Inhibition of HDAC6 Attenuates Tumor Growth of Non-Small Cell Lung Cancer

Histone deacetylase 6 (HDAC6) regulates cytoplasmic signaling networks through deacetylation of various cytoplasmic substrates and serves as a key member of the ubiquitin proteasome system (UPS). This study is focused on HDAC6 regulation of the Notch1 receptor that plays a crucial role in tumor growth in NSCLC. A series of cell culture experiments were employed using A549, Lewis lung carcinoma 2 (LL2), and H1299 NSCLC cell lines to investigate HDAC6-mediated regulation of the Notch1 receptor through the UPS. HDAC6 was inhibited with small molecule inhibitors tubacin and ACY1215 in vitro and in vivo. Inhibition of HDAC6 led to reduced levels of Notch1 receptor in a dose-dependent manner in all three NSCLC cell lines tested. HDAC6 inhibition with ACY1215 led to G2 arrest, increased apoptosis, and increased levels of cleaved PARP1 in A549, LL2, and H1299 cell lines. In vivo inhibition of HDAC6 with ACY1215 significantly reduced LL2 tumor growth rate. Our data show that HDAC6 in NSCLC cells supports Notch1 signaling and promotes cell survival and proliferation. Our results support clinical investigation of HDAC6 inhibitors as a potential therapeutic option for treatment of NSCLC patients.     

Prognostic impact of vitamin B6 metabolism in lung cancer

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.     

Global detection of molecular changes reveals concurrent alteration of several biological pathways in nonsmall cell lung cancer cells

To identify the molecular changes that occur in non-small cell lung carcinoma (NSCLC), we compared the gene expression profile of the NCI-H292 (H292) NSCLC cell line with that of normal human tracheobronchial epithelial (NHTBE) cells. The NHTBE cells were grown in a three-dimensional organotypic culture system that permits maintenance of the normal pseudostratified mucociliary phenotype characteristic of bronchial epithelium in vivo. Microarray analysis using the Affymetrix oligonucleotide chip U95Av2 revealed that 1,683 genes showed a >1.5-fold change in expression in the H292 cell line relative to the NHTBE cells. Specifically, 418 genes were downregulated and 1,265 were upregulated in the H292 cells. The expression data for selected genes were validated in several different NSCLC cell lines using quantitative real-time PCR and Western analysis. Further analysis of the differentially expressed genes indicated that WNT responses, apoptosis, cell cycle regulation and cell proliferation were significantly altered in the H292 cells. Functional analysis using fluorescence-activated cell sorting confirmed concurrent changes in the activity of these pathways in the H292 line. These findings show that (1) NSCLC cells display deregulation of the WNT, apoptosis, proliferation and cell cycle pathways, as has been found in many other types of cancer cells, and (2) that organotypically cultured NHTBE cells can be used as a reference to identify genes and pathways that are differentially expressed in tumor cells derived from bronchogenic epithelium.     

Arctigenin Enhances Chemosensitivity to Cisplatin in Human Nonsmall Lung Cancer H460 Cells through Downregulation of Survivin Expression

Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin‐mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin‐treated non‐small‐cell lung cancer (NSCLC) H460 cells. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and annexin‐V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose‐dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase‐3 and poly(ADP‐ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin‐induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell‐cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combina‐tion with chemotherapeutic agents for NSLC.     

LukS-PV induces cell cycle arrest and apoptosis through p38/ERK MAPK signaling pathway in NSCLC cells

