Fibroblasts weaken the anti-tumor effect of gefitinib on co-cultured non-small cell lung cancer cells

Background Non-small cell lung cancer （NSCLC） is the most common lung malignancy worldwide.The metastatic potential of NSCLC cells has been shown to be associated with the tumor microenvironment,which consists of tumor cells,stroma,blood vessels,immune infiltrates and the extracellular matrix.Fibroblasts can produce numerous extraceilular matrix molecules and growth factors.Gefitinib has been evaluated as a first-line treatment in selected patients,and it has shown favorable efficacy especially in NSCLC,but it is not effective for everyone.Methods In this study,we examined the antitumor activity of gefitinib on lung fibroblasts co-cultured of lung cancer cells.A series of co-culture experiments that employed cell counting kit-8 （CCK8）,transwells,real-time polymerase chain reaction （RT-PCR） and Western blotting with HFL-1 fibroblasts and A549 human lung carcinoma cells were performed to learn more about tumor cell proliferation,migration and invasion; and to determine any change of epithelial mesenchymal transition （EMT）-associated tumor markers vimentin,matrix metallopro-teinase 2 （MMP2） and chemotaxis cytokines receptor 4 （CXCR4） mRNA levels.Results A549 cell proliferation in the presence of HFL-1 cells was not significantly increased compared with A549 cells alone,but A549 cell spheroid body formation was increased after co-culture,and treatment with gefitinib increased further.Our study also revealed that fibroblasts attenuated the lung cancer cell inhibition ratio of migration and invasion after gefitinib treatment in vitro.To further study this mechanism,RT-PCR analysis showed that vimentin,MMP2 and CXCR4 mRNA levels were more highly expressed in the lung cancer cells after co-culture,but did not obviously decrease compared with the control cells following gefitinib treatment.This suggests the mechanism by which fibroblasts attenuate gefitinib-induced expression of EMT-associated tumor markers.Finally,our results demonstrated that co-culture with A549 lung cancer cells does not alter the cell cycle distribution of HFL-1 fibroblasts.Furthermore,HFL-1 fibroblasts had no effect on the cell cycle distribution of HFL-1 cells treated with gefitinib.Conclusion Gefitinib has lower anti-tumor activity on A549 lung cancer cells when co-cultured with HFL-1 fibroblasts.     

Hypoxia-Inducible Factor-lalpha Suppressing Apoptosis and Increasing Tolerance of Lung Cancer Cells to Chemotherapy

In order to construct plasmid of hypoxia-inducible factor-lalpha （HIF-1α）, and transfect into human lung cancer cells A549, the change in sensitivity of lung cancer cells A549 to chemotherapy was observed. HIF-1α mRNA structure region was amplified by RT-PCR and inserted into plasmid pcDNA3. The expression plasmid pcDNA3/HIF-1α was transfected into A549 with LipofectAMINE^TM2000. The expression of HIF-1α protein was detected by Western blot. After A549 cells were transfected with HIF-1α prior to addition of 5-Fu, the growth activity was measured by growth curve, apoptosis was detected by flow cytometry at 48 h, and the levels of caspase3 and MDR-1 were determined by Western blot. The results showed that the constructed expression plasmid was analyzed with restriction enzymes and gel electrophoresis. Two DNA lanes at 2.55 kb and 5.4 kb respectively were found, which were consistent with that expected. The growth rate in 5-Fu group was significantly inhibited, and the apoptosis index and caspase3 activity were increased significantly as compared with control group. After HIF-1α being transfected into A549, the activity of MDR-1 was increased and the effect of 5-Fu was weakened. In conclusion, HIF-1α can promote chemoresistance by increasing the activation of MDR1 and suppressing apoptosis during lung cancer cells A549 in- duced with 5-Fu.     

Morinda citrifolia (Noni) Juice Suppresses A549 Human Lung Cancer Cells via Inhibiting AKT/Nuclear Factor-κ B Signaling Pathway

