Chemoresistance In Lung Cancer Cells Research Articles

Reversing Multidrug Resistance in Chemo-resistant Human Lung Adenocarcinoma (A549/DOX) Cells by Algerian Propolis Through Direct Inhibiting the P-gp Efflux-pump, G0/G1 Cell Cycle Arrest and Apoptosis Induction.

Lung cancer is one of the most common malignancies with the highest incidence and mortality rate worldwide. Multidrug Resistance (MDR) continues to pose a major challenge for the clinicians and pharmacologists to effectively treat this disease. A new approach using natural substances with moderate or low cytotoxic properties become a promising hope for reversing multidrug resistance due to pgp- overexpression. This study aims to explore the efficacy of Algerian propolis in reversing multidrug resistance and sensitizing chemo-resistant lung cancer cells (A549/DOX) to chemotherapy with DOX. Resistant lung adenocarcinoma A549/DOX cell line was developed and used as in vitro model for MDR. Cell viability, Annexin V-PI apoptosis assay and cell cycle progression were tested to evaluate the reversal effect of propolis alone or in combination with DOX. Caspases 3, 8 and 9 assays were conducted to determine the type of apoptotic pathway. To investigate the mechanisms of MDR reversal agents, intracellular accumulation of DOX and P-gp-pump activity were investigated. Our results showed that the obtained chemo-resistant cells were 13-fold more resistant to DOX than the parental A549 cells. Propolis showed dramatically cell growth inhibition on A549/DOX cells (The IC50 was 50.44± 0.07µg/ml). The killing effect of propolis was due to G0/G1 cell cycle arrest and apoptosis induction. After 24hours treatment, propolis at 100 µg/ml caused cells accumulation in G0/G1 phase and increased with 50, 65-fold the percentage of apoptotic population sub-G. Annexin V-PI assay showed that propolis induces apoptosis with 53.57-fold at 100 µg/ml. It induced intrinsic apoptotic pathway by increasing caspase-3 (22.15-fold) and caspase-9 (16.73-fold) activities. The direct approach to investigate the mechanisms of reversal agents is to detect the accumulation of P-gp substrates in resistant cells. Our results indicated that resistant cells poorly accumulated Doxorubicin and rhodamine 123 (7-fold lower) when compared to parental A549 cells, suggesting that chemo-resistant cells overexpress P-gp which pump DOX out of cells. Propolis inhibited in a concentration-dependent manner, the pgp efflux-pump, enhancing thereby the intracellular level of DOX with 5.48- fold against 3.33 fold obtained with verapamil, the conventional P-gp inhibitor. Taken together, Algerian propolis reverses multidrug resistance in resistant human lung adenocarcinoma cells through direct inhibiting the transport function of pgp-pump resulting in enhancing intracellular DOX-accumulation, G0/G1 cell cycle arrest and apoptosis induction. Thus, propolis could be developed as a chemotherapeutic agent for reversing multidrug resistance.

Induction of apoptosis and suppression of tumor growth by Nur77-derived Bcl-2 converting peptide in chemoresistant lung cancer cells.

Resistance to chemotherapy is a major cause of treatment failure and poor overall survival in patients with lung cancer. Identification of molecular targets present in resistant cancer cells is essential for addressing therapeutic resistance and prolonging lung cancer patient survival. Members of the B-cell lymphoma 2 (Bcl-2) family of proteins are associated with chemotherapeutic resistance. In this study, we found that pro-survival protein Bcl-2 is upregulated in paclitaxel resistant cells, potentially contributing to chemotherapy resistance. To exploit the increase in Bcl-2 expression for targeting therapy resistance, we investigated the effects of a peptide derived from the nuclear receptor Nur77 that converts Bcl-2 from an anti-apoptotic protein to a pro-apoptotic protein. The Nur77 derived peptide preferentially induced apoptosis in paclitaxel-resistant cancer cells with high expression of Bcl-2. This peptide also induced apoptosis of multidrug resistant H69AR lung cancer cells that express Bcl-2 and inhibited their growth in 3D spheroids. The Nur77 peptide strongly suppressed the growth of paclitaxel-resistant lung cancer cells in a zebrafish xenograft tumor model. Taken together, our data supports a new strategy for treating lung cancers that acquire resistance to chemotherapy through overexpression of Bcl-2.