Non-small-cell lung cancer (NSCLC) accounts for nearly 85% of lung cancer cases. LukS-PV, one of the two components of Panton-Valentine leucocidin (PVL), is produced by Staphylococcus aureus. The present study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) lines (THP-1 and HL-60). However, the role of LukS-PV in NSCLC is unclear. In this study, we treated NSCLC cell lines A549 and H460 and a normal lung cell line, 16HBE, with LukS-PV and investigated the biological roles of LukS-PV in NSCLC. Cells were treated with varying concentrations of LukS-PV and cell viability was evaluated by CCK8 and EdU assay. Flow cytometry was used to detect cell apoptosis and analyze the cell cycle, and the expression of apoptosis and cell cycle-associated proteins and genes were identified by western blotting analysis and qRT-polymerase chain reaction, respectively. We found that LukS-PV inhibited the proliferation of NSCLC cells but had little cytotoxicity in normal lung cells. LukS-PV induced NSCLC cell apoptosis and increased the BAX/BCL-2 ratio, triggering S-phase arrest in A549 and H460 cells while increasing P21 expression and decreasing CDK2, cyclin D1, and cyclin A2 expression. We also observed increased P-p38 and P-ERK in NSCLC cells treated with LukS-PV. Treatment of NSCLC with LukS-PV combined with p38 and ERK inhibitors reversed the pro-apoptotic and pro-cell cycle arrest effects of LukS-PV. Overall, these findings indicate that LukS-PV has anti-tumor effects in NSCLC and may contribute to the development of anti-cancer agents.     

p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells

This study examined the effects of p53 gene status on DNA damage-induced cell death and chemosensitivity to various chemotherapeutic agents in non-small cell lung cancer (NSCLC) cells. A mutant p53 gene was introduced into cells carrying the wild-type p53 gene and also vice versa to introduce the wild-type p53 gene into cells carrying the mutant p53 gene. Chemosensitivity and DNA damage-induced apoptosis in these cells were then examined. This study included five cell lines, NCI-H1437, NCI-H727, NCI-H441 and NCI-H1299 which carry a mutant p53 gene and NCI-H460 which carries a wild-type p53 gene. Mutant p53-carrying cells were transfected with the wild-type p53 gene, while mutant p53 genes were introduced into NCI-H460 cells. These p53 genes were individually mutated at amino acid residues 143, 175, 248 and 273. The representative cell line NCI-H1437 cells transfected with wild-type p53 gene (H1437/wtp53) showed a dramatic increase in susceptibility to three anticancer agents (7-fold to cisplatin, 21-fold to etoposide, and 20-fold to camptothecin) compared to untransfected or neotransfected H1437 cells. An increase in chemosensitivity was also observed in wild-type p53 transfectants of H727, H441, H1299 cells. The results of chemosensitivity were consistent with the observations on apoptotic cell death. H1437/wtp53 cells, but not H1437 parental cells, exhibited a characteristic feature of apoptotic cell death that generated oligonucleosomal-sized DNA fragments. In contrast, loss of chemosensitivity and lack of p53-mediated DNA degradation in response to anticancer agents were observed in H460 cells transfected with mutant p53. These observations suggest that the increase in chemosensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In addition, our results also suggest that p53 gene status modulates the extent of chemosensitivity and the induction of apoptosis by different anticancer agents in NSCLC cells.     

Baicalein inhibits cell growth and increases cisplatin sensitivity of A549 and H460 cells via miR‐424‐3p and targeting PTEN/PI3K/Akt pathway

Lung cancer is the leading cause of death in individuals with malignant disease. Non‐small‐cell lung cancer (NSCLC) is the most common type of lung cancer, and chemotherapy drugs such as cisplatin are the most widely used treatment for this disease. Baicalein is a purified flavonoid compound that has been reported to inhibit cancer cell growth and metastasis and increase sensitization to chemotherapeutic drugs via different pathways. Therefore, we assessed the effects of baicalein on the proliferation, apoptosis and cisplatin sensitivity in the NSCLC A549 and H460 cell lines and determined the pathways through which baicalein exerts its effects. Baicalein was slightly toxic to normal human bronchial NHBE cells but inhibited growth, induced apoptosis and increased cisplatin sensitivity in A549 and H460 cells. Baicalein down‐regulated miR‐424‐3p, up‐regulated PTEN expression and down‐regulated expression of PI3K and p‐Akt in A549 and H460 cells. Dual‐luciferase reporter assay demonstrated that PTEN is a target gene of miR‐424‐3p, and overexpression of miR‐424‐3p or silencing of PTEN partially attenuated the effects of baicalein on A549 and H460 cells. Taken together, we concluded that baicalein inhibits cell growth and increases cisplatin sensitivity to A549 and H460 cells via down‐regulation of miR‐424‐3p and targeting the PTEN/PI3K/Akt pathway.     