Objective: To study the mechanism of the anti-tumor effect of Morinda citrifolia (noni). Methods: The influences of noni juice on cell proliferation, apoptosis, invasion, migration and the activity of AKT/nuclear factor-κ B (NF-κ B) signaling pathway in A549 human lung cancer cells were detected by MTT, cell counting kit-8, colony formation, Annexin V/PI double labeling, transwell, scratch test and immunoblotting assay, respectively. A549 cells were inoculated into the right axilla of nude mice, followed by noni juice treatment. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell proliferation and expression of apoptosis-related proteins were measured by immunohistochemistry, and the activity of NF-κ B signaling pathway was measured by immunoblotting. Results: The in vitro studies showed that noni juice inhibited the A549 cells proliferation, migration and invasion. Noni juice also promoted cells apoptosis in A549 cells. Immunoblotting assay showed that the phosphorylation level of AKT, p50, and STAT3 proteins was inhibited to different extents after noni juice treatment. The in vivo studies showed that noni juice effectively suppressed tumor formation of A549 cells in nude mice. Noni juice treatment inhibited the expression of Ki67, PCNA, and Bcl-2 protein in the tumor; while promoted the expression of caspase-3 protein. Additionally, we also found that noni juice treatment could restrain the activity of AKT/NF-κ B signaling pathway in the tumor tissue. Conclusion: Noni juice inhibited the proliferation of A549 lung cancer cells, induced apoptosis, and inhibited cell invasion and migration via regulating AKT/NF-κ B signaling pathway.     

rmhTNF-αCombined with Cisplatin Inhibits Proliferation of A549 Cell Line In Vitro

Objective To explore the inhibitory effect of recombinant mutant human tumor necrosis factor-α （rmhTNF-α in combination with cisplatin on human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma cell line A549 was treated with varying concentrations of rmhTNF- （0.38, 0.75, 1.50, 6.00 and 12.00 IU/ml） or cisplatin （3.91, 7.81, 15.63, 31.25 and 62.50 μg/ml） for 24 hours. Viable cell number was analyzed by using crystal violet staining. The inhibitory rates of A549 cells growth by the two drugs were calculated. For analyzing whether there was a synergistic effect of rmhTNF-α with cisplatin, A549 cells were treated with 0.75 IU/ml rmhTNF-α and increased concentrations of cisplatin. Results rmhTNF-α or cisplatin inhibited the growth of A549 cell lines in a dose-dependent manner. The inhibitory effect of rmhTNF-α combined with cisplatin was significantly greater than cisplatin alone at the same concentration （all P〈0.01）. Conclusion rmhTNF-α combined with cisplatin might have synergistic inhibitory effect on human lung adenocarcinoma cell line A549.     

The Effect of RhoC siRNA on the Invasiveness and Proliferation of Human Cervical Cancer Cell Line SiHa Cells

This study investigated the effect of RhoC GTPase on the proliferation and metastasis of cervical cancer cells, SiHa cells, in vitro. RhoC siRNA was introduced into SiHa cells to silence the RhoC gene. The mRNA and protein expression of RhoC, before and after RhoC siRNA transfection, was examined by RT-PCR and Western blotting, respectively. The proliferation and apoptosis of SiHa cells were examined by MTT assay and flow cytometry （FACS）, respectively. Adhesive rate was evaluated by Matrigel adhesive assay, and the invasive capability and migration capability were assessed by transwell invasive assay and migration assay, respectively. The results showed that after the RhoC siRNA transfection, the mRNA and protein expression of RhoC was down-regulated in SiHa cells. The down-regulation of RhoC GTPase did not affect the cell proliferation and apoptosis （P〉0.05）, but it did suppress SiHa cells＇ adhesion to matrigel （P〈0.01）, the invasive capability （P〈0.01） and the migration capability （P〈0.01）. It was concluded that RhoC obviously promotes the adhesion, invasion and migration of SiHa cells in vitro, but not proliferation and apoptosis, suggesting that RhoC plays an important role in the progression in cervical cancer.     

Study on Expression of HERG Protein in A549 Cell and Their Clinical Significance

Objective To study the expression of HERG protein in A549 cell （a kind of non-small-cell lung cancer cells, NSCLC） and the relationship with biological behavior of NSCLC. Methods Expression of the HERG protein was detected by RT-PCR, and was evaluated by immunohistochemical polymer detection in 32 cases of non-small-cell lung cancer. Results The positive expression rate of HERG was 68.89% （22/32） in 32 cases of non-small-cell lung cancer, which is related to histological differentiation and surviving time （P〈0.05）, and the expression of HERG protein in A549 was highly detected by RT-PCR. Conclusion HERG protein expression may play an important role in judgment of prognosis of non small cell lung cancer, further study will likely clarify the molecular mechanism of tumor angiogenesis, it is also helpful toe the development of target-therapy to select the suitable target-gene.     

