Malignant Transformation Of BEAS-2B Cells Research Articles

Effects of the SEMA4B gene on hexavalent chromium [Cr(VI)]-induced malignant transformation of human bronchial epithelial cells.

Our previous study identified the potential of SEMA4B methylation level as a biomarker for hexavalent chromium [Cr(VI)] exposure. This study aimed to investigate the role of the SEMA4B gene in Cr(VI)-mediated malignant transformation of human bronchial epithelial (BEAS-2B) cells. In our population survey of workers, the geometric mean [95% confidence intervals (CIs)] of Cr in blood was 3.80 (0.42, 26.56) μg/L. Following treatment with various doses of Cr(VI), it was found that 0.5μM had negligible effects on the cell viability of BEAS-2B cells. The expression of SEMA4B was observed to decrease in BEAS-2B cells after 7days of treatment with 0.5μM Cr(VI), and this downregulation continued with increasing passages of Cr(VI) treatment. Chronic exposure to 0.5μM Cr(VI) enhanced the anchorage-independent growth ability of BEAS-2B cells. Furthermore, the use of a methylation inhibitor suppressed the Cr(VI)-mediated anchorage-independent growth in BEAS-2B cells. Considering that Cr levels exceeding 0.5μM can be found in human blood due to occupational exposure, the results suggested a potential carcinogenic risk associated with occupational Cr(VI) exposure through the promotion of malignant transformation. The in vitro study further demonstrated that Cr(VI) exposure might inhibit the expression of the SEMA4B gene to promote the malignant transformation of BEAS-2B cells.

Screening of the differentially expressed proteins in malignant transformation of BEAS-2B cells induced by coal tar pitch extract.

Coal tar pitch extract (CTPE) was carcinogenic and could cause occupational lung cancer. Hence, we explored the changes of protein molecules during CTPE-induced malignant transformation (MT) of immortalized human bronchial epithelial (BEAS-2B) cells and provided clues for screening early biomarkers of CTPE-associated occupational lung cancer. The MT model of BEAS-2B cells induced by CTPE with 15.0μg/mL. Subsequently, the MT of the BEAS-2B cells was verified by morphological observation, cell proliferation test, plate colony formation assay, and cell cycle assay. At the end of the experiment, we explored the differentially expressed proteins (DEPs) by total protein tandem mass tags quantitative proteomics technique between DMSO40 cells and CTPE40 cells. It was found that the proliferation ability, and colony formation rate were enhanced, and the cell cycle was changed. Then, bioinformatics analysis showed that a total of 107 DEPs were screened between CTPE40 and DMSO40 cells, of which 74 were up-regulated and 33 were down-regulated. As a result, 6 hub proteins were screened by protein-protein interaction network analysis. The expression levels of COX7A2, COX7C, MT-CO2, NDUFB4, and NDUFB7 were up-regulated as well as the expression of RPS29 protein was down-regulated. In summary, we established an MT model in vitro and explored the changes in protein molecules. As a result, this study suggested that changes of protein molecules, including COX7A2, COX7C, NDUFB7, MT-CO2, NDUFB4, and RPS29, occurred at the stage of BEAS-2B cell malignancy following CTPE exposure, which provided key information for screening biomarkers for CTPE-related occupational lung cancer.

Abstract A010: Chemopreventive effects of carvedilol and nebivolol in benzo(a)pyrene-induced transformation of non-tumorous human bronchial epithelial cell

