lithium-chloride and Lung-Neoplasms

The Wnt/β‑catenin signaling pathway has been reported to regulate cisplatin resistance in several types of cancer cell. The present study aimed to investigate the role and underlying mechanism of Wnt/β‑catenin signaling in cisplatin resistance of lung adenocarcinoma cells. Wild‑type and cisplatin‑resistant A549 human lung adenocarcinoma cells (A549/WT and A549/CDDP, respectively) were cultured in vitro and exposed to different cisplatin concentrations. Cells were incubated with 10 mM lithium chloride (LiCl) to activate β‑catenin signaling. Cell proliferation was determined using the MTS assay. Cell apoptosis was evaluated using Annexin V/propidium iodide double staining, followed by flow cytometry. β‑catenin was knocked down using small interfering RNA (siRNA). The intracellular distribution of β‑catenin was determined by immunocytochemistry, and the mRNA and protein expressions of target genes were examined by reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. β‑catenin and B‑cell lymphoma‑extra large (Bcl‑xl) were significantly upregulated in A549/CDDP cells compared with A549/WT cells (P<0.05). LiCl reduced the sensitivity of A549/WT cells to cisplatin (P<0.01); and upregulated, increased phosphorylation (P<0.05) and enhanced nuclear translocation of β‑catenin. LiCl also significantly elevated the mRNA and protein expression levels of Bcl‑xl (P<0.05). Notably, silencing of β‑catenin with siRNA decreased the mRNA and protein expression of Bcl‑xl, and sensitized A549/WT cells to cisplatin (P<0.01). The findings of the current study suggest that upregulation of β‑catenin signaling may contribute to cisplatin resistance in lung adenocarcinoma cells by upregulating Bcl‑xl. Therefore, molecular targeting of Wnt/β‑catenin signaling may sensitize lung cancer cells to cisplatin.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; bcl-X Protein; Cisplatin; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Lithium Chloride; Lung Neoplasms; Up-Regulation; Wnt Signaling Pathway

Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Lithium Chloride; Lung Neoplasms; MAP Kinase Kinase 4; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Up-Regulation

Thymosin beta-4 (Tβ4), actin-sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase (GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK-3 activity is regulated by Tβ4 and it is associated with Tβ4-mediated migration in gastric cancer cells. Various expression level of Tβ4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tβ4. Cell migration was higher in SNU668 with a higher expression level of Tβ4 than that in SNU638 with a lower Tβ4. Although the level of phosphorylated(p)-GSK-3α (inactive), β-catenin, E-cadherin and E-cadherin:β-catenin complex was relatively higher, p-GSK-3β (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK-3α/β inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference (si)RNA of GSK-3α increased SNU638 cell migration in accordance with the reduction of E-cadherin:β-catenin complex formation through a decrease in β-catenin and E-cadherin. Expression level of GSK-3α/β, β-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tβ4-siRNA and by the treatment with acetylated-serine-aspartic acid-lysine-proline (SDKP) tetrapeptide of Tβ4, respectively. E-cadherin expression in SNU638 cells was decreased by β-catenin-siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p-GSK-3α, β-catenin and E-cadherin. Taken together, data indicated that the expression of GSK-3α, β-catenin and E-cadherin could be negatively regulated by Tβ4-induced ERK phosphorylation. It suggests that Tβ4 could be a novel regulator to control Wnt signaling pathways.

