Amiloride and guggulsterone suppression of esophageal cancer cell growth in vitro and in nude mouse xenografts

Esophageal adenocarcinoma is increasing in the US and Western countries and frequent gastresophageal reflux or gastresophageal reflux disease carrying gastric acid and bile acid could contribute to esophageal adenocarcinogenesis. This study was designed to detect the expression of gastric acid-inducing gene Na＋/H＋ exchanger-1 （NHE-1） ex vivo and then to explore targeting of NHE-1 expression or activity to control esophageal cancer cell viability in vitro and in nude mouse xenografts. The data showed that NHE-1 was highly expressed in esophageal adenocarcinoma tissues （66 of 101 cases [65.3%｜, but not in normal esophageal squamous cell epithelium （1 of 26 cases [3.8~0]）. Knockdown of NHE-1 expression using NHE-1 shRNA or inhibition of NHE-1 activity using the NHE-1 inhibitor amiloride suppressed viability and induced apoptosis in esophageal cancer cells. Molecularly, amiloride inhibited expression of cyclooxygenase-2 and matrix metallopeptidase-9 but not NHE-1 mRNA in esophageal cancer cells. A combination of amiloride and guggulsterone （a natural bile acid receptor inhibitor） showed more than additive effects in suppressing esophageal cancer cell growth in vitro and in nude mouse xenografts. This study suggests that inhibition of NHE-1 expression or activity or combination of amiloride and guggulsterone could be useful in control of esophageal adenocarcinoma.     

MiRNA-218, a new regulator of HMGB1, suppresses cell migration and invasion in non-small cell lung cancer

MieroRNAs （miRNAs） function as negative regulators of gene expression involved in cancer metastasis. The aim of this study is to investigate the potential roles of miR-218 in non-small cell lung cancer and validate its regulation mech- anism. Functional studies showed that miR-218 overexpres- sion inhibited cell migration and invasion, but had no effect on cell viability. Enhanced green fluorescent protein reporter assay, real-time polymerase chain reaction and western blot analysis confirmed that miR-218 suppressed the expression of high mobility group box-1 （HMGB1） by directly targeting its 31-untranslated region. Accordingly, silencing of HMGBI accorded with the effects of miR-218 on cell migration and invasion, and overexpression of HMGB1 can restore cell migration and invasion which were reduced by miR-218. In conclusion, these findings demon- strate that miR-218 functions as a tumor suppressor in lung cancer. Furthermore, miR-218 may act as a potential thera- peutic biomarker for metastatic lung cancer patients.     

Combined therapy of p53-wt and drug in an orthotopic multidrug-resistant human lung cancer model

Balb/c nu/nu mice were inoculated intratracheally with multidrug-resistant human lung cancer cells GLK containing p53 mutation at codon 245 and treated with intratracheal instillation of p53-wt retroviral vector (pDOR53W) to increase cell chemosensitivity, and then with intraperitoneal injection of doxorubicin. 30 d after tumor cell inoculation, 75% of the control mice showed macroscopic tumors in the lung. Sole pDOR53W suppressed GLK tumor formation in 68 % of mice; sole doxorubicin 33. 3 % , but the combination of pDOR53W and doxorubicin 88.9%. The exogenous p53 sequence was detected and confirmed in the tumor that grew after treatment with pDOR53W retroviral vector by PCR and Southern blot hybridization with p53 cDNA. These results suggested that di-rect administration of a retroviral vector expressing p53-wt combined with treatment of anticancer agent was an effec-tive therapeutic method for multidrug-resistant human lung cancer.     

Suppression of type 1 Insulin-like growth factor receptor expression by small interfering RNA inhibits A549 human lung cancer cell invasion in vitro and metastasis in xenograft nude mice

Cancer invasion and metastasis, involving a variety of pathological processes andcytophysiological changes,contribute to the high mortality of lung cancer.The type 1 insulin-like growthfactor receptor (IGF-1R),associated with cancer progression and invasion,is a potential anti-invasion andanti-metastasis target in lung cancer.To inhibit the invasive properties of lung cancer cells,we successfullydown-regulated IGF-1R gene expression in A549 human lung cancer cells by small interfering RNA (siRNA)technology,and evaluated its effects on invasion-related gene expression,tumor cell in vitro invasion,andmetastasis in xenograft nude mice.A549 cells transfected with a plasmid expressing hairpin siRNA forIGF-1R showed a significantly decreased IGF-1R expression at the mRNA level as well as the proteinlevel.In biological assays,transfected A549 cells showed a significant reduction of cell-matrix adhesion,migration and invasion.Consistent with these results,we found that down-regulation of IGR-1Rconcomitantly accompanied by a large reduction in invasion-related gene expressions,including MMP-2,MMP-9,u-PA,and IGF-1R specific downstream p-Akt.Direct tail vein injections of plasmid expressinghairpin siRNA for IGF- 1R significantly inhibited the formation of lung metastases in nude mice.Our resultsshowed the therapeutic potential of siRNA as a method for gene therapy in inhibiting lung cancer invasionand metastasis.     