CCL2 influences the sensitivity of lung cancer A549 cells to docetaxel.

Lung cancer is one of the most common malignant tumor types globally. Acquisition of chemoresistance in lung cancer cells is the primary cause of chemotherapy failure. Inflammatory chemokine C-C motif chemokine ligand 2 (CCL2) has been reported to be involved in the progression of cancer and drug resistance. However, its function in docetaxel (DTX) resistance of lung cancer remains unclear. In the present study, the mechanism underlying DTX-induced drug resistance was investigated. Reverse transcription-quantitative polymerase chain reaction and western blot analysis revealed that DTX treatment increased the mRNA and protein expression of CCL2 in lung cancer A549 cells. CCL2 was knocked down by small interfering RNA or was overexpressed by recombinant CCL2 lentivirus, and cell viability was determined. An MTT assay indicated that CCL2 downregulation decreased the viability of A549 cells and augmented the DTX-induced cytotoxicity, whereas CCL2 upregulation protected A549 cells from DTX-induced cytotoxicity. Additionally, it was revealed that CCL2 overexpression activated phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling and inhibited apoptosis-associated protein caspase-3 activation and B-cell lymphoma 2 (Bcl-2) phosphorylation at Ser70 induced by DTX, and enhanced DTX-induced Bcl-2-associated death promoter phosphorylation at Ser112. PI3K/AKT inhibitor LY294002 restored DTX-induced caspase-3 activation and Bcl-2 phosphorylation, reversed the effect of CCL2 on the viability of A549 cells and enhanced DTX-induced cytotoxicity. These results demonstrated that chemoresistance may be mediated by cell stress responses involving CCL2 expression, suggesting that CCL2 may be a potential target for enhancing the therapeutic effect of DTX in lung cancer.

Aminoglucose-functionalized, redox-responsive polymer nanomicelles for overcoming chemoresistance in lung cancer cells

BackgroundChemotherapeutic drugs used for cancer therapy frequently encounter multiple-drug resistance (MDR). Nanoscale carriers that can target tumors to accumulate and release drugs intracellularly have the greatest potential for overcoming MDR. Glucose transporter-1 (GLUT-1) and glutathione (GSH) overexpression in cancer cells was exploited to assemble aminoglucose (AG)-conjugated, redox-responsive nanomicelles from a single disulfide bond-bridged block polymer of polyethylene glycol and polylactic acid (AG-PEG-SS-PLA). However, whether this dual functional vector can overcome MDR in lung cancer is unknown.ResultsIn this experiment, AG-PEG-SS-PLA was synthetized successfully, and paclitaxel (PTX)-loaded AG-PEG-SS-PLA (AG-PEG-SS-PLA/PTX) nanomicelles exhibited excellent physical properties. These nanomicelles show enhanced tumor targeting as well as drug accumulation and retention in MDR cancer cells. Caveolin-dependent endocytosis is mainly responsible for nanomicelle internalization. After internalization, the disulfide bond of AG-PEG-SS-PLA is cleaved in the presence of high intracellular glutathione levels, causing the hydrophobic core to become a polar aqueous solution, which subsequently results in nanomicelle disassembly and the rapid release of encapsulated PTX. Reduced drug resistance was observed in cancer cells in vitro. The caspase-9 and caspase-3 cascade was activated by the AG-PEG-SS-PLA/PTX nanomicelles through upregulation of the pro-apoptotic proteins Bax and Bid and suppression of the anti-apoptotic protein Bcl-2, thereby increasing apoptosis. Furthermore, significantly enhanced tumor growth inhibition was observed in nude mice bearing A549/ADR xenograft tumors after the administration of AG-PEG-SS-PLA/PTX nanomicelles via tail injection.ConclusionsThese promising results indicate that AG-PEG-SS-PLA/PTX nanomicelles could provide the foundation for a paradigm shift in MDR cancer therapy.

