Activation of p53/miR-34a Tumor Suppressor Axis by Chinese Herbal Formula JP-1 in A549 Lung Adenocarcinoma Cells

Lung cancer is the leading cause of cancer death worldwide; the most common pathologic type is lung adenocarcinoma (LADC). In spite of the recent progress in targeted therapy, most LADC patients eventually expired due to the inevitable recurrence and drug resistance. New complementary agent with evidence-based molecular mechanism is urgently needed. MiR-34a is an important p53 downstream tumor suppressor, which regulates apoptosis, cell-cycle, EMT (epithelial mesenchymal transition), and so forth. Its expression is deficient in many types of cancers including LADC. Here, we show that a Chinese herbal formula JP-1 activates p53/miR-34a axis in A549 human LADC cells (p53 wild-type). Treatment with JP-1 induces p53 and its downstream p21 and BAX proteins as well as the miR-34a, resulting in growth inhibition, colony formation reduction, migration repression, and apoptosis induction. Accordingly, the decreases of miR-34a downstream targets such as CDK6, SIRT1, c-Myc, survivin, Snail, and AXL were observed. Moreover, JP-1 activates AMPKα and reduces mTOR activity, implying its inhibitory effect on the energy-sensitive protein synthesis and cell proliferation signaling. Our results show that JP-1 activates p53/miR-34a tumor suppressor axis and decreases proteins related to proliferation, apoptosis resistance, and metastasis, suggesting its potential as a complementary medicine for LADC treatment

Diverse cellular transformation capability of overexpressed genes in human hepatocellular carcinoma

For isolation of novel cellular transforming genes that potentially participated in hepatocarcinogenesis,we conducted anchorage- independent growth (AIG)assays on 10 human liver cancer cell lines and observed strong AIG capabilities in PLC5 and Huh7 but negligible in Tong cells.After cloning of genes by di fferential subtractive chain reactions (DSC)from strong AIG to AIG negative cells,we sequenced 2304 clones and identi fied 245 genes.After four stringent criteria for selection of transforming genes among DSC clones,our results of quantitative RT-PCR analysis indicated that six genes,DDX3 ,EIF3S2 ,CLIC1 ,HDGF ,GPC3 ,and HSPCA were overexpressed in 64%,62%,60%,58%,49%,and 47%,respectively,of 45 hepatocellular carcinoma (HCC)tissues.The results of cellular transformation capability by AIG assays indicated that the transfectants of EIF3S2 showed the strongest (>100-fold), DDX3 and CLIC1 were moderate,GPC3 and HSPCA were weak,and HDGF was none in forming colonies in soft agar.Together, our results suggested that Tong is a suitable human cell line for screening of overexpressed and/or cellular transforming genes.In addition,our results suggested that diverse functions of cellular transforming genes in various biological pathways could transform human Tong cells and potentially reveal new targets for drug development of HCC

Oxidative stress enhances Axl-mediated cell migration through an Aktl/Racl-dependent mechanism

Persistent oxidative stress is common in cancer cells because of abnormal generation of reactive oxygen species (ROS) and has been associated with malignant phenotypes, such as chemotherapy resistance and metastasis. Both overexpression of Axl and abnormal ROS elevation have been linked to cell transformation and increased cell migration. However, the relationship between Axl and ROS in malignant cell migration has not been previously evaluated. Using an in vitro human lung cancer model, we examined the redox state of lung adenocarcinoma cell lines of low metastatic (CL1-0) and high metastatic (CL1-5) potentials. Here we report that Axl activation elicits ROS accumulation through the oxidase-coupled small GTPase Racl. We also observed that oxidative stress could activate Axl phosphorylation to synergistically enhance cell migration. Further, Axl signaling activated by H2O2 treatment results in enhancement of cell migration via a PI3K/Akt-dependent pathway. The kinase activity of Axl is required for the Axl-mediated cell migration and prolongs the half-life of phospho-Akt under oxidative stress. Finally, downregulation of Akt1, but not Akt2, by RNAi in Axl-overexpressing cells inhibits the amount of activated Rac1 and the ability to migrate induced by H2O2 treatment. Together, these results show that a novel Axl-signaling cascade induced by H2O2 treatment triggers cell migration through the PI3K/Akt1/Racl pathway. Elucidation of redox regulation in Axl-related malignant migration may provide new molecular insights into the mechanisms underlying tumor progression. (C) 2013 Elsevier Inc. All rights reserved

Diverse Cellular Transformation Capability of Overexpressed Genes in Human Hepatocellular Carcinoma