Abstract Lung cancer is among the most common cancers worldwide. The primary risk factor for the development of lung cancer is cigarette smoking and there are a large amount of studies has confirmed the association between smoking and lung cancer. Chemopreventive drug would be beneficial among patients who are in high risk due to chronic exposure to carcinogens. In the previous study of chemopreventive activity of carvedilol, it significantly delayed and reduced skin squamous cell carcinoma development. The aim of the present study was to explore the role of carvedilol and nebivolol in benzo(a)pyrene induced transformation of the non-tumorous human bronchial epithelial cells. To evaluate the effects of carvedilol and nebivolol on chemopreventive activity against BPDE-induced lung carcinogenesis, anchorage-independent growth assay in soft agar was used to determine the degree of malignant transformation of BEAS-2b cells. Before the investigation of carvedilol and nebivolol, the appropriate drug concentration aiming minimum cytotoxicity on normal BEAS-2b cells were determined by MTT assay. The MTT assay was used to measure cellular metabolic activity as an indicator of cytotoxicity. Carvedilol at 10 μM or lower and Nebivolol at 1 μM or lower did not cause significant cytotoxicity. In non-tumorigenic human bronchial epithelial cell culture BEAS-2B vitro assays, carvedilol and nebivolol exert a protective effect against BPDE-indued transformation at non-toxic concentrations in a dose dependent manner. As shown in the figure below, Nebivolol (1 μM and 0.5 μM) and Carvedilol (1 μM and 0.5 μM) both significantly attenuated colony formation compared with BPDE control. However, the anti-transformation mechanism is unknown, further studies of carvedilol and nebivolol are needed to investigate their chemoprevention activity. Citation Format: Carol Lin. Chemopreventive effects of carvedilol and nebivolol in benzo(a)pyrene-induced transformation of non-tumorous human bronchial epithelial cell [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A010.

Involvement of m6A regulatory factor IGF2BP1 in malignant transformation of human bronchial epithelial Beas-2B cells induced by tobacco carcinogen NNK

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a Group 1 human carcinogen, as classified by the International Agency for Research of Cancer (IARC), and plays a significant role in lung carcinogenesis. However, its carcinogenic mechanism has not yet been fully elucidated. In this study, we performed colony formation assays, soft-agar assays, and tumor growth in nude mice to show that 100 mg/L NNK facilitates the malignant transformation of human bronchial epithelial Beas-2B cells. Transcriptome sequencing showed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a post-transcriptional regulator, was differentially expressed in NNK-induced malignant transformed Beas-2B cells (2B-NNK cells). Small interfering RNA (SiRNA) was used to downregulate the expression of the IGF2BP1 gene. The reduction in protein expression, cell proliferation rate, and colony-forming ability and the increase in the apoptosis rate of Beas-2B cells transfected with the SiRNA indicated a role for IGF2BP1 in NNK-induced malignant transformation. IGF2BP1 is an N6-methyladenosine (m6A) regulatory factor, but it is not known whether its association with m6A mediates the malignant transformation of cells. Therefore, we measured the overall levels of m6A in Beas-2B cells. We found that the overall m6A level was lower in 2B-NNK cells, and knocking down IGF2BP1, the overall level of m6A was restored. Hence, we concluded that IGF2BP1 is involved in the NNK-induced malignant transformation of Beas-2B cells, possibly via m6A modification. This study therefore contributes novel insights into the environmental pathogenesis of lung cancer and the gene regulatory mechanisms of chemical carcinogenesis.

Postchronic Single-Walled Carbon Nanotube Exposure Causes Irreversible Malignant Transformation of Human Bronchial Epithelial Cells through DNA Methylation Changes.

As environmental pollutants and possible carcinogens, carbon nanotubes (CNTs) have recently been found to induce carcinogenesis and tumor metastasis after long-term pulmonary exposure. However, whether CNT-induced carcinogenesis can be inherited and last for generations remains unclear. Herein, postchronic single-walled carbon nanotubes (SWCNTs) exposed human lung cell model (BEAS-2B cells) are established to investigate SWCNT-induced carcinogenesis. At a tolerated sublethal dose level, postchronic SWCNT exposure significantly increases the migration and invasion abilities of BEAS-2B cells, leading to malignant cell transformation. Notably, the malignant transformation of BEAS-2B cells is irreversible within a 60 day recovery period after SWCNT exposure, and the malignant transformation activities of cells gradually increase during the recovery period. Moreover, these transformed cells promote carcinogenesis in vivo, accompanied by a raised level of biomarkers of lung adenocarcinoma. Further mechanism analyses reveal that postchronic exposure to SWCNTs causes substantial DNA methylation and transcriptome dysregulation of BEAS-2B cells. Subsequent enrichment and clinical database analyses reveal that differentially expressed/methylated genes of BEAS-2B cells are enriched in cancer-related biological pathways. These results not only demonstrate that postchronic SWCNT-exposure-induced carcinogenesis is heritable but also uncover a mechanism from the perspective of DNA methylation.

