Berberine enhances cisplatin sensitivity in non-small cell lung cancer H1299 cells via modulation of the p38-MAPK signaling pathway

Advanced glycosylation end-product specific receptor (AGER) is a multi-ligand cell surface receptor abnormally expressed in lung cancer, and is a member of the immunoglobulin superfamily. Therefore, this study aimed to explore the effect of AGER on the biological behavior of non-small cell lung cancer (NSCLC) H1299 cell line. A microarray-based gene expression profiling analysis of the GSE27262 dataset from the Gene Expression Omnibus (GEO) database was conducted to identify differentially expressed genes, which were verified using The Cancer Genome Atlas (TCGA) database. The expression of AGER in the normal human lung BEAS-2B cell line and NSCLC H1299 cell line was examined using reverse transcription-quantitative PCR. Lentiviral interference and overexpression vectors of AGER were constructed and transfected into H1299 cells using Lipofectamine®. AGER expression and biological properties, including cell viability, apoptosis, migration and invasion abilities, in H1299 cells were investigated using MTT, flow cytometry, wound healing and Transwell assays. AGER was expressed at a low level in NSCLC tissues and H1299 cells (P<0.05). Compared with control cells, AGER overexpression cells displayed decreased cell viability, proliferation, migration and invasion abilities, and significantly increased levels of apoptosis. Furthermore, AGER overexpression increased the expression of Bax and decreased the expression of Bcl-2 in H1299 cells (P<0.05), and AGER knockdown displayed the opposite effects on H1299 cells. Therefore, AGER overexpression decreased the proliferation, invasion and migration abilities of H1299 cells, and increased apoptosis. The present study suggested that AGER might serve as a potential molecular marker for NSCLC.

Background/Aims: Caffeic acid (CA) is known to possess multiple biological activities including anti-cancer activities. However, the molecular mechanisms underlying these activities in non-small-cell lung cancer (NSCLC) cells are not fully understood. We attempted to clarify whether CA could enhance paclitaxel (PTX)-induced cytotoxicity in H1299 cells. Methods: First, we tested the cytotoxic effects in both H1299 cells and normal human Bease-2b cells by cell proliferation experiments. Next, we use Annexin V/propidium iodide apoptosis analysis and flow cytometric analysis to investigate apoptosis and cell cycle arrest under the treatments mentioned above. To further pinpoint changes in apoptosis, we tested the caspase-associated apoptotic pathway, which involves the activities of caspase-3 and caspase-9. Moreover, apoptosis-related proteins and MAPK pathway proteins were examined by western blot. An H1299 xenograft nude mice model was used to further evaluate the tumor-suppressing effects of CA and PTX in vivo. Results: Combination treatment with low-dose CA and PTX decreased the proliferation of NSCLC H1299 cells but not normal Beas-2b cells. Flow cytometry showed that H1299 cells were arrested in the sub-G1 phase and apoptosis was significantly increased in H1299 cells after CA treatment. Caspase-3 and caspase-9 activities were both increased after CA treatment. Furthermore, CA increased the PTX-induced activation of Bax, Bid, and downstream cleaved PARP, and phosphorylation of extracellular signal regulated kinase1/2 and c-Jun NH2-terminal protein kinase1/2. An in vivo tumor-suppression assay demonstrated that CA and PTX combined treatment exerted a more effective suppressive effect on tumor growth in H1299 xenografts without causing significant adverse effects. Conclusions: Our results indicated that CA inhibited NSCLC H1299 cell growth by inducing apoptosis and CA and PTX combined produced a synergistic anti-cancer effect in H1299 cells.

Derlin-1 Is Overexpressed in Non-Small Cell Lung Cancer and Promotes Cancer Cell Invasion via EGFR-ERK–Mediated Up-Regulation of MMP-2 and MMP-9