P2.02-032 Interplaying between Gamma-H2AX and Autophagy in A549 Cells Treated with Cisplatin and Etoposide

DNA damage-repair and autophagy may contribute to the efficacy of anticancer therapy. The gamma-H2AX is phosphorylated to repair DNA after the double stranded breakdown. Thus, this study was aimed to check the expression of gamma-H2AX and autophagy in cisplatin- and etoposide-treated lung cancer cell line. A549 cells were cultured within Dulbecco’s Modified Essential Medium with 10% of fetal bovine serum. Cisplatin and etoposide were treated into the cells with time and dose dependent manners. MTT assay and light microscopy were performed to determine the LD50 of these drugs. Western blotting was performed to define the expression of proteins related to LC3A/B-I/II, NBR1 and DNA damage (gamma-HA2X). Based on MTT assay, LC50’s were determined as the followings: LC50 of CDDP was 14.4 micromole/L and LC50 of etoposide was approximately 25 micromole/L. Chemoresistant cancer cell islets were defined the surviving cells under light microscopy with the treatment of cisplatin and etoposide. In 0-, 5-, 10- and 20-h time points, gamma HA2X showed increasing expressional pattern. This pattern was also similar in etoposide with the peak time point of 10 hour. LC3A/B-I/II and NBR1 expressions were increased by 25 micromole/L of CDDP and 100 micromole/L of etoposide whose patterns were also similar to gamma H2AX. Similar expressional patterns of DNA breakdowns (gamma H2AX) and autophagy (LC3A/B-I/II, NBR1) by cisplatin and etoposide may suggest the linkage between autophagy and DNA breakdown. Therefore, this tentative conclusion must be furthermore defined by the research for molecular networks in the chemoresistant lung cancer cells.

Synergistic Effect of Notch-3-Specific Inhibition and Paclitaxel in Non-Small Cell Lung Cancer (NSCLC) Cells Via Activation of The Intrinsic Apoptosis Pathway.

BackgroundLung cancers are resistant to conventional chemotherapeutic interventions such as paclitaxel. Notch signaling is crucial in the chemoresistance of lung cancer cells. The Notch inhibitor gamma-secretase inhibitor (GSI) inhibits the Notch signaling pathway.Material/MethodsHere, we evaluated how Notch-3 inhibition by GSI can enhance the sensitivity of lung cancer cells to paclitaxel. To study how Notch-3-specific inhibition affects non-small cell lung cancer (NSCLC), we compared the cell viability, apoptosis, and colony formation of A549 and H1299 cells treated with Notch-3 siRNA and GSI.ResultsThe expression levels of Notch-3 or Notch intracellular domain 3 (NICD3) and apoptosis-related proteins were measured and compared between different groups. Notch-3 was significantly overexpressed in both cell lines, and Notch-3 expression was elevated after paclitaxel treatment, indicating activation of the Notch signaling pathway. Inhibition of the Notch signaling pathway by GSI and Notch-3 siRNA reduced cell proliferation and induced apoptosis in A549 and H1299 cells, thereby boosting sensitivity of the cell lines to paclitaxel. Concomitant treatment with paclitaxel and GSI or siRNA downregulated Bcl-2 expression and upregulated Bax expression levels.ConclusionsThese results indicate a synergistic effect of Notch-3-specific inhibition and paclitaxel through alteration of the intrinsic apoptosis pathway, which was involved in Notch-3-induced chemoresistance in NSCLC cells, and GSI inhibited Notch-3-induced chemoresistance in a concentration-dependent manner. This approach that combines Notch-3-specific inhibition and paclitaxel would be likely to apply in NSCLC.

Downregulation of PKCζ/Pard3/Pard6b is responsible for lung adenocarcinoma cell EMT and invasion

Atypical protein kinase C ζ (PKCζ) forms an apico-basal polarity complex with Partitioning Defective (Pard) 3 and Pard6 to regulate normal epithelial cell apico-basolateral polarization. The dissociation of the PKCζ/Pard3/Pard6 complex is essential for the disassembly of the tight/adherens junction and epithelial-mesenchymal transition (EMT) that is critical for tumor spreading. Loss of cell polarity and epithelial organization is strongly correlated with malignancy and tumor progression in some other cancer types. However, it is unclear whether the PKCζ/Pard3/Pard6 complex plays a role in the progression of non-small-cell lung cancer (NSCLC). We found that hypoxia downregulated the PKCζ/Pard3/Pard6 complex, correlating with induction of lung cancer cell migration and invasion. Silencing of the PKCζ/Pard3/Pard6 polarity complex components induced lung cancer cell EMT, invasion, and colonization in vivo. Suppression of Pard3 was associated with altered expression of genes regulating wound healing, cell apoptosis/death and cell motility, and particularly upregulation of MAP3K1 and fibronectin which are known to contribute to lung cancer progression. Human lung adenocarcinoma tissues expressed less Pard6b and PKCζ than the adjacent normal tissues and in experimental mouse lung adenocarcinoma, the levels of Pard3 and PKCζ were also decreased. In addition, we showed that a methylation locus in the gene body of Pard3 is positively associated with the expression of Pard3 and that methylation of the Pard3 gene increased cellular sensitivity to carboplatin, a common chemotherapy drug. Suppression of Pard3 increased chemoresistance in lung cancer cells. Together, these results suggest that reduced expression of PKCζ/Pard3/Pard6 contributes to NSCLC EMT, invasion, and chemoresistance.

