Reversibility of retinoid effect on sialyltransferase activity, sialic acid content and invasive ability of human lung carcinoma cells

The effect of retinoid induced suppression of in vitro invasive ability of A549 human lung carcinoma cells on sialyltransferase activity and sialic acid content was investigated. Inhibition by retinol acetate of cell invasive potential was accompanied by a significant decrease in the enzyme activity of intact cells as well as total and cell surface neuraminidase-releasable sialic acid contents. Moreover, reversibility of the invasion-suppressed A549 cell phenotype resulted in a return of invasion potential, sialyltransferase activity and surface sialic acid content to invasive cell levels. These findings suggest that membrane-bound sialic acid plays a role in invasiveness of A549 cells.     

Inhibition of invasion activity in vitro by a novel class of antitumor agents: silatrane derivatives

The effect of several silatranes on in vitro invasion of the human amnion basement membrane (BM) by A549 human lung carcinoma cells was examined. Cells treated for two days with the derivatives were examined for invasive activity in the absence of the compounds. From silatrane dose-invasion response curves, an 80% inhibition of invasiveness compared to untreated cells was obtained with 40 micrograms/mg of 1-vinyl silatrane, 50 micrograms/ml of 1-(p-aminophenyl) silatrane, 80 micrograms/mg of 1-(3-phenylthiocarbamidopropyl) silatrane, 66 micrograms/ml of parent silatrane or 171 micrograms/ml of 1-bromosilatrane. Treatment with these doses had no effect on viability, growth or BM attachment of A549 cells.     

Inhibition of N-(4-hydroxyphenyl)retinamide-induced apoptosis in breast cancer cells by galectin-3

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) induces apoptosis in a variety of human cancer cells including breast carcinoma and this property may be important for its chemopreventive and therapeutic effects. Resistance to 4HPR has been described, however, the molecular mechanisms underlying sensitivity or resistance to this retinoid are not clear. Recently, it has been shown that the carbohydrate-binding protein galectin-3, which has been implicated in tumor progression, contains the anti-death motif NWGR present in the anti-apoptotic protein Bcl-2. To determine whether galectin-3 expression can abrogate the effect of 4HPR, we tested the effects of 4HPR on apoptosis of cell clones derived from the galectin-3 deficient human BT549 breast carcinoma cells after transfection with either wild type galectin-3 (BT549Gal-3Wt), galectin-3 inactivated by a point mutation in the NWGR motif (BT549Gal-3Mu), or empty vector control (BT549Vec). Both BT549Vec and BT549Gal-3Mu cells showed a marked decrease in survival after treatment with 4HPR principally due to induction of apoptosis. 4HPR-induced apoptosis in these cells was associated with stimulation of reactive oxygen species generation, decreased levels of Bcl-2 protein, release of cytochrome c into the cytosol, increased caspase-3 activity, and poly(ADP-ribose) polymerase cleavage. In contrast, 4HPR failed to exert any of these effects in the BT549Gal-3Wt cells. The demonstration that galectin-3 suppresses 4HPR-induced apoptosis in human breast carcinoma cells suggests that the increased expression of galectin-3 during cancer progression may be associated with 4HPR resistance.     

Inhibition of retinoid toxicity by rat liver S9 fraction in cultured mammalian cells

