Migration Of H1299 Cells Research Articles

Glycine max Merr. leaf extract possesses anti-oxidant properties, decreases inflammatory mediator production in murine macrophages, and inhibits growth, migration, and adhesion in human cancer cells.

The present study aimed to investigate the in vitro anti-oxidant, anti-inflammatory, and anticancer properties of the ethanol extract of soybean (Glycine max Merr.) leaves (SLE). The total polyphenol and flavonoid levels were 142.0±14.0mg gallic acid equivalent/g and 104.9±2.0 mg quercetin equivalent/g, respectively. The radical scavenging activity and ferric-reducing anti-oxidant power of SLE at the concentrations of 125-500 μg/mL were 5-61%. In lipopolysaccharide-treated RAW 264.7 macrophages, treatment with SLE at concentrations of 62.5-500 μg/mL dose-dependently decreased the production of nitric oxide and prostaglandin E2. In both HCT116 human colon cancer cells and H1299 human lung cancer cells, treatment with SLE inhibited the growth and anchorage-independent colony formation. SLE was also effective in inhibiting the migration of H1299 cells and the adhesion of both HCT116 and H1299 cells. These results suggest that SLE exerts anti-oxidant, antiinflammatory, and anti-cancer activities in vitro. It needs to be determined whether similar effects are reproduced in vivo.

Anti-Metastatic Effect of Dehydrocorydaline on H1299 Non-Small Cell Lung Carcinoma Cells via Inhibition of Matrix Metalloproteinases and B Cell Lymphoma 2.

Though Dehydrocorydaline, an alkaloid isolated from Corydalis turtschaninovii tuber, was known to have anti-coronary artery disease, anti-inflammatory, apoptotic, anti-allergic, anti-acetylcholinesterase, and antitumor effects, the underlying anti-metastatic mechanism of Dehydrocorydalin was never elucidated in lung cancer cells so far. Thus, in the present study, the anti-metastatic effect of Dehydrocorydaline was examined in non-small cell lung carcinoma (NSCLC) cells, mainly targeting matrix metalloproteinases (MMPs) and B cell lymphoma-2 (Bcl-2) signaling. Here, Dehydrocorydaline exerted weak cytotoxicity and attenuated the protein expression of Bcl-2 and activated Bax in a concentration-dependent manner in NSCLC cells, such as A549, H460, H1299, and H596 cells. Also, Dehydrocorydaline suppressed the migration of H1299 cells by wound healing assay and transwell migration assay. Consistently, Dehydrocorydaline attenuated mRNA and protein levels of MMP7 and MMP9 as metastasis biomarkers in H1299 cells by quantitative reverse transcription polymerase chain reaction. Of note, Bcl-2 overexpression reduced the cytotoxic and anti-metastatic effects of Dehydrocorydaline on pCDNA-Bcl-2 transfected H1299 cells. Overall, our findings provide scientific evidence that Dehydrocorydaline exerts anti-metastatic potential via suppression of MMPs and Bcl-2 signaling in NSCLC cells. Copyright © 2017 John Wiley & Sons, Ltd.

Fibroblast growth factor 18 promotes proliferation and migration of H460 cells via the ERK and p38 signaling pathways.

Recently, fibroblast growth factor 18 (FGF18) expression was reported to be upregulated in colon cancer and ovarian cancer, and increased expression of FGF18 mRNA and protein is associated with tumor progression and poor overall survival in patients; however, its role in lung cancer remains to be explored. In the present study, the effect and underlying molecular mechanisms of FGF18 on H460 cells were investigated. Cell proliferation and cell cycle alterations were detected using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and flow cytometry. A wound healing assay was conducted to detect cell migration. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to measure extracellular signal-regulated kinase (ERK), p38 and matrix metalloproteinase26 (MMP26) expression. Knockdown of FGF18 using short interfering RNA (siRNA-FGF18) suppressed H460 cell proliferation, inhibited cell migration via the downregulation of MMP26 levels, with siRNA-FGF18 additionally inhibiting the ERK and p38 signaling pathway. The present study indicates that FGF18 serves an essential role in the growth and migration of non-small cell lung cancer (NSCLC) cells by regulating the ERK, p38 signaling pathways and MMP26 protein levels, suggesting that FGF18 may be a potential molecular drug target for the treatment NSCLC.