A Combination of Pterostilbene with Autophagy Inhibitors Exerts Efficient Apoptotic Characteristics in Both Chemosensitive and Chemoresistant Lung Cancer Cells.

The emergence of multidrug resistance (MDR), meaning that cancer cells develop simultaneous resistance to different drugs, has limited the clinical efficacy and application of chemotherapy. Pterostilbene, a naturally occurring phytoalexin exerts a variety of pharmacologic activities, including cancer prevention, cytotoxicity, and antioxidant activity. In this study, results proved the capability of pterostilbene to effectively inhibit the cell viability of docetaxel-induced MDR human lung cancer cell lines through cell cycle arrest and apoptosis. Meanwhile, the observation of LC3-II production and formation of acidic vesicular organelles revealed an induction of autophagy at an early stage by pterostilbene, which was triggered by an inhibition of the AKT and JNK pathways and activation of ERK1/2. Furthermore, pretreatment with the autophagy inhibitors 3-methyladenine and bafilomycin A1 or with beclin-1 small interfering RNA was able to enhance pterostilbene-triggered apoptosis. In conclusion, this study demonstrated that pterostilbene causes autophagy and apoptosis in lung cancer cells. Furthermore, pterostilbene in combination with autophagy inhibitors may strengthen the efficiency of chemotherapeutic strategies in both chemosensitive and chemoresistant lung cancer cells, which may be of immense value for the clinical management of lung cancer patients with MDR.

UPregulated single-stranded DNA-binding protein 1 induces cell chemoresistance to cisplatin in lung cancer cell lines.

Cisplatin and its analogues are widely used as anti-tumor drugs in lung cancer but many cisplatin-resistant lung cancer cases have been identified in recent years. Single-stranded DNA-binding protein 1 (SSDBP1) can effectively induce H69 cell resistance to cisplatin in our previous identification; thus, it is necessary to explore the mechanism underlying the effects of SSDBP1-induced resistance to cisplatin. First, SSDBP1-overexpressed or silent cell line was constructed and used to analyze the effects of SSDBP1 on chemoresistance of lung cancer cells to cisplatin. SSDBP1 expression was assayed by real-time PCR and Western blot. Next, the effects of SSDBP1 on cisplatin sensitivity, proliferation, and apoptosis of lung cancer cell lines were assayed by MTT and flow cytometry, respectively; ABC transporters, apoptosis-related genes, and cell cycle-related genes by real-time PCR, and DNA wound repair by comet assay. Low expression of SSDBP1 was observed in H69 cells, while increased expression in cisplatin-resistant H69 cells. Upregulated expression of SSDBP1 in H69AR cells was identified to promote proliferation and cisplatin resistance and inhibit apoptosis, while downregulation of SSDBP1 to inhibit cisplatin resistance and proliferation and promoted apoptosis. Moreover, SSDBP1 promoted the expression of P2gp, MRP1, Cyclin D1, and CDK4 and inhibited the expression of caspase 3 and caspase 9. Furthermore, SSDBP1 promoted the DNA wound repair. These results indicated that SSDBP1 may induce cell chemoresistance of cisplatin through promoting DNA repair, resistance-related gene expression, cell proliferation, and inhibiting apoptosis.