Identification of four methylation-driven genes as candidate biomarkers for monitoring single-walled carbon nanotube-induced malignant transformation of the lung

Long-term exposure to carbon nanotubes (CNTs) has been reported to induce malignant transformation. This study aimed to screen candidate biomarkers for monitoring occupational workers to prevent the development of lung cancer. mRNA (GSE56104) and methylation (GSE153246) profiles of lung epithelial BEAS-2B cells exposed to malignant transformation dose of single-walled CNTs or control medium were downloaded from Gene Expression Omnibus database. A total of 1513 differentially expressed genes (DEGs) and 912 differentially methylated genes (DMGs) were identified using LIMMA method. The weighted correlation network analysis identified blue and turquoise modules were associated with malignant transformation of BEAS-2B cells, 124 DMGs of which were overlapped with DEGs. The mRNA and methylation levels of four methylation-driven DEGs were validated in both lung adenocarcinoma (LUAD) and squamous cell carcinomas (LUSC) of The Cancer Genome Atlas dataset and they were associated with overall survival of LUAD patients. Downregulation of PXMP4 and MCOLN2, while upregulation of MET was confirmed in both LUSC and LUAD via Human Protein Atlas analysis. PXMP4 and MET protein levels were also supported in the proteomic analysis of LUAD. Receiver operating characteristic (ROC) curve analysis showed the combination of four genes may be the optimal biomarker for predicting lung cancer, with the area under ROC curve >0.9. Function analysis revealed BARX2 may interact with CCND1 to regulate cell cycle; MET and PXMP4/MCOLN2 may positively correlate with CCR5/IL-6 or GATA3/HLA-DPB1/HLA-DPA1 to involve immune regulation. In conclusion, these four methylation-driven genes may be candidate prognostic and diagnostic biomarkers for single-walled CNT-related lung cancer.

Epigenetic-Based Biomarkers in the Malignant Transformation of BEAS-2B Cells Induced by Coal Tar Pitch Extract.

Background and Objectives: The carcinogenicity of coal tar pitch (CTP) to occupational workers has been confirmed by the International Agency for Research on Cancer, especially for lung cancer. Herein, we explored the dynamic changes of epigenetic modifications in the malignant transformation process of CTP-induced BEAS-2B cells and also provided clues for screening early biomarkers of CTP-associated occupational lung cancer. Material and Methods: BEAS-2B cells treated with 3.0 μg/mL CTP extract (CTPE) were cultured to the 30th passage to set up a malignant transformation model, which was confirmed by platelet clone formation assay and xenograft assay. DNA methylation levels were determined by ultraviolet-high performance liquid chromatography. mRNA levels in cells and protein levels in supernatants were respectively detected by Real-Time PCR and enzyme-linked immunosorbent assay. Results: The number of clones and the ability of tumor formation in nude mice of CTPE-exposed BEAS-2B cells at 30th passage were significantly increased compared to vehicle control. Moreover, genomic DNA methylation level was down-regulated. The mRNA levels of DNMT1, DNMT3a and HDAC1 as well as the expression of DNMT1 protein were up-regulated since the 10th passage. From the 20th passage, the transcriptional levels of DNMT3b, let-7a and the expression of DNMT3a, DNMT3b, and HDAC1 proteins were detected to be higher than vehicle control, while the level of miR-21 increased only at the 30th passage. Conclusion: Data in this study indicated that the changes of epigenetic molecules including DNMT1, DNMT3a, DNMT3b, HDAC1, and let-7a occurred at the early stages of BEAS-2B cell malignant transformation after CTPE exposure, which provided critical information for screening early biomarkers of CTP-associated occupational lung cancer.