Inhibitory effect of trans-ferulic acid on proliferation and migration of human lung cancer cells accompanied with increased endogenous reactive oxygen species and β-catenin instability.

BackgroundTrans-ferulic (FA) acid exhibits antioxidant effects in vitro. However, the underlying mechanism of trans-FA activity in cellular physiology, especially cancer physiology, remains largely unknown. This study investigated the cellular physiological effects of trans-FA on the H1299 human lung cancer cell line.MethodsThe 2,2-diphenyl-1-picrylhydrazyl assay was used to determine free radical scavenging capability. Assessment of intracellular reactive oxygen species (ROS) was evaluated using oxidized 2ʹ,7ʹ-dichlorofluorescin diacetate and dihydroethidium staining. Trypan blue exclusion, colony formation, and anchorage-independent growth assays were used to determine cellular proliferation. Annexin V staining assay was used to assess cellular apoptosis by flow cytometry. Wound healing and Boyden’s well assays were used to detect the migration and invasion of cells. Gelatin zymography was used to detect matrix metalloproteinase (MMP-2 and MMP-9) activity. Western blotting was used to detect expression levels of various signaling pathway proteins.ResultsDPPH assay results indicated that trans-FA exerted potent antioxidant effects. However, trans-FA increased intracellular ROS levels, including hydrogen peroxide and superoxide anion, in H1299 cells. Trans-FA treatment inhibited cellular proliferation and induced moderate apoptotic cell death at the highest concentration used (0.6 mM). Furthermore, trans-FA moderately inhibited the migration of H1299 cells at the concentrations of 0.3 and 0.6 mM and attenuated MMP-2 and MMP-9 activity. Trans-FA caused the phosphorylation of β-catenin, resulting in proteasomal degradation of β-catenin. Conversely, trans-FA treatment increased the expression of pro-apoptotic factor Bax and decreased the expression of pro-survival factor survivin.ConclusionVarious concentrations (0.06–0.6 mM) of trans-FA exert both anti-proliferation and anti-migration effects in the human lung cancer cell line H1299.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0116-7) contains supplementary material, which is available to authorized users.

Effect of metastasis suppressor1 on H1299 cells and its clinical significance in non-small cell lung cancer.

The present study aimed to investigate the effect of metastasis suppressor1 (MTSS1) on the proliferation, migration and invasion of human H1299 non-small cell lung cancer cells and its clinical significance in non‑small cell lung cancer. The target gene MTSS1-overexpressing lentivirus (LV-MTSS1) was transfected into H1299 cells and expression of MTSS1 was detected at the mRNA and protein levels. Cell Counting Kit-8, wound healing and Transwell assays revealed that the migration and invasion activities were significantly suppressed by MTSS1, but that it had no effect on cell proliferation. In addition, MTSS1 expression in tissue microarrays including samples from 223cases of non-small cell lung cancer was tested by immunohistochemistry to explore the correlation between MTSS1 and clinicopathological characteristics and prognosis. MTSS1 suppressed H1299 cell migration and invasion, and its expression level can be used as a new independent factor for determining the prognosis of non-small cell lung cancer.

Punica granatum (pomegranate) leaves extract induces apoptosis through mitochondrial intrinsic pathway and inhibits migration and invasion in non-small cell lung cancer in vitro.