Sox2 inhibits Wnt-β-catenin signaling and metastatic potency of cisplatin-resistant lung adenocarcinoma cells

Lung cancer remains one of the most common cancer-associated mortalities worldwide, and platinum-based doublet chemotherapies are recommended as the first-line treatment for advanced non-small cell lung cancer (NSCLC). However, the frequent development of multidrug resistance, to cisplatin regimens in particular, is a major cause of chemotherapy failure in patients with aggressive NSCLC. Wnt/β-catenin signaling and sex-determining region Y box 2 (Sox2) have been implicated in the development and progression and resistance to epidermal growth factor receptor-targeting therapy in lung cancer. The present study aimed to explore the effects of Wnt/β-catenin and Sox2 signaling on the chemoresistance of cisplatin-resistant lung cancer cells by assessing the effects of Sox2 on Wnt/β-catenin signaling activity, cell migration, invasion and clonogenicity, and susceptibility to cisplatin in lung adenocarcinoma A549 cells and cisplatin-resistant A549/DDP cells. The results demonstrated that an enforced expression of Sox2 led to inhibition of Wnt/β-catenin signaling activity, potentially by upregulating glycogen synthase kinase 3 β in A549 and A549/DDP cells. An overexpression of Sox2 promoted cell migration and invasion, in addition to enhancing the clonogenic capacity in A549 cells. Notably, knockdown Sox2 using short hairpin RNA led to an enhanced susceptibility of A549 and A549/DDP cells to cisplatin, along with increased cell apoptosis. The present study thus suggests that Sox2 may be an important regulator in development of chemoresistance of lung cancer cells and may be a novel therapeutic target for treatment chemoresistant lung cancer.

P2.01-086 Luteolin is a Novel Target of Axl Receptor Tyrosine Kinase to Inhibit Cell Proliferation and Circumvent Chemoresistance in Lung Cancer Cells

Axl receptor tyrosine kinase (RTK) plays a critical role in cell growth, proliferation, and anti-apoptosis. In this study, we demonstrated the effect of luteolin, a non-toxic flavonoid widely found in various plants, on expression and activation of Axl RTK in NSCLC, H460, and its cisplatin-resistant cell, H460/CisR. 1. Cell viability measurement & Clonogenic assay. 2. Western blot analysis. 3. Promoter activity test. 4. Ectopic expression of Axl. 5. siRNA trasnfection for Axl knockdown. Luteolin treatment of H460 and H460/CisR cells was found to cause a dose-dependent decline of Axl protein as well as mRNA levels. Axl promoter activity was also reduced by luteolin, suggesting the transcriptional down-regulation of Axl expression by luteolin. Axl phosphorylation upon its ligand, Gas6, was inhibited by luteolin, indicating that luteolin also abrogates Gas6-induced Axl phosphorylation. Next, it was found that treatment of both H460 and H460/CisR cells with luteolin decreased the cell viability and clonogenic ability in dose-dependent manner. We further observed the synergistic anti-proliferative effect of luteolin in cells transfected with Axl specific siRNA, while the reduction of its cytotoxic effect in Axl RTK overexpressing cells, confirming that luteolin exerts its anticancer potential via interference of Axl expression. In addition, luteolin was found to result in the increase of p21, a cyclin-dependent kinase inhibitor, in H460 and H460/CisR cells. In summary, our data demonstrate that luteolin inhibits Axl expression and the activation which are associated with its anti-proliferative activity in both parental and cisplatin-resistant NSCLC cells. Thus, Axl seems to be a potent therapeutic target of luteolin to inhibit cell proliferation and to overcome chemoresistance of NSCLC cells.

Chemotherapy-Induced IL34 Enhances Immunosuppression by Tumor-Associated Macrophages and Mediates Survival of Chemoresistant Lung Cancer Cells.

The ability of tumor cells to escape immune destruction and their acquired resistance to chemotherapy are major obstacles to effective cancer therapy. Although immune checkpoint therapies such as anti-PD-1 address these issues in part, clinical responses remain limited to a subpopulation of patients. In this report, we identified IL34 produced by cancer cells as a driver of chemoresistance. In particular, we found that IL34 modulated the functions of tumor-associated macrophages to enhance local immunosuppression and to promote the survival of chemoresistant cancer cells by activating AKT signaling. Targeting IL34 in chemoresistant tumors resulted in a remarkable inhibition of tumor growth when accompanied with chemotherapy. Our results define a pathogenic role for IL34 in mediating immunosuppression and chemoresistance and identify it as a tractable target for anticancer therapy. Cancer Res; 76(20); 6030-42. ©2016 AACR.

Pellino-1 confers chemoresistance in lung cancer cells by upregulating cIAP2 through Lys63-mediated polyubiquitination.