Inhibition of the growth of human hepatoma cell line both in vitro and in vivo by transducing CKI gene p21~(WAF-1) with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous gene pCEP-p21WAF-1 into human hepatocellular carcinoma cell both in vitro and in vivo. After in vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. The in vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untrans-fected control. The average tumor weight of the experiment group was (0.083 ?0.043) g, while that of the control group was (0.28110.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous gene pCEP-p21WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growt     

Target genes involved in antiproliferative effect of modified ginseng extracts in lung cancer A549 cells

Lung cancer is the most common cancer and the leading cause of cancerrelated deaths. Panax ginseng has long been used to treat cancer and other diseases worldwide. Most of the pharmacological actions of ginseng are attribu ted to a variety of ginsenosides, which are often metabo lized by intestinal bacteria into more effective forms. In this study, we found that the antiproliferative activity of ginseng was increased after enzymatic processing of ginseng saponin （50% inhibitory concentration, 〉70μg/ml）. To elucidate the mechanism by which modified ginseng extract （MGX） induced cell death in human lung cancer cells, the gene expression profiles ofA549 cells regulated by MGX were assayed using Agilent PrimeView Human Gene Expression Arrays. The expression of 17 genes involved in the regulation of cell signaling, cell metabolism, transport, and cytoskeletonregulation was upregulated, whereas the expression of 16 genes implicated in invasion and metastasis and cellular metabolism was downregulated in MGX treated A549 cells. Moreover, nuclear staining with 4：6dia midino2phenyHndole revealed that MGX clearly caused nuclear condensation and fragmentation which are observed in apoptosis cell. These results elucidate crucial antieancer mechanisms of MGX and provide potential new targets for the assessment of anticancer activity of MGX.     

XPA A23G polymorphism is associated with the elevated response to platinum-based chemotherapy in advanced non-small cell lung cancer

DNA repair capacity （DRC） is correlated with sensitivity of cancer cells toward platinum-based chemotherapy. We hypothesize that genetic polymorphisms in DNA repair gene XPA （xeroderma pigmentosum group A） and XPG （xeroderma pigmentosum group G） （ERCC5, excision repair cross-complementation group 5）, which result in inter-individual differences in DNA repair efficiency, may predict clinical response to platinum agents in advanced non-small cell lung cancer （NSCLC） patients. In this study, we find that the A → G change of XPA A23G polymorphism significantly increased response to platinum-based chemotherapy. Polymorphism in XPG His46His was associated with a decreased treatment response, but was not statistically significant.     

The expression of core fucosylated E-cadherin in cancer cells and lung cancer patients： prognostic implications

It is well documented that the glycosylation of E-cadherin is correlated with cancer metastasis, but whether E-cadherin could be core fucosylated remains largely unknown. We found that E-cadherin was core fucosylated in highly metastatic lung cancer cells while absent in lowly metastatic lung cancer cells. Sinceα-1,6 Fucosyltransferase (α-1,6 FucT) is known to catalyze the reaction of core fucosylation, we investigated the biological function of core fucosylation on E-cadherin by α-1,6 FucT targeted RNAi and transfecting α-1,6 FucT expression vector. As a result, calcium dependent cell-cell adhesion mediated by E-cadherin was strengthened with the reduction of core fucosylation on E-cadherin after RNAi and was weakened with the elevated core fucosylation on E-cadherin after α-1,6 FucT over expression. Our data indicated that α-1,6 FucT could regulate E-cadherin mediated cell adhesion and thus play an important role in cancer development and progression. Computermodeling showed that core fucosylation on E-cadherin could significantly impair three-dimensional conformation of N-glycan on E-cadherin and produce conformational asymmetry so as to suppress the function of E-cadherin. Furthermore, the relationship between the expression of core fucosylated E-cadherin and clinicopathological background of lung cancer patients was explored in lung cancer tissue of patients. It turns out to demonstrate that core fucosylated E-cadherin could serve as a promising prognostic indicator for lung cancer patients.     