MicroRNA and mRNA Interaction Network Regulates the Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cigarette Smoke.

This study analyzes the correlation and interaction of miRNAs and mRNAs and their biological function in the malignant transformation of BEAS-2B cells induced by cigarette smoke (CS). Normal human bronchial epithelial cells (BEAS-2B) were continuously exposed to CS for 30 passages (S30) to establish an in vitro cell model of malignant transformation. The transformed cells were validated by scratch wound healing assay, transwell migration assay, colony formation and tumorigenicity assay. The miRNA and mRNA sequencing analysis were performed to identify differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) between normal BEAS-2B and S30 cells. The miRNA-seq data of lung cancer with corresponding clinical data obtained from TCGA was used to further identify lung cancer-related DEMs and their correlations with smoking history. The target genes of these DEMs were predicted using the miRDB database, and their functions were analyzed using the online tool “Metascape.” It was found that the migration ability, colony formation rate and tumorigenicity of S30 cells enhanced. A total of 42 miRNAs and 753 mRNAs were dysregulated in S30 cells. The change of expression of top five DEGs and DEMs were consistent with our sequencing results. Among these DEMs, eight miRNAs were found dysregulated in lung cancer tissues based on TCGA data. In these eight miRNAs, six of them including miR-96-5p, miR-93-5p, miR-106-5p, miR-190a-5p, miR-195-5p, and miR-1-3p, were found to be associated with smoking history. Several DEGs, including THBS1, FN1, PIK3R1, CSF1, CORO2B, and PREX1, were involved in many biological processes by enrichment analysis of miRNA and mRNA interaction. We identified the negatively regulated miRNA-mRNA pairs in the CS-induced lung cancer, which were implicated in several cancer-related (especially EMT-related) biological process and KEGG pathways in the malignant transformation progress of lung cells induced by CS. Our result demonstrated the dysregulation of miRNA-mRNA profiles in cigarette smoke-induced malignant transformed cells, suggesting that these miRNAs might contribute to cigarette smoke-induced lung cancer. These genes may serve as biomarkers for predicting lung cancer pathogenesis and progression. They can also be targets of novel anticancer drug development.

Inflammatory and tumorigenic effects of environmental pollutants found in particulate matter on lung epithelial cells

Exposure to environmental pollutants is a major public health concern. This study investigated the inflammatory and tumorigenic effects of environmental pollutants (benzene, benzo[a]pyrene, cadmium, and diisononyl phthalate) on transformed A549 and H292 lung alveolar epithelial cells and non-transformed BEAS-2B lung bronchial epithelial cells. The cytotoxic effects of the pollutants were analyzed by the methyl thiazolyl tetrazolium assay. The anchorage-independent soft agar assay demonstrated that treatment with benzene, cadmium, and diisononyl phthalate for 4 weeks induced malignant transformation of BEAS-2B cells and tumorigenesis of A549 and H292 cells. mRNA expression of the inflammation-related genes tenascin-C, matrix metalloproteinase (MMP)-9, and MMP-2, as well as inhibitors of MMPs (TIMP-1 and TIMP-2), was analyzed by RT-PCR. The pollutants largely upregulated expression of MMP-9 and MMP-2, but suppressed expression of their inhibitors TIMP-1 and TIMP-2. Measurement of transepithelial electrical resistance revealed that cadmium and diisononyl phthalate significantly increased cell permeability. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor of inflammatory genes, including MMP-9 and MMP-2, while signal transducer and activator of transcription (STAT) 3 is a key regulator of malignant transformation. All the pollutants activated the NF-κB promoter, while only cadmium induced activation of the STAT3 promoter in HEK293T cells. Moreover, all the pollutants increased the phospho-NF-κB level, but only cadmium and diisononyl phthalate increased the phospho-STAT3 level in A549 and BEAS-2B cells. These findings suggest that specific environmental pollutants enhance inflammation, cell permeability, and malignant transformation in lung epithelial cells by activating the oncogenic transcription factors STAT3 and NF-κB.

