Effect Of Blockade Of NF-?B Signaling Pathway On Cell Apoptosis And The Research Of Molecular Mechanisms

Introduction:Lung cancer is the most common malignant tumor with an incidence of 12.8%rate among all cancers with the leading cause of cancer-related deaths in the world.With the increase in air pollution in the developing countries,lung cancer incidence rate is increasing.Most lung cancers are diagnosed at the advanced stage of disease,making curable surgery impossible.Chemo-and radiotherapy is currently the major therapeutic option in management of advanced inoperable lung cancer patients in clinical practice,although chemotherapy often generates the drug resistance and toxicity.Lung cancer prognosis is generally poor and to date only approximately 15%patients survive for five years after lung cancer diagnosis.Therefore,there is an urgent need on effective prevention and develop effective therapy to reduce the morbidity and mortality of lung cancer.Cancer development and progression rely on the balance between tumor cell survival and apoptosis.Nuclear factor-kappaB(NF-Kb)is an important transcriptional factor in regulation of a variety of pathophysiologic process involved in cell survival and death,especially during carcinogenesis and cancer progression by inhibiting the apoptosis thereby enhancing the tumor growth.Level and activity of NF-?B protein have been found constitutively increased in a variety of human cancer.Recent in vivo and in vitro studies suggested that NF-?B was closely associated with lung carcinogenesis.Thus,targeting of the NF-?B signaling pathway has significant implication in treatment and chemoprevention of lung cancer.For example,inhibition of NF-?B activity is associated with increased sensitivity of lung cancer cells to chemotherapeutic agents.NF-?B protein consisted of a DNA binding subunit p50 and a transactivation subunit RelA/p65.These two subunits form a heterodimer to function as a transcriptional factor,i.e.,when it is not activated,NF-?B dimer is localized in the cytoplasm as a latent complex by binding specifically to I?B inhibitor proteins.After activated,I?B inhibitor proteins are phosphorylated and subsequently degraded,leading to NF-?B translocation into the nucleus and activates the transcription of NF-?B downstream genes which are related to cell proliferation,survival and anti-apoptosis.In the present study,we utilized siRNAduplex to knockdown expression of NF-?B p65 subunit to assess whether knockdown of NF-?B can inhibit growth of lung cancer cell in vitro and in vivo nude mouse xenografts and understand the underlying molecular events.This study expects to provide evidence whether targeting the NF-?B activity could be an effective therapeutic strategy in future clinical control of lung cancer.Instrumentation:instrument model origin company Thermostatic drum wind drying oven QH01-9030A Shanghai Jinghong-30 ? constant cold slicer in low CM69001 Germany Leica temperature electro-heating standing-temperature DH36001B Tianjin Tianjin tiger cultivator Ultrapurewater system NW10LVF Hong Heal Force Kong Asana microscope BA400 Xiamen Motic The level of shaking table WD-9405B Beijing Beijingliuyi Trace pipetting device Proline Suzhou BIOHIT Electrophoresis system DYY-10C Beijing Beijingliuyi(electrophoresis)Electrophoresis system(transfer)DYCZ-24DN Beijing Beijing liuyi Gel imaging system WD-9413B Beijing Beijing liuyi Speed refrigerated centrifuge H-2050R Changsha Hunan xiangyi Enzyme standard instrument ELX-800 USA BIOTEK Vacuum drying oven DZF-6050 Shanghai SYSBERY Ultraviolet spectrophotometer NANO 2000 USA Thermo Fluorescence quantitative PCR Exicycler 96 South BIONEER KoreaMaterials and methods:1.Cell line and cultureA Lewis murine lung carcinoma cell line was obtained from Jin Zijing Company(Beijing,China)and maintained in RPMI1640(Invitrogen,Carlsbad,CA,USA)containing 10%fetal bovine serum(Hyclone,Beijing,China),100 U/mL penicillin,and 100 pg/mL streptomycin in a humidified incubator with 5%CO2 at 37?.2.In vivo tumorigenicityTo assess the effects of NF-?B p65 subunit knockdown,we performed a nude mouse xenograft assay by using 18 healthy male C57BL/6 nude mice(age of 6-8 weeks and body weight of 20±2g,maintained in the animal facility at China Medical University).The care and use of laboratory animals was in accordance with the principles and standards set forth in the Principles for Use of Animals at China Medical University.All animal procedures were approved by the Institutional Animal Care and Use Committee.In brief,the Lewis lung carcinoma cells were grown to 75%confluence,trypsinized,washed twice in phosphate buffered saline(PBS),and resuspended in the growth medium.Next,0.5 ml cell suspension at a density of 2×107/mL was subcutaneously injected into forelimbs of each of three nude mice.Two weeks after tumor cell inoculation,three mice were sacrificed and the subcutaneous tumors were removed for tumor cell suspension.After that,0.2 ml of tumor cell suspension at a density of 6×106 cells was subcutaneously injected into forelimbs of each of 15 mice.The mice were randomly divided into different treatment groups,i.e.,i).PBS control group,intraperitoneal injection of PBS;ii).siRNA negative control group,intraperitoneal injection of negative control siRNA with the final concentration of 100 nmol/L;and iii).p65 siRNA group,intraperitoneal injection of p65 siRNA with the final concentration of 100 nmol/L.p65 siRNA sequence was 5'-GATCAATGGCTACACAGGA-3' and negative