Snail Research Articles

Axon guidance repulsant SEMA3F increases chemosensitivity to oxaliplatin and inhibits epithelial-mesenchymal transition of colorectal cancer cells

Background: Our previous study has shown that down-regulation of axon guidance repulsive Semaphorin-3F (SEMA3F) promotes growth and metastasis of colorectal cancer (CRC) cells. However, the role of SEMA3F in chemosensitivity and epithelial-mesenchymal transition of CRC cells remains unknown. Methods: The expression of SEMA3F, P-gp, GST-π and TOPO-II in CRC tissues from 94 patients was determined by immunohistochemical staining. Knock-down of SEMA3F was achieved by lentivirus transfection. Functional assays were done to evaluate the invasion, apoptosis and cell viability. Results: We found that the expression of SEMA3F is negatively correlated with the expression of P-gp and GST-π in CRC tissues from 94 patients. Knock-down of SEMA3F significantly decreased apoptosis of HCT116 and SW480 cells, accompanying with up-regulation of anti-apoptotic BCL-2 and down-regulation of pro-apoptotic BAD and BAX. In addition, knock-down of SEMA3F attenuated chemosensitivity to oxaliplatin of CRC cells. Moreover, we found that down-regulation of SEMA3F promotes epithelial-mesenchymal transition of HCT116 and SW480 cells by decreasing the expression of epithelial marker E-cadherin, and increasing the expression of mesenchymal marker Slug, Snail and Vimentin. Finally, knock-down of SEMA3F increased the tumor volume and decreased overall survival of nude mice using colorectal cancer orthotopic xenograft model under the treatment of oxaliplatin. Conclusions: SEMA3F increases chemosensitivity to oxaliplatin and inhibits epithelial-mesenchymal transition of CRC cells.

CCL2 induces epithelial-mesenchyme transition in breast cancer MCF-7 cells

Objective To explore the effect and molecular mechanism of CC chemokine ligand 2 (CCL2) on epithelial-mesenchymal transition in human breast cancer cells. Methods Breast cancer Michigan cancer foundation-7 (MCF-7) cells were treated with 50 ng/ml CCL2. The abilities of invasion were detected by Transwell assay. The expression of epithelial-mesenchymal transition (EMT)-associated markers, E-cadherin and vimentin were detected by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of Snail, protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), phosphorylated glycogen synthase kinase 3β (p-GSK3β) and GSK3β were detected by Western blot. Snail nuclear localization was detected by immunoflurescence staining. Results We found that CCL2 treatment could induce morphological alteration of MCF-7 cells from epithelial morphology to mesenchymal morphology. CCL2 significantly increased the migration of MCF-7 cells, and increased the expression of mesenchymal maker vimentin and decreased epithelial marker E-cadherin. More important, treatment of CCL2 significantly increased the expression of Snail, and promoted the nuclear localization of Snail. Knockdown of Snail significantly reverse the effects of CCL2 on the EMT in MCF-7 cells. Moreover, treatment of CCL2 significantly increased the phosphorylation levels of p-AKT and p-GSK3β, and AKT inhibitor LY294002 significantly inhibited CCL2-induced Snail and p-GSK3β expression. Conclusion CCL2 might induce EMT in MCF-7 cells, by which mechanism is related to activate AKT/GSK3β Snail pathway. Key words: Chemokine CCL2; Breast neoplasms; Tumor cells, cultured; Biotransformation

Localization of oestrogen hormone receptors in the reproductive tract of giant African land snail (Archachatina marginata) and potential role of Mucuna pruriens on level of expression