Pellino-1 is an E3 ubiquitin ligase that mediates immune receptor signaling pathways. The role of Pellino-1 in oncogenesis of lung cancer was investigated in this study. Pellino-1 expression was increased in human lung cancer cell lines compared with non-neoplastic lung cell lines. Pellino-1 overexpression in human lung cancer cells, A549 and H1299 cells, increased the survival and colony forming ability. Pellino-1 overexpression in these cells also conferred resistance to cisplatin- or paclitaxel-induced apoptosis. In contrast, depletion of Pellino-1 decreased the survival of A549 and H1299 cells and sensitized these cells to cisplatin- and paclitaxel-induced apoptosis. Pellino-1 overexpression in A549 and H1299 cells upregulated the expression of inhibitor of apoptosis (IAP) proteins, including cIAP1 and cIAP2, while Pellino-1 depletion downregulated these molecules. Notably, Pellino-1 directly interacted with cIAP2 and stabilized cIAP2 through lysine63-mediated polyubiquitination via its E3 ligase activity. Pellino-1-mediated chemoresistance in lung cancer cells was dependent on the induction of cIAP2. Moreover, a strong positive correlation between Pellino-1 and the cIAP2 expression was observed in human lung adenocarcinoma tissues. Taken together, these results demonstrate that Pellino-1 contributes to lung oncogenesis through the overexpression of cIAP2 and promotion of cell survival and chemoresistance. Pellino-1 might be a novel oncogene and potential therapeutic target in lung cancer.

80P XRCC6BP1: A key DNA repair gene in platinum-resistant NSCLC

Background In the absence of specific treatable mutations, platinum-based doublet chemotherapy remains the gold standard treatment for NSCLC patients. However, its clinical efficacy is hindered in many patients due to both intrinsic and acquired resistance to these agents, in particular, cisplatin. Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. Methods DNA Repair Pathway RT2 Profiler Arrays were used to elucidate key DNA repair genes in H460 cisplatin resistant (CisR) and corresponding parental (PT) NSCLC cell lines previously generated in our laboratory. DNA repair genes significantly altered in CisR cells were validated using RT-PCR and western blot analysis, respectively. Loss of function studies were carried out using siRNA technology. The effect of gene knockdown on apoptosis was assessed by Annexin V/propidium iodide (PI) staining using the Cytell® Imaging System. DNA damage and repair in response to cisplatin following gene knockdown was investigated using the γH2AX foci formation assay. The translational relevance of these genes was examined in a cohort of chemo-naive matched normal and tumour lung tissues (n = 20). Results We identified a number of important DNA repair genes differentially regulated between H460 PT and CisR NSCLC cells. These included XRCC6BP1, TOP3A, XPA, PMS1 and hSSB1. XRCC6BP1 mRNA was significantly increased (19.4-fold) in H460 CisR cells relative to their PT counterparts. Gene silencing of XRCC6BP1 re-sensitized CisR lung cancer cells to the pro-apoptotic effects of cisplatin and significantly reduced the DNA repair capacity of these cells. Relative to matched normal lung tissues, XRCC6BP1 mRNA was significantly increased in adenocarcinoma tissues (AD), while hSSB1 mRNA was significantly increased in both AD and squamous cell carcinoma (SCC) tissues. Conclusions We have identified XRCC6BP1, in addition to hSSB1, as key DNA repair genes implicated in cisplatin resistant NSCLC. Our data highlights the potential of targeting XRCC6BP1, at least in part, in re-sensitizing chemoresistant lung cancer cells to the cytotoxic effects of cisplatin chemotherapy.

MicroRNA-7 Compromises p53 Protein-dependent Apoptosis by Controlling the Expression of the Chromatin Remodeling Factor SMARCD1

