Ailanthone Inhibits The Growth Of Non-small Cell Lung Cancer And Induces Autophagy And Ferroptosis In Vivo And In Vitro

According to the latest cancer statistics of the International Agency for Research on Cancer in 2020,there were an estimated 2.2 million new lung cancer cases and 1.8 million lung cancer deaths worldwide.The death toll of lung cancer far exceeds that of other cancer types,ranking first in the number of cancer deaths.Non-small cell lung cancer（NSCLC）accounts for～85%of all lung cancer cases.Although the treatment of surgical operation,chemotherapy,radiotherapy,and targeted therapy is carried out,some patients still relapse or metastasize,accompanied by various side effects,leading to poor prognosis.Therefore,exploring new anticancer targets and new drugs remains a problem in NSCLC treatmentAutophagy,a complex but promising target in cancer therapy,plays a key role in cancer progression,metastasis,and treatment resistance.It has been found that drugs that induce high levels of autophagy can cause the collapse of tumor cells and induce activation of cell death procedures.Recently,ferroptosis,a new form of cell death,has been shown to play an anticancer role in tumor model experiments,which has attracted much attention from scholars at domestic and abroad.There are abundant resources of traditional Chinese medicine,among which many active ingredients have regulatory effects on autophagy and ferroptosis,and the purpose of tumor inhibition can be achieved by searching for corresponding drugs.Ailanthone（AIL）,one of the active components of the Chinese herbal medicine ailanthus altissima has been proven to have significant anticancer activity,but its specific mechanism in the treatment of lung cancer remains unclear.This study aims to investigate the anti-tumor effect of AIL on NSCLC cells and its effect on autophagy and ferroptosis in vivo and in vitro,and to evaluate the preventive and therapeutic effects of traditional Chinese medicine monomer AIL on lung cancer.Part 1 Antitumor effect of ailanthone in vitroObjective:To explore the effects of AIL on the proliferation,autophagy and ferroptosis of LLC cells in vitroMethods:1.LLC cells treated with gradient AIL were detected by MTT assay,computing IC₅₀ value concentration,and subsequent experimental concentrations were determined.2.LLC cells were treated with AIL at concentrations of 0,2.5,5,and10μM for 24h,and the cell morphological changes were observed under an optical microscope.3.LLC cells were treated with AIL at concentrations of 0,2.5,5,and10μM for 24h,and the clone-forming ability of LLC cells was detected by colony-forming assay.4.Immunofluorescence assay was used to detect the protein expression levels of autophagy-related genes P62 and LC3B in LLC cells treated with 0,2.5,5,and 10μM AIL for 24h.5.Western blot was performed to detect the protein expression levels of autophagy-related genes P62,Beclin1,ATG5,and LC3B in LLC cells treated with 0,2.5,5,and 10μM AIL for 24h.6.DCFH-DA probe was used to detect the ROS accumulation of LLC cells treated with 0,2.5,5,and 10μM AIL for 24h.7.The expression levels of Fe²⁺,LPO,MDA,GSH,T-SOD,and CAT in LLC cells treated with AIL at 0,2.5,5,and 10μM for 24h were detected by colorimetric assay.8.Western blot was used to detect the protein expression levels of ferroptosis markers x CT,GPX4,FTH,and TFRC in LLC cells treated with 0,2.5,5,and 10μM AIL for 24h.Results:1.AIL inhibited the viability of LLC cells:MTT assay showed that AIL inhibited the viability of LLC cells in a dose-dependent and time-dependent manner,with an IC₅₀value of7.696μM.2.Effect of AIL on the morphology of LLC cells:After AIL treatment,LLC cells showed reduced adhesion,morphological shrinkage,uneven size,and poor refractive ability under a light microscope.With the increase in drug concentration,the changes in cell morphology became more obvious.3.AIL inhibited the ability of LLC cell clone formation:Compared with the 0μM control group,the clone-forming capacity of LLC cells（F=0.680,P<0.001）was significantly decreased with the increasing dose of AIL concentrations.4.Immunofluorescence assay showed that AIL promoted LLC cellsautophagy:AIL increased the green fluorescence intensity of LC3B protein（F=152.179,P<0.001）and decreased the expression of P62 protein（F=255.494,P<0.001）in the cells in a dose-dependent manner.5.Western blot showed that AIL promoted LLC cells autophagy:The results showed that the expression levels of Beclin1（F=456.014,P<0.001）,ATG5（F=247.187,P<0.001）,and LC3B（F=152.740,P<0.001）increased,while P62（F=1234.348,P<0.001）protein expression decreased with the increasing of AIL concentration.6.AIL increased ROS accumulation in LLC cells:Fluorescence microscope observation showed that compared with the0μM control group,the intracellular green fluorescence signal representing ROS expression level increased after AIL treatment,and the fluorescence effect was more obvious with the increase of drug concentration（F=182.200,P<0.001）.The same results were obtained in observation with a fluorescent microplate reader（F=15.863,P<0.001）.7.AIL increased intracellular Fe²⁺,LPO,and MDA levels and decreased GSH,T-SOD,