The Anti-tumor Effect And Mechanism Of PolyI:C Complex On NSCLC In Vivo And In Vitro

Non-small cell lung cancer(NSCLC)is the leading cause of death in cancer patients.It is now recognized that the immune system has the potential to destroy cancer cells and inhibit the growth of cancer cells through their innate and adaptive immune responses,which alters the prospects for NSCLC treatment.Toll-like receptors(TLRs).as the most important trans-membrane receptors in innate immunity,and involved in the induction and regulation of immune responses.Latest researches have suggested that TLRs found in various tumor cells,which relates to cancer development and progression.Some types of TLRs detect tumor antigens and arouse primary anti-tumor immune response.TLRs can further prevent the formation of inflammatory tumor microenvironment by eliminating tumor antigens.TLR3 is located in the membrane of endosomes,utilizes a MyD88-independent pathway,recruiting TRIF to activate AP-1,NF-?B and IRFs.Recent evidences suggested that TLR3 worked as a possible therapeutic target in pharyngeal,breast,ovarian,head and neck,prostate,oral sqaumous cancer.As a ligand for TLR3,PolyI:C activates TLR3,PKR,RIG-1,and MDA5,induces cancer cell apoptosis directly and destroyed tumor microenvironment.In addition,PolyI:C enhanced the immunogenicity of tumor antigens by affecting DC maturation and type I IFN.These evidences indicate that TLR3 may be used as a therapeutic target in immunotherapy of lung cancer.PI3K/Akt plays an essential role in regulation of cell proliferation,inhibition of apoptosis,promotion of metastasis,and correlating with poor prognosis of cancer.Accordingly,blocking the Akt signaling pathway has been the target of anti-cancer therapies.And p53 was known as a tumor suppressor gene downstream of Akt.Current new insights into p53 and TLR suggest that p53 regulates TLR signaling in cancer cells,inhibits proliferation of cancer cells.A recent study showed that PolyI:C can cause prostate cancer apoptosis through the PI3K/Akt signaling pathway.However,little information of ability of PolyI:C to affect signaling proteins of PI3K/Akt/p53 pathway in NSCLC.Therefore,we hypothesized that TLR3 agonist PolyI:C may interfere PI3K/Akt/p53 signaling in lung cancer.To this aim,we have investigated the anti-tumor effect of PolyI:C by using LL/2 and A549 cells in vitro and in tumor-bearing mice,and explored whether its mechanism is related to interference with Akt phosphorylation.Part One The inhibitory effect of PoIyI:C complex on non-small cell lung cancer cells in vitroObjective:To investigate inhibitory effect of PolyI:C complex on cell proliferation and cell migration in mouse LL/2 and human A549 cell lines in vitro.Methods:The expression of TLR3 mRNA of LL/2 and A549 cells with or without PolyI:C complex treated were dectected by RT-QPCR.The influence of different concentration of PolyI:C complex on the viability of LL/2 and A549 cell lines evaluated by CCK-8 cell viability assay.Monolayer cell proliferation assay for the influence of PolyI:C complex on ploidization time of LL/2 and A549 cells.Effect of PolyI:C complex on apoptosis of LL/2 and A549 cells was detected by Annexin V-FITC/PI double staining assay.The influence of PolyI:C complex on cell cycle arrest of LL/2 and A549 cells was confirmed by flow cytometry.Wound scratch assay used to evaluate the healing ability of LL/2 and A549 cells before and after PolyI:C complex intervention.Transwell migration assay for investigating the longitudinal motor ability of LL/2 and A549 cells before and after PolyI:C complex intervention.Transwell invasion assay was used to evaluate the invasive potential of LL/2 and A549 cells before and after PolyI:C complex intervention.Results:Under normal conditions.LL/2 and A549 cells expressed TLR3 mRNA,and the expression of TLR3 mRNA was significantly increased when PolyI:C complex was administered(P<0.001;P<0.01).When the PolyI:C complex was administered at a concentration of 10 ?g/mL,50 ?g/mL,100 ?g/mL,and 200 ?g/mL to intervene LL/2,the proliferation rate of LL/2 cells decreased from 94%to 76%at 24 hours,cell proliferation rate decreased from 48%to 32%at 48 hours(P<0.05).When interfering with A549 cells,the proliferation rate decreased from 92%to 61%at 24h,and the cell proliferation rate decreased from 55%to 37%at 48h(P<0.05).The monolayer proliferation rate of LL/2 decreased by 1.46 times after stimulation with PolyI:C complex for 24 hours,and decreased by 2.87 times at 48 hours(P<0.01;P<0.001).The proliferation rate of A549 monolayer cells decreased by 1.49 times at 24h,and decreased by 2.07 times at 48h(P<0.01).After 