Study On The Antitumor Mechanism Of Green Tea Polyphenol EGCG And The Investigation On EGCG-NPs Reduce The Resistance Of EGFR-TKIs In Non-small Cell Lung Cancer

Purpose In recent years,the incidence of lung cancer has been increasing,and lung cancer has become the leading cause of cancer-related death.Approximately 85% of patients with lung cancer are diagnosed with non-small-cell lung cancer(NSCLC),one of the most predominant pathological types of lung cancer.The 5-year overall survival rate of patients with NSCLC remains poor.Moreover,resistance and the recurrence rate of this disease is high.Many drugs have been designed to target specific signaling molecules,however these are affiliated with serious adverse effects,which limit their utility in the clinic.Thus,it is critical to consider other therapeutic measures for lung cancer.Green tea is a popular beverage worldwide.Tea consumption may be linked to low incidences of lung cancer.Out of all the constituents of green tea,commonly referred to as catechins,(-)-epigallocatechin-3-gallate(EGCG)is the most abundant and biologically active component.EGCG inhibits multiple signal transduction pathways in cancer cells including NF-?B.As NF-?B regulates a panel of key oncogenes and pro-survival genes,this pathway has also been implicated in tumor initiation,progression,and resistance to chemotherapy.And NF-?B is overexpressed in lung cancer.In spite of promising preclinical findings,the applicability of tea in human cancer therapy is limited owing to a lack of efficient systemic delivery,bioavailability and stability.Nanoparticles as drug carriers have drawn considerable attention since nanoparticles possess unique physical properties,such as higher tissue permeability,colloidal stability,and drug bioavailability,as well as relatively lower cost.Many researches have demonstrated the great potential of PLGA as a carrier for cancer treatment.We plan to investigate the molecular mechanism of EGCG-mediated inhibition of proliferation in lung cancer cells and to explore the effects of combined treatment with low concentration of EGCG and an NF-?B inhibitor,BAY11-7082,lung cancer cells both in vitro and in vivo.Meanwhile,we aimed to establish a nanoparticle strategy to overcome the shortcomings of EGCG in the treatment of lung cancer.The efficacy of optimized nano-formulations was evaluated by constructing a patient-derived tumor xenograft(PDX)model,providing the experimental basis for improving the stability of the drug and the clinical application of EGCG in treating lung cancer.Finally,the EGFR-TKIs acquired resistant NSCLC cell lines were established in vivo.And the key genes and the signal pathway were detected by RNA-sequence,so as to explore the molecular mechanism of EGFR-TKIs acquired resistance.Then to determine if EGCG in combination with targeting strategies would be more effective than treatment with single agents and providing potential therapeutic strategies for patients with clinical drug resistance.Methods 1 EGCG exerts antitumor effect in lung cancer cells by inhibiting NF-?B signaling MTT assay and colony formation assay were used to evaluate the anti-proliferation effect of EGCG and BAY11-7082 either administrated alone or in combination.To more accurately determine the effects(additive,synergistic or antagonistic)of combination therapy,these results were evaluated mathematically using the method of CI.And the interaction between protein-NF-?B and EGCG was detected by bio-layer interferometry(BLI).The apoptotic cells were detected by flow cytometry after Annexin V/PI staining.Scratch wound and Transwell migration assays were used to detect cell mobility.The expression of related genes was detected by RT-PCR and Western blot.The NF-?B promoter activity was measured by Luciferase Reporter Assay.The in vitro anti-cancer effects were validated in vivo in xenograft model.2 EGCG-NPs enhance the therapeutic effect of EGCG on human lung cancer tissue Poly lactic-co-glycolic acid(PLGA)nanoparticles(NPs)loaded with EGCG were prepared by the oil-in-water emulsion solvent evaporation technique.The characteristics of NPs,entrapment efficiency,and in vitro release were systematically evaluated.The cellular uptake and cytotoxic activity of free-from EGCG and the NPs were compared.The effect of the formulation on cellular apoptosis was evaluated by flow cytometry.NF-?B signaling was evaluated by western blotting and RT-PCR.The efficacy of the optimized nanoformulation was evaluated using a PDX model.3 EGCG-NPs reduce the resistance of