Role of autophagy in inhibiting the proliferation of A549 cells by type III interferon

Interferons (IFNs) have anti‐viral and anti‐tumour effects. Type III interferon, as a member of the recently discovered interferon family, has been proved to inhibit tumour proliferation and promote the apoptosis of various tumour cells. However, whether type III IFN could inhibit the proliferation of lung cancer was not clear. In this study, we found that interferon λ (IFN λ) could inhibit the proliferation of A549 cells and induce autophagy and apoptosis of A549 cells. IFN λ could promote the expression of autophagy gene Beclin1 and interfere the expression of autophagy gene Beclin1 with small interfering RNA, thus inhibiting the effect of type III interferon on anti‐proliferation and promoting apoptosis of lung cancer cell. These results suggested that IFN λ could inhibit the proliferation of A549 cells by activating autophagy pathway, and IFN λ might be one of the potential therapeutic drugs for lung cancer.     

Afatinib Decreases P‐Glycoprotein Expression to Promote Adriamycin Toxicity of A549T Cells

We investigated the reversal effect of afatinib (AFT) on activity of adriamycin (ADR) in A549T cells and clarified the related molecular mechanisms. A549T cells overexpressing P‐glycoprotein (P‐gp) were resistant to anticancer drug ADR. AFT significantly increased the antitumor activity of ADR in A549T cells. AFT increased the intracellular concentration of ADR by inhibiting the function and expression of P‐gp at mRNA and protein levels in A549T cells. Additionally, the reversal effect of AFT on P‐gp mediated multidrug resistance (MDR) might be related to the inhibition of PI3K/Akt pathway. Cotreatment with AFT and ADR could enhance ADR‐induced apoptosis and autophagy in A549T cells. Meanwhile, the co‐treatment significantly induced cell apoptosis and autophagy accompanied by increased expression of cleaved caspase‐3, PARP, LC3B‐II, and beclin 1. Apoptosis inhibitors had no significant effect on cell activity, while autophagy inhibitors decreased cell viability, suggesting that autophagy may be a self protective mechanism of cell survival in the absence of chemotherapy drugs. Interestingly, when combined with AFT and ADR, inhibition of apoptosis and/or autophagy could enhance cell viability. These results indicated that in addition to inhibit P‐gp, ADR‐induced apoptosis, and autophagy promoted by AFT contributed to the antiproliferation effect of combined AFT and ADR on A549T cells. These findings provide evidence that AFT combined ADR may achieve a better therapeutic effect to lung cancer in clinic. J. Cell. Biochem. 119: 414–423, 2018. © 2017 Wiley Periodicals, Inc.     

Cigarette smoke alters the secretome of lung epithelial cells

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label‐free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.     

肿瘤相关成纤维细胞对非小细胞肺癌恶性生物学行为影响

目的:探讨肿瘤相关成纤维细胞(Tancer Associated Fibroblast,TAF)对非小细胞肺癌(Non-small Cell Lung Cancer,NSCLC)恶性生物学行为的影响。方法:选取在本院肿瘤科住院手术的非小细胞肺癌患者,收集术后肺癌标本,马松三色染色(Masson Trichrome Stain)和天狼星红染色(Sirius Red Stain)观察肺癌组织(Lung Cancer Tissue,LCT)、癌旁组织(Pericarcinomatous Tissue,PCT)和正常组织(Normal Tissue,NT)中TAF的表达情况;体外将非小细胞肺癌细胞A549与非小细胞肺癌成纤维细胞P-gp共培养,CCK-8检测共培养前后A549细胞增殖能力;细胞划痕和Trans-well实验分别检测A549细胞迁移和侵袭能力;q RT-PCR和Western blot检测A549细胞上皮间质转化(Epithelial Mesenchymal Transition,EMT)标志蛋白E-cadherin、N-cadherin和Vimentin的表达。结果:Masson和Sirius染色结果显示:肺癌组织中纤维的表达明显高于癌旁组织;与P-gp共培养的A549细胞的增殖、迁移和侵袭能力及上皮间质转化相关蛋白N-cadherin和Vimentin表达均明显高于阴性对照组(P0.05),而E-cadherin的表达明显降低(P0.05)。结论:TAF可能通过诱导非小细胞肺癌细胞EMT的发生从而促进非小细胞肺癌的增殖、迁移和侵袭等恶性生物学行为。     