Most conventional treatments on non-small cell lung carcinoma always accompany with awful side effects, and the incidence and mortality rates of this cancer are increasing rapidly worldwide. The objective of this study was to examine the anticancer effects of extract of Punica granatum (pomegranate) leaves extract (PLE) on the non-small cell lung carcinoma cell line A549, H1299 and mouse Lewis lung carcinoma cell line LL/2 in vitro, and explore its mechanisms of action. Our results have shown that PLE inhibited cell proliferation in non-small cell lung carcinoma cell line in a concentration- and time-dependent manner. Flow cytometry (FCM) assay showed that PLE affected H1299 cell survival by arresting cell cycle progression in G2/M phase in a dose-dependent manner and inducing apoptosis. Moreover, PLE could also decrease the reactive oxygen species (ROS) and the mitochondrial membrane potential (ΔYm), indicating that PLE may induce apoptosis via mitochondria-mediated apoptotic pathway. Furthermore, PLE blocked H1299 cell migration and invasion, and the reduction of matrix metalloproteinase (MMP) MMP-2 and MMP-9 expression were also observed in vitro. These results suggested that PLE could be an effective and safe chemotherapeutic agent in non-small cell lung carcinoma treatment by inhibiting proliferation, inducing apoptosis, cell cycle arrest and impairing cell migration and invasion.

MiR-373-3p Promotes Invasion and Metastasis of Lung Adenocarcinoma Cells

背景与目的肺癌位居全球癌症相关死亡率的首位，其中肿瘤转移是导致肺癌患者死亡的主要原因，研究表明miR-373与多种肿瘤细胞的侵袭转移有密切关系。本研究旨在探讨miR-373-3p在非小细胞肺癌(non-small cell lung cancer, NSCLC)中的表达情况及其对肺腺癌细胞侵袭转移能力的影响。方法利用qRT-PCR法检测miR-373-3p在NSCLC组织和肺腺癌细胞株中的表达。瞬时转染hsa-miR-373-3p的mimics和inhibitor至肺腺癌H1299和A549细胞株中，利用Transwell小室检测转染后肺腺癌细胞侵袭转移能力的改变，Western blot检测转染后肺腺癌细胞中基质金属蛋白酶-9(matrix metalloproteinase-9, MMP-9)及MMP-14蛋白水平的改变。结果miR-373-3p在51例NSCLC组织和5种肺腺癌细胞株中均明显高表达。在miR-373-3p低表达的H1299细胞中过表达miR-373-3p，细胞的侵袭转移能力明显提高，同时MMP-9及MMP-14的表达上调；在miR-373-3p高表达的A549细胞中抑制miR-373-3p表达，细胞的侵袭转移能力下降，并且下调MMP-9和MMP-14的表达。结论miR-373-3p可能通过正向调节MMP-9、MMP-14的表达而促进肺腺癌细胞的侵袭转移能力。

들깨 추출물의 항산화 활성과 암세포 기본 특성에 대한 억제 효과

The aim of the study was to investigate the antioxidant activities of ethanol extract of perilla seed (PSE) and its inhibitory effects against major characteristics of cancer cells, such as unrestricted growth and activated metastasis in vitro. The total polyphenol and flavonoid levels of PSE were 222.6 mg gallic acid equivalent/100 g and 285.7 mg quercetin equivalent/100 g, respectively. The radical scavenging activity and ferric reducing antioxidant power of PSE at concentration of 87.5 to 350 μg/mL were 24∼45% and 28∼62%, respectively. Treatment of HCT116 colorectal carcinoma cells and H1299 non-small cell lung carcinoma cells with PSE dose-dependently inhibited growth by 18∼94% (at concentration range of 87.5 to 350 μg/mL) and completely abolished colony formation (at concentration of 175 μg/mL). PSE was also effective in inhibiting migration of H1299 cells (by 30∼37% at concentration range of 87.5 to 350 μg/mL) and adhesion of both HCT116 and H1299 cells (by 14∼16% at concentration of 350 μg/mL). These results indicate that PSE exerts antioxidant and anti-cancer activities in vitro. It needs to be determined whether or not similar effects can be reproduced in vivo.

MicroRNA-99a is downregulated and promotes proliferation, migration and invasion in non-small cell lung cancer A549 and H1299 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.

Inhibitory activities of Perilla frutescens britton leaf extract against the growth, migration, and adhesion of human cancer cells.