Implication of EMT Induced by TGF-β1 in Pancreatic Cancer

This study examined the implication of EMT induced by TGF-β1 in pancreatic cancer invasion. TGF-β1 expression was determined in 29 cases of human pancreatic carcinoma （PC） by immunohistochemistry and the results were compared with those of pathological examination. Moreover, the effects of TGF-β1 on the phenotype and invasion of pancreatic cancer cell line Panc-1 were also investigated. TGF-β1 was detected in 12 cases （41.4 %） of PC. Significant correlation was found between the expression of TGF-β1 and lymph node involvement （P=0.047） and the depth of invasion （P=0.035）. TGF-β1 obviously promoted EMT of Panc-1 cell lines and their invasion ability was substantially enhanced. TGF-β1 may promote the malignancy of pancreatic cancer by triggering EMT.     

Construction of Eukaryotic Expression Vector of Human CC10 Gene and Expression of CC10 Protein in Lung Adenocarcinoma A549 Cell Line

Summary： A mammalian expression plasmid pcDNA3. 1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3. 1, and the recombinant plasmid was identified by employing double digestion restriction enzymes HindⅢ and BanH 1 and the cDNA sequence was assayed by the Sanger dideoxymediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot. Our results showed that the cDNA fragment included the entire coding region （273 bp）. The re combinant eukaryotic cell expression vector of pcDNA3. 1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3. 1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1- hCC10 may serve as an effecnve tool for the study of tumorogenesis and tumor treatment.     

Inhibitory activity of nuclear factor-κB potentiates cisplatin-induced apoptosis in A549 cells

Whether inhibiting the activity of nuclear factor （NF）-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1（＋）/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were transfected with pcDNA3.1 （＋）/IκBα alone, or pcDNA3.1（＋）/IκBα combined with cisplatin. The mitochondrial membrane potential （△ψm） was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V/propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1（＋）/IκBα. Transfection of pcDNA3.1（＋）/IκBα alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1（＋）/IκBα combined with cisplatin treatment significantly induced apoptosis of A549 ceils. It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Effects of Exogenous p16^ink4a Gene on Biological Behaviors of Human Lung Cancer Cells

The effects of exogenous p16ink4a gene on biological behaviors of human lung cancer cell line with homozygous deletion of p16ink4a gene were investigated. Exogenous p16ink4a gene was transfected by lipofectin into human lung cell line A549, in which p16ink4a gene was homozygously deleted. The expression of p16ink4a mRNA and protein was detected by RT-PCR and immunocyto-chemistry, respectively. The changes in the behaviors of the transfected cell lines in vitro and in vivo were observed. In the transfected cell line A549, the exogenous p16ink4a gene could be stably ex-pressed. The growth of A549 cells transfected with p16ink4a gene was obviously slowed down. Flow cytometry revealed that transfection of the exogenous p16ink4a gene resulted in A549 cell lines arrest in G1 phase of cell cycle. The tumorigenicity of these transfected cells in nude mice could be inhib-ited, and the tumor growth of nude mice was significantly suppressed. It was concluded that exoge-nous p16ink4a gene may be stably expressed in human lung cancer cell line A549. The expression of the introduced p16ink4a could block lung cancer cells to entry into S phase of cell cycle and inhibit tumor malignant growth both in vitro and in vivo.     

Human decorin regulates proliferation and migration of human lung cancer A549 cells

Background Decorin is a small leucine-rich proteoglycan and it plays an important role in regulation of cell growth and migration in various tumor cell lines. Decorin was found down-regulated in non-small cell lung cancer tissue and may be involved in regulation of lung cancer development. Methods In this study, lentivirus-mediated RNA interference and over expression were employed to change the expression levels of decorJn Jn lung cancer A549 cells. We tested the cell cycle of A549 cells and the expression of transforming growth factor （TGF）-[31, cyclin D1, epidermal growth factor receptor （EGFR）, P53, and P21. Results We found that up-regulation of decorin could inhibit proliferation, block cell cycle at G1 and decrease invasive activity of A549 cells. Moreover, we also show that up-regulation of decorin induced significant decreases of TGF-131, cyclin D1 expression, phosphorylation of EGFR, and increases of P53 and P21 expression. Opposite results were observed in A549 cells with down-regulation of decorin. Conclusion Our results suggest that decorin is a key regulator involved in proliferation and migration ofA549 cells.     