Spherical silica nanoparticles promote malignant transformation of BEAS-2B cells by stromal cell-derived factor-1α (SDF-1α).

ObjectiveThis study aimed to examine the role of spherical silica nanoparticles (SiNPs) on human bronchial epithelial (BEAS-2B) cells through inflammation.MethodsHuman mononuclear (THP-1) cells and BEAS-2B cells were co-cultured in transwell chambers and treated with 800 mmol/L benzo[a]pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE) and 12.5 µg/mL SiNPs for 24 hours. For controls, cells were treated with BPDE alone. Subcutaneous tumorigenicity and epithelial-mesenchymal transition (EMT) of BEAS-2B cells were measured. The cells were blocked with a stromal cell-derived factor-1α (SDF-1α)-specific antibody. EMT was analyzed in cells treated with 800 mmol/L BPDE and 12.5 µg/mL SiNPs relative to matched control cells and xenografts in vivo. Serum SDF-1α levels were measured in 23 patients with lung adenocarcinoma in Xuanwei, in 25 with lung adenocarcinoma outside Xuanwei, and in 22 with benign pulmonary lesions in Xuanwei.ResultsSiNPs significantly promoted tumorigenesis and EMT, induced the release of SDF-1α, and activated AKT (ser473) in BEAS-2B cells. EMT and phosphorylated AKT (ser473) and glycogen synthase kinase-3β levels were decreased when blocked by SDF-1α antibody in BEAS-2B cells. SDF-1α was mainly secreted by THP-1 cells.ConclusionSiNPs combined with BPDE promote EMT of BEAS-2B cells via the AKT pathway by inducing release of SDF-1α from THP-1 cells.

NLRP3 inflammasome activation involved in LPS and coal tar pitch extract-induced malignant transformation of human bronchial epithelial cells.

Inflammatory microenvironment has been found as a new characteristic of cancer; however, the mechanisms of inflammation-related lung cancer remain unclear. To explore the role of NLRP3 inflammsome activation in inflammation-related lung carcinogenesis, a cell model was set up. Human bronchial epithelial cells (BEAS-2B) were stimulated with 1 μg/mL lipopolysaccharide (LPS) for 24 hours, and then treated with 2.4 μg/mL coal tar pitch extract (CTPE) for 24 hours, after removal of LPS and CTPE, the cells were numbered passage 1 and were passaged and treated in this way until passage 30, which was called LPS + CTPE group. DMSO and Saline were used as vehicle controls. Malignant transformation of cells in passage 30 was evaluated by morphological change, platelet clone formation assay, and tumor formation in nude mice. The mRNA levels of NLRP3 and IL-1β were detected by real time-PCR. The combination of NLRP3 and caspase-1 were determined using immunofluorescence and confocal. The protein expression of NLRP3, cleaved caspase-1(p10), and cleaved IL-1β was detected using Western blot. It was shown that CTPE, LPS + CTPE-stimulated BEAS-2B cells of passage 30 changed a lot morphologically. The clone formation rates, the rates of positive cells of NLRP3 and caspase-1 combination, the mRNA levels of NLRP3 and IL-1β, the protein expression of NLRP3, cleaved caspase-1(p10) and cleaved IL-1β of cells exposed with CTPE and LPS + CTPE at passage 30 were significantly increased compared to vehicle controls. Furthermore, the ability of tumor formation in nude mice, the rates of clone formation and positive cells, mRNA and protein levels of NLRP3 inflammasome activation-related factors in LPS + CTPE-induced cells were all higher than those in cells stimulated with CTPE alone. In conclusion, the cell model of inflammation-related lung cancer is set up successfully, and NLRP3 inflammasome activation may be involved in the malignant transformation of BEAS-2B cells which induced by CTPE alone or LPS combined with CTPE.