Dihydroartemisinin Sensitizes Human Lung Adenocarcinoma A549 Cells to Arsenic Trioxide via Apoptosis

Recent studies have shown that arsenic trioxide (ATO) is an effective anti-cancer drug for treatment of acute promyelocytic leukemia and other types of human cancer. However, we have found that lung cancer cells constantly develop a high level of resistance to ATO. In this study, we have explored a possibility of combination of dihydroartemisinin (DHA) and ATO treatments to reduce ATO resistance of lung cancer cells. We determined the combinatory effects of DHA and ATO on cytotoxicity of human lung adenocarcinoma (A549) cells. We showed that co-exposure to DHA and ATO of A549 cells synergistically increased the cytotoxicity and apoptotic cell death in the cells. We found that the synergistic effect of DHA and ATO in promoting apoptosis mainly resulted from increased cellular level of reactive oxygen species (ROS) and DNA damage. ATO alone only exerted moderate growth inhibitory effects on A549 cells. The results indicate that DHA can significantly sensitize ATO-induced cytotoxicity of A549 lung cancer cells through apoptosis mediated by ROS-induced DNA damage. Interestingly, we found that the combinatory treatment of DHA and ATO did not result in significant adverse effects in normal human bronchial epithelial (HBE) cells. Our results further provide evidence for the potential application of combinatory effects of DHA and ATO as a safe therapy for human lung cancer.     

Autophagy induction and antiproliferative effect of a novel curcumin derivative MOMI‐1 on the human lung cancer cells A549

To date, there are some chemically synthesized curcumin derivatives which were produced and identified to evade the disadvantages of physiochemical stability and solubility of curcumin. Here, one novel curcumin derivative, (2‐(3‐{(1E)‐{(E)‐3‐(4‐hydroxy‐3‐methoxybenzylidene)‐2‐oxocyclohexylidene)methyl)‐1H‐indol‐1‐yl)acetic acid}, (abbreviated as MOMI‐1) was first used to detect the antiproliferation activity with MTT assays in different cancer cells including A549 lung cancer cells, MCF‐7, and HEPG2 cell lines, and exhibited its wide inhibition spectrum. Next, we found that MOMI‐1 could induce autophagic genesis of A549 cells by acridine orange or monodansylcadaverine (MDC) staining and green fluorescent protein‐light chain 3 (GFP‐LC3) recombinant plasmid transfection analysis, respectively. Western blot analysis confirmed the LC3‐I/II conversion, beclin‐1 increase and p62 reduction of A549 cells after exposure of MOMI‐1, which suggested the typical autophagy induction. The following cell cycle test showed that MOMI‐1 could block A549 cells in G0/G1 phase. Furthermore, wounding healing experiment and transwell assays demonstrated that MOMI‐1 also possessed the antimigration ability of A549 cells. Our current results confirmed that MOMI‐1 could inhibit the proliferation and induce autophagy of A549 cells, which provide a new potential chemical candidate of antigrowth of A549 lung cancer cells. Future work needs to focus on the mechanism of autophagy pathway of A549 cells.     

Synthesis and Cytotoxicity Evaluation of Panaxadiol Derivatives

In this study, 13 panaxadiol (PD) derivatives were synthesized via reactions with aromatic compounds and amino acids. Following this, the cytotoxicity of these compounds was evaluated against four cancer cell lines (human hepatoma cells HepG‐2, human lung cancer cells A549, human breast cancer cells MCF‐7, and human colon cancer cells HCT‐116) and one normal cell lines (human gastric epithelial cells GES‐1). The results showed that the panaxadiol derivatives 3 , 12 , and 13 showed significant inhibition of cellular proliferation against cancer cells compared with PD, and the panaxadiol derivative 12 had the lowest IC₅₀ value for A549 (IC₅₀=18.91±1.03 μm ). For MCF‐7 cells, most compounds exhibited good inhibition of cellular proliferation, and the panaxadiol derivative 13 showed the strongest inhibitory effect (IC₅₀=8.62±0.23 μm ), which significantly increased the cytotoxicity of PD and was stronger than the positive control (mitomycin). For normal cells, all compounds exhibited low or no toxic effects; thus, these derivatives can be used to develop novel antiproliferative agents.     