Breast cancer associated fibroblasts promote MCF-7 invasion in vitro by secretion of HGF

This study was aimed to explore the influence of breast cancer associated fibroblasts (CAFs) in migration and invasion of breast cancer cell line MCF-7,and investigate whether hepatocyte growth factor (HGF) is involved in this process.Primary breast CAFs and their corresponding normal breast fi-broblasts (NFs) were obtained by collagenase digestion.On the basis of the co-culture,the migration and invasion capacity of MCF-7 cells was compared between CAFs and NFs by Transwell.The differ-ence in the HGF expression between them was detected by ELISA.The secretion of HGF was knocked down by using RNA interference technology in CAFs.Then the changes of migration and invasion ca-pacity of MCF-7 cells were investigated by Transwell.Eventually,we isolated high-purity CAFs and NFs,and the CAFs had a stronger ability in promoting MCF-7 migration and invasion than the NFs.ELISA results demonstrated that CAFs secreted higher HGF,and the capacity of MCF-7 migration and invasion was declined after knocking down the secretion of HGF in CAFs by RNA interference.It is suggested that CAFs can promote MCF-7 migration and invasion through HGF in vitro.     

消积饮通过Akt 信号可对人肺癌A549细胞株产生抑制增生及促进凋亡作用

Objective:To investigate the inhibitive effect and the underlying mechanism of Xiaoji Decoction(消积饮,XJD)in human lung cancer A549 cells.Methods:A549 cells in logarithmic proliferation were cultivated in RPMI-1640 containing 10%low,medium or high dosages of XJD serum.The inhibitive effect of XJD in A549 cell proliferation was assessed by methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay.The pro-apoptotic effect of XJD in A549 cells was observed by fluorescence microscope via Hoechst 33258 staining.The role of the Akt signaling pathway was observed by examining the presence of p-Akt protein by Western blot and the mRNA expression of downstream proteins such as Bcl-2/Bcl-XL-associated death promoter(BAD)and caspase-9 by real time polymerase chain reaction.Results:MTT assay revealed that XJD could inhibit A549 proliferation in a dose-and time-dependent manner.Hoechst 33258 staining showed that XJD induced the typical nuclear apoptotic morphology after XJD treatment.Moreover,XJD could reduce the phosphorylation of Akt and increase the mRNA expression of BAD and caspase-9.Conclasions:XJD can inhibit the proliferation of A549 cells in a dose-and time-dependent manner through signaling Akt pathway via up-regulating the expression of BAD and caspase-9.XJD may provide a novel therapeutic model for lung cancer and deserve further study.     

Vascular endothelial growth inhibitor affects the invasion, apoptosis and vascularisation in breast cancer cell line MDA-MB-231

Background Breast cancer is one of the most common malignant female diseases worldwide.It is a significant threat to every woman＇s health.Vascular endothelial growth inhibitor （VEGI） is known to be abundant in endothelial cells.According to previous literature,overexpression of VEGI has been shown to inhibit tumor neovascularisation and progression in cellular and animal models,but there has been limited research on the significance of VEGI in the breast cancer.Methods In our study,cell lines MDA-MB-231 were first constructed in which VEGI mediated by lentivirus over-expressed.The effects of VEGI over-expression on MDA-MB-231 cells were investigated both in vitro and in vivo.The expression of VEGI in the MDA-MB-231 cells after infection of lentivirus was analyzed using real-time PCR and Western blotting.The effect of the biological characteristics of MDA-MB-231 cells was assessed by growth,invasion,adhesion,and migration assay with subcutaneous tumor-bearing nude mice models.Then the growth curves of the subcutaneous tumors were studied.Expressions of VEGI,CD31 and CD34 in the tumors were analyzed by immunohistochemistry and apoptosis was detected by flow cytometry and immunohistochemistry.Results Infection of MDA-MB-231 cells within the lentivirus resulted in approximately a 1 000-fold increase in the expression of VEGI.As can be seen in the invasion,adhesion and migration assay,the over-expression of VEGI can inhibit the ability of MDA-MB-231 cells during migration,adhesion and invasion.The volume of the subcutaneous tumor in the over-expression group was distinctly and significantly less than that of the control groups.Immunohistochemistry analysis of the tumor biopsies cleady showed the expression of VEGI in the over-expression group increased while CD31 and CD34 decreased significantly.In vitro and in vivo,the early apoptosis rate and the apoptosis index were increased within the VEGI over-expression group as compared with the control group.Conclusions Taken together,recombinant lentivirus that were successfully constructed,demonstrated up-regulated VEGI gene expression in breast cancer cells.Lentivirus-mediated over-expression of VEGI weakened the ability of the breast cancer cell migration,adhesion and invasion.Over-expression of VEGI diminished the tumorigenic capacity of breast cancer cells in vivo.Up-regulation of VEGI gene expression however inhibited breast cancer MDA-MB-231 cell in the early apoptosis.     