The effects of retinol (Rol), retinyl acetate (RAc) or retinoic acid (RA) on growth of Chinese hamster V79 cells were studied. In addition, the effect of Rol on cell growth in the human lymphoid cell line RPMI no. 1788 was examined. Cultures were treated with retinoid alone, with retinoid plus S9 mix and with retinoid plus the supernatant of S9 fraction. Treatment of Rol, RAc or RA alone inhibited the growth of both cell lines as evidenced by a dose-dependent decrease of viable cell counts. Concurrent treatment of a retinoid with the well-known metabolic activation system S9 mix containing the rat liver S9 fraction plus enzyme cofactors resulted in a total elimination of the toxic effect of a retinoid. For instance, V79 cells treated with Rol, RAc or RA alone at the highest dose of 32 micrograms/ml, and human lymphoid cells treated with Rol at the highest dose of 24 micrograms/ml resulted in killing of over 90% of the cells, while addition of S9 mix to the cultures treated with such high doses of a retinoid showed no reduction of viable cells as compared with the controls. The supernatant of S9 fraction after high speed centrifugation also had dose-dependent protective effects against the toxicity of retinol in V79 cells. Finally, experiments using sucrose gradient centrifugation and [3H] Rol suggests that binding of a retinoid to its specific binding protein in the S9 mixture and supernatant greatly decreases or abolished the toxicity of free retinoid in cultured mammalian cells.     

miR-145通过靶向抑制SMAD3的表达抑制非小细胞肺癌细胞侵袭能力

目的:探讨miR-145靶向调控SMAD3的表达对非小细胞肺癌细胞侵袭能力的影响。方法:采用qRT-PCR检测miR-145和SMAD3在30例非小细胞肺癌组织和癌旁组织中的表达水平并分析其相关性。利用靶基因预测网站预测miR-145的潜在靶基因SMAD3,利用双荧光素酶报告基因实验验证。利用细胞转染实验对非小细胞肺癌A549细胞进行了miR-145 mimics、miR-145 inhibitor、siRNA SMAD3及其相关对照的细胞转染,采用Transwell实验检测转染后A549细胞侵袭能力的变化。使用Western blot检测上调或下调miR-145表达对A549细胞中SMAD3蛋白表达的影响。结果:qRT-PCR结果显示,非小细胞肺癌组织中miR-145低表达,SMAD3高表达,且两者表达水平呈负相关。靶基因预测及验证实验结果显示miR-145可以与SMAD3 的 3'-UTR特异性结合,SMAD3是miR-145的靶基因。Western blot 结果显示,转染miR-145 mimics可以显著降低SMAD3蛋白的表达水平（P<0.001）,转染miR-145 inhibitor则显著提高SMAD3蛋白的表达水平（P<0.01）。Transwell体外侵袭实验结果显示,与对照组相比,转染miR-145 mimic显著降低了A549细胞的侵袭能力（P<0.001）,转染miR-145 inhibitor显著提高了A549细胞的侵袭能力（P<0.05）;与对照组相比,敲低SMAD3的表达A549细胞的侵袭能力减弱（P<0.001）;与单独敲低SMAD3相比,共同转染siRNA SMAD3和miR-145 mimics A549细胞的侵袭能力减弱（P<0.05）,共同转染siRNA SMAD3和miR-145 inhibitor A549细胞的侵袭能力无显著差异（P>0.05）。结论:miR-145在非小细胞肺癌组织中低表达,miR-145通过靶向抑制SMAD3的表达发挥对非小细胞肺癌细胞侵袭能力的抑制作用,为临床综合治疗提供了新的作用靶点。     

Retinol metabolism and lecithin:retinol acyltransferase levels are reduced in cultured human prostate cancer cells and tissue specimens