NF-E2-related factor 2 serves a key function in resistance to malignant transformation of BEAS-2B cells induced by coal tar pitch.

Coal tar pitch (CTP) is a key factor in the development of occupational lung cancer. In order to investigate the function of the anti-oxidative signaling pathway regulated by NF-E2-related factor 2 (Nrf2) during cancer development, BEAS-2B cells were cultured with CTP extract for 30 passages. It was revealed that malignant transformation occurred in cells between the 20 and 30th passage. The expression levels of Nrf2 and NAD(P)H:quinone oxidoreductase 1 (NQO1) were promoted throughout the CTP exposure culture, and there was a positive linear correlation between the expression levels of Nrf2 and NQO1. Following knockdown of Nrf2 expression, the level of NQO1 decreased markedly and malignant transformation was more likely to occur. It was hypothesized that CTP may be toxic to BEAS-2B cells, which may lead to malignant transformation. Nrf2 was a quick response factor: Counteracting cytotoxicity by promoting the expression of anti-oxidative genes. Thus, Nrf2 was associated with the malignant transformation of BEAS-2B cells exposed to CTP and may be a potential therapeutic target.

Inactivation of miR-100 combined with arsenic treatment enhances the malignant transformation of BEAS-2B cells via stimulating epithelial -mesenchymal transition

ABSTRACTChronic arsenic treatment induces epithelial-mesenchymal transition (EMT) and promotes tumorigenicity, but the mechanism is unclear. MiR-100 has been shown to be involved in this biologic process. In this study, we hypothesize that inactivation of miR-100 combined with low concentration of arsenic exposure could promote the malignant transformation of human bronchial epithelial cells (BEAS-2B cell) by promoting EMT. To test this hypothesis, BEAS–2B cells were treated with low-dose of As2O3 chronically, and lentiviral vectors were used to mediate the inhibition of miR-100 expression. Flow cytometry, cloning formation, and transwell assays were used to examine cell cycle progression, cell proliferation, and cell migration, respectively. The mouse xenograft model was used to investigate the cell malignant growth in vivo, and western blot was used to detect EMT related marker expressions. Our results showed that, the inactivation of miR-100 combined with arsenic treatment significantly promoted the proliferation, viability, and migration of BEAS-2B cells in vitro, and tumorigenesis in vivo. Consistently, the EMT related marker expressions were also significantly increased in corresponding groups. Our data indicate that inactivation of miR-100 combined with chronic arsenic treatment promotes tumorigenicity of BEAS-2B cells via activation of EMT. This novel insight may help us to better understand the pathogenesis of arsenic carcinogenesis.

Aldehyde dehydrogenase 1A1 up-regulates stem cell markers in benzo[a]pyrene-induced malignant transformation of BEAS-2B cells.

Recently, Aldehyde dehydrogenase 1A1 (ALDH1A1) has been proposed to be a common marker of cancer stem cells and can be induced by benzo[a]pyrene (B[a]P) exposure. However, the underlying mechanism of how ALDH1A1 contributes to B[a]P-induced carcinogenesis in human bronchial epithelial cells remains unclear. Here, we found that B[a]P up-regulated expression levels of stem cell markers (ABCG2, SOX2, c-Myc and Klf4), epithelial-mesenchymal transition (EMT) associated genes (SNAIL1, ZEB1, TWIST and β-CATENIN) and cancer-related long non-coding RNAs (lncRNAs; HOTAIR and MALAT-1) in malignant B[a]P-transformed human bronchial epithelial cells (BEAS-2B-T cells), and these up-regulations were dependent on increased expression of ALDH1A1. The inhibition of endogenous ALDH1A1 expression down-regulated expression levels of stem cell markers and reversed the malignant phenotype as well as reduced the chemoresistance of BEAS-2B-T cells. In contrast, the overexpression of ALDH1A1 in BEAS-2B cells increased the expression of stem cell markers, facilitated cell transformation, promoted migratory ability and enhanced the drug resistance of BEAS-2B cells. Overall, our data indicates that ALDH1A1 promotes a stemness phenotype and plays a critical role in the BEAS-2B cell malignant transformation induced by B[a]P.