Overexpression of geranylgeranyl diphosphate synthase contributes to tumour metastasis and correlates with poor prognosis of lung adenocarcinoma

This study aimed to evaluate the biological role of geranylgeranyl diphosphate synthase (GGPPS) in the progression of lung adenocarcinoma. GGPPS expression was detected in lung adenocarcinoma tissues by qRT‐PCR, tissue microarray (TMA) and western blotting. The relationships between GGPPS expression and the clinicopathological characteristics and prognosis of lung adenocarcinoma patients were assessed. GGPPS was down‐regulated in SPCA‐1, PC9 and A549 cells using siRNA and up‐regulated in A549 cells using an adenoviral vector. The biological roles of GGPPS in cell proliferation, apoptosis, migration and invasion were determined by MTT and colony formation assays, flow cytometry, and transwell and wound‐healing assays, respectively. In addition, the regulatory roles of GGPPS on the expression of several epithelial‐mesenchymal transition (EMT) markers were determined. Furthermore, the Rac1/Cdc42 prenylation was detected after knockdown of GGPPS in SPCA‐1 and PC9 cells. GGPPS expression was significantly increased in lung adenocarcinoma tissues compared to that in adjacent normal tissues. Overexpression of GGPPS was correlated with large tumours, high TNM stage, lymph node metastasis and poor prognosis in patients. Knockdown of GGPPS inhibited the migration and invasion of lung adenocarcinoma cells, but did not affect cell proliferation and apoptosis. Meanwhile, GGPPS inhibition significantly increased the expression of E‐cadherin and reduced the expression of N‐cadherin and vimentin in lung adenocarcinoma cells. In addition, the Rac1/Cdc42 geranylgeranylation was reduced by GGPPS knockdown. Overexpression of GGPPS correlates with poor prognosis of lung adenocarcinoma and contributes to metastasis through regulating EMT.     

bFGF 诱导肺腺癌细胞系A549 上皮间质转化

目的:探讨上皮间质转化(epithelial-mesenchymal transition,EMT)过程在肺癌侵袭转移中的作用。方法:体外培养A549细胞,以bFGF(10ng/ml)进行干预后,倒置相差显微镜观察细胞形态学变化;间接免疫荧光观察上皮细胞标志物E-cadherin和间质细胞标志物vimentin蛋白表达的变化;采用细胞划痕试验检测bFGF对A549细胞迁移能力的影响;采用transwell小室试验检测bFGF对A549细胞侵袭能力的影响。结果:bFGF(10ng/ml)干预后,在倒置相差显微镜下观察,A549细胞形态变成了梭形,形态如同成纤维细胞。间接免疫荧光显示A549细胞E-cadherin表达随时间延长逐渐减弱,而vimentin表达逐渐增强。细胞划痕试验显示,bFGF干预后细胞迁移能力提高。Transwell小室试验显示,bFGF干预后细胞侵袭能力提高。结论:bFGF在体外诱导肺腺癌细胞系A549细胞发生上皮间质转化,上皮间质转化是肺癌侵袭转移的重要机制之一。     

Low levels of AMPK promote epithelial‐mesenchymal transition in lung cancer primarily through HDAC4‐ and HDAC5‐mediated metabolic reprogramming

AMP‐activated protein kinase (AMPK) serves as a “supermetabolic regulator” that helps maintain cellular energy homeostasis. However, the role of AMPK in glucose metabolism reprogramming in lung cancer remains unclear. Here, our study shows that low AMPK expression correlates with metastasis and clinicopathologic parameters of non–small‐cell lung cancer. Low AMPK significantly enhances the Warburg effect in HBE and A549 cells, which in turn induces the expression of mesenchymal markers and enhances their invasion and migration. At the mechanistic level, low AMPK up‐regulates HK2 expression and glycolysis levels through HDAC4 and HDAC5. Collectively, our findings demonstrate that low AMPK‐induced metabolism can promote epithelial‐mesenchymal transition progression in normal bronchial epithelial cells and lung cancer cells, and increase the risk for tumour metastasis.     