Recent studies from our laboratory have indicated that the metabolism of vitamin A (retinol) to retinyl esters, carried out primarily by the enzyme lecithin:retinol acyltransferase (LRAT), is greatly reduced in human carcinoma cell lines of the oral cavity, skin, breast, and kidney as compared with their normal epithelial counterparts. These studies suggest that human carcinoma cells are retinoid-deficient relative to normal epithelial cells. In this study, we examined the metabolism of [(3)H]retinol and [(3)H]retinoic acid (RA) in human prostate cancer lines and in primary cultures of human prostate epithelial cells. Normal cells esterified all of the [(3)H]retinol added to the cultures. In contrast, all seven prostate cancer cell lines and four primary cultures derived from prostatic adenocarcinomas metabolized only trace amounts of [(3)H]retinol to [(3)H]retinyl esters. Correlated with this relative lack of esterification of [(3)H]retinol by the cancer cells was loss of expression of LRAT protein, whereas normal cells expressed abundant levels of LRAT protein by Western analysis. The metabolism of [(3)H]RA was also examined in these prostatic cells. Two of the prostate cancer tumor lines, DU 145 and PJ-1, exhibited rapid metabolism of [(3)H]RA; in contrast, the other tumor lines or primary cultures metabolized [(3)H]RA at a much slower rate. We also found that the immortalization of normal human prostatic epithelial cells by SV40 T antigen led to a reduction in LRAT protein expression and esterification of [(3)H]retinol. Further transformation to tumorigenicity with the ras oncogene resulted in loss of detectable LRAT expression. Finally, we analyzed LRAT protein expression in tissue sections from six prostatectomy specimens by immunohistochemistry. LRAT protein was predominantly expressed in the basal cells of normal prostatic epithelium, whereas its expression was lost in prostate cancer. Collectively, these data implicate aberrant retinoid metabolism in the process of prostatic carcinogenesis.     

MicroRNA-328 is associated with (non-small) cell lung cancer (NSCLC) brain metastasis and mediates NSCLC migration

Brain metastasis (BM) can affect ～ 25% of nonsmall cell lung cancer (NSCLC) patients during their lifetime. Efforts to characterize patients that will develop BM have been disappointing. microRNAs (miRNAs) regulate the expression of target mRNAs. miRNAs play a role in regulating a variety of targets and, consequently, multiple pathways, which make them a powerful tool for early detection of disease, risk assessment, and prognosis. We investigated miRNAs that may serve as biomarkers to differentiate between NSCLC patients with and without BM. miRNA microarray profiling was performed on samples from clinically matched NSCLC from seven patients with BM (BM+) and six without BM (BM-). Using t-test and further qRT-PCR validation, eight miRNAs were confirmed to be significantly differentially expressed. Of these, expression of miR-328 and miR-330-3p were able to correctly classify BM+ vs. BM- patients. This classifier was used on a validation cohort (n = 15), and it correctly classified 12/15 patients. Gene expression analysis comparing A549 parental and A549 cells stably transfected to over-express miR-328 (A549-328) identified several significantly differentially expressed genes. PRKCA was one of the genes over-expressed in A549-328 cells. Additionally, A549-328 cells had significantly increased cell migration compared to A549 cells, which was significantly reduced upon PRKCA knockdown. In summary, miR-328 has a role in conferring migratory potential to NSCLC cells working in part through PRKCA and with further corroboration in additional independent cohorts, these miRNAs may be incorporated into clinical treatment decision making to stratify NSCLC patients at higher risk for developing BM.     

Inhibitory Effects of Syk Transfection on Lung Cancer Cell Invasion

Objective: Spleen tyrosine kinase (Syk) is closely related to tumor invasion and metastasis, and has been shownto have potential inhibitory effects in tumors. In this study, we constructed a eukaryotic expression vector forSyk and analyzed its effects on invasive ability of the A549 non-small cell lung cancer cell line in vitro. Methods:A fragment of Syk was obtained by RT-PCR from human lung cancer cells and cloned into the expression vectorpLNCXSyk. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinantSyk expression plasmid was transfected into A549 human lung cancer cells using lipofectamine protocols. Afterselection, the cells stably expressed Syk. Detection of Syk expression of the cells by RT-PCR, and invasive abilitywere examined. Results: The eukaryotic expression plamid pLNCXSyk was constructed and expressed stablyin the A549 human lung cancer cells. The RT-PCR results showed that Syk mRNA expression was upregulatedsignificantly (P<0.05). Lower invasion through a basal membrane were apparent after transfection (P<0.05).Conclusions: A eukaryotic expression plasmid to cause Syk expression in lung cancer cells can obviously inhibittheir invasive ability in vitro.     