miR-200c inhibits metastasis of breast cancer cells by targeting HMGB1

miR-200c has been shown to regulate the epithelial-mesenchymal transition （EMT） by inhibiting ZEB1 and ZEB2 expression in breast cancer cells. This study further examined the role of miR-200c in the invasion and metastasis of breast cancer that goes beyond the regulation on ZEB1 and ZEB2 expression. In this study, the bioinformatics software （miRanda） was used to predict the target gene of miR-200c and Renilla luciferase assay to verify the result. The metastatic breast cancer cells MDA-MB-231 were cultured and transfected with the miR-200c mimic or inhibitor. The expressions of miR-200c and HMGB 1 were detected by RT-PCR and Western blotting, respectively. Transwell assay and wound healing assay were employed to examine the invasive and migrating ability of transfected cells. Target prediction and Renilla luciferase analysis revealed that HMGB1 was a putative target gene of＇miR-200c. After transfection of MDA-MB-231 cells with the miR-200c mimic or inhibitor, the expression of miR-200c was significantly increased or decreased when compared with cells transfected with the miR-200c mimic NC or inhibitor NC. Moreover, the expression of HMGB1 was reversely correlated with that of miR-200c in transfected cells. Tranwell assay showed that the number of invasive cells was significantly reduced in miR-200c mimic group when compared with miR-200c inhibitor group. It was also found that the migrating ability of cells transfected with miR-200c mimics was much lower than that of cells transfected with miR-200c inhibitors. It was suggested that miR-200c can suppress the invasion and migration of breast cancer cells by regulating the expression of HMGB1. miR-200c and HMGB1 may become useful biomarkers for progression of breast cancer and targets of gene therapy.     

Silencing MTA1 by RNAi Reverses Adhesion,Migration and Invasiveness of Cervical Cancer Cells （SiHa） via Altered Expression of p53, and E-cadherin/β-catenin Complex

It has been reported that metastasis-associated gene 1 (Mta1) is overexpressed in many malignant tumors with high metastatic potential. In addition, some studies indicated that MTA1 participated in invasion, metastasis, and survival of cancer cells by regulating cell migration, adhesion and proliferation. But the role of MTA1 is unclear in vitro in the development of cervical cancer cells. This study investigated whether and how MTA1 mediated cell proliferation, migration, invasion and adhesion in cervical cancer. MTA1 expression level was detected by Western blot in two cervical cancer cell lines of different invasion potentials. The effects of MTA1 expression on SiHa cell apoptosis, cycle, proliferation, migration, invasion and adhesion were tested by flow cytometry, MTT, wound-healing assay, Transwell assay and adhesion assay, respectively. The expression levels of p53, E-cadherin, and β-catenin activity were evaluated in untreated and treated cells. The results showed that MTA1 protein expression was significantly higher in SiHa than in HeLa, which was correlated well with the potential of migration and invasion in both cell lines. Furthermore, the cell invasion, migration and adhesion capabilities were decreased after inhibition of MTA1 expression mediated by Mta1-siRNA transfection in SiHa. However, no significant differences were found in cell apoptosis, cycle, and proliferation. In addition, E-cadherin and p53 protein levels were significantly up-regulated, while β-catenin was significantly down-regulated in SiHa transfected with the siRNA. These results demonstrated that MTA1 played an important role in the migration and invasion of cervical cancer cells. It was speculated that the decreased migration and invasion capability by inhibiting the MTA1 expression in the SiHa cell line may be mediated through the altered expression of p53, and E-cadherin/β-catenin complex. MTA1 could serve as a potential therapeutic target in cervical cancer.     

Inhibitory Activity of Nuclear Factor-κB Potentiates Cisplatin-induced Apoptosis in A549 Cells

Whether inhibiting the activity of nuclear factor(NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated.The recombinant plasmid pcDNA3.1( )/IκBα expressing IκBα was constructed.The in vitro cultured A549 cells were transfected with pcDNA3.1( )/IκBα alone,or pcDNA3.1( )/IκBα combined with cisplatin.The mito-chondrial membrane potential(?ψm) was determined by rhodamine 123,the activity of caspase-3 was tested by colorimetric assay,and cell apoptosis was detected by flow cytometry with the annexin Ⅴ/propidium iodide assay.The results showed that the activity of NF-κΒ in A549 cells was inhibited by transfecting pcDNA3.1( )/IκΒα.Transfection of pcDNA3.1( )/IκΒα alone did not promote apoptosis.Treatment of cisplatin alone had a little effect on cell apoptosis.Transfection of pcDNA3.1( )/IκΒα combined with cisplatin treatment significantly induced apoptosis of A549 cells.It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.