This study determines localization of oestrogen hormone receptors in selected parts of the reproductive tract of giant African land snail (Archachatina marginata) and potential role of Mucuna pruriens on levels of expression. Thirty (30) snails were used for this experiment, with average weight of 100-150g. The snails were allotted to three dietary treatments which includes concentrate (CON), concentrate + Mucuna pruriens (CON+MSP) and Mucuna pruriens seed powder only (MSP). Each treatment contains 10 Replicate each. After eight weeks, five snails were randomly selected from each dietary treatment and dissected. Organs removed were oviduct, albumen gland, ovo-testis and the spermatheca. RNA and DNA extractions were carried out with commercial kits. Gel electrophoresis on 1% agarose was also carried out to evaluate the expression. Primers sequence used were the forward and reverse β oestrogen primer which was designed to detect the expression of the gene encoding oestrogen receptor in the reproductive tract of the giant African Land Snail (Archachatina marginata) were: Forward: 5’-GCT TCG AGC TCA GCC TG-3’ Reverse: 5’-AGG ATC ATG GCC TTG ACA CAG A-3’. Result showed that receptors for estrogen were present in oviduct, albumen gland, ovo-testis and spermatheca for both RNA and DNA analysis considering CON and MSP. While oviduct was also moderately expressed for CON and MSP. But combination of concentrate and Mucuna seed powder (CON+MSP) showed no visible expression for estrogen receptor. It was however concluded that combination of concentrate and mucuna seed powder (CON+MSP) down-regulate expression of estrogen receptor gene expression. It was recommended that feed to be combined for snail production must be free from substance(s) that has the potential to block the expression of reproductive hormone function.

THE ANTIGENIC RELATIONSHIP BETWEEN SCHISTOSOMA MANSONI AND ITS INTERMEDIATE SNAIL HOST.

Schistosomiasis is a public health problem, in many developing: countries including Egypt, Determination of the antigenic relationship between S mansoni and its intermediate snail host (IMH) Biomphalaria alexandrina can open a new field for diagnosis and control of the dis- ease. In the present study infected and non-infected B. alexandrina foot and visceral hump tissue as well as S. mansion crude Ag (SWAg) were fractionated using SDS-PAGE. It's specific and cross reacted protein fractions were determine using EITB versus experimentally prepared mice hyper immune sera (HIS) versus each antigen. After treatment of fractionated S.mansoni crude worm antigens (SWAg) versus HIS produced after vaccination of mice by the same Ag, 8 kda protein fractions ranged from 35-140 kda were reacted specifically. Treatment of fractionated B.alexandrina infected and non-infected foot and visceral hump Ag versus previous HIS revealed presence of common polypeptides bands between SWAg and non-infected snail antigens. The fraction at 135 kda, 68 kda, were detected in all cases, while that at 40-42 kda and that at 35 kda was diagnosed in SWAg and that of infected snails only. The fraction at 68 kda was reacted specifically between SWAg and all tested fractionated snail antigens either that of foot or visceral hump when they treated separately by HIS of mice vaccinated by each snail A eparately. The fraction at 135 kda was common between SWAg and snail (infected and non-infected) visceral hump antigen. The fraction at the level of 110 kda was diagnosed inSWAg, in non-infected foot antigen and visceral hump Ag. The fraction at the level of 46-48 (da are common between SWAg and snail foot and visceral hump Ag after treated by HIS of mice vaccinated by foot Ag, Presence of common antigenic fractions between snail tissues and Schistosoma species can prefer an easily source of antigen valuable for diaguosis or vaccination as well as can be considered as new tool for determination to the snail IMH of new discovered trematode Darasites.

EPIDEMIOLOGICAL SITUATION OF URINARY SCHISTOSOMIASIS IN TAMWAH AREA, GIZA, EGYPT: ASSESSMENT AND CONTROL.