Differential effects of retinoids on nitric oxide production by promonocytic U937 cells and ZR-75-1 human breast cancer cells

We demonstrated the presence of inducible and endothelial nitric oxide synthases in histiocytic lymphoma U937 cells by staining with anti-iNOS and anti-eNOS antibodies. We also investigated the effects of retinol and retinoic acid on nitric oxide production by both U937 cells and ZR-75-1 human breast cancer cells. U937 cells which had been treated with either retinol or retinoic acid (10-10-10-6 M) exhibited no significant difference in nitric oxide secretion into conditioned medium. Conversely, for ZR-75-1 cells, both retinol and retinoic acid (10-10-10-6 M) caused a significant (p<0.001) increase in the amount of nitrite secreted. Our results indicate that retinoid induced growth inhibition of breast cancer cells is associated with an increase in NO production, however, an increase in NO synthesis does not mediate retinoid induced differentiation of U937 cells.     

Connexin 32 expression reduces malignant phenotype in human A549 adenocarcinoma cells: Implication of Src involvement

Recent evidence suggests that a member of the gap junction protein family, connexin (Cx) 32, acts as a tumor suppressor gene against lung adenocarcinoma. However, the precise mechanism remains unclear. In this study, we tried to explore the mechanism for the Cx32-dependent tumor-suppressive effect in lung adenocarcinoma. To perform this study, we established a stable clone of the human lung adenocarcinoma cell line, A549 in which the Cx32 gene was expressed. Cx32 expression in A549 cells reduced anchorage-independent growth and development of tumors in a xenograft model. Additionally, Cx32 induced contact inhibition of growth and reduced invasive activity in A549 cells. The tumor-suppressive effects of Cx32 depended on the inhibition of Src activity. These events were confirmed by an Src inhibitor (PP1) and siRNA for Cx32. These results suggest that the Cx32-dependent tumor-suppressive effect in A549 cells is explained by the inhibition of Src activity.     

针对layilin的shRNA抑制透明质酸诱导人肺腺癌A549细胞体外黏附和侵袭

背景与目的 透明质酸参与了多种肿瘤细胞黏附侵袭行为。layilin是一种新发现的与细胞骨架有密切联系的透明质酸受体。本研究的目的是观察针对layilin的短发夹RNA（shorthairpinRNA，shRNA）对体外培养的人肺腺癌A549细胞layilin表达及受透明质酸诱导黏附侵袭行为的影响。方法 设计和构建带有U6启动子的能产生针对layilin的shRNA的RNA干扰质粒，转染至A549细胞，以RT—PCR、Westernblot和免疫荧光检测A549细胞转染前后layilin表达，以平板黏附模型和Boyden小室模型检测A549细胞转染前后黏附和侵袭能力。结果和转染前相比，转染了RNA干扰表达质粒的A549细胞layilin表达明显减少（P〈0．01），而且受透明质酸诱导而黏附于平板和穿过Boyden小室隔膜的A549细胞数皆明显减少（P〈0．01）。结论 针对layilin的shRNA能明显抑制透明质酸诱导的人肺腺癌A549细胞体外黏附和侵袭能力。     

Retinol decreases phosphatidylinositol 3-kinase activity in colon cancer cells

Previously, we showed that retinol inhibited all-trans-retinoic acid (ATRA)-resistant human colon cancer cell invasion via a retinoic acid receptor-independent mechanism. Because phosphatidylinositol 3-kinase (PI3K) regulates cell invasion, the objective of the current study was to determine if retinol affected PI3K activity. Following 24 h of serum starvation, the ATRA resistant human colon cancer cell lines HCT-116 and SW620 were treated with 0, 1, or 10 microM retinol. Thirty minutes of retinol treatment resulted in a significant decrease in PI3K activity in both cell lines. To determine the mechanism by which retinol reduces PI3K activity, the levels and heterodimerization of the regulatory subunit, p85, and the catalytic subunit, p110, of PI3K were examined. Retinol treatment did not alter p85 or p110 protein levels or the heterodimerization of these subunits at any time point examined. To determine if retinol affected the ability of PI3K to phosphorylate the substrate, phosphatidylinositol (PI), PI3K was immunoprecipitated from control cells and incubated with 10 microg PI and increasing concentrations of retinol or 10 microg retinol and increasing concentrations of PI. Retinol decreased PI3K activity in a dose-responsive manner and increased PI suppressed the inhibitory effect of retinol on PI3K activity. Finally, the PI3K inhibitor, LY294002, mimicked the ability of retinol to decrease cell invasion. Computational modeling revealed that retinol may inhibit PI3K activity in a manner similar to that of wortmannin. Thus, a decrease in PI3K activity due to retinol treatment may confer the ability of retinol to inhibit ATRA-resistant colon cancer cell invasion.     