For isolation of novel cellular transforming genes that potentially participated in hepatocarcinogenesis, we conducted anchorage-independent growth (AIG) assays on 10 human liver cancer cell lines and observed strong AIG capabilities in PLC5 and Huh7 but negligible in Tong cells. After cloning of genes by differential subtractive chain reactions (DSC) from strong AIG to AIG negative cells, we sequenced 2304 clones and identified 245 genes. After four stringent criteria for selection of transforming genes among DSC clones, our results of quantitative RT-PCR analysis indicated that six genes, DDX3, EIF3S2, CLIC1, HDGF, GPC3, and HSPCA were overexpressed in 64%, 62%, 60%, 58%, 49%, and 47%, respectively , of 45 hepatocellular carcinoma (HCC) tissues. The results of cellular transformation capability by AIG assays indicated that the transfectants of EIF3S2 showed the strongest (>100-fold), DDX3 and CLIC1 were moderate, GPC3 and HSPCA were weak, and HDGF was none in forming colonies in soft agar. Together, our results suggested that Tong is a suitable human cell line for screening of overexpressed and/or cellular transforming genes. In addition, our results suggested that diverse functions of cellular transforming genes in various biological pathways could transform human Tong cells and potentially reveal new targets for drug development of HCC. ( C) 2004 Elsevier Inc. All rights reserved

AXL phosphorylates and up-regulates TNS2 and its implications in IRS-1-associated metabolism in cancer cells

Abstract Background TNS2 is a focal adhesions protein and a binding partner for many proteins, including the receptor tyrosine kinase Axl. Although TNS2 can bind with Axl, the details of their interactions have not been elucidated. TNS2 is involved in IRS-1 signaling pathway. In this study, we confirmed the relationship between TNS2 expression and the expression of Axl, IRS-1, PDK1 and Glut4 in pancreatic cancer patients. Methods The expression levels of TNS2, Axl, IRS-1, PDK1 and Glut4 in human cancer cells were measured by Western blot and/or IP-Western blot assays. Paired samples of pancreatic cancer and non-cancer tissues were obtained from 33 patients and were used to construct tissue microarrays. The expression levels of these markers in the tissue microarrays were measured by enzyme-linked Immunohistochemistry assay, and the relationships were analyzed by Pearson’s chi-square test and two-tailed t-test analysis. Results We demonstrated for the first time that TNS2 is a phosphorylation substrate of Axl. Moreover, we found a positive relationship between TNS2 expression and the expression of Axl, IRS-1, PDK1 and Glut4 in pancreatic cancer patients. Based on these results, we suggest that Axl modulates glucose metabolism potentially through TNS2 and IRS-1. We hypothesize that there exists a novel mechanism whereby Axl binds to and phosphorylates TNS2, releasing TNS2 from interaction with IRS-1 and resulting in increased stability of IRS-1. The two key enzymes of aerobic glycolysis (Glut4 and PDK1) were found to be up-regulated by Axl/TNS2/IRS-1 cross-talk and may play a critical role in glucose metabolism of cancer cells. Conclusions Our results revealed for the first time that Axl binds to and phosphorylates TNS2 and that Axl/TNS2/IRS-1 cross-talk may potentially play a critical role in glucose metabolism of cancer cells

Negative feedback regulation of AXL by miR-34a modulates apoptosis in lung cancer cells

[[abstract]]The AXL receptor tyrosine kinase is frequently overexpressed in cancers and is important in cancer invasion/metastasis and chemoresistance. Here, we demonstrate a regulatory feedback loop between AXL and microRNA (miRNA) at the post-transcriptional level. Both the GAS6-binding domain and the kinase domain of AXL, particularly the Y779 tyrosine phosphorylation site, are shown to be crucial for this autoregulation. To clarify the role of miRNAs in this regulation loop, approaches using bioinformatics and molecular techniques were applied, revealing that miR-34a may target the 3' UTR of AXL mRNA to inhibit AXL expression. Interestingly and importantly, AXL overexpression may induce miR-34a expression by activating the transcription factor ELK1 via the JNK signaling pathway. In addition, ectopic overexpression of ELK1 promotes apoptosis through, in part, down-regulation of AXL. Therefore, we propose that AXL is autoregulated by miR-34a in a feedback loop; this may provide a novel opportunity for developing AXL-targeted anticancer therapies

Additional file 1: Figure S1. of Honokiol inhibits sphere formation and xenograft growth of oral cancer side population cells accompanied with JAK/STAT signaling pathway suppression and apoptosis induction

Honokiol (5 μM) and siSTAT3 did not affect the cell viability of SAS cells in the 24-h wound healing assay. (A) SAS cells were treated with honokiol (5 μM) for 24 h in the same culture condition shown in Fig. 6b for wound healing assay. The cell viability was then determined by the quantitative staining of cellular proteins by sulforhodamine B. (B) After transfection with siSTAT3 and analysis for the expression of STAT3, the SAS cells were seeded into 6-well plate in the same condition shown in Fig. 6b for wound healing assay. After 24 h of incubation, the cell viability was then determined by the quantitative staining of cellular proteins by sulforhodamine B. (TIFF 1240 kb