Preliminary studies were carried out on schistosomiasis in Giza Governorate for the last three years. These studies revealed that Tamwah village was one of areas afflicted by the highest number of Schistosoma haematobium infection cases. 'The study assessed the epidemiological situation of S. haematobium by parasitological and snail surveys. During April 2016, urine samples of 1285 children collected from three primary schools were centrifuged for microscopic examination. Also a snail survey was done along the shore (700m length). The snail were classified and examined for cercariae by light exposure and crushing. The results revealed that S. haematobiwn was 4.04% (52 cases). Majority were males (76.9% out of positive cases), with highly significant. There was a strong correlation between age of children and infection (44.2%) among oldest children (11 years) lowest (3.8%) was among the youngest group (6 years). Enterobius verinicularis ova in females' urine samples was 0.54% (7 cases). One was mixed infection with S. haematobium. The highest percentage among children infected with S. haematobium had pus cells 6- 30/HPF and RBCs less than 100/HPF in urine. There were crystals of uric acid, Ca oxalates and triple phosphate 5.8 %, 1.8% & 0.8% respectively. The snails were Lanistes carinatus, Bellamya unicolor, Physa micropleura, Succinia cleopatra, Cleopatra bulmnoides, Bulinus trancatus & Lymnaea natalensis. The commonest was B. trancatus followed by L. natalensis. Builinus spp. were positive for schistosomiasis -cercaria and Lymnaea spp were positive for virgulate xiphidiocercariae; parasites of bates, birds and amphibians.

The association between snail mRNA expression and airway remodeling in elderly patients with acute asthma attack

Objective This study is aimed at demonstrating the relationship between the Snail mRNA expression and airway remodeling in the elderly patients with acute asthma. Methods The used animal model was elderly rat with asthma.The expression levels of Snail mRNA were detected in the lung tissues of asthma rats by RT-PCR.The corresponding trachea tensions were also determined.The sputum samples of 60 asthma patients and 20 health controls were collected, in which the expression level of Snail mRNA was detected and analyzed. Results In asthma rats, mRNA expression level of Snail was up-regulated corresponding to the alleviated asthma progression(0.65±0.04、0.91±0.05、1.25±0.04、1.47±0.07). In asthma patients, similar result was obtained(0.63±0.02、0.85±0.06、1.05±0.04、1.24±0.07). Conclusions Snail expression is positively correlated with asthma progression in both rats and human.It might be an important marker indicating asthma progression, which is a potential therapeutic target for asthma. Key words: Asthma; Lung diseases, interstitial

Insulin signaling: At a snail's pace.

Insulin signaling: At a snail's pace.

Silencing Twist gene leads to mesenchymal-to-epithelial transition and loss of stemlike properties in breast cancer

Objective To observe the effect of silencing Twist gene on mesenchymal-to-epithelial transition (MET) and stem-like properties in breast cancer. Methods The small interfering RNA (siRNA) vectors targeting Twist gene were transfected into MDA-MB-231 cells. The mRNA and protein expression levels of Twist and MET-associated genes (Slug, Snail and Vimentin) were detected by using real-time quantitative polymerase chain reaction(Real-time PCR) and Western blotting. Cell morphology was observed under microscope. The proportion of CD44+ CD24- cells was measured by flow cytometry. In vitro self-renewal capacity was evaluated by sphere formation assay. Tumor-initiating ability was measured by limited dilution assays. Results Compared to the control group, the Twist mRNA (0.24±0.04, P<0.01) and protein expression in siRNA group was significantly decreased. Cells with Twist silencing underwent MET reversal, showing an epithelial-like appearance, decreased mRNA expression of mesenchymal markers, including Slug (0.25±0.05, P<0.01), Snail (0.34±0.03, P<0.01) and Vimentin (0.44±0.04, P<0.01), increased mRNA expression of epithelial marker, E-cadherin (58.71±1.27, P<0.01), down-regulated protein levels of Slug, Snail and Vimentin, and up-regulated protein level of E-cadherin. The proportion of CD44+ CD24- cells in the si-Twist group and control group was (63.30±0.80)% and (86.50±0.70)% respectively (P<0.01). The sphere number in the si-Twist and control groups was (46.67±2.52) and (109.67±2.52) respectively (P<0.01). Twist silencing led to the decrease of about 6-folds in tumor-initiating ability in nude mice compared to the control (P<0.01). Conclusion Silencing Twist gene leads to MET and loss of self-renewal capacity and tumor-initiating ability in breast cancer. Key words: Breast cancer; Twist; Small interfering RNA; Mesenchymal-to-epithelial transition; Tumor stem cells

Effect of vasoactive intestinal peptide on cell migration and invasion and epithelial-mesenchymal transition in prostate cancer DU145 cells