We previously demonstrated that the epidermal growth factor receptor (EGFR) up-regulated miR-7 to promote tumor growth during lung cancer oncogenesis. Several lines of evidence have suggested that alterations in chromatin remodeling components contribute to cancer initiation and progression. In this study, we identified SMARCD1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 1) as a novel target gene of miR-7. miR-7 expression reduced SMARCD1 protein expression in lung cancer cell lines. We used luciferase reporters carrying wild type or mutated 3'UTR of SMARCD1 and found that miR-7 blocked SMARCD1 expression by binding to two seed regions in the 3'UTR of SMARCD1 and down-regulated SMARCD1 mRNA expression. Additionally, upon chemotherapy drug treatment, miR-7 down-regulated p53-dependent apoptosis-related gene BAX (BCL2-associated X protein) and p21 expression by interfering with the interaction between SMARCD1 and p53, thereby reducing caspase3 cleavage and the downstream apoptosis cascades. We found that although SMARCD1 sensitized lung cancer cells to chemotherapy drug-induced apoptosis, miR-7 enhanced the drug resistance potential of lung cancer cells against chemotherapy drugs. SMARCD1 was down-regulated in patients with non-small cell lung cancer and lung adenocarcinoma cell lines, and SMARCD1 and miR-7 expression levels were negatively correlated in clinical samples. Our investigation into the involvement of the EGFR-regulated microRNA pathway in the SWI/SNF chromatin remodeling complex suggests that EGFR-mediated miR-7 suppresses the coupling of the chromatin remodeling factor SMARCD1 with p53, resulting in increased chemo-resistance of lung cancer cells.

Mucin 1-mediated chemo-resistance in lung cancer cells.

Paclitaxel (PTX) is a commonly used drug to treat diverse cancer types. However, its treatment can generate resistance and the mechanisms of PTX-resistance in lung cancers are still unclear. We demonstrated that non-small cell lung cancers (NSCLCs) survive PTX treatment. Compared with the progenitor NSCLC A549 cells, the PTX-resistant A549 cells (A549/PTX) displayed enhanced sphere-formation ability. The proportion of the cancer stem cell marker, aldehyde dehydrogenase-positive cells, and epithelial–mesenchymal transition signaling protein levels were also elevated in A549/PTX. Importantly, the levels of oncoproteins phosphoinositide-3 kinase/Akt, mucin 1 cytoplasmic domain (MUC1-C) and β-catenin were also significantly elevated in A549/PTX. Furthermore, nuclear translocation of MUC1-C and β-catenin increased in A549/PTX. The c-SRC protein, an activator of MUC1-C, was also overexpressed in A549/PTX. These observations led to the hypothesis that enhanced expression of MUC1-C is associated with stemness and PTX resistance in NSCLCs. To test this, we knocked down or overexpressed MUC1-C in A549/PTX and found that inhibition of MUC1-C expression coupled with PTX treatment was sufficient to reduce the sphere-forming ability and survival of A549/PTX. In summary, our in vitro and in vivo studies have revealed a potential mechanism of MUC1-C-mediated PTX resistance and provided insights into a novel therapeutic measure for lung cancers.

ERK3 regulates TDP2-mediated DNA damage response and chemoresistance in lung cancer cells.

Posttranslational modifications (PTMs), such as phosphorylation and ubiquitination, play critical regulatory roles in the assembly of DNA damage response proteins on the DNA damage site and their activities in DNA damage repair. Tyrosyl DNA phosphodiesterase 2 (TDP2) repairs Topoisomerase 2 (Top2)-linked DNA damage, thereby protecting cancer cells against Top2 inhibitors-induced growth inhibition and cell death. The regulation of TDP2 activity by post-translational modifications in DNA repair, however, remains unclear. In the current study, we have found that ERK3, an atypical MAPK, phosphorylates TDP2 at S60 and regulates TDP2's phosphodiesterase activity, thereby cooperatively protecting lung cancer cells against Top2 inhibitors-induced DNA damage and growth inhibition. As such, our study revealed a post-translational regulation of TDP2 activity and discovered a new role of ERK3 in increasing cancer cells’ DNA damage response and chemoresistance to Top2 inhibitors.

Abstract 1841: A miR551b/catalase/MUC1/EGFR cascade for acquired apoptosis resistance and chemoresistance in lung cancer cells