BackgroundLung cancer is the major cause of cancer-related death worldwide, and 80% patients of lung cancer are non-small-cell lung cancer (NSCLC) cases. MicroRNAs are important gene regulators with critical roles in diverse biological processes, including tumorigenesis. Studies indicate that sphingosine kinase 2 (SphK2) promotes tumor progression in NSCLC, but how this occurs is unclear. Thus, we explored the effect of miR-338-3p targeting SphK2 on proliferation and apoptosis of NSCLC cells.MethodsExpression of miR-338-3p and SphK2 in NSCLC A549 and H1299 cell lines was measured using qRT-PCR and Western blot. CCK-8 and colony formation assays were used to assess the effect of miR-338-3p on NSCLC cell line proliferation. Flow cytometry was used to study the effect of miR-338-3p on NSCLC apoptosis. Luciferase reporter assay and Western blot were used to confirm targeting of SphK2 by miR-338-3p. Finally, in vivo tumorigenesis studies were used to demonstrate subcutaneous tumor growth.ResultsmiR-338-3p expression in 34 NSCLC clinical samples was downregulated and this was correlated with TNM stage. miR-338-3p significantly suppressed proliferation and induced apoptosis of NSCLC A549 and H1299 cells in vitro. SphK2 was a direct target of miR-338-3p. Overexpression of miR-338-3p significantly inhibited SphK2 expression and reduced luciferase reporter activity containing the SphK2 3′-untranslated region (3′-UTR) through the first binding site. SphK2 lacking 3′-UTR restored the effects of miR-338-3p on cell proliferation inhibition. miR-338-3p significantly inhibited tumorigenicity of NSCLC A549 and H1299 cells in a nude mouse xenograft model.ConclusionsCollectively, miR-338-3p inhibited cell proliferation and induced apoptosis of NSCLC cells by targeting and down-regulating SphK2, and miR-338-3p could inhibit NSCLC cells A549 and H1299 growth in vivo, suggesting a potential mechanism of NSCLC progression. Therapeutically, miR-338-3p may serve as a potential target in the treatment of human lung cancer.

Ulmus davidiana Nakai induces apoptosis and autophagy on non-small cell lung cancer cells

There is increasing evidence that microRNAs (miRNAs) are able to play a key role in the diagnosis and therapy of cancer. miRNA-99a (miR-99a), which is downregulated in several human malignancies, has been reported as a potential tumor suppressor. However, to the best of our knowledge, the expression and function of miR-99a has not been investigated in human non-small cell lung cancer (NSCLC) at present. The aim of the current study was to evaluate the association between NSCLC and miR-99a. miR-99a expression was analyzed in 15 pairs of NSCLC and non-cancerous tissue samples by reverse transcription-quantitative polymerase chain reaction. In addition, the NSCLC A549 and H1299 cell lines were transfected with miR-99a mimics, and the effect of miR-99a on the cell cycle, cell proliferation, migration and colony formation of A549 and H1299 cells was investigated. It was found that the level of miR-99a expression was significantly downregulated in NSCLC tissues and that ectopic overexpression of miR-99a significantly inhibited the growth of A549 and H1299 cells. Additionally, ectopic overexpression of miR-99a inhibited A549 and H1299 cell migration and invasion by inhibiting epithelial to mesenchymal transition. The downregulation of insulin-like growth factor 1 receptor (IGF-1R) by miR-99a and knockdown of IGF-1R mediated by siRNA were each found to phenocopy the effect of miR-99a overexpression in NSCLC. To the best of our knowledge, the present study demonstrated for the first time that, in NSCLC, miR-99a is downregulated and thus regulates proliferation, colony formation and migration through the IGF-1R pathway, which indicates that miR-99a is a diagnostic biomarker for NSCLC.

DMH4, a VEGFR2 inhibitor, effectively suppresses growth and invasion of lung cancer cells

Although protein kinase C (PKC) has been widely implicated in the positive and negative control of proliferation, the underlying cell cycle mechanisms regulated by individual PKC isozymes are only partially understood. In this report, we show that PKCdelta mediates phorbol ester-induced G1 arrest in lung adenocarcinoma cells and establish an essential role for this novel PKC in controlling the expression of the cell cycle inhibitor p21. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) in early G1 phase impaired progression of lung adenocarcinoma cells into S phase, an effect that was completely abolished by specific depletion of PKCdelta, but not PKCalpha. Although the PKC effect was unrelated to the inhibition of cyclin D1 expression, PKC activation significantly up-regulated p21 and down-regulated Rb hyperphosphorylation and cyclin A expression. Elevations in p21 mRNA and protein by PMA were mediated by PKCdelta but not PKCalpha. Studies using luciferase reporters also revealed an essential role for PKCdelta in the PMA-induced inhibition of Rb-dependent cyclin A promoter activity. Finally, we showed that the cell cycle inhibitory effect of PKCdelta is greatly attenuated by RNA interference-mediated knock-down of p21. Our results identify a novel link between PKCdelta and G1 arrest via p21 up-regulation and highlight the complexities in the downstream effectors of PKC isozymes in the context of cell cycle progression and proliferation.