Objective To observe the effect of intestinal peptide on cell migration and invasion of the DU145 cells, and explore the mechanism of epithelial-mesenchymal transition. Methods DU145 cells were treated with VIP at concentrations of 0, 25, 50 and 100 μmol/L, respectively. E-cadherin, Vimentin, and Snail mRNA and protein were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting respectively at 24 h. The cell mobility, migration and invasion ability of DU145 cells were respectively measured by wound-healing assay, Transwell migration and invasion assays at 48 h. Results With the increases of concentrations of VIP for 24 h, the expression levels of E-cadherin, Vimentin, and Snail mRNA in PC-3 cells were increased at 24 h (0.897±0.053, 0.715±0.041, 0.521±0.036, 0.343±0.029; 0.586±0.061, 0.827±0.089, 1.329±0.107, 1.764±0.135; 0.645±0.055, 0.917±0.074, 1.383±0.097, 1.725±0.124; P<0.05). Meanwhile, the cell mobility was increased, the number of migrative cells and invasive cells was significantly increased gradually with the increased concentrations of VIP [(76.7±5.8), (104.5±6.7), (128.3±10.4), (147.7±12.6) cells; (84.2±6.4), (118.3±10.3), (142.1±13.7), (173.6±15.4) cells; P<0.01]. Conclusion VIP can increase ability of movement, migration and invasion of prostate cancer DU145 cells by epithelial-mesenchymal transition. Key words: Vasoactive intestinal peptide; Prostate carcinoma; Tumor metastasis; Epithelial-mesenchymal transition

Expression and clinical biology behavior of thymidine kinase 1 and snail in non-small cell lung carcinoma

Objective To investigate the expression of thymidine kinase 1 (TK1) and snail in non-small cell lung carcinoma (NSCLC), and their correlation with the clinicopathological features. Methods Envision immunohistochemical method was used to assess the expression of TK1 and Snail in 87 cases of human NSCLC tissues and non-cancerous adjacent lung tissues. Results The expression rate of TK1 in breast carcinoma tissues was higher than that of non-cancerous adjacent breast tissues (71.26% vs. 21.84%, P<0.01). The expression rate of Snail in breast carcinoma tissues was higher than that of non-cancerous adjacent breast tissues (85.06% vs. 18.39%, P<0.01). The expression levels of TK1 were correlated histologic grade, TNM stage and lymph node metastasis of NSCLC (P<0.05). There was a close correlation between the expression of Snail and histologic grade and TNM stage (P<0.05). Conclusion TK1 and Snail may participate in the development and progression of NSCLC, and may be used to provide information for predicting prognosis of patients with NSCLC. Key words: Non-small cell lung carcinoma; Thymidine Kinase 1; Snail; Immunohistochemical method

Radiation-induced expression of HOTAIR, Snail and E-cadherin in esophageal squamous cell carcinoma cells

目的：探索X线诱导食管鳞癌细胞内HOX转录反义RNA（HOX transcript antisense RNA,HOTAIR）及上皮间质化相关因子Snail,E-钙黏蛋白（E-cadherin）表达的时间剂量效应,方法：0、2、4、6、8 Gy X射线分别照射食管鳞癌Eca109细胞后,分别于0、2、4、8、16、24 h提取细胞内总RNA及蛋白,实时定量PCR检测（real-time quantitative PCR,qRTPCR）及Western blot检测相应指标mRNA及蛋白的表达水平.结果：Eca109细胞接受X线照射后继续培养24 h后发现,随X线照射剂量渐增大（0→2→4→6→8 Gy）,HOTAIR与Snail表达水平逐渐升高：HOTAIR mRNA,Snail mRNA在6 Gy与8 Gy照射组表达水平显著高于0 Gy组,Snail蛋白在4 Gy照射组即有显著升高;而E-cadherin mRNA与其蛋白表达水平逐渐降低,6 Gy与8 Gy照射组显著低于0 Gy组,均P〈0.01.细胞接受8 Gy X线照射后,伴随照射后培养时间延长（0→2→4→8→16→24 h）细胞内HOTAIR,Snail表达水平逐渐升高：与0 Gy组相比,细胞培养至第8小时HOTAIR mRNA表达水平显著升高,培养至第16小时Snail mRNA与其蛋白表达水平显著升高;而E-cadherin mRNA与其蛋白伴随照射后培养时间延长表达水平逐渐降低,培养至第8小时E-cadherin与其蛋白表达水平即显著低于0 Gy组（均P〈0.01）.结论：X射线诱导HOTAIR,Snail及E-cadherin的异常表达呈时间和剂量依赖性,三者可能共同参与肿瘤细胞放疗抵抗形成.