48h administration with PolyI:C complex for 48 hours,the late apoptotic rate of LL/2 cells was(11.43±0.46)%(P<0.001),while A549 was(20.14±0.85)%(P<0.001).The G1 phase of LL/2 and A549 cells were blocked by PolyI:C complex at 48 h,and the percentage was significantly increased to(32.01±0.09)%and(55.16±0.97)%(P<0.001).PolyI:C complex can inhibit the parallel movement ability of non-small cell lung cancer cells,the scratch gap area of LL/2 cells at 24h and 48h were(12.19±0.72)×105?m2 and(9.03±0.73)×105?m2(P<0.05;P<0.01).The scratch gap area of A549 cells stimulated at 24h and 48h were(7.50±0.98)×105?m2 and(5.25±0.71)×105?m2(P<0.01).The PolyI:C complex can effectively inhibit the vertical migration and invasion of both cells to a certain extent(P<0.01).Conclusions:1.PolyI:C complex up-regulated the expression of TLR3 mRNA in LL/2 and A549 cells.2.PolyI:C complex achieves anti-tumor effect in LL/2 and A549 cells by promoting apoptosis and inhibiting cell proliferation in vitro.3.PolyI:C complex can achieve anti-metastasis effect by interfering with the parallel movement ability,longitudinal movement ability and invasion potential of LL/2 and A549 cells in vitro.Part Two The inhibitory effect of PolyI:C complex on non-small cell lung cancer cells in vivoObjective:To investigate the anti-tumor and anti-metastatic function of PolyI:C complex in mice.Methods:The lung cancer transplantation model mice were randomly divided into four groups(n=6):PBS group,PolyI:C complex nasal administration group,PolyI:C complex intramuscular injection group and PD1 group.C57BL/6 Mice were injected subcutaneously with LL/2 cells into the right flank.When the tumor volume reached 100 mm3,100?L(200?g)of PolyI:C complex was given by nasal administration or intramuscular injection every other day,or 100?g of PD1 was given once a week by celiac injection,or 100?.L of PBS was given by nasal administration every other day.When the average tumor volume of PBS group reached 2500mm3 the animals were quickly killed by cervical dislocation.All tumor tissues were excised and weighed for quantitative comparison,and the tumor inhibition rate was calculated.Then,tumor,lung,liver,brain tissues were fixed in formalin.The TUNEL assay was used to detect apoptosis and necrosis in tumor tissues.H&E staining was used to observe the metastasis in organs.Results:There was no significant difference in the volume of transplanted tumors between the four groups on the 7th day(P>0.05).The volume of transplanted tumors on the 9th,11th,13th.15th,17th,19th and 21st day of the intramuscular injection group was significantly lower than that of the PBS group(P<0.001).The volume of transplanted tumors on the 11th,13th,15th,17th,19th and 21st day of the intramuscular injection group was significantly lower than that of the PD1 group(P<0.05).The volume of transplanted tumors on the 11th,13th,15th,17th,19th and 21st days of the nasal administration group was significantly lower than that of the PBS group(P<0.01).There was no significant difference in the volume of transplanted tumor between the nasal administration group and the PD1 group(P>0.05).The tumor masses of PBS group,nasal administration group,intramuscular injection group and PD 1 group were(5.35±0.93),(3.2810.57),(2.65±0.47),(4.55±0.87)g,respectively.Compared with the PBS group,P<0.01 in the nasal administration group and P<0.001 in the intramuscular group;compared with the PD1 group,P<0.05 in the nasal administration group and P<0.01 in the intramuscular group.Monitoring the body weight of the mice showed that the weight loss of the mice in the nasal administration group was not obvious(P>0.05).At the beginning of the 7th administration,the body weight of the mice in the intramuscular injection group decreased significantly(P<0.05).The median survival of the intramuscular and nasal administration groups of the PolyI:C complex was extended to 44 days and 31 days,compared with 25 days in the PBS group,and 42 days in the PD1 group.When the TUNEL apoptosis kit was detected and the image was magnified 200 times,more brown cells were found in the nasal administration group,intramuscular injection group and PD1 group than PBS group.The intramuscular injection group had more apoptosis cells than the nasal administration group and the PD1 group.In terms of lung tissue,there existed a huge tumor metastasis loci in the PBS group and a smaller one in the both of PolyI:C complex nasal administration and PD1 groups while there was none in the lung of PolyI:C complex intramuscular injection group.PolyI:C complex intramuscular injection group has precursor of tumor formation.Pulmonary