Lung cancer is the most frequently diagnosed malignancy and leading cause of cancer-related deaths worldwide. The 5-year survival rate of 14% has shown little improvement over the last 30 years even with the development of molecularly targeted therapies. Non-small-cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers, suggesting that additional approaches and strategies are needed to control this malignancy. For the last few decades, major emphasis has also been placed on identifying natural dietary and/or non-dietary products for both prevention and intervention of several malignancies including lung cancer. One such naturally occurring polyphenolic product is grape seed extract (GSE); which is especially rich in proanthocyanidins. In recent studies, GSE has shown promising cancer chemo-preventive and anti-cancer effects against various epithelial cancers. Accordingly, here we evaluated the efficacy of GSE against a panel of human NSCLC cell lines, namely A549, H460, H1299 and H322 cells. GSE (50-100 µg/ml) treatment for 24 and 48 hrs significantly suppressed cell proliferation and induced cell death in all the four cell lines studied; however, H460 and H322 cells were more sensitive to GSE treatment even at comparatively lower doses (20-50 µg/ml). Additional studies showed that GSE causes a dose- and a time-dependent apoptotic cell death in A549, H460, H322 and H1299 cells which was confirmed by the Annexin V-PI staining, activation of caspase-3 and −9, and cleavage of PARP. In detailed mechanistic studies, to determine the cause of GSE-induced apoptotic cell death, pre-treatment of cells with N-acetylcysteine (NAC) strongly inhibited GSE-caused apoptotic cell death, suggesting the involvement of reactive oxygen species (ROS). Interestingly, GSE treatment also resulted in a strong dose- and time-dependent phosphorylation of ERK1/2 in these cell lines, which was inhibited by pre-treatment of cells with NAC, further supporting the role of GSE-caused oxidative stress in the activation of ERK1/2. Based on these results, next we assessed whether GSE causes oxidative stress in these NSCLC cell lines. Dihydroethidium staining followed by flow cytometry analysis of cells showed that indeed GSE treatment results in ROS generation. Next we assessed the intracellular reduced glutathione levels, which were decreased by 82% and 60% in A549 and H460 cells, respectively, after GSE treatment. Together, these results confirm that GSE generates ROS causing oxidative stress that leads to apoptotic death in NSCLC cells. Based on these findings, more studies are needed in future to evaluate both anti-cancer and chemopreventive efficacy of GSE in relevant pre-clinical lung cancer models to establish its potential usefulness against human lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5687.

BACKGROUND: Cisplatin and tyrosine kinase inhibitors (TKIs) are recommended to treat non-small-cell lung cancer (NSCLC). However, ubiquitous acquired drug resistance in NSCLC patients diminishes their therapeutic efficacy. Overcome cisplatin and TKIs resistance is an unmet medical need. DZ-191, a novel synthetic statin derivative, is capable of targeting specifically tumor cells, bypassing the liver. We observed this agent inhibited tumor cell growth in vitro, tumor growth in vivo and accelerated tumor regression in mice. METHODS: Cell viability was examined by crystal violet assay in human NSCLC A549, A549DDP, H1650, and H1975 cells. DZ-191-induced apoptosis in NSCLC cells was detected by Annexin V-FITC/PI staining followed by FACS and confirmed with western blot of apoptosis-associated proteins. Mitochondrial membrane potential was determined by rhodamine-123 followed by flow cytometry. H1650 tumor growth in mice was assessed and autophagy markers were analyzed by western blotting. RESULTS: Treatment with DZ-191 alone inhibited the growth of A549, H1650 and H1975 NSCLC cells, with IC50 values in the range of 8±1μM. In the cisplatin-resistant A549DDP cells, DZ-191 exposure at the IC10, markedly sensitized the cisplatin-resistant A549DDP cells by lowering its IC50 value from 87.6±1.0 to 13.6±1.2 μM. We observed DZ-191, but not statins, significantly increased the apoptosis of A549DDP and H1650 cells. These results are consistent with the observation that DZ191, but not statins, accumulates in mitochondria and lysosomes, resulting in depolarized mitochondrial membrane potential, decreased autophagy and lysosomal protein degradation. We observed decreased autophagy markers, assessed by decreased LC3 conversion and increased p62 accumulation. Combining with our RNA-seq data, DZ191 could induce NSCLC cell death by reducing the removal of damaged mitochondria through mitophagy. Furthermore, we observed DZ-191, but not statins, could resensitize anti-tumor responses of geftinib in a TKI-resistant NSCLC tumor xenografts in nude mice. CONCLUSIONS: These results demonstrated that DZ-191 is superior to statins in inhibiting and resensitizing the anti-tumor responses of cisplatin- and geftinib-resistant NSCLC cells by targeting mitochondria-mediated autophagy and downstream lysosome-related protein degradation. DZ-191 can be employed as a promising sensitizer to overcome cisplatin- and geftinib-resistance in NSCLC patients. Key words: DZ-191, statins, cisplatin- geftinib-resistance, autophagy, NSCLC, mitochondria, lysosomes This work is support in part by US NCI P01 CA098912 and a Board of Governors Chair of Cancer Research fund from Cedars-Sinai Medical Center to LWK Chung and China National 863 Lung Cancer Research Grant (2012AA02A502) and International Technology Targeted Therapeutics Grant (2016YEE0103400) to Q. Zhou Citation Format: Liyuan Yin, Gina Chia-Yi Chu, Ruoxiang Wang, Anisha Madhav, Lijuan Yin, Neil Bhowmick, Haiyen E. Zhau, Qinghua Zhou, Leland W.K. Chung. A novel mitochondria-based targeting to restore therapeutic responsiveness in cisplatin- and geftinib-resistant human lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1267.