BACKGROUND/OBJECTIVESPerilla frutescens Britton leaves are a commonly consumed vegetable in different Asian countries including Korea. Cancer is a major cause of human death worldwide. The aim of the current study was to investigate the inhibitory effects of ethanol extract of perilla leaf (PLE) against important characteristics of cancer cells, including unrestricted growth, resisted apoptosis, and activated metastasis, using human cancer cells.MATERIALS/METHODSTwo human cancer cell lines were used in this study, HCT116 colorectal carcinoma cells and H1299 non-small cell lung carcinoma cells. Assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were performed for measurement of cell growth. Soft agar and wound healing assays were performed to determine colony formation and cell migration, respectively. Nuclear staining and cell cycle analysis were performed for assessment of apoptosis. Fibronectin-coated plates were used to determine cell adhesion.RESULTSTreatment of HCT116 and H1299 cells with PLE resulted in dose-dependent inhibition of growth by 52-92% (at the concentrations of 87.5, 175, and 350 µg/ml) and completely abolished the colony formation in soft agar (at the concentration of 350 µg/ml). Treatment with PLE at the 350 µg/ml concentration resulted in change of the nucleus morphology and significantly increased sub-G1 cell population in both cells, indicating its apoptosis-inducing activity. PLE at the concentration range of 87.5 to 350 µg/ml was also effective in inhibiting the migration of H1299 cells (by 52-58%) and adhesion of both HCT116 and H1299 cells (by 25-46%).CONCLUSIONSThese results indicate that PLE exerts anti-cancer activities against colon and lung cancers in vitro. Further studies are needed in order to determine whether similar effects are reproduced in vivo.

TGFβ can stimulate the p(38)/β-catenin/PPARγ signaling pathway to promote the EMT, invasion and migration of non-small cell lung cancer (H460 cells).

Signaling pathway(s) responsible for transforming growth factor β (TGFβ)-induced epithelial mesenchymal transition (EMT), invasion and migration of H460 cells (non-small cell lung cancer/NSCLC) was identified in the study. The results showed that TGFβ-induced p(38)/β-catenin/PPARγ signaling pathway played a critical role in the promotion of EMT, invasion and migration of H460 cells. All these pathological outcomes attributed to PPARγ-increased expression of p-EGFR, p-c-MET and Vimentin and the decrease of E-cadherin. Transforming growth factor β and p(38)-induced β-catenin not only stimulated the expression of PPARγ but also physically interacted with it. Blocking the ligand binding domain of PPARγ (with GW9662) could significantly interfere the binding between PPARγ and β-catenin, and interrupt the nuclear infiltration of both factors. These findings suggested that β-catenin was an upstream regulator and a ligand of PPARγ, and the binding between these two molecules was critical for their nuclear infiltration. Transforming growth factor β-induced tumor invasion and migration was also seen in U373 cells (brain glioma, with high inducible PPARγ) in a PPARγ-dependent manner, but not in CH27 cells (squamous NSCLC, with low PPARγ). PPARγ shRNA, GW9662, JW67 and 2,4-diaminoquinazoline were all revealed to have important values in the control of the intrinsic and TGFβ-induced EMT, tumor invasion and migration of H460 cells. The results further suggested that PPARγ and β-catenin may be the potential markers for the early diagnosis and/or treatment of metastatic tumors.

MicroRNA-26a involved in Toll-like receptor 9‑mediated lung cancer growth and migration.