RING finger protein 38 induces the drug resistance of cisplatin in non-small-cell lung cancer

Cisplatin resistance of non-small-cell lung cancer (NSCLC) needs to be well elucidated. RING finger protein (RNF38) has been proposed as a biomarker of NSCLC poor prognosis. However, its role in drug resistance in NSCLC is poorly understood. RNF38 expression was detected in normal lung epithelial cell and four NSCLC cell lines. RNF38 was stably overexpressed in A549 and H460 cells or silenced in H1975 and cisplatin-resistant A549 cells (A549-CDDP resistant) using lentiviral vectors. RNF38 expression levels were determined using quantitative real-time polymerase chain reaction and western blotting analysis. Cell viability in response to different concentrations of cisplatin was evaluated by Cell Counting Kit-8 assay. RNF38 expression levels were markedly elevated in NSCLC cells and cells harboring high RNF38 were less sensitive to cisplatin. Overexpression of RNF38 reduced, while RNF38 silencing increased the drug sensitivity of cisplatin in NSCLC cells. Cisplatin-resistant cells expressed high RNF38 level. RNF38 silencing promoted cell apoptosis and enhanced the drug sensitivity of cisplatin in cisplatin-resistant NSCLC cells. These findings indicate that RNF38 might induce cisplatin resistance of NSCLC cells via promoting cell apoptosis and RNF38 could be a novel target for rectify cisplatin resistance in NSCLC cases.     

Prediction of a non-small cell lung cancer sensitivity to cisplatin and paclitaxel based on the marker genes expression

The goal of the present study was to define gene expression signatures that predict a chemosensitivity of non-small cell lung cancer (NSCLC) to cisplatin and paclitaxel. To generate set of candidate genes likely to be predictive a current knowledge of the pathways involved in resistance and sensitivity to individual drugs was used. Forty four genes coding proteins belonging to following categories: ATP-dependent transport proteins, detoxification system proteins, reparation system proteins, tubulin and proteins responsible for its synthesis, cell cycle and apoptosis proteins were considered. Eight NSCLC cell lines (A549, Calul, H1299, H322, H358, H460, H292, and H23) were used in our study. For each NSCLC cell line a cisplatin and paclitaxel chemosensitivity as well as an expression level of 44 candidate genes were evaluated. To develop a chemosensitivity prediction model based on selected genes expression level a multiple regression analysis was performed. The model based on the expression level of 11 genes (TUBB3, TXR1, MRP5, MSH2, ERCC1, STMN, SMAC, FOLR1, PTPN14, HSPA2, GSTP1) allowed us to predict the paclitaxel cytotoxic concentration with high level of correlation (r = 0.91, p < 0.01). However, none model developed was able to reliably predict a sensitivity of the NSCLC cells to cisplatin.     

Cyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type‐Iα (PKG‐Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG‐Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti‐apoptotic Bcl‐2‐related proteins in NCI‐H460 and A549 non‐small cell lung cancer (NSCLC) cells. 1H‐(1,2,4)oxadiazolo(4,3‐a)quinoxalin‐1‐one (ODQ), which blocks endogenous NO‐induced activation of cGMP/PKG‐Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c‐IAP1, livin, and survivin. DT‐2 (inhibitor of PKG‐Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c‐IAP1, livin, and survivin expression. Gene knockdown of PKG‐Iα expression using small‐interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c‐IAP1, livin, survivin, and Mcl‐1 expression. Inhibition of PKG‐Iα kinase activity with DT‐2 dramatically enhanced pro‐apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT‐2 and cisplatin increased apoptosis compared with cisplatin or DT‐2 alone, showing a synergistic effect. The data suggest that the PKG‐Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c‐IAP1, livin, survivin, and Mcl‐1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin. J. Cell. Biochem. 113: 3587–3598, 2012. © 2012 Wiley Periodicals, Inc.     