Paclitaxel is one of the chemotherapeutic drugs widely used for the treatment of nonsmall cell lung cancer (NSCLC) patients. Here, we tested the ability of alpha-tocopheryl succinate (TOS), another promising anticancer agent, to enhance the paclitaxel response in NSCLC cells. We found that sub-apoptotic doses of TOS greatly enhanced paclitaxel-induced growth suppression and apoptosis in the human H460 NSCLC cell lines. Our data revealed that this was accounted for primarily by an augmented cleavage of poly(ADP-ribose) polymerase (PARP) and enhanced activation of caspase-8. Pretreatment with z-VAD-FMK (a pan-caspase inhibitor) or z-IETD-FMK (a caspase-8 inhibitor) blocked TOS/paclitaxel cotreatment-induced PARP cleavage and apoptosis, suggesting that TOS potentiates the paclitaxel-induced apoptosis through enforced caspase 8 activation in H460 cells. Furthermore, the growth suppression effect of TOS/paclitaxel combination on human H460, A549 and H358 NSCLC cell lines were synergistic. Our observations indicate that combination of paclitaxel and TOS may offer a novel therapeutic strategy for improving paclitaxel drug efficacy in NSCLC patient therapy as well as for potentially lowering the toxic side effects of paclitaxel through reduced drug dosage.

There is increasing evidence that functional crosstalk between GPCRs and EGFR contributes to the progression of colon, lung, breast, ovarian, prostate and head and neck tumors. In this study, we performed multiple analyses of GPCR expression in a gefitinib-resistant non-small cell lung cancer (NSCLC) cell line, H1975, which harbors an L858R/T790M mutation. To determine the expression profile of mRNAs encoding 384 GPCRs in normal human lung fibroblast (NHLF) and H1975 cells, a GPCR-specific microarray analysis was performed. A heat-map of the microarray revealed considerable differences in the expression of GPCRs between NHLF and H1975 cells. From the GPCR expression list, we selected some GPCR agonists/antagonist to investigate whether the respective ligands could affect the growth of H1975 cells. Among them, treatment with either a selective antagonist of adenosine A2a receptors, which were highly expressed in H1975 cell and another gefitinib-resistant NSCLC cells, HCC827GR cells or “small interfering RNA” (siRNA) targeting adenosine A2a receptors produced a significant decrease in cell viability of both H1975 and HCC827GR cells. Among up-regulated GPCRs in H1975 cells, Gs-, Gi- and Gq-coupled GPCRs were expressed almost equally. Among down-regulated GPCRs, Gi-coupled GPCRs were dominantly expressed in H1975 cells. The present results suggest that multilayered crosstalk between GPCRs and EGFR may play an important role in orchestrating downstream signaling molecules that are implicated in the development of gefitinib-resistant NSCLC.