Toll-like receptor 9 (TLR9) has been shown to have a significant role in cancer. MicroRNAs (miRNAs), a group of small non-coding RNAs that fine tune translation of multiple target mRNAs, are involved in the development and progression of human cancers. The present study was undertaken to determine the roles of TLR9 on lung cancer and whether miR-26a is involved in TLR9‑mediated lung cancer growth and migration. The lung cancer models were elicited by injecting human lung cancer cells into the left ventricle. The expression of TLR9 and miR-26a in lung cancer tissues obtained from lung cancer patients was increased. TLR9 ligand CpG-oligodeoxynucleotides (CpG-ODN) caused an increase in the mean tumor weight and the size of tumor mass in nude mice, and the proliferation and migration of H460 human lung cancer cells. CpG-ODN also induced an increase in the expression of miR-26a in H460 cells. The overexpression of miR-26a increased the weight and size of the tumor mass in the nude mice, and the proliferation and migration of H460 cells. Expression of phosphoinositide 3 kinase (PI3K) and phosphorylation of protein kinase B (Akt) was increased after miR-26a overexpression in the H460 cells. PI3K inhibitor wortmannin (WM) or Akt inhibitor triciribine hydrate (TCN) eliminated the increase in the proliferation and migration induced by the overexpression of miR-26a in H460 cells. These results suggested that miR-26a is involved in the TLR9‑mediated growth and migration of lung cancer through the PI3K-Akt signaling pathway.

Influence on the behavior of lung cancer H1299 cells by silencing SLC35F2 expression

BackgroundTo investigate the effects of RNA interference-mediated downregulation of Human Solute Carrier Family 35 member F2 (SLC35F2) expression on the biological behavior of lung cancer H1299 cells.MethodsThe lentiviral vector of small interfering RNA targeting SLC35F2 was introduced into H1299 cells by liposome-mediated transfection. Expression of the SLC35F2 protein was measured by western blot. The proliferation of H1299 cells was determined by Cell Counting Kit-8 assay. The migration of H1299 cells was measured by Transwell migration assay. Cell cycle analysis used fluorescence-activated cell sorting.ResultsSLC35F2 expression was markedly downregulated in H1299 cell clone (transfected with the lentiviral vector harboring small interfering RNA targeting SLC35F2). Proliferation decreased significantly compared with that of non-transfected H1299 cells. Transwell migration assay showed that fewer cells moved through the artificial basement membrane compared with untransfected H1299 cells (38.3 ± 5.7 vs. 113.5 ± 8.5, P < 0.05). The cell cycle of H1299 cells was changed, the percentage of H1299 cells in S and G2/M phases being significantly decreased compared with untransfected H1299 cells (S phase: 15.3% ± 3.0% vs. 27.0% ± 5.4%, P > 0.05; G2/M phase; 3.0% ± 1.1% vs. 10.5% ± 1.7%, P < 0.05), whereas the percentage of H1299 cells in G0/G1 phase increased markedly (81.7% ± 4.0% vs. 62.5% ± 1.9%, P < 0.05).ConclusionRNA interference-mediated downregulation of SLC35F2 expression by lentiviral vector can attenuate the proliferation, migration and invasion of H1299 cells.

Inhibition of Cellular Growth and Migration by Suppression of Endothelial Protein C Receptor (EPCR) in Lung Carcinoma Cells

Increasing evidence shows that beyond its role in coagulation, thrombosis interferes with carcinogenesis. Endothelial cell protein C receptor (EPCR) is a cellular receptor for protein C and activated protein C (APC). Such EPCR-induced signal transduction promotes cancer cell migration, invasion, and angiogenesis and inhibits cancer cell apoptosis. However, its role in lung carcinoma biology is yet to be demonstrated. Here, the recombinant EPCR siRNA plasmids were constructed to investigate the effects of inhibition of EPCR on human lung cancer H1299. EPCR siRNA led to inhibition of endogenous EPCR mRNA and protein expression as determined by RT-PCR and Western blotting analysis. EPCR siRNA significantly inhibited cell growth, blocked entry into the S phase of the cell cycle, and reduced the migration of H1299 cells. EPCR siRNA also decreases MMP-2 and cyclin E expression in H1299 cells. In addition, siRNA targeting of EPCR inhibited the growth of H1299 cells and decreased MVD in SCID mice tumor models. Taken together, EPCR was involved in regulating progression of human lung cancer cells. Manipulation of EPCR expression may be a potential therapeutic strategy for lung cancer.