Lobetyolin suppressed lung cancer in a mouse model by inhibiting epithelial-mesenchymal transition

Traditional Chinese medicines are gaining more attention as promising adjuvant agents for conventional chemotherapy. Recent studies have shown that lobetyolin (LBT) is one of the main bioactive compounds of traditional Chinese medicines and it exhibits anticancer activity in several types of cancer. Therefore, this study aimed to investigate the mechanism by which LBT inhibits lung cancer. A549 human lung cancer cells were treated with LBT. In addition, A549 cells were injected into Balc/b nude mice to establish model of lung cancer. The mice were treated with cisplatin (DDP) or LBT alone or in combination, and tumor growth was monitored. Protein levels of E-cadherin, vimentin and matrix metalloproteinase 9 (MMP9) were detected. We found that the combination of LBT and DDP showed stronger effect to inhibit the proliferation of A549 cells compared to LBT or DDP treatment alone. Wound healing assay showed that the ratio of wound healing was significantly lower in LBT group and DDP group and was the lowest in LBT+DDP group. Transwell invasion assay showed that the invasion ability of A549 cells was the weakest in LBT+DDP group. Protein levels of E-cadherin were the highest while those of vimentin and MMP9 were the lowest in A549 cells treated with LBT+DDP. Nude mouse xenograft tumor model showed that the combination of LBT with DDP had the highest efficacy to inhibit the growth of lung cancer, and tumor tissues of mice treated with LBT+DDP had the lowest expression of vimentin and MMP9 and the highest expression of E-cadherin. In conclusion, LBT significantly enhances the efficacy of chemotherapy on lung cancer, and the mechanism may be related to the inhibition of epithelial-mesenchymal transition.Key words: Lobetyolin, lung cancer, cisplatin, epithelial-mesenchymal transition, Chinese traditional medicine     

Protein arginine methyltransferase 5 is essential for growth of lung cancer cells

PRMT5 (protein arginine methyltransferase 5) is an enzyme that catalyses transfer of methyl groups from S-adenosyl methionine to the arginine residues of histones or non-histone proteins and is involved in a variety of cellular processes. Although it is highly expressed in some tumours, its direct role in cancer growth has not been fully investigated. In the present study, in human lung tissue samples we found that PRMT5 was highly expressed in lung cancer cells, whereas its expression was not detectable in benign lung tissues. Silencing PRMT5 expression strongly inhibited proliferation of lung adenocarcinoma A549 cells in tissue culture, and silencing PRMT5 expression in A549 cells also abolished growth of lung A549 xenografts in mice. In vitro and in vivo studies showed that the cell growth arrest induced by loss of PRMT5 expression was partially attributable to down-regulation of fibroblast growth factor receptor signalling. These results suggest that PRMT5 and its methyltransferase activity is essential for proliferation of lung cancer cells and may serve as a novel target for the treatment of lung cancer.     

Production of CCL20 from lung cancer cells induces the cell migration and proliferation through PI3K pathway

Tumour inflammatory microenvironment is considered to play a role in the sensitivity of tumour cells to therapies and prognosis of patients with lung cancer. The expression of CCL20, one of the critical chemoattractants responsible for inflammation cells recruitment, has been shown overexpressed in variety of tumours. This study aimed at investigating potential mechanisms of CCL20 function and production in human non‐small cell lung cancer (NSCLC). Expression of CCL20 gene and protein in lung tissues of patients with NSCLC and NSCLC cells (A549) were determined. The interleukin (IL)‐1β‐induced signal pathways in A549 and the effect of CCL20‐induced A549 cell migration and proliferation were determined using migration assays and cell‐alive monitoring system. Mechanisms of signal pathways involved in the migration of CCL20 were also studied. We initially found that NSCLC tumour tissues markedly overexpressed CCL20 in comparison with normal lung samples. In addition, IL‐1β could directly promote CCL20 production in lung cancer cells, which was inhibited by extracellular signal‐regulated kinase (ERK)1/2 inhibitor, p38 mitogen‐activated protein kinase (p38 MARP) inhibitor or PI3K inhibitors. CCL20 promoted lung cancer cells migration and proliferation in an autocrine manner via activation of ERK1/2‐MAPK and PI3K pathways. Our data indicated that IL‐1β could stimulate CCL20 production from lung cancer cells through the activation of MAPKs and PI3K signal pathways, and the auto‐secretion of CCL20 could promote lung cancer cell migration and proliferation through the activation of ERK and PI3K signal pathways. Our results may provide a novel evidence that CCL20 could be a new therapeutic target for lung cancer.     