Effect of L-arginine on pulmonary artery smooth muscle cell apoptosis in rats with hypoxic pulmonary vascular structural remodeling

This study investigated the effect of L-arginine (L-Arg) on the apoptosis of pulmonary arterysmooth muscle cells (PASMC) in rats with hypoxic pulmonary vascular structural remodeling,and itsmechanisms.Seventeen Wistar rats were randomly divided into a control group (n=5),a hypoxia group(n=7),and a hypoxia L-Arg group (n=5).The morphologic changes of lung tissues were observed underoptical microscope.Using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay,the apoptosis of PASMC was examined.Fas expression in PASMC wasexamined using immunohistochemistry.The results showed that the percentage of muscularized artery insmall pulmonary vessels,and the relative medial thickness and relative medial area of the small and medianpulmonary muscularized arteries in the hypoxic group were all significantly increased.Pulmonary vascularstructural remodeling developed after hypoxia.Apoptotic smooth muscle cells of the small and median pul-monary arteries in the hypoxia group were significantly less than those in the control group.After 14 d ofhypoxia,Fas expression by smooth muscle cells of median and small pulmonary arteries was significantlyinhibited.L-Arg significantly inhibited hypoxic pulmonary vascular structural remodeling in association withan augmentation of apoptosis of smooth muscle cells as well as Fas expression in PASMC.These resultsshowed that L-Arg could play an important role in attenuating hypoxic pulmonary vascular structural remod-eling by upregulating Fas expression in PASMC,thus promoting the apoptosis of PASMC.     

Matrine reduces the proliferation and invasion of colorectal cancer cells via reducing the activity of p38 signaling pathway

Matrine has been used in anti-inflammatory and anti-cancer therapies for a long time. However, the anti-metastatic effect and related mechanism（s） in colorectal cancer （CRC） are still unclear. In this study, we investigated whether the adminis- tration of matrine could inhibit the proliferation, motility, and invasion of human CRC cells via regulating p38 signal- ing pathway. Results showed that matrine inhibited migration and invasion of CRC cells in vitro and in vivo. Additionally, after being treated with matrine for 24 h, the expression levels of matrix metalloproteinase-2 （MMP-2） and MMP-9 as well as proteinase activity in CRC cells were reduced in a dose-dependent manner. Moreover, matrine reduced the phosphorylation level of p38 obviously. Combined treatment with p38 inhibitor （SB203580） and matrine resulted in a syn- ergistic reduction of invasion as well as MMP-2/-9 expression in CRC cells. It was also found that matrine inhibited the pro- liferation and metastasis of CRC tumor in vivo. In conclusion, p38 signaling pathway may involve in matrine＇s inhibitory effects on migration and invasion of CRC cells by reducing the expression of MMP-2/-9, suggesting that matrine may be a potential therapeutic agent for CRC.     

Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung cancer     

Inhibition of metastasis to lung of a human nasopharyngeal carcinoma cell line CNE-2L2 transfected with pRc/CMV-antisense 6A8 cDNA in nude mice

The growth of CNE-2L2 cell, a cloned line of human nasopharyngeal carcinoma with a high potentiality of metastasis to lung was inhibited to a certain extent after transfection with a recombinant antisense expression vector of a cDNA encoding a human α-mannosidase (pRc/CMV-antisense 6A8 cDNA)( the Genbank accession number of 6A8 cDNA is U37248) in comparison with that of the cell transfected with the Mock and of the wild cell. Two months after a subcutaneous inoculation of CNE-2L2 cell into the axilla of nude mice metastatic lesions in the lung were observed in 9/10 mice (90%) with grade Ⅲ in 8 mice and grade Ⅱ in one mouse in the wild cell group, in 6/8 mice (75%) with grade Ⅲ in one mouse, grade Ⅱ in 2 mice and grade Ⅰ in 3 mice in the Mock-transfection group, in only 3/10 mice (30%) with all grade Ⅰ in pRc/CMV-antisense 6A8 cDNA-transfection group.     

Casticin suppresses self-renewal and invasion of lung cancer stem-like cells from A549 cells through down-regulation of pAkt