EPS8 Inhibition Increases Cisplatin Sensitivity in Lung Cancer Cells

Cisplatin, a commonly used chemotherapeutic, is associated with ototoxicity, renal toxicity and neurotoxicity, thus identifying means to increase the therapeutic index of cisplatin may allow for improved outcomes. A SNP (rs4343077) within EPS8, discovered through a genome wide association study of cisplatin-induced cytotoxicity and apoptosis in lymphoblastoid cell lines (LCLs), provided impetus to further study this gene. The purpose of this work was to evaluate the role of EPS8 in cellular susceptibility to cisplatin in cancerous and non-cancerous cells. We used EPS8 RNA interference to determine the effect of decreased EPS8 expression on LCL and A549 lung cancer cell sensitivity to cisplatin. EPS8 knockdown in LCLs resulted in a 7.9% increase in cisplatin-induced survival (P = 1.98×10⁻⁷) and an 8.7% decrease in apoptosis (P = 0.004) compared to control. In contrast, reduced EPS8 expression in lung cancer cells resulted in a 20.6% decrease in cisplatin-induced survival (P = 5.08×10⁻⁵). We then investigated an EPS8 inhibitor, mithramycin A, as a potential agent to increase the therapeutic index of cisplatin. Mithramycin A decreased EPS8 expression in LCLs resulting in decreased cellular sensitivity to cisplatin as evidenced by lower caspase 3/7 activation following cisplatin treatment (42.7%±6.8% relative to control P = 0.0002). In 5 non-small-cell lung carcinoma (NSCLC) cell lines, mithramycin A also resulted in decreased EPS8 expression. Adding mithramycin to 4 NSCLC cell lines and a bladder cancer cell line, resulted in increased sensitivity to cisplatin that was significantly more pronounced in tumor cell lines than in LCL lines (p<0.0001). An EGFR mutant NSCLC cell line (H1975) showed no significant change in sensitivity to cisplatin with the addition of mithramycin treatment. Therefore, an inhibitor of EPS8, such as mithramycin A, could improve cisplatin treatment by increasing sensitivity of tumor relative to normal cells.     

The effects on cell growth and chemosensitivity by livin RNAi in non-small cell lung cancer

Livin is highly expressed in most tumor tissues and could inhibit the tumor cells apoptosis. Knockdown of endogenous livin expression in non-small cell lung cancer (NSCLC) cells could inhibit cell growth. But it is still unclear if knockdown of endogenous livin expression combined with conventional chemotherapy could play a positive role in NSCLC treatment. In this article, the efficient RNA interferences (RNAi) of livin were constructed, and then we transfected them into A549 cells and 103H cells to study their influence on cell cycle and apoptosis index. At last, we detected the cell's sensitivity to conventional chemotherapeutic drugs after knockdown endogenous livin expression in A549 cells and 103H cells. Our results showed that knockdown livin expression could inhibit cell growth and induce apoptosis in A549 cells and 103H cells. A549 cells and 103H cells had an increased chemosensitivity to adriamycin and cisplatin after transfection of livin RNAi constructs. The results indicated that cell cycle redistribution and increased apoptosis index after knockdown livin expression might provide the main explanation for the enhanced chemosensitivity. Proper combination of livin RNAi and some conventional chemotherapeutic drugs may entail potential benefits in the treatment of NSCLC.     