Background: Mutations in the EGFR kinase are common in NSCLC. Patients with first-line EGFR mutations (exon 19 deletions, L858R mutations, exon 20 variants, codon 719 variants) initially respond to the reversible EGFR inhibitors erlotinib and gefitinib, but then relapse as additional mutations emerge. EGFR T790M is the most common of these mutations and is found in &amp;gt; 50% of refractory tumors. We assessed the in vitro and in vivo activity of CPD 24, a newly developed, covalent, small-molecule inhibitor of both first-line and T790M mutant EGFR, but not WT EGFR. Inhibition of WT EGFR in normal tissues can be a source of dose-limiting toxicities. Methods: The effects of CPD 24 were evaluated on H1975 (EGFR T790M/L858R), HCC827 (EGFR exon 19 deletion), and A431 (WT) cell lines. Inhibition of EGFR phosphorylation was measured in serum-starved cells using an MSD assay. Viability was assessed by measuring ATP in cells using a CellTiter-Glo® assay. Dose- and time-dependent pharmacodynamic effects were evaluated by treating mice bearing matrigel plugs containing H1975 cells. Drug levels were measured in plasma samples by LC-MS. CPD 24 was administered to athymic nude mice bearing H1975, HCC827, or A431 xenografts (3, 10, and 30 mg/kg, QD, PO) and tumor growth was measured twice per week with digital calipers. CPD 24 was also formulated in PLGA microspheres and administered subcutaneously (SC) as a sustained release formulation to mice bearing H1975 or A431 xenografts (30 and 100 mg/kg, Q3D, SC). Proliferation of H1975 and HCC827 cells in the presence of CPD 24, growth factors, and a MET inhibitor was measured with an IncuCyte live-cell imaging system. Results: The IC50 of CPD 24 for p-EGFR inhibition was 4 nM on L858R/T790M mutant EGFR (H1975 cells), 17 nM on the exon 19 deletion EGFR (HCC827 cells), and 510 nM on WT EGFR (A431 cells). CPD 24 inhibited the growth of tumor cells in vitro and led to both dose- and time-dependent p-EGFR inhibition in vivo. Phosphorylation of EGFR in H1975 cells growing in matrigel plugs was inhibited by &amp;gt; 75% for 12 hours by a single 30 mg/kg dose. Tumor growth inhibition (TGI) was observed in two EGFR mutant NSCLC xenograft models (H1975, 89% TGI; HCC827, 139% TGI) but not in a WT EGFR model (A431). CPD 24 formulated in PLGA microspheres achieved drug levels above the IC50 for 72 hours after a single SC dose. Tumor regression was observed with this formulation in H1975 xenografts but not in WT tumors. HGF was able to confer resistance to CPD 24 inhibition in both H1975 and HCC827 cells in vitro. This resistance was prevented by the addition of a selective MET inhibitor. Conclusion: CPD 24 is a covalent inhibitor of both first-line and T790M mutant EGFR while maintaining &amp;gt; 100-fold selectivity over WT EGFR. The antiproliferative effects of CPD 24 can be reversed by HGF, and this resistance can be prevented by treatment with a MET inhibitor. Citation Format: Pedro J. Beltran, Jinghui Zhan, Petia Mitchell, Ryan P. Wurz, Liping Pettus, Tian Wu, Mary Chaves, Darren L. Reid, Robert Radinsky, Keegan Cooke, Andrew Tasker. A novel covalent inhibitor of mutant but not wild-type (WT) epidermal growth factor receptor (EGFR) has activity in vitro and in vivo in non-small cell lung cancer (NSCLC) models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2587. doi:10.1158/1538-7445.AM2015-2587

MKP1 repression is required for the chemosensitizing effects of NF-κB and PI3K inhibitors to cisplatin in non-small cell lung cancer

Mitotic slippage, which enables cancer cells to bypass cell death by transitioning from mitosis to the G1 phase without undergoing normal cytokinesis, is one likely mechanism of paclitaxel (PTX) resistance. DNA double-strand breaks (DSBs) in the G1 phase are mainly repaired through non-homologous end joining (NHEJ). Therefore, inhibiting NHEJ could augment the PTX-induced cytotoxicity by impeding the repair of PTX-induced DSBs during the G1 phase following mitotic slippage. We aimed to evaluate the effects of NHEJ inhibition on mitotic slippage after PTX treatment in non-small cell lung cancer (NSCLC). H1299, A549, H1975, and H520 NSCLC cell lines were employed. In addition, A-196 and JQ1 were used as NHEJ inhibitors. H1299 cells were PTX-resistant and exhibited an increased frequency of mitotic slippage upon PTX treatment. NHEJ inhibitors significantly augmented the PTX-induced cytotoxicity, DSBs, and apoptosis in H1299 cells. The newly generated PTX-resistant cells were even more prone to mitotic slippage following PTX treatment and susceptible to the combined therapy. Docetaxel further demonstrated synergistic effects with the NHEJ inhibitor in PTX-resistant cells. NHEJ inhibition may overcome intrinsic or acquired PTX resistance resulting from mitotic slippage by synergistically increasing the cytotoxic effects of antimitotic drugs in NSCLC.