CD44v3在透明质酸诱导人肺腺癌A549细胞体外粘附和侵袭中的作用

目的:研究CD44v3在透明质酸诱导A549细胞体外粘附和侵袭中的作用。方法:设计和构建能产生针对CD44v3的短发夹RNA(shorthairpinRNA,shRNA)的RNA干扰质粒,转染A549细胞,以RT-PCR和Westernblot检测A549细胞转染前后CD44v3表达,以平板粘附模型和波登氏小室模型检测A549细胞转染前后粘附和侵袭能力。结果:和转染前相比,转染了RNA干扰质粒的A549细胞CD44v3mRNA和蛋白表达以及受透明质酸诱导而粘附于平板和穿过波登氏小室隔膜的细胞数皆明显减少(P<0.01)。结论:CD44v3介导了透明质酸诱导人肺腺癌A549细胞体外粘附和侵袭行为。     

Antisense-mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells.

Heparan sulfate proteoglycans is a major component of the cell surface and extracellular matrix and functions as a barrier against cationic molecules and macromolecules. Heparanase is an endoglucuronidase capable of specifically degrading heparan sulfate, and its activity is associated with the metastatic potential of tumor cells. To inhibit human heparanase expression in human cancer cells, we constructed an adenoviral vector carrying a full-length human heparanase cDNA in an antisense orientation (Ad-AS/hep). Increased heparanase expression in T.Tn human esophageal cancer cells and A549 human lung cancer cells after infection with an adenovirus vector expressing the human heparanase gene (Ad-S/hep) was specifically inhibited by simultaneous infection with Ad-AS/hep in a dose-dependent manner. A modified Boyden chamber assay demonstrated that infection with Ad-AS/hep significantly inhibited in vitro invasion of A549 cells after Ad-S/hep infection. Moreover, intrathoracic administration of Ad-AS/hep reduced the number and size of heparanase-expressing A549 tumors implanted intrathoracically into BALB/c-nu/nu mice. Our results suggest that heparanase contributes to the invasive phenotype of tumor cells, and that antisense-mediated inhibition of heparanase activity may be efficacious in the prevention of pleural dissemination.     

Retinoic acid and epidermal growth factor binding in retinoid-mediated invasion suppressed human lung carcinoma cells

The effect of retinoic acid (RA)-induced suppression of in vitro invasive ability of A549 human lung carcinoma cells on cellular binding of RA and epidermal growth factor (EGF) was investigated. RA inhibition of cell invasive potential was accompanied by a significant increase in specific high affinity cellular retinoic acid binding protein (CRABP) level. An approximately 2.7-fold increase in cytosolic CRABP was found in RA-treated cells (450 fm/mg protein compared to 167 fm/mg control cell protein). In contrast, 125I-EGF ligand binding was similar for control and treated cells.     