Selective cytotoxicity of synthesized procyanidin 3-O-Galloylepicatechin-4b,8-3-O-galloylcatechin to human cancer cells

Cocoa-derived flavanols and procyanidins have been previously reported to exhibit anti-oxidant and anti-tumor properties. In this study, we have investigated the cellular growth inhibitory effect of chemically-synthesized procyanidin 3-O-Galloylepicatechin-4b,8-3-O-galloylcatechin (GECGC) on a variety of human cancer cell lines. Among 16 human cancer cell lines tested, GECGC selectively inhibited proliferation of a subset of human cancer cell lines, especially those of short doubling time. In contrast, all 6 normal cell lines tested including human mammary epithelial cells and skin fibroblast were resistant to GECGC’s cytotoxicity. Cell cycle analysis and apoptosis assay showed that GECGC increased sub-G1 population and increased the population of propidium iodide and Annexin V staining cells in GECGC-sensitive cell lines, suggesting that cell growth inhibition by GECGC may be mediated through both apoptotic and non-apoptotic mechanisms. Further characterization of GECGC cytotoxicity on 30 genetically modified cell lines with overexpression or depletion of key proteins involved in cell cycle regulation and signal transduction pathways suggested that GECGC-mediated cell death involves IKK&alpha; and IKK&gamma;. Collectively, our observations indicate that synthesized GECGC has selective anti-proliferative effect on human cancer cells and warrant further evaluation as a preventive and chemotherapeutic reagent to human malignancies.     

A functional role for nicotine in Bcl2 phosphorylation and suppression of apoptosis

Nicotine is not only a major component in tobacco but is also a survival agonist that inhibits apoptosis induced by diverse stimuli including chemotherapeutic drugs. However, the intracellular mechanism(s) involved in nicotine suppression of apoptosis is unclear. Bcl2 is a potent antiapoptotic protein and tumor promotor that is expressed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells. It is possible that nicotine may regulate Bcl2 to stimulate cell survival. Here we report that nicotine can induce Bcl2 phosphorylation exclusively at the serine 70 site in association with prolonged survival of SCLC H82 cells expressing wild-type but not the phosphorylation-deficient S70A mutant Bcl2 after treatment with chemotherapeutic agents (i.e. cisplatin or VP-16). Nicotine induces activation of PKC alpha and the MAPKs ERK1 and ERK2, which are physiological Bcl2 kinases. Furthermore, ET-18-OCH3, a specific phospholipase C (PLC) inhibitor, blocks nicotine-stimulated Bcl2 phosphorylation and promotes apoptosis, suggesting that PLC may be involved in nicotine activation of Bcl2 kinases. Using a genetic approach, the gain-of-function S70E mutant, which mimics Ser(70) site phosphorylation in the flexible loop domain, potently enhances chemoresistance in SCLC cells. Thus, nicotine-induced cell survival results, at least in part, from a mechanism that involves Bcl2 phosphorylation. Therefore, novel therapeutic strategies for lung cancer in which Bcl2 is expressed may be used to abrogate the anti-apoptotic activity of Bcl2 by inhibiting multiple upstream nicotine-activated pathways.     

Prediction of nonsmall cell lung cancer sensitivity to cisplastin and paclitaxel based on marker gene expression

The goal of the present study was to define gene expression signatures that predict a chemosensitivity of nonsmall cell lung cancer (NSCLC) to cisplatin and paclitaxel. To generate a set of candidate genes likely to be predictive, current knowledge of the pathways involved in resistance and sensitivity to individual drugs was used. Forty-four genes coding proteins belonging to the following categories—ATP-dependent transport proteins, detoxification system proteins, reparation system proteins, tubulin and proteins responsible for its synthesis, cell cycle, and apoptosis proteins—were considered. Eight NSCLC cell lines (A549, Calu1, H1299, H322, H358, H460, H292, and H23) were used in our study. For each NSCLC cell line, a cisplatin and paclitaxel chemosensitivity, as well as an expression level of 44 candidate genes, were evaluated. To develop a chemosensitivity prediction model based on selected genes’ expression level, a multiple regression analysis was performed. The model based on the expression level of 11 genes (TUBB3, TXR1, MRP5, MSH2, ERCC1, STMN, SMAC, FOLR1, PTPN14, HSPA2, GSTP1) allowed us to predict the paclitaxel cytotoxic concentration with a high level of correlation (r = 0.91, p < 0.01). However, no model developed was able to reliably predict sensitivity of the NSCLC cells to cisplatin.