Turismo de risco para esquistossomose mansônica em Porto de Galinhas, Estado de Pernambuco, Brasil

Conselho Nacional de Desenvolvimento Cientifico e Tecnologico e Secretaria de Vigilância em Saude do Ministerio da Saude.

Lizards evolved at snail's pace

Lizards evolved at snail's pace

Associations between interleukin-17A expression and epithelial-mesenchymal transition in patients with hepatocellular carcinoma

To detect the expression of interleukin-17A(IL-17A) in hepatocellular carcinoma (HCCs) tissues, and to analyze its relationship with clinicopathological characteristics and epithelial-mesenchymal transition (EMT). The expression of IL-17A, E-cadherin, vimentin proteins and Snail mRNA were detected by immunohistochemistry and in situ hybridization histochemistry in the hepatocellular carcinoma (HCC) tissues of 74 patients. IL-17A staining was detected in 54.1% (40/74) specimens of human HCCs, but only 25.0% (5/20) in corresponding peritumoral tissues (P<0.05). The positive rate of IL-17A expression in HCC patients with grade III+IV and UICC stage III+IV tumors was significantly higher than those with grade I+II and UICC stage I+II tumors. The expression of IL-17A was positively correlated with portal vein tumor thrombus and microvascular invasion (all P<0.05). The 1- and 2-year recurrence-free survival rates were 27.6% and 17.2% in the patients with positive IL-17A expression, but 79.3% and 58.5% in IL-17A-negative HCCs. The 1- and 2-year overall survival rates were 69.0% and 27.8% in the cases with positive IL-17A expression, while 91.3% and 87.0% in IL-17A-negative cases. Patients with IL-17A-positive HCCs showed significantly shorter recurrence-free and overall survival compared with the patients with IL-17A-negative HCCs (P<0.05). Multivariate analysis indicated that IL-17A expression was an independent factor for recurrence-free and overall survival of HCCs. IL-17A-positive HCCs were characterized by increased expression of vimentin (r=0.492, P<0.01) or Snail (r=0.410, P<0.05) and loss of E-cadherin expression (r=-0.404, P<0.05). Our results suggest that IL-17A is closely related to epithelial-mesenchymal transition in hepatocellular carcinoma. IL-17A-positive hepatocellular carcinoma demonstrates more aggressive biological behavior, and IL-17A may serve as a potential prognostic marker for this cancer.

Impact on prevalence of schitosomiasis after runs of Three Gorges Reservoir Project in the section of Anhui province