Src羧基端激酶结合蛋白影响线粒体分裂及NK细胞杀伤肺癌细胞活性的分子机制探讨

目的:探究Src羧基端激酶结合蛋白（csk-binding protein,CBP）在人肺癌组织中的表达水平,及其对线粒体分裂和自然杀伤（natural killer,NK)细胞杀伤肺癌细胞活性的影响。方法:免疫组织化学染色和实时荧光定量PCR（qRT-PCR）检测肺癌组织及癌旁组织中CBP的表达;含CBP慢病毒感染人肺癌细胞株A549,通过荧光倒置显微镜、qRT-PCR 和Western blot检测细胞感染效果;CCK-8法检测各组A549细胞活性,Mito-Tracker染色观察各组A549细胞内线粒体形态和长度变化,Western blot检测各组A549细胞线粒体动态相关蛋白Mfn1、Mfn2、Drp1的表达,免疫荧光染色检测各组A549细胞内细胞色素C（Cyt C）的表达,流式细胞术检测各组A549细胞凋亡情况;从人外周血单个核细胞（peripheral blood mononuclear cell,PBMC）中分离NK细胞并进行鉴定,乳酸脱氢酶释放实验检测NK细胞对各组A549细胞的杀伤率。结果:肺癌组织中CBP的表达水平较癌旁组织中明显下降（P＜0.01）;感染CBP过表达慢病毒的A549细胞中CBP mRNA和蛋白的相对表达量均升高,培养48 h、72 h、96 h后细胞增殖活性下降,线粒体呈椭圆状或短杆状,长度明显缩短,线粒体分裂相关蛋白Mfn1、Mfn2蛋白表达减少,Drp1蛋白表达增加,有大量Cyt C从线粒体释放,细胞凋亡率升高,同时NK细胞对A549细胞的杀伤率明显提高,差异均具有统计学意义（P＜0.01）;而在使用线粒体分裂蛋白抑制剂Mdivi-1预先处理A549细胞后再感染CBP过表达慢病毒,线粒体分裂受到抑制,Mfn1、Mfn2蛋白表达增加,Drp1蛋白表达减少,Cyt C释放减少,细胞凋亡率降低,NK细胞对A549细胞的杀伤率也受到抑制,差异均具有统计学意义（P＜0.01）。结论:CBP在人肺癌组织中低表达,在A549细胞中过表达CBP能够促进线粒体分裂与细胞凋亡,并提高NK细胞对A549细胞的杀伤率。     

Retinoid receptor mRNA expression profiles in human bladder cancer specimens

Retinoids, which include vitamin A (retinol) and its derivatives, have previously been investigated as potential chemopreventive and chemotherapeutic agents in bladder cancer. We examined mRNA expression of the retinoid receptors RARalpha, RARbeta2, RARgamma and RXRalpha, as well as two putative RARbeta2 target genes, DAB2 and Midkine, in normal and malignant bladder tissue specimens from human patients. We evaluated 24 normal and malignant bladder specimens for retinoid receptor, DAB2 and Midkine mRNA expression using RT-PCR. We also examined the effects of retinoic acid and retinol on the expression of these genes in five human bladder cancer cell lines. Expression of RARalpha, RARbeta2, RARgamma and RXRalpha mRNA was detected in all of the non-neoplastic patient bladder specimens. RARbeta2 mRNA expression was undetectable in 7/13 tumors, RARalpha in 3/13, RARgamma in 1/13 and RXRalpha in 2/13. DAB2 mRNA was expressed in all non-neoplastic and all tumor specimens, while Midkine mRNA was detected in 8/11 non-neoplastic specimens versus 11/13 tumors. Two of the five bladder cancer cell lines expressed RARbeta2 independent of retinoid exposure; in three cell lines RARbeta2 expression was induced by retinoids. RARalpha, RARgamma and RXRalpha mRNA expression was detected in 5/5 cell lines, independent of retinoid exposure. We found a reduction in retinoid receptor mRNA expression, particularly for RARbeta2, in human bladder cancer specimens. We also demonstrated induction of RARbeta2 mRNA expression in some of the retinoid-treated bladder cancer cell lines. We suggest that restoration of RARbeta2 expression may be a reasonable biomarker for developing bladder cancer preventive and/or therapeutic drugs.