Silencing of SLC35F2 gene influences the biologic behavior of human lung cancer cell line H1299.

e13512 Background: SLC35F2 is a member of solute carrier family 35 which encode nucleotide sugar transporters localizing in the Golgi apparatus and/or the endoplasmic reticulum, and transport nucleotide sugars that have pooled in the cytosol into the lumen of this organelle, where most of the glycoconjugate synthesis occurs. However, the SLC35F2 subfamily has not been completely characterized with regard to its biological activity. In this study, we tested the hypothesis that SLC35F2 influences the biological behavior of lung cancer cell line. We examined the effect of the inhibition of SLC35F2 expression on proliferation and invasion by establishing the SLC35F2 knockdown lung cancer cell line using lentivirus-mediated RNA interference (RNAi). Methods: The lentiviral vector of small interfering RNA targeting SLC35F2 was introduced into H1299 cells by liposome-mediated transfection. The expression of SLC35F2 protein was measured by Western blot. The proliferation of H1299 cells was determined by cell counting kit-8 (CCK-8) assay. The migration and invasion ability of H1299 cells was evaluated by Transwell migration and invasion assay. Cell cycle analysis was also performed by fluorescence-activated cell sorting (FACS). Results: SLC35F2 expression was markedly down-regulated in H1299 cell clone transfected with the lentiviral vector harboring small interfering RNA targeting SLC35F2. The proliferation of H1299 cells decreased significantly compared with untransfected H1299 cells. Transwell migration assay showed that less H1299 cells could move through the artificial basement membrane when compared with untransfected H1299 cells (38.3±5.7 vs 113.5±8.5, P <0.05). The cell cycle of H1299 cells was changed. Compared with untransfected H1299 cells, the percentage of H1299 cells in S and G2/M phase decreased significantly (S phase: F15.31%±3.04% vs 27.00%±5.41%, P >0.05; GG2/M phase: F3.02%±1.10% vs 10.47%±1.72%, P <0.05), whereas the percentage of H1299 cells in G0/G1 phase increased remarkably (81.67%±3.98% vs 62.53%±1.89%, P <0.05). Conclusions: RNA interference-mediated down-regulation of SLC35F2 by lentiviral vector can attenuate the proliferation and migration of H1299 cells.

Abstract 3693: Berry anthocyanidins inhibit key events of lung cancer metastasis: Effects on miRNA and protein targets

Abstract Lung cancer continues to represent the largest cause of mortality in the world claiming over 1.3 million lives every year. The majority of patients who succumb to lung cancer die from cancer relapse and/or metastatic disease progression making the search for new anti-cancer and anti-metastasis agents imperative. Berry phytochemicals have received increasing attention lately for their various biological effects. We previously demonstrated notable synergistic anti-tumor effects of blueberry anthocyanidins (cyanidin, malvidin, peonidin, petunidin and delphinidin) against human lung cancer cells in culture and in vivo in nude mouse model. Herein, we show the anti-metastatic effects of all the 5 anthocyanidins individually and in combination in the highly aggressive and invasive human NSCLC H1299 cells. Using wound-healing and Boyden chamber assays, all the five anthocyanidins (25 µM each) were found to exhibit varied inhibitory effects on H1299 cell migration and invasion. However, an equimolar mixture containing either 3.125 or 6.25 µM of each anthocyanidin elicited similar inhibition of H1299 cell migration and invasion indicating a synergistic effect. Given the multi-complexity of the metastasis process, the effect of anthocyanidins on a plethora of molecular targets mediating of the metastatic process was analyzed by western blotting. We found that berry anthocyanidins decreased the expression of several metastatic and angiogenic mediators, viz; matrix metalloproteinases (MMP-2 and MMP-9), vascular endothelial growth factor (VEGF), c-MYC, NOTCH-1, WNT1. Furthermore, the anthocyanidins effectively inhibited TNF-α-induced NF-κB activation and suppressed of ERK-1/2 activation, which could further explain their anti-invasive capabilities. Recently, emerging evidence also suggests the involvement of miRNAs in regulating various processes of cancer cell invasion and metastasis. Effect of anthocyanidins on four such miRNAs frequently downregulated in NSCLC and implicated in lung cancer invasion and metastasis (miR-126, miR125a-5p, miR200c) and lung cancer relapse (miR-34a) was analyzed by qPCR. We observed a significant increase in the expression of all the four miRNAs by the anthocyanidins. Target prediction scan for gene targets revealed several angiogenic signaling molecules to be regulated by these miRNA's. Our results thus establish that protective properties of anthocyanidins may arise, at least, in part, from its capability to manipulate some distinct and some overlapping miRNA and protein targets that may potentiate the anti-cancer and anti-metastatic effects of the berry anthocyanidins. Collectively, our data suggest that berry anthocyanidins may provide a safe, effective, unconventional and user-friendly approach to prevent or delay the onset of lung cancer recurrence and metastasis (Supported from KLCRP and Agnes Brown Duggan Endowment). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3693. doi:10.1158/1538-7445.AM2011-3693