To investigate the changes of water level and the distribution of snails in Anhui province before and after runs of the Three Gorges Reservoir Project, and to determine the relationship between the two factors and schistosomiasis transmission. The hydrologic data of Datong hydrologic station and the data of snail status and schistosomiasis morbidity in Anhui Province were collected. The data from 1991 to 2002 and 2003 to 2012 were considered as before and after the impoundment of the Three Gorges Reservoir Project. Based on the prevalence of schistosomiasis, the cases of people and cattle were speculated, and the average infection rate of people and cattle were calculated. The t-test was used to compare the difference of snail area and the density of living snails before and after the impoundment of the Three Gorges Project. The pearson method was used to analyze the relationship between water level and snail area. The spearman method was used to analyze the relationship between the water level and the distribution of snails. From 1991 to 2012, the range of the highest water level, the lowest water level, the difference between the highest and lowest water level, the mean in the abundant water seasons, the mean in the dry water seasons, and the difference between the abundant water seasons and the dry water seasons was 11.40-16.30, 3.68-5.20, 6.70-12.12, 9.92-14.40, 4.77-7.64 and 4.13-8.93 m, respectively. The snail areas was (28 613 ± 362) hm² and (29 477 ± 918) hm² (t = -3.00, P = 0.007), the density of living snails was 1.51 (1.15-2.43) and 0.43 (0.29-1.10) snails/0.11 m² (H = 4.28, P < 0.001) before and after the impoundment of the Three Gorges Project, respectively. The average infection rate of people and cattle was 1.68% (99 482/5 935 147) and 4.62% (13 923/3 011 33), and the average number of acute schistosomiasis cases was 328, before the impoundment of the Three Gorges Project; 0.60% (39 747/6 649 380), 1.65% (1 291/783 224) and 71 after the impoundment of the Three Gorges Reservoir Project, respectively. The snail areas had negative correlation with the highest water level, the difference between the highest and lowest water level, the mean in the abundant water seasons (r value was -0.514, -0.509 and -0.477; P value was 0.014, 0.015 and 0.025, respectively). The infection rate of people had positive correlation with the highest water level, the difference between the highest and lowest water level, the mean in the abundant water seasons (r value was 0.532, 0.587 and 0.446; P value was 0.011, 0.004 and 0.038, respectively). The infection rate of cattle had positive correlation with the highest water level, the difference between the highest and lowest water level (r value was 0.507 and 0.553; P value was 0.016 and 0.008, respectively). The number of acute schistosomiasis cases had positive correlation with the highest water level, the difference between the highest and lowest water level (r value was 0.481 and 0.486; P value was 0.023 and 0.022, respectively). Following the runs of the Three Gorges Reservoir Project, the change of water level in the section of Anhui Province affected the distribution of snails and the infection of people and cattle to some extent. The snail areas showed an upward trend, and the density of living snails, the infection rate of people and cattle showed a downward trend. The runs of Three Gorges Reservoir Project has certain role to reduce flood and helpful for schistosomiasis control.

Efecto de la espirulina (Arthrospira platensis) (Oscillatoriales: Cyanobacteria) en la cría experimental de Pseudosuccinea columella (Basommatophora: Lymnaeidae)

&lt;p&gt;Snails of the family Lymnaeidae, as &lt;em&gt;Pseudosuccinea columella&lt;/em&gt;, are the intermediate hosts of &lt;em&gt;Fasciola hepatica&lt;/em&gt;, the causative agent of fasciolosis in human and livestock all over the world. A thorough knowledge of snail biology is essential for describing the transmission dynamics and for controlling this disease. Since food quality has had a significant effect on snail growth, fecundity and fertility, in this study we evaluated the use of spirulina (&lt;em&gt;Arthrospira platensis&lt;/em&gt;) as a food resource for the artificial breeding of &lt;em&gt;P. columella&lt;/em&gt;, an invasive snail and the main intermediate host of &lt;em&gt;F. hepatica&lt;/em&gt; in Northeastern Argentina. The main purpose was to measure the effect of spirulina on fitness parameters such as survival rate, growth rate, size at first reproduction, lifetime fecundity and viable offspring. A total of 20 676 newly-laid F&lt;sub&gt;2&lt;/sub&gt; eggs were used; half of them were fed with lettuce (treatment L) and the other half with lettuce plus spirulina (treatment L+S). In comparison with &lt;em&gt;P. columella&lt;/em&gt; snails fed only with lettuce, we found that &lt;em&gt;P. columella&lt;/em&gt; fed with lettuce plus spirulina: &lt;strong&gt;1)&lt;/strong&gt; showed higher survival rates, &lt;strong&gt;2)&lt;/strong&gt; grew faster and showed higher growth increments, &lt;strong&gt;3)&lt;/strong&gt; attained sexual maturity earlier in time (L+S:60 days vs. L:120 days) and at a smaller size (L+S:4.8mm vs. L:8.2mm), &lt;strong&gt;4)&lt;/strong&gt; had a longer reproductive period (L+S:150 days vs. L:90 days), &lt;strong&gt;5)&lt;/strong&gt; produced a higher number of eggs/snail (L+S:29.6 vs. L:13.3), and &lt;strong&gt;6)&lt;/strong&gt; showed a higher offspring hatching rate (L+S:70% vs. L:40%). The supplementation of &lt;em&gt;P. columella&lt;/em&gt; diet with commercial spirulina enhances it fitness and improved the artificial breeding of this species. Spirulina may have a direct positive effect on &lt;em&gt;P. columella&lt;/em&gt; development by consuming it, along with an indirect positive effect by improving the water quality. This rearing technique provided large number of reproducing adults and a continuous production of offspring, which are essential for developing future experimental studies in order to improve our knowledge on &lt;em&gt;P. columella&lt;/em&gt; biology.&lt;/p&gt;