MiR-138-1* regulates aflatoxin B1-induced malignant transformation of BEAS-2B cells by targeting PDK1.

Environmental carcinogens-induced lung cancer and potential mechanisms have attracted widespread attention. Currently, microRNAs (miRNAs) have been recognized as key players in development of cancer, among which guide strand of miRNA has been well documented rather than its passenger strand (miRNA*). Our previous study showed that treatment of 0.1nM AFB1 for 50 passages could induce malignant transformation of immortalized human bronchial epithelial cells stably expressing CYP2A13 (P50 B-2A13 cells). However, the role of miRNAs in this carcinogenic proceeding is still unclear. In present study, 36 upregulated and 27 downregulated miRNAs in P50 B-2A13 cells were first identified by miRNA microarray, and miR-138-1* was selected as a candidate miRNA by RT-qPCR and pilot experiments. Functional studies revealed that miR-138-1* could inhibit proliferation, colony formation, migration and invasion of P50 B-2A13 cells. Further, target analysis and dual-luciferase reporter gene assay identified that miR-138-1(*) was consequentially paired with 3'-UTR of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and decreased the luciferase activity. miR-138-1* could decrease the expressions of PDK1 and its downstream proteins in PI3K/PDK/Akt pathway but not vice versa, indicating that miR-138-1* might affect AFB1-induced malignant transformation through targeting PDK1. As predicted, interference of PDK1 showed the similar effects to miR-138-1* in the proliferation, colony formation, migration and invasion of P50 B-2A13 cells. Our study demonstrated that miR-138-1* played a critical role in AFB-induced malignant transformation of B-2A13 cells by targeting PDK1. Still, the study provides a novel insight into the roles of miRNA* during carcinogenesis, particularly airborne carcinogens-induced lung cancer.

Evidence for p53 Expression as a Target for Lung Cancer Early Diagnosis

Obiective: To study the change of p53 expression in BEAS-2B cells malignant transformation process, and detect the role of p53 expression for lung cancer early diagnosis. Method: BEAS-2B cells acute expose NNK at 500 μg/ml for 24 hours, and these cells (BEAS-2BNNK cells) were subcultured continuously in vitro, Biological characteristics and ultrastructure of them were studied with Colony formation assay and electron microscopy. The change p53 expression of BEAS-2B cells were detected by immunohistochemical method. Results: The serum resistance was appeared in the 5th passage of BEAS-2BNHK cells and these cells could not grow into tumor in nude micei¼›The plating efficiency of the 15th passage of BEAS-2BNNK cells in soft agar (0.032%) increased 13.9 fold Compared with that of control group cells(0.0023%)P<0.001, the cells had the biological characteristic of transformation cells but could not grow into tumor in nude mice; The 25th passage of BEAS-2BNNK cells could grow into tumor in nude micei¼›The tumor cells were confirmed cancer cells by histopathology. The ultrastructrure also showed that BEAS-2BNNK cells were Transformed into cancer cells. p53 expression of BEAS- 2B cells were 10.7 ± 2.3%, but p53 expression of the 5th passage of BEAS-2BNNK cells were 43.3 ± 5.7%, the 15th passage of BEAS-2BNNK cells were 73.8 ± 5.2%, the 25th passage of BEAS-2BNNK cells were 92.4 ± 6.5%. The 5th,15th, 25th passage BEAS-2BNNK cells.VS. p53 expression of BEAS-2B cells, P<0.001. Conclusion: The model of malignant transformation of BEAS-2B cells induced by NNK(500 μg/ml) established successfully. The change of biological characteristic and ultrastructure indicated that the malignant transformation of BEAS-2BNNK cell is a Chronic, multiple-step development process. p53 overexpression appeared early stage in BEAS-2BNNK cells which did not change into cancer cells. It indicated that p53 expression would be as a target for lung cancer early diagnosis, and it was beneficial to early-warning and mass screening of lung cancer in high-risk smoking population.