Abstract The oncoprotein Mucin-1 (MUC1) plays multiple roles in the development of different human tumors. However, whether MUC1 is involved in acquired chemoresistance, a major challenge in cancer therapy, has not been determined. Using a lung cancer cell model with acquired apoptosis resistance (AR)-associated chemoresistance, we investigated the role and mechanism of MUC1 in acquired chemoresistance. MUC1 expression was substantially increased in cells with acquired apoptosis resistance and knockdown of MUC1 expression effectively increased sensitivity of the AR cells to apoptotic cytotoxicity induced by anticancer therapeutics, substantiating a role for MUC1 in acquired chemoresistance. Decreased hydrogen peroxide-reducing enzyme catalase and increased cellular reactive oxygen species (ROS) were found to be associated with MUC1 overexpression. Pharmacological ROS scavengers or exogenous catalase dramatically suppressed MUC1 expression, suggesting catalase suppression-mediated ROS accumulation contributes to MUC1 overexpression. Further, we found miR-551b expression was increased in the AR cells. Knockdown of miR-551b restored catalase expression, and suppressed ROS accumulation and MUC1 expression. Finally, with manipulation of MUC1 expression, we found MUC1 promotes EGFR-mediated activation of the cell survival Akt/c-FLIP/Cox2/Mcl-1 cascade and stabilization of anti-apoptosis factors c-IAP1 and c-IAP2. Thus, our results establish a role for MUC1 in acquired chemoresistance and a pathway consisting of miR-551b/catalase/ROS for MUC1overexpression, and interventions against this pathway may be exploited for overcoming acquired chemoresistance. Citation Format: Xiuling Xu, Alexandria Wells, Mabel T. Padilla, Kwang Chul Kim, Yong Lin. A miR551b/catalase/MUC1/EGFR cascade for acquired apoptosis resistance and chemoresistance in lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1841. doi:10.1158/1538-7445.AM2014-1841

Reversal of Cisplatin Resistance by Epigallocatechin Gallate Is Mediated by Downregulation of Axl and Tyro 3 Expression in Human Lung Cancer Cells

Lung cancer is still the number one cause of death from cancer worldwide. The clinical effect of platinum-based chemotherapy for non-small cell lung cancer is constrained by the resistance to drug. To overcome chemo-resistance, various modified treatment including combination therapy has been used, but overall survival has not been improved yet. In this study, chemo-resistant lung cancer cells, A549/Cis and H460/Cis, were developed by long-term exposure of cells to cisplatin and the proliferative capability of these resistant cells was verified to be reduced. We found cytotoxic effect of epigallocatechin gallate (EGCG), a major catechin derived from green tea, on both the parental lung cancer cells, A549 and H460, and their cisplatin resistant cells, A549/Cis and H460/Cis. ELISA and Western blot analysis revealed that EGCG was able to increase interlukine-6 (IL-6) production per cell, whereas its downstream effector Signal transducers and activators of transcription 3 (STAT3) phosphorylation was not changed by EGCG, indicating that IL-6/STAT3 axis is not the critical signaling to be inhibited by EGCG. We next found that EGCG suppresses the expression of both Axl and Tyro 3 receptor tyrosine kinases at mRNA and protein level, explaining the cytotoxic effect of EGCG on lung cancer cells, especially, regardless of cisplatin resistance. Taken together, these data suggest that EGCG impedes proliferation of lung cancer cells including their chemo-resistant variants through downregulation of Axl and Tyro 3 expression.

A Combination of Pterostilbene With Autophagy Inhibitors Exerts Efficient Apoptotic Characteristics in Both Chemosensitive and Chemoresistant Lung Cancer Cells

The emergence of multidrug resistance (MDR), meaning that cancer cells develop simultaneous resistance to different drugs, has limited the clinical efficacy and application of chemotherapy. Pterostilbene, a naturally occurring phytoalexin exerts a variety of pharmacologic activities, including cancer prevention, cytotoxicity, and antioxidant activity. In this study, results proved the capability of pterostilbene to effectively inhibit the cell viability of docetaxel-induced MDR human lung cancer cell lines through cell cycle arrest and apoptosis. Meanwhile, the observation of LC3-II production and formation of acidic vesicular organelles revealed an induction of autophagy at an early stage by pterostilbene, which was triggered by an inhibition of the AKT and JNK pathways and activation of ERK1/2. Furthermore, pretreatment with the autophagy inhibitors 3-methyladenine and bafilomycin A1 or with beclin-1 small interfering RNA was able to enhance pterostilbene-triggered apoptosis. In conclusion, this study demonstrated that pterostilbene causes autophagy and apoptosis in lung cancer cells. Furthermore, pterostilbene in combination with autophagy inhibitors may strengthen the efficiency of chemotherapeutic strategies in both chemosensitive and chemoresistant lung cancer cells, which may be of immense value for the clinical management of lung cancer patients with MDR.