edema,capillary hyperplasia around the metastasis,alveolar septal thickening and alveolar structure destruction were severe in lungs of all groups.It can be seen from the liver tissue H&E staining small nodular cancer metastases were present in all groups except PolyI:C complex intramuscular injection group.The liver metastasis of the PD1 group was less than that of the PBS and the PolyI:C complex nasal administration groups,and the PolyI:C complex nasal administration group was less than the PBS group.No obvious metastases were seen in the four groups of brain tissues.Conclusions:1.The PolyI;C complex has a significant anti-tumor effect in tumor-bearing mice.2.Polyl:C complex has obvious anti-metastatic effect in tumor-bearing mice.3.The side reaction of the PolyI:C complex is weight loss.4.Polyl: complex can significantly prolong the median survival of tumor-bearing mice.Part Three The mechanism of PolyI:C complex anti-tumor effect in non-small cell lung cancerObjective:To investigate the effects of PolyI:C complex on cytokines and spleen DC cell populations and PI3K/Akt/p53 pathway in vitro and in vivo.Methods:In order to detect changes in cytokines in the body before and after administration,we used the Multiplex immunoassay.Normal healthy C57BL/6 were randomly divided into two groups:PBS and PolyI:C complex intramuscular injection group.2h after administration,blood was taken from the posterior venous plexus of the mouse eye,separated serum.The cytokines in serum was detected by Multiplex immunoassay using a ProcartaPlexTM kit according to the manufacturer's protocol.The lung cancer transplantation model mice were randomly divided into four groups(n=6):PBS group,PolyI:C complex nasal administration group,PolyI:C complex intramuscular injection group and PD1 group.C57BL/6 Mice were injected subcutaneously with LL/2 cells into the right flank.When the tumor volume reached 100 mm3 100?L(200?g)of PolyI:C complex was given by nasal administration or intramuscular injection every other day,or 100?g of PD1 was given once a week by celiac injection,or 100?L of PBS was given by nasal administration every other day.When the average tumor volume of PBS group reached 2500mm3 the animals were quickly killed by cervical dislocation.All tumor tissues and spleen tissues were excised,tumor tissues were fixed in formalin,spleen tissues were saved in PBS.The contents of CD 11c,CD80 and CD86 in spleen DC cells were measured by flow cytometry.The expression of Akt and its downstream channel proteins p53,p21,cyclinDl and caspase3 were detected by immunohistochemistry.LL/2 and A549 cell lines were selected and treated with PolyI:C complex for 48h.The expression of Akt and its downstream channel proteins p53,p21,cyclinD1 and caspase3 were detected by western blot.Results:The secretion of IL-2,IL-4,IL-6 and TNF-a in healthy mice 2 hours after intramuscular injection of PolyI:C complex were(30.81±11.34),(3.62±1.02),(1010.84±162.09),(301.33±19.16)pg/mL,had significantly higher than PBS group(P<0.01,P<0.05,P<0.001,P<0.001).The CD11c positive rate of spleen tissue of the tumor-bearing mice PolyI:C complex group was 7.85%,CD80 positive rate and MFI were 20.16%and 1210.76,CD86 positive rate and MFI were 4.01%and 353.20,respectively.Both were significantly higher than the control group(P<0.01;P<0.01;P<0.01;P<0.001).The results of immunoblotting showed that the PolyI:C complex inhibited the activity of p-Akt and CyclinDl in LL/2 and A549 cells(P<0.01;P<0.001),and effectively upregulated the expression of p53,p21 and cleaved caspase3(P<0.001).There was no significant difference in the expression of Aktl/2 between LL/2 and A549 cells before and after treatment(P>0.05).After PolyI:C complex stimulation,the expression of caspase3 was increased in LL/2 cells(P<0.05),and there was no significant difference in A549(P>0.05).The expression of p53,p21 and caspase3 in the intramuscular injection group of PolyI:C complex was higher than that in PBS group,while the expression of p-Akt and cyclinDl was decreased compared with the control group.There was no significant difference in the expression of Aktl/2 after treatment.Conclusions:1.Polyl:C complex stimulates the secretion of serum inflammatory cytokines in normal mice.2.Polyl:C complex stimulation exerts anti-tumor effect by up-regulating CD80 and CD86 molecules of DC cells of spleen tissue in tumor-bearing mice.3.PolyI:C complex through PI3K/Akt/p53 pathway,down-regulates p-Akt and cyclinD1 affects cell cycle,up-regulates p53,p21,caspase3 to induce apoptosis of NSCLC cells in vitro and in vivo,furthermore,inhibits cell proliferation and exerts an anti-tumor effect.