Long non‐coding RNA H19 is responsible for the progression of lung adenocarcinoma by mediating methylation‐dependent repression of CDH1 promoter

Lung adenocarcinoma is a common histologic type of lung cancer with a high death rate globally. Increasing evidence shows that long non‐coding RNA H19 (lncRNA H19) and CDH1 methylation are involved in multiple tumours. Here, we tried to investigate whether lncRNA H19 or CDH1 methylation could affect the development of lung adenocarcinoma. First, lung adenocarcinoma tissues were collected to detect CDH1 methylation. Then, the regulatory mechanisms of lncRNA H19 were detected mainly in concert with the treatment of overexpression of lncRNA H19, siRNA against lncRNA H19, overexpression of CDH1 and demethylating agent A‐5az in lung adenocarcinoma A549 cell. The expression of lncRNA H19 and epithelial‐mesenchymal transition (EMT)‐related factors as well as cell proliferation, sphere‐forming ability, apoptosis, migration and invasion were detected. Finally, we observed xenograft tumour in nude mice so as to ascertain tumorigenicity of lung adenocarcinoma cells. LncRNA H19 and methylation of CDH1 were highly expressed in lung adenocarcinoma tissues. A549 cells with silencing of lncRNA H19, overexpression of CDH1 or reduced CDH1 methylation by demethylating agent 5‐Az had suppressed cell proliferation, sphere‐forming ability, apoptosis, migration and invasion, in addition to inhibited EMT process. Silencing lncRNA H19 could reduce methylation level of CDH1. In vivo, A549 cells with silencing lncRNA H19, overexpression of CDH1 or reduced CDH1 methylation exhibited low tumorigenicity, reflected by the smaller tumour size and lighter tumour weight. Taken together, this study demonstrates that silencing of lncRNA H19 inhibits EMT and proliferation while promoting apoptosis of lung adenocarcinoma cells by inhibiting methylation of CDH1 promoter.     

RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells

Background

Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance.In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin.

Result

Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage.

Conclusion

Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.     

Involvement of TRPC Channels in Lung Cancer Cell Differentiation and the Correlation Analysis in Human Non-Small Cell Lung Cancer

The canonical transient receptor potential (TRPC) channels are Ca²⁺-permeable cationic channels controlling the Ca²⁺ influx evoked by G protein-coupled receptor activation and/or by Ca²⁺ store depletion. Here we investigate the involvement of TRPCs in the cell differentiation of lung cancer. The expression of TRPCs and the correlation to cancer differentiation grade in non-small cell lung cancer (NSCLC) were analyzed by real-time PCR and immunostaining using tissue microarrays from 28 patient lung cancer samples. The association of TRPCs with cell differentiation was also investigated in the lung cancer cell line A549 by PCR and Western blotting. The channel activity was monitored by Ca²⁺ imaging and patch recording after treatment with all-trans-retinoic acid (ATRA). The expression of TRPC1, 3, 4 and 6 was correlated to the differentiation grade of NSCLC in patients, but there was no correlation to age, sex, smoking history and lung cancer cell type. ATRA upregulated TRPC3, TRPC4 and TRPC6 expression and enhanced Ca²⁺ influx in A549 cells, however, ATRA showed no direct effect on TRPC channels. Inhibition of TRPC channels by pore-blocking antibodies decreased the cell mitosis, which was counteracted by chronic treatment with ATRA. Blockade of TRPC channels inhibited A549 cell proliferation, while overexpression of TRPCs increased the proliferation. We conclude that TRPC expression correlates to lung cancer differentiation. TRPCs mediate the pharmacological effect of ATRA and play important roles in regulating lung cancer cell differentiation and proliferation, which gives a new understanding of lung cancer biology and potential anti-cancer therapy.     

Simulated microgravity alters the metastatic potential of a human lung adenocarcinoma cell line

Simulated microgravity (SM) has been implicated in affecting diverse cellular pathways. Although there is emerging evidence that SM can alter cellular functions, its effect in cancer metastasis has not been addressed. Here, we demonstrate that SM inhibits migration, gelatinolytic activity, and cell proliferation of an A549 human lung adenocarcinoma cell line in vitro. Expression of antigen MKI67 and matrix metalloproteinase-2 (MMP2) was reduced in A549 cells stimulated by clinorotation when compared with the 1×g control condition, while overexpression of each gene improves ability of proliferation and migration, respectively, under SM conditions. These findings suggest that SM reduced the metastatic potential of human lung adenocarcinoma cells by altering the expression of MKI67 and MMP2, thereby inhibiting cell proliferation, migration, and invasion, which may provide some clues to study cancer metastasis in the future.     