control siRNA sequence was 5'-TTCTCCGAACGTGTCACGT-3'(Both were purchased from Santa Cruz biotechnology,Santa Cruz,CA,USA).The tumor volume and inhibition of tumor growth were recorded every other day from the beginning of the siRNA treatment.The nude mice were weighted and tumor dimension was measured with calipers.The tumor volume was calculated by using the formula:Volume(mm3)=1/2AB2.A represents the long diameter(mm)and B represents the short diameter(mm).After two weeks following the treatment,the mice were sacrificed and the tumor mass was resected and weighed.Tumor weigh and inhibition rate were calculated and plotted using the formula:tumor inhibitive rate =[(the average tumor weight of control group-the average tumor weight of siRNA group)/the averagetumor weight of control group]× 100%.3.In situ TUNEL apoptosis assayTo determine whether inhibition of NF-?B by siRNA induces tumor cells to undergo apoptosis,we measured apoptosis levels in tumor xenograft tissue specimens by using the TUNEL detection kit(Tiangen,Beijing,China).In particular,formalin-fixed,paraffin-embedded tumor xenografts were cut into 4-mm-thick sections.After deparaffinized in xylene and rehydrated through graded ethanol,the tissue sections were then stained according to the kit protocols.At the end,apoptotic cells from randomly selected five visual fields(X 200)in each slide were counted under a double-headed light microscope by two investigators who didn't know about the identity of the slides,which obtained approximately 500 TUNEL positive cells in each slide(Cells with brown granules in the nucleus were TUNEL positive cells).4.Quantitative RT-PCRTotal cellular RNA was isolated from tumor xenograft tissue sections using a Trizol(Tiangen)and reversely transcribed into cDNA using an RT kit(Tiangen)according to the manufacturers' instructions.qPCR was performed in triplicates,i.e.,the reaction system includes 1 ?L cDNA template,0.5 ?L of 10 ?M primers,10 ?L SYBR GREEN master mix,up to 20 ?ddH2O.The reaction conditions were 950C for 10 min and then 40 cycles of 95? for 10 s,60? for 20 s,and 72? for 30 s and stored at 4?.An ExicyclerTM fluorescence quantitative analysis system(BIONEER,Korea)was used.All primers were synthesized in SANGON Biological Engineering(Shanghai)Co.,Ltd.(Shanghai,China).5.Protein extraction and Western blotTotal cellular protein was extracted from 20 mg tumor xenograft tissue using 100 ?L protein lysis buffer containing 50 mM Tris(pH 7.4),150 mM NaCl,1%Triton X-100,1%sodium deoxycholate,0.1%SDS,sodium orthovanadate,sodium fluoride,EDTA,and leupeptin.40 ?g of protein sample was resolved in 10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and transferred onto a nitrocellulose membrane(Millipore,Billerica,MA,USA).After that,the membrane was blocked in 5%skimmed milk for 2 h,and blotted with an anti-p65 antibody at a dilution of 1:400 or anti-Bax antibody at a dilution of 1:1000)(all from Santa Cruz biotechnology)at 4 ? overnight.The membrane was washed with PBS-T and incubated with a second antibody at room temperature for 1 h.After washing in PBS-T,the protein bands were visualized using an ECL kit(Biyuntian,Shanghai,China)and exposed to x-ray film(Fujifilm,Tokyo,Japan)and quantified using Gene Tools software(Media Cybernetics,MD,USA).6.Statistical analysisSPSS version 13 software(SPSS,Chicago,IL,USA)was used for statistical analysis.Each experiment was repeated thrice,the results were expressed as Mean ± SD.P<0.05 was considered as statistically significant.Results:1.Knockdown of NF-?B p65 expression inhibits growth of Lewis lung carcinoma cell xenograft in nude mice.To evaluate the effect of NF-?B p65 knockdown on inhibition of growth of nude mouse lung cancer cell xenografts,we first established this animal model and treated these mice with p65 siRNA and controlled with PBS and negative control(NC)siRNA treatment.After two weeks,the animals were sacrificed and the data showed that p65 siRNA treatment significant suppressed tumor xenograft volume and weight comparing to either the PBS treated or NC siRNA treated mice(Figure 1A and 1B).The inhibition rate of tumor growth was nearly 30-fold more than that in the PBS group(*P<0.05;Figure 1C).2.Knockdown of NF-?B p65 expression induces apoptosis of tumor xenograftsThe TUNEL assay data showed that apoptotic cells in tumor xenograft sections were 45± 5 in PBS-treated mice and 38 ± 3 in NC siRNA-treated mice,but increased to 271 ± 11 in p65 siRNA-treated mice(p<0.05;Figure 2D).NF-?B p65 siRNA significantly inhibited p65 expression(P<0.01;Figure 3)but induced the increase in levels of Bax mRNA and protein,an important pro-apoptotic protein compared to the controls(P<0.01;3.our current data showed that knockdown of NF-?B p65 subunit expression was able to significantly up-regulate the Bax expression and down-regulate Bcl-2 expression in tumor xenograft tissue at both protein and mRNA levels.Conclusion:1.NF-KB/NF-?B/p65 siRNA restrains the growth of the tumor transplanted in mice.2.NF-?B/p65 siRNA significantly induced lung tumor cell apoptosis transplanted in mice apoptosis.3.NF-?B/p65 siRNA can significantly reduce the protein and mRNA expression of the apoptosis suppressor gene the Bcl-2,but improve on the level of Bax promoting apoptosis gene.4.NF-?B/p65 may play an important role in the apoptosis of lung cancer,in which NF-?B/p65 higher apoptosis suppressor BCL-2 and lower promoting apoptosis gene Bax to inhibit cancer cell apoptosis may be a molecular mechanism.