Topics: Animals; beta Catenin; Butadienes; Cadherins; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Glycogen Synthase Kinase 3; Humans; Lithium Chloride; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Nitriles; Peptides; Phosphorylation; RNA Interference; RNA, Small Interfering; Stomach Neoplasms; Thymosin; Wnt Signaling Pathway

Recent studies have revealed that microRNAs have a strong association with cancer in humans. The miRNA let-7 is highly expressed in normal lung tissue, but frequently expressed at reduced levels in lung cancers. Let-7a2 is a member of the let-7 family. So far, little is known about the transcriptional regulation of let-7a2. Our study is focused on the characterization and functional analysis of the promoter of the human miRNA let-7a2 in A549 cell lines. Firstly, 5' rapid amplification of cDNA ends (5' RACE) was carried out and a 2.8 kb fragment in the upstream of let-7a2 gene was then cloned into pGL3-basic vector. Sequence analysis with the MatInspector database revealed that there were putative binding sites for some important transcriptional factors in the promoter region of let-7a2, such as p53, c-Myc, Ras, CEBPα, RORA, RXR, TCF, and GR. Additionally, a series of transfection and luciferase reporter assays were carried out to test let-7a2 promoter activity. RT-PCR and transfection of let-7a target sequence-reporter plasmid were performed to detect transcription levels of the let-7a2 gene in A549 cells treated with 9-cis-RA, all-trans-RA, lithium chloride or dexamethasone. Our results showed that the recombinant pGL3-p7a2 could acts as a promoter. The promoter activity of the 2.8 kb fragment could be downregulated by transfection with CEBPα or treatment with lithium chloride and enhanced by 9-cis-RA or all-trans-RA treatment. Furthermore, the results of RT-PCR analysis and transfection of let-7a target sequence-reporter plasmid showed that 9-cis-RA and all-trans-RA both upregulated let-7a2 expression, while lithium chloride downregulated its expression. Our results suggest that 9-cis-RA, all-trans-RA,lithium chloride and CEBPα might play important regulatory roles in let-7a2 gene expression in A549 cells.

Topics: Base Sequence; Binding Sites; Cell Line, Tumor; Dexamethasone; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Lithium Chloride; Luciferases; Lung Neoplasms; MicroRNAs; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

Wnt/beta-catenin signaling plays an important role not only in cancer, but also in cancer stem cells. In this study, we found that beta-catenin and OCT-4 was highly expressed in cisplatin (DDP) selected A549 cells. Stimulating A549 cells with lithium chloride (LiCl) resulted in accumulation of beta-catenin and up-regulation of a typical Wnt target gene cyclin D1. This stimulation also significantly enhanced proliferation, clone formation, migration and drug resistance abilities in A549 cells. Moreover, the up-regulation of OCT-4, a stem cell marker, was observed through real-time PCR and Western blotting. In a reverse approach, we inhibited Wnt signaling by knocking down the expression of beta-catenin using RNA interference technology. This inhibition resulted in down-regulation of the Wnt target gene cyclin D1 as well as the proliferation, clone formation, migration and drug resistance abilities. Meanwhile, the expression of OCT-4 was reduced after the inhibition of Wnt/beta-catenin signaling. Taken together, our study provides strong evidence that canonical Wnt signaling plays an important role in lung cancer stem cell properties, and it also regulates OCT-4, a lung cancer stem cell marker.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Gene Knockdown Techniques; Humans; Lithium Chloride; Lung Neoplasms; Neoplastic Stem Cells; Octamer Transcription Factor-3; Signal Transduction; Wnt Proteins