Bufalin reverses hepatocyte growth factor-induced resistance to afatinib in H1975 lung cancer cells

To investigate the effects of bufalin in reversing hepatocyte growth factor (HGF)-induced resistance to afatinib in H1975 lung cancer cells, and explore its possible mechanism. The afatinib-resistant H1975 lung cancer cells (H1975AR) were induced by exogenous HGF and transfected with recombinant adenoviral vector Ad-HGF-GFP. The cytostatic effects of bufalin, afatinib and bufalin plus afatinib on H1975AR cells were evaluated by MTT assay. The impact of combined therapy with bufalin and afatinib on invasion of H1975AR cells was determined by transwell migration assay. The concentrations of HGF in the culture supernatants of H1975/Vec and H1975/HGF cells were determined by ELISA. The expression of EGFR, cMET and EMT signal pathway-related proteins in H1975AR cells treated with bufalin, afatinib and bufalin plus afatinib were detected by Western blot. The results of MTT assay showed that afatinib did not inhibit the growth of H1975 cells, but after 72 h of the combined treatment with bufalin and afatinib and in the presence of HGF, the growth rate of H1975 cells was (38.67 ± 8.76)%, significantly lower than the growth rate of (63.45 ± 12.65)% in the H1975 cells treated with HGF alone (P < 0.05). The results of transwell migration assay showed that in the presence of HGF, afatinib plus bufalin combination therapy markedly decreased the number of invaded H1975 cells through the Matrigel chamber (48.98 ± 11.43), significantly lower than the 118.92 ± 37.29 of afatinib-treated or the 88.84 ± 19.53 of bufalin-treated cells (P < 0.05 for all). The result of ELISA showed that H1975/HGF cells secreted high levels of HGF, and afatinib and bufalin had no effect on the HGF secretion in H1975/HGF cells. The results of Western blot analysis showed that the expression of p-EGFR, p-cMet, p-AKT, p-ERK, vimentin and snail in H1975AR cells treated with bufalin puls afatinb was down-regulated markedly, and the expression of E-cadherin was up-regulated markedly. Combination of bufalin and afatinib strongly inhibits the growth of H1975AR lung cancer cells and decreases their invasion ability. The possible mechanism of combined treatment with bufalin and afatinib may be related to the blocking of cMet/PI3K/AKT and cMet/MAPK/ERK pathways and inhibiting of epithelial-mesenchymal transition.

NDRG1 suppresses TGF-β-induced epithelial-mesenchymal transition in lung cancer A549 cells