A subpopulation of cancer stem cells is recognized as the cause of tumorigenesis and spreading. To investigate the effects of casticin （5,3＇-dihydroxy-3,6,7,4＇-tetramethoxyflavone）, derived from Fructus Viticis Simplicifoliae, on lung cancer stem cells, we isolated and identified a subpopulation of lung cancer stem-like cells （LCSLCs） from non-small-cell lung carcinoma A549 cells with the features including self- renewal capacity and high invasiveness in vitro, elevated tumorigenic activity in vivo, and high expression of stemness markers CD133, CD44, and aldehyde dehydrogenase 1 （ALDH1）, using serum-free suspension sphere-forming culture method. We then found that casticin could suppress the proliferation of LCSLCs in a concentration-dependent manner with an IC50 value of 0.4 μmol/L, being much stronger than that in parental A549 cells. In addition, casti- cin could suppress the self-renewal and invasion of LCSLCs concomitant with decreased CD133, CD44, and ALDH1 protein expression and reduced MMP-9 activity. Further experiments showed that casticin suppressed self-renewal and invasion at least partly through down-regulation of Akt phosphorylation. In conclusion, casticin suppressed the characteristics of LCSLCs, suggesting that casticin may be a candidate compound for curing lung cancer via elim- inating cancer stem cells.     

Combined chemotherapy of platinum and fluorouracil promotes T cell–mediated antitumor immunity

The two-drug combined chemotherapy of platinum and fluorouracil has been reported to efficiently kill tumor cells as the first-line treatment for advanced gastric cancer.However,the effect of these drugs on T cells remains unclear.Here,we showed that T cells including CD4+T cells and CD8+T cells of the patients with advanced gastric cancer after platinum and fluorouracil chemotherapy exhibited enhanced ex vivo proliferation ability as compared to that before chemotherapy.In addition,platinum and fluorouracil also promoted the differentiation of human T cells into Th1 and Th9 subtypes and cytotoxic T lymphocytes(CTLs)in vitro and in vivo.Accordingly,the combination therapy greatly suppressed tumor growth with increased tumor infiltration of Th1,Th9,and CTL cells in a mouse tumor model.Moreover,in activated T cells,long-term treatment with these two drugs further facilitates T cell activation along with promoted nuclear factor-κB(NF-κB)activation.Our findings demonstrate a previously unidentified function of platinum and fluorouracil combination chemotherapy in promoting T cell–mediated antitumor immunity.     

Choline Transporters in Human Lung Adenocarcinoma： Expression and Functional Implications

Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN 1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H 1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential ＂choline-starvation＂ strategy of cancer interference through targeting choline transporters, especially CTL1.     

A mesenchymal-like subpopulation in non-neuroendocrine SCLC contributes to metastasis

Small cell lung cancer (SCLC) is the most aggressive lung cancer with high heterogeneity.Mouse SCLC cells derived from the Rb1~(L/L)/Trp53~(L/L)(RP) autochthonous mouse model grew as adhesion or suspension in cell culture,and the adhesion cells are defined as non-neuroendocrine (non-NE) SCLC cells.Here,we uncover the heterogenous subpopulations within the non-NE cells and referred to them as mesenchymallike (Mes) and epithelial-like (Epi) SCLC cells.The Mes cells have increased capability to form colonies in soft agar and harbored stronger metastatic capability in vivo when compared with the Epi cells.Gene Set Enrichment Analysis reveals that the transforming growth factor (TGF)-β signaling is enriched in the Mes cells.Importantly,inhibition of the TGF-β signaling through ectopic expression of dominant-negative Tgfbr2(Tgfbr2-DN) or treatment with Tgfbr1 inhibitor SD-208 consistently abrogates tumor metastasis in nude mouse allograft assays.Moreover,genetic deletion of Tgfbr2 or Smad4,the key components of the TGF-β signaling pathway,dramatically attenuates SCLC metastasis in the RP autochthonous mouse model.Collectively,our results uncover the high heterogeneity in non-NE SCLC cells and highlight an important role of TGF-β signaling in promoting SCLC metastasis.     

Effects of laminin glycopeptides on metastasis—related behaviors of cancer cells

Our previous reports have shown that lamininglycopeptides (LN-GPs),the total glycopeptides prepared from laminin (LN),can prevent the experimental lung metastasis and liver metastasis of mouse cancer cells.In order to explore the anti-metastatic mechanism of LN-GPs,we studied the effects of LN-GPs on metastasisrelated behaviors of cancer cells in vitro.LN-GPs did not affect cell survival.However,LN-GPs inhibited cell attachment and spreading of S180 cells on LN-and Matrigelsubstrate in dose-dependent and time-dependent manners.Moreover,inhibition of cell attachment and spreading on Matrigel substrates were much greater on Matrigel substrate than on LN substrate.In the gresence of LN-GPs,S180 cells on LN substrate changed from a flattened polygonal shape to a round one,the migration of S180 cells on LN substrate decreased,and the number of a highly invasive human pulmonary giant carcinoma PG cells invading Matrigel filter in a Boyden chamber was reduced.LN-GPs thus have multiple inhibitory effects on cancer metastasisrelated behaviors.