We previously reported that overexpression of Axin downregulates T cell factor-4 (TCF-4) transcription. However, the mechanism(s) by which Axin downregulates the transcription and expression of TCF-4 is not clear. It has been reported that beta-catenin promotes and p53 inhibits TCF-4 transcription, respectively. The aim of this study was to investigate whether beta-catenin and/or p53 is required for Axin-mediated downregulation of TCF-4.. Axin mutants that lack p53/HIPK2 and/or beta-catenin binding domains were expressed in lung cancer cells, BE1 (mutant p53) and A549 (wild type p53). Expression of Axin or AxinDeltap53 downregulates beta-catenin and TCF-4, and knock-down of beta-catenin upregulates TCF-4 in BE1 cells. However, expression of AxinDeltabeta-ca into BE1 cells did not downregulate TCF-4 expression. These results indicate that Axin downregulates TCF-4 transcription via beta-catenin. Although overexpression of wild-type p53 also downregulates TCF-4 in BE1 cells, cotransfection of p53 and AxinDeltabeta-ca did not downregulate TCF-4 further. These results suggest that Axin does not promote p53-mediated downregulation of TCF-4. Axin, AxinDeltap53, and AxinDeltabeta-ca all downregulated beta-catenin and TCF-4 in A549 cells. Knock-down of p53 upregulated beta-catenin and TCF-4, but cotransfection of AxinDeltabeta-ca and p53 siRNA resulted in downregulation of beta-catenin and TCF-4. These results indicate that p53 is not required for Axin-mediated downregulation of TCF-4. Knock-down or inhibition of GSK-3beta prevented Axin-mediated downregulation of TCF-4. Furthermore, expression of Axin and AxinDeltap53, prevented the proliferative and invasive ability of BE1 and A549, expression of AxinDeltabeta-ca could only prevented the proliferative and invasive ability effectively.. Axin downregulates TCF-4 transcription via beta-catenin and independently of p53. Axin may also inhibits the proliferation and invasion of lung cancer cells via beta-catenin and p53.

Topics: Animals; Axin Protein; beta Catenin; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Lung Neoplasms; Mice; Mutant Proteins; Neoplasm Invasiveness; Repressor Proteins; RNA, Messenger; RNA, Small Interfering; TCF Transcription Factors; Transcription Factor 7-Like 2 Protein; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53

In carcinoid cell lines, the histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) activate the Notch1 pathway, whereas lithium inhibits glycogen synthase kinase-3beta (GSK-3beta). These compounds limit growth and decrease hormonal secretion in vitro. We hypothesized that lower-dose combination therapy of HDAC inhibitors and lithium chloride could achieve similar growth inhibition to that of the drugs alone. Gastrointestinal and pulmonary carcinoid cells were treated with either VPA or SBHA and lithium chloride for up to 48 hours. Western blot analysis was used to measure the effects on the Notch1 and GSK-3beta pathways and the neuroendocrine tumor marker chromogranin A (CgA). Growth was measured by a cellular proliferation assay. With lower-dose combination therapy, a decrease in CgA was observed. The HDAC inhibitors increased the amount of active Notch1 protein, whereas treatment with lithium was associated with inhibition of GSK-3beta. Moreover, growth was inhibited with lower-dose combination therapy. Treatment of carcinoid cells with either VPA or SBHA and lithium chloride suppresses the neuroendocrine marker CgA while upregulating Notch1 and inhibiting GSK-3beta. This combination effectively reduces growth. Thus, lower-dose combination therapy may be a viable therapeutic approach for carcinoid tumors.

Topics: Adjuvants, Immunologic; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Carcinoid Tumor; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromogranin A; Enzyme Inhibitors; Gastrointestinal Neoplasms; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Lithium Chloride; Luciferases; Lung Neoplasms; Receptor, Notch1; Valproic Acid

The immunomodulatory effect of lithium chloride was studied. The IL-2 and IFNr production by peripheral blood mononuclear cells(PBMC) were assayed in 52 normal subjects and 156 cancer patients. IL-2 and IFN-r levels in lymphoma patients were lower than in normal subjects. The IFNr level in the majority of patients with lung and esophageal cancers was abnormal but the IL-2 level was within the normal range. When lithium, IL-2 was incubated with PBMC in vitro, upregulation of IL-2 and IFNr production was observed in normal subjects and cancer patients. The IL-2 and IFNr levels were significantly increased by both lithium and rIL-2 but the effect of lithium was more potent. Lithium upregulated IFNr in 70% of patients with low levels whereas it did so in only 10-20% of patients with high levels. Therefore, lithium is a promising new immunoregulatory agent for clinical use.

Topics: Chlorides; Esophageal Neoplasms; Humans; Interferon-gamma; Interleukin-2; Leukocytes, Mononuclear; Lithium; Lithium Chloride; Lung Neoplasms; Lymphoma