Effects And Mechanisms Of SAHA Combined With Erlotinib On The Growth Of H1975 Lung Cancer Xenograft In Nude Mice

Background:Lung cancer is one of the most common malignant tumors that poses serious hazards to human health.Most patients lose the chance of surgery due to the late stage of the first diagnosis.EGFR-TKI can be used as a first-line treatment for advanced lung cancer.However,with the extension of time,almost all patients with effective initial treatment are resistant within about 1 year,and drug resistance greatly limits the clinical application of EGFR-TKI in lung cancer.HDACIs are a new class of targeted epigenetic drugs that affect the transcription and expression of genes by altering the acetylation level of histones and non-histones in specific regions of the chromosome.In hematological tumors,HDACIs alone can show significant efficacy,while in solid tumors,HDACIs have synergistic anti-tumor effects combinied with traditional chemotherapeutic drugs,targeted drugs and radiotherapy.SAHA is a broad-spectrum hydroxamic acid HDACI,which is the first HDACI approved by FDA for the treatment of PTCL.In this study,a xenograft model of H1975 lung cancer nude mice was established to explore whether SAHA combined with erlotinib has synergistic anti-tumor effects in vivo and its mechanisms.Objectives:By observing the effect of SAHA combined with erlotinib on the growth of H1975 lung cancer xenografts in nude mice,and exploring the possible mechanism of SAHA reversing EGFR-TKI resistance,which provided a theoretical basis for the clinical application of HDACIs in lung cancer.Methods:（1）Animal modeling and group administration:5′10⁶ H1975 cells were injected into the right axilla of Balb/c nude mice,and the tumor size was measured 2 to 3 times per week.After successful modeling,the moderate and uniformity tumor-burdened nude mices were selected and randomly divided into 4 groups,8 in each group:the control group（PBS）,SAHA group（SAHA 50 mg/kg）,erlotinib group（erlotinib 30 mg/kg）,the combination group（SAHA 50 mg/kg+erlotinib 30 mg/kg）,and administered continuously by intragastric administration for 21 days.（2）Tumor size and relative tumor proliferation rate:The tumor size was measured 2 to3 times per week with a vernier caliper,and calculate the tumor volume（TV）,relative tumor volume（RTV）and relative tumor proliferation rate（T/C）.TV=1/2ab²（a is the long diameter,b is the short diameter）,the average value is taken and the tumor growth curve is drawn;RTV=V_t/V₀,V₀ is the tumor volume measured before administration（ie,d₀）,and V_t is the tumor volume measured after administration;T/C is the percentage of RTV in the experimental group and the control group(T/C=T_RTV/C_RTV′100%);（3）Tumor weight and tumor growth inhibition rate:At the end of the experiment,the tumor tissue was taken,and the tumor weight（TW）and the tumor growth inhibition rate（TGI）was calculated.TGI=（1-the average tumor weight of the experimental group/the average tumor weight of the control group）′100%;Using the Jin’s formula to calculate the Q value to evaluate the effect of the combination of the two drugs,Q=E_A+B/（E_A+E_B-E_A×E_B）,E_A+B is the tumor inhibition rate of the combination group,E_A and E_B is the tumor inhibition rate of the single drug group.（4）Western blot:Western blot was used to detect the expressions of related proteins in PI3K/AKT/mTOR and MAPK/ERK signaling pathways in the tumor tissues of nude mice in each group.（5）IHC:IHC was used to detect the expression of p-AKT,p-mTOR and p-ERK in tumor tissues of nude mice in each group.（6）Proliferation rate and apoptosis rate:HE staining was used to observe the gross morphological changes of tumor tissues.Immunohistochemical Ki-67 staining method was used to detect the proliferation of tumor cells and TUNEL staining was used to detect the apoptosis of tumor cells.Image J software was used to count the proliferation rate and apoptosis rate of tumor cells.Results:（1）H1975 lung cancer xenograft model was successfully established,and the rate of tumor formation was 100%.（2）Tumor size and tumor weight:TV:the control group was（1838±469）mm³,SAHA group was（1669±428）mm³,erlotinib group was（1648±355）mm³,and the combination group was（1061±205）mm³;TW:the control group was（2.41±1.02）g,SAHA group was（2.11±0.69）g,erlotinib group was（1.95±0.67）g,and the combination group was（1.01±0.30）g.Compared with the single drug groups and the control group,the difference of the combination group was statistically significant（F=7.58,8.59,P<0.05）.（3）T/C and TGI:T/C:the control group was 100%,SAHA group was 90.8%,erlotinib group was 89.7%,and the combination group was 57.7%;TGI:the control group was 0,SAHA group was 12.5%,erlotinib group was 19.1%and the combination group was54.4%.T/C≤60%and TGI≥40%（P<0.05）,and the Q value of the combination group was 1.86.（4）Western blot:SAHA combined with erlotinib can significantly down-regulate the expression of p-AKT,p-mTOR and p-ERK without affecting the expression of total protein of AKT,mTOR and ERK,compared with the single drug groups and the control group,the difference of the combination group was statistically significant（F=8.64,25.19,46.12,P<0.05）.（5）IHC:Compared with the control group,the single-agent groups had no significant effect on the expression of p-AKT,p-mTOR and p-ERK in H1975 tumor tissues（P>0.05）,while the combination group could significantly inhibit the expression of p-AKT,p-mTOR,p-ERK in tumor tissues.（F=5.28,9.67,10.44,P<0.05）.（6）Proliferation rate and apoptotic rate:proliferation rate:the control group was（29.12±7.24）%,SAHA group was（26.36±7.27）%,erlotinib group was（27.15±3.40）%,and the combination group was（17.98±5.03）%;apoptotic rate:the control group was（2.35±1.25）%,SAHA group was（6.39±1.79）%,erlotinib group was（6.44±5.07）%,and the combination group was（12.15±5.43）%,compared with the control group and the single drug groups,the difference of the combination group was statistically significant（F=5.44,8.67,P<0.05）.Conclusions:SAHA combined with erlotinib can partially reverse EGFR-TKI resistance and significantly enhance the efficacy of H1975 lung cancer xenografts in nude mice.SAHA combined with erlotinib has synergistic anti-tumor effects,which may be related to inhibit the activation of related proteins in PI3K/AKT/mTOR and MAPK/ERK signaling pathways.