcinoma, epidermoid, and large cell), but not to small cell lung cancer (0/11) tumors tested. The epitopes these antibodies recognized are also expressed on human melanomas (7/8), two other tumors (osteogenic sarcoma, renal cell carcinoma), but not on many other human tumors (breast, colon, neuroblastoma, lymphoid), and not on a panel of normal adult human tissues. Because the antigen(s) are preserved after fixation and because of their ability to distinguish lung cancer types from each other and normal tissues, they should be of clinical, as well as of biologic interest.