Synergistic Antitumor Activity of Sorafenib in Combination with Epidermal Growth Factor Receptor Inhibitors in Colorectal and Lung Cancer Cells

Cancer cell survival, invasion, and metastasis depend on cancer cell proliferation and on tumor-induced angiogenesis. We evaluated the efficacy of the combination of sorafenib and erlotinib or cetuximab. Sorafenib, erlotinib, and cetuximab, alone or in combination, were tested in vitro in a panel of non-small cell lung cancer (NSCLC) and colorectal cancer cell lines and in vivo in H1299 tumor xenografts. Epidermal growth factor receptor (EGFR) ligand mRNAs were expressed in all NSCLC and colorectal cancer cell lines with variable levels ranging from 0.4- to 8.1-fold as compared with GEO colorectal cancer cells. Lung cancer cells had the highest levels of vascular endothelial growth factors (VEGF) A, B, and C, and of VEGF receptors as compared with colorectal cancer cells. Combined treatments of sorafenib with erlotinib or cetuximab produced combination index values between 0.02 and 0.5, suggesting a significant synergistic activity to inhibit soft agar colony formation in all cancer cell lines, which was accompanied by a marked blockade in mitogen-activated protein kinase and AKT signals. The in vitro migration of H1299 cells, which expressed high levels of both VEGF ligands and receptors, was inhibited by treatment with sorafenib, and this effect was significantly increased by the combination with anti-EGFR drugs. In nude mice bearing established human H1299 xenografts, treatment with the combination of sorafenib and erlotinib or cetuximab caused a significant tumor growth delay resulting in 70 to 90 days increase in mice median overall survival as compared with single-agent sorafenib treatment. Combination treatment with sorafenib and erlotinib or cetuximab has synergistic antitumor effects in human colorectal and lung cancer cells.

Urokinase plasminogen activator receptor activation of invasion and migration in H460 cells requires Na+/H+ transporter activity and expression

Non‐small cell lung cancer (NSCLC) is an aggressive cancer where 10% of patients survive beyond five years of initial diagnosis. One of the reasons for the poor survival rate is the robust invasion and metastatic properties of NSCLC. Urokinase plasminogen activator (uPA) and its receptor are directly associated with a poor prognosis in NSCLC. Na+/H+ transporter (NHE1) has been implicated in regulation of cell motility in a number of cancer cells. We wished to identify the role NHE1 plays in uPA activation of tumor progression in NSCLC. We used both uPA and the inactive‐receptor binding amino terminal fragment of uPA (ATF) to stimulate human lung H460 carcinoma cells. 10 nM uPA or ATF stimulated proliferation 1.4 ‐ 1.2 fold over non‐treated cells. Inhibition of NHE1 with 10 μM EIPA abrogated agonist‐stimulated proliferation. Tumor formation size and number was significantly increased by addition of either uPA or ATF, while NHE1 inhibition blocked the ability of either agonist to induce tumors in soft agar assays. Migration of H460 cells was inhibited in the presence of EIPA. Both uPA and ATF enhanced MMP9 activity but not in cells treated with EIPA. Only ATF increased MMP9 expression. We also show the effect of uPA in NHE1 and uPAR knockdown cell lines where we investigated MMP9 activity, and the location of NHE1 and uPAR in lipid rafts. Our results indicate that uPAR activation of cancer cell properties requires NHE1. Support from NIH‐1‐R15‐CA135616‐01