The effects of monocyte-macrophages on malignant transformation of human bronchial epithelial cells induced by extracts from coal tar pitch

To study the effects of monocyte-macrophages (THP-1) in malignant transformation of human bronchial epithelial cells (BEAS-2B) cells induced by coal tar pitch (CTP) and the expression of TNF-α in the process of the cell malignant transformation. BEAS-2B cells and THP-1 Cells were divided into four groups: coal tar pitch (CTP) group, benzo(a)pyrene [B(a)P] group, dimethyl sulfoxide (DMSO) group, BEAS-2B and THP-1 co-culture (co-culture group) group. Carcinogenesis model was established. The soft agar colony formation, chromosome aberrations and cell cycle tests were used to detect the cellular malignant transformation. The ELISA assay was utilized to measure the levels of TNF-α in the supernatant of CTP group and co-culture group. The chromosome number abnormalities could be observed in early stage of the experiment (the 10th generation cells), which showed the increased ratio of aneuploid to polyploid, and the decreased number of diploid. The colony formation rate of co-culture group (the 20th generation cells) was 17.63‰ ± 0.97‰, which was significantly higher than that (13.94‰ ± 0.84‰) of CTP group and that (12.96‰ ± 1.62‰) of B(a)P group (P < 0.05). The proportion of S phase cells in the co-culture group was 44.49% ± 0.68%, which was significantly higher than that (38.19% ± 1.26%) of CTP group and that (36.41% ± 1.19%) of B(a)P group (P < 0.05). The TNF-α level in the co-culture group was significantly higher than that in CTP group (P = 0.001). Monocyte-Macrophages can accelerate the malignant transformation of BEAS-2B cells induced by CTP and increase the expression level of TNF-α.

FHIT protein expression during malignant transformation of human bronchial epithelial cells induced by NNK

Most studies about FHIT protein expression were performed in normal tracheal epithelium, precancerous lesions and lung cancer tissues respectively, but not in the course of malignant transformation of lung cancer. The aim of this study is to detect the changes of FHIT protein expression during malignant transformation of immortalized human bronchial epithelial cells (BEAS-2B) induced by tobacco-specific nitrosamine (NNK), and to explore its significance. BEAS-2B cells were induced to malignantly transform (BEAS-2B NNK) by 500mg/L NNK, and FHIT protein expression was detected in the different passages of BEAS-2B NNK and BEAS-2B cells by SP immunocytochemistry. Part 1: Model of malignant transformation of BEAS-2B cells induced by NNK was established. (1) The serum resistance was significantly increased in the 5th passage of BEAS-2B NNK cells. (2) The anchorage independent growth (soft agar colony formation) appeared in the 15th passage of BEAS-2B NNK cells. (3) The ultrastructure of the 20th passage of BEAS-2B NNK cells showed obvious heteromorphy characterization. (4) The 25th passage of BEAS-2B NNK cells developed into tumors in nude mice, which were well differentiated squamous cell carcinoma. Part 2: FHIT protein was steadily expressed in the different passages of BEAS-2B cells (P > 0.05). FHIT protein expression was obviously decreased from 5th to 15th passage of BEAS-2B NNK cells, but it was unexpectedly overexpressed in the 25th passage. (1) The model of malignant transformation of BEAS-2B cells induced by NNK (500mg/L) can be established successfully and may be used for investigation of molecular biological mechanisms of lung cancer, especially for smoking-related cases. (2) Decrease of FHIT protein expression might be an early event, however, its overexpression in the late passages should be further studied.