Objective To explore the effect and molecular mechanism of N-myc downstream regulated gene 1 (NDRG1) on transforming growth factor-beta (TGF-β)-induced epithelial-mesenchymal transition (EMT) in human lung cancer cells. Methods Lung cancer A549 cells were transfected with NDRG1 overexpressed vector, and then treated with 5 μg/L TGF-β1. The abilities of invasion were detected by Transwell assay. The expressions of NDRG1 mRNA and protein were analyzed by teal-time reverse transcription polymerase chain reaction (RT-PCR) were examined with Western blot. The expressions of EMT-associated markers E-cadherin and Vimentin, and EMT-associated signaling molecules Snail, AKT and Smad were detected with Western blot. Results We found that TGF-β1 treatment could induce morphological alteration of A549 cells from epithelial morphology to mesenchymal morphology. TGF-β1 significantly increased the migration of A549 cells, and increased the expression of mesenchymal maker vimentin and decreased epithelial marker E-cadherin. More importantly, overexpression of NDRG1 significantly reversed the effects of TGF-β1 on A549 cells. Moreover, NDRG1 significantly decreased the levels of phospho-AKT, and suppressed the expression of EMT-related transcription factor Snail. Conclusions NDRG1 could reverse the effects of TGF-β1 on EMT in A549 cells, by which mechanism is related to reduction of the expressions of phospho-AKT and Snail. Key words: Intercellular signaling peptides and proteins/PD/ME; Transforming growth factor beta/AE; Lung neoplasms/PA; Cell transformation, neoplastic/DE; Epithelial cells/DE/PA; Stromal cells/DE/PA

Phosphatase of regenerating liver-3 regulates the expression of Snail in human colon cancer cells LoVo through tumor microenvironment

Objective To investigate the interaction between colon cancer cells and hepatic cell (L02), and it’s influence to Snail expression in tumor cells. Methods Lo Vo cells which transfected with phosphatase of regenerating liver- 3(PRL- 3)and human hepatic cells(L02)simulated tumor microenvironment were cocultured by 0- 5 day, reverse transcriptase- polymerase chain reaction (RT- PCR)and Western blotting were used to examine Snail expression of Lo Vo cells, and the migration of Lo Vo cells also be detected. Results Used by RT- PCR and Western blotting it was found out that Lo Vo cells(transfected with PRL- 3) were reduced Snail express after cocultured with hepatic cells, the inhibition rates were 34. 06%, 37. 82%, 39. 91%, 64. 07%, 73. 58%(P< 0. 05), LoVo cells(without transfected PRL- 3) decreased expression of Snail was not obvious.Besides, coculture also depress the migration of Lo Vo cells. Conclusion PRL- 3 depress the migration of Lo Vo cells through reduced Snail expression in liver metastasis of colon cancer microenvironment. Key words: Phosphatase of regenerating liver-3; LoVo; Hepatic cell; Snail

The effects of pseudomonasaeruginosa mannose sensitive hamemagglutination vaccine on epithelial-mesenchymal transition of hepatocellular carcinoma

Objective To investigate the effects of pseudomonasaeruginosa mannose sensitive hamemagglutination vaccine(PA-MSHA) on the epithelial-mesenchymal transition(EMT) of hepatocellular carcinoma(HCC). Methods MHCC97L and HepG2 cells were cultured and treated with different dose of PA - MSHA. After that, morphological changes of cells were observed under a light microscope. Immunofluorescence and Western blot analysis were used to evaluate the expression of EMT related genes and transcription factors. Immunohistochemistry and reverse transcription polymerase chain reaction(RT- PCR)analysis were used to evaluate the expression of E- cadherin,Snail and Slug in the tumor tissue after the mice were treated with PA- MSHA. Results Western blot and immunofluorescence analysis reveled that the expression of E-cadherin was significantly elevated while expression of Vimentin was significantly inhibited by PA- MSHA,in a dose dependent manner. Moreover, the expression of fibronectin, nuclear factor- kappa B(NF-κB),Snail and Slug were also inhibited by PA- MSHA, but theses effects of PA- MSHA were blocked by mannose. After PA- MSHA treatment, HCC cells have altered from irregular shape to epithelial cobblestone appearance.In vivo study, PA- MSHA also significantly increased the expression of E- cadherin and inhibited the expression of Snail mRNA and Slug mRNA. Conclusion PA-MSHA can significantly inhibit EMT of HCC in a mannose-mediated manner. Key words: Hepatocellular carcinoma; Pseudomonas aeruginosa; Mannose; Epithelialmesenchymal transition