Abstract C19: C2-O-sLeX glycans on human carcinoma cells are high-affinity selectin ligands that mediate tumor adhesion to vascular endothelium and invasion

Abstract The family of selectin adhesion molecules, CD62-E and P-selectin expressed on vascular endothelium, and L-selectin expressed on leukocytes, bind sialyl-Lewis X (sLeX)-related glycans and are critical for leukocyte trafficking to lymphoid tissues and sites of inflammation. The sLeX-modified core 2 O-glycans (C2-O-sLeX) on leukocytes promote significantly higher affinity selectin binding than sLeX. Our objectives were to determine whether C2-O-sLeX glycans conferred carcinoma cells with high binding activity to the selectins and participated in metastasis. We investigated C2-O-sLeX expression in human colorectal adenocarcinomas and metastatic liver tumors by immunohistochemistry and RT-PCR. C2-O-sLeX was abundantly expressed in adenocarcinomas and mRNA expression of C2GnT1, the enzyme responsible for C2-O-sLeX synthesis, was greater than 15-fold upregulated. C2-O-sLeX was also present in metastatic liver tumors but not normal colorectal or liver tissues. We evaluated human lung (H1299), pancreatic (AsPC-1), colon (LS174T), and hepatic (HepG2) carcinoma cell lines for their expression of C2-O-sLeX or sLeX glycans and ability to bind to selectins. We also examined mRNA expression of genes coding for the enzymes alpha-2,3 fucosyltransferases (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1). FucT-III is responsible for sLeX synthesis and the formation of the core 2 branched O-glycan structures is dependent on C2GnT1. The activity of both enzymes is necessary for C2-O-sLeX synthesis. All four carcinoma cell lines expressed similar levels of C2GnT1 mRNA but FucT-III mRNA levels were lower in H1299 and AsPC-1 cells by RT-PCR. LS174T and HepG2 cell lines were 70 to 90% positive for cell surface expression of C2-O-sLeX and between 70 to 97% positive for sLeX by flow cytometry. C2-O-sLeX and sLeX were also detected in these cells by Western blotting. H1299 and AsPC-1 cells did not express these glycans. H1299 and AsPC-1 cells stably transfected with FucT-III were 43% and 7% positive for C2-O-sLeX respectively. Transfected (H1299/FucT-III and AsPC-1/FucTIII) and LS174T and HepG2 cell lines bound avidly to E-selection but not P- and L-selectin. We used shRNA vectors to silence gene expression of C2GnT1 in H1299/FucT-III and LS174T cells. We observed a 40–90% decrease in C2-O-sLeX but not sLeX expression and a 30–50% decrease in binding to E-selectin on cells containing C2GnT1 shRNA compared to unmanipulated cells. Migration of H1299/FucT-III cells across a reconstituted basement membrane was significantly increased compared to untransfected cells using a matrigel invasion assay. In contrast, the migration of H1299 cells containing C2GnT1 shRNA was reduced. Similarly, LS174T cells transfected with C2GnT1 shRNA exhibited significantly reduced migration and proliferation. Our findings demonstrate that C2-O-sLeX glycans are tumor-associated antigens expressed on carcinoma cells. Importantly, C2-O-sLeX glycans are high affinity ligands for E-selectin and play a role in the adhesion of carcinoma cells to endothelium and tumor invasiveness. These findings are relevant to the development of novel biomarkers and allow further insight into the role of sLeX-containing glycans in carcinoma metastasis. C2-O-sLeX may prove useful to predict patient prognosis and survival. This work was supported in part by funds from the National Institutes of Health/National Cancer Institute grant K08 CA111829-04. Citation Information: Cancer Res 2009;69(23 Suppl):C19.