NSCLC to EGFR-TKIs NCI-PC9 cell line containing EGFR del 19 mutation was induced to establish the three generation EGFR-TKIs(Erlotinib,Afatinib and Osimertinib)resistant cell line(PC9/ER,PC9/AR and PC9/OR)by low-concentration addition method in vitro.To explore the NF-?B role in resistance,the expression of P-NF-?B and NF-?B were detected by Western Blot in both parental and resistant cells.And the key genes and pathway which may induce to EGFR-TKIs acquired resistance were detected by RNA-sequence.The result of RNA-sequence was verified by RT-PCR.MTT assay and colony formation assay were used to evaluate the anti-proliferation effect of EGCG-NPs plus EGFR-TKIs.Western Blot was used to examine the effect of EGCG combined with EGFR-TKIs on P-NF-?B protein expression.The expression of NF-?B related genes was detected by RT-PCR.Results 1 EGCG exerts antitumor effect in lung cancer cells by inhibiting NF-?B signaling EGCG inhibits cell proliferation and migration and induces apoptosis in A549 and H1299 cells at relatively high concentrations(IC50=86.4 ?M for A549 cells and 80.6 ?M for H1299 cells).Molecular interaction assays demonstrated that EGCG binds to NF-?B with high a nity(KD=4.8×10-5 M).According to it,the anti-tumor effects are partially achieved via inhibition of the NF-?B signaling pathway.Combined treatment with EGCG and BAY11-7082,a potent NF-?B inhibitor,shows significant synergistic effects at relatively low concentrations.The inhibition rate reached approximately 50% in cells treated for 72 h with 20 ?M EGCG and 5 ?M(A549 cells)or 2.5 ?M BAY11-7082(H1299 cells).And co-treatment with EGCG and BAY11-7082 can inhibits lung cancer cells migration and invasion significantly.This synergistic anti-tumor effect was also observed in a xenograft model and no significant difference was observed in body weight.2 EGCG-NPs enhance the therapeutic effect of EGCG on human lung cancer tissue EGCG-loaded NPs(175.8±3.8 nm in size)demonstrated optimal efficacy,with approximately 86.0±1.5% encapsulation efficiency and ~14.2±0.3% loading efficiency.And it is with an excellent particle size distribution(PDI=0.096±0.015).Additionally,EGCG encapsulated PLGA-NPs offered a ~3-4-fold dose advantage compared to free EGCG in terms of exerting antiproliferative effects and inducing apoptosis at lower doses(12.5,25 ?M).EGCG-NPs were more effective in inhibiting NF-?B activation and suppressing the expression of NF-?B-regulated genes involved in cell proliferation and metastasis than free EGCG.Furthermore,EGCG-NPs demonstrated superior results in terms of inhibiting proliferation and expression of phospho-NF-?B,inducing apoptosis in tumors harvested from the treated mice.EGCG-NPs showed superior anticancer activity in the PDX model than free EGCG.3 EGCG-NPs reduce the resistance of NSCLC to EGFR-TKIs The drug low concentration gradient method was used for 6 months to successfully established the EGFR-TKIs resistant cell lines: PC9/ER,PC9/AR and PC9/OR.The drug resistance index is 180.051,128.15 and 269.38,respectively.The expression of P-NF-?B was higher than parental cell,and may be a reason of TKIs acquired resistance.Next,the RNA-sequence showed that 362 common genes were differentially expressed by at least 2-fold(P < 0.05)among the 3 EGFR-TKIs resistant cells.And the NF-?B signaling pathway was activated in resistant cells.The NF-?B pathway related genes were higher than parental cell.The role of NF-?B in the resistance of EGFR-TKIs can be confirmed.EGCG-NPs potentiates the antiproliferative of EGFR-TKIs in the resistant cells.And they can significantly decrease the expression of P-NF-?B.In addition,EGFR-TKIs and EGCG-NPs also have synergistic anti-proliferative effects in H1650 and H1975 cells.Conclusions Firstly,co-administration of EGCG and BAY11-7082 has a synergistic effect both in vitro and in vivo by suppressing NF-?B pathway,and it may serve as a novel therapeutic strategy for lung cancer.Secondly,the prepared EGCG-NPs were more effective than free EGCG in inhibiting lung cancer tumors in the PDX model.This clearly indicates that by using this formulation strategy,EGCG could be delivered in physiologically active concentrations.Thus,the outcome of this study could have direct practical implication,with translational relevance in human lung cancer patients.Thirdly,EGCG-NPs combined with EGFR-TKIs could reduce the resistance of EGFR-TKIs acquired resistant NSCLC cell lines via down-regulation of P-NF-?B and providing potential therapeutic strategies for patients with clinical drug resistance.