Targeting lung cancer stem‐like cells with TRAIL gene armed oncolytic adenovirus

Lung cancer stem cell (LCSC) is critical in cancer initiation, progression, drug resistance and relapse. Disadvantages showed in conventional lung cancer therapy probably because of its existence. In this study, lung cancer cell line A549 cells propagated as spheroid bodies (named as A549 sphere cells) in growth factors‐defined serum‐free medium. A549 sphere cells displayed CSC properties, including chemo‐resistance, increased proportion of G0/G1 cells, slower proliferation rate, ability of differentiation and enhanced tumour formation ability in vivo. Oncolytic adenovirus ZD55 carrying EGFP gene, ZD55‐EGFP, infected A549 sphere cells and inhibited cell growth. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) armed oncolytic adenovirus, ZD55‐TRAIL, exhibited enhanced cytotoxicity and induced A549 sphere cells apoptosis through mitochondrial pathway. Moreover, small molecules embelin, LY294002 and resveratrol improved the cytotoxicity of ZD55‐TRAIL. In the A549 sphere cells xenograft models, ZD55‐TRAIL significantly inhibited tumour growth and improved survival status of mice. These results suggested that gene armed oncolytic adenovirus is a potential approach for lung cancer therapy through targeting LCSCs.     

Protein Network Analysis and Functional Studies of Calcium Oxalate Crystal‐Induced Cytotoxicity in Renal Tubular Epithelial Cells

Our previous expression study has reported a set of proteins with altered levels in renal tubular cells after exposure to calcium oxalate monohydrate (COM) crystals, which are the main composition of kidney stones. However, their functional significance remained largely unknown. In this study, protein network analysis revealed that the significantly altered proteins induced by COM crystals were involved mainly in three main functional networks, including i) cell proliferation and wound healing; ii) oxidative stress and mitochondrial function; and iii) cellular junction complex and integrity. Cell proliferation and wound healing assays showed that the COM‐treated cells had defective proliferation and tissue healing capability, respectively. Oxyblot analysis demonstrated accumulation of the oxidized proteins, whereas intracellular ATP level was significantly increased in the COM‐treated cells. Additionally, level of zonula occludens‐1 (ZO‐1), a tight junction protein, was significantly decreased, consistent with the significant declines in transepithelial resistance (TER) and level of RhoA signaling molecule in the COM‐treated cells. These findings indicate significant perturbations in mitochondrial and oxidative stress axis that cause defective cell proliferation, tissue healing capability, junctional protein complex, and cellular integrity of renal tubular epithelial cells exposed to COM crystals that may play important roles in kidney stone pathogenesis.     

GFP Stable Transfection Facilitated the Characterization of Lung Cancer Stem Cells

Cancer stem cells (CSCs) are a subset of cancer cells that play key roles in metastasis and cancer relapse. The elimination of CSCs is very important during cancer therapy. To develop drugs that target CSCs, the isolation and identification of putative CSCs are required. Some of the characteristics of CSCs are assessed by cell survival assays. In such experiments, the density of the cells seeded on the plates may affect the experimental results, leading to potentially inaccurate conclusions. In this study, a new assay to facilitate the characterization of CSCs has been developed by stable transfection of GFP, using the A549 lung cancer cell line as a model. A putative CSC line, A549 sphere cells, was obtained by culturing A549 cells in ultra-low dishes in serum-free medium. To ensure that the putative CSCs were grown under the same conditions as the A549–GFP cells and were not affected by the number of cells seeded, A549 sphere cells were mixed with GFP stably transfected A549 (A549–GFP) cells. The mixture was subjected to flow cytometry assay and inverted fluorescence microscopy to detect changes in the proportion of GFP-positive cells after treatment. A549 sphere cells had a slower proliferation rate and an improved chemoresistance. They also showed differentiation ability. This work suggests that mixing GFP stably transfected cancer cells with putative CSCs may facilitate the identification of CSCs, making it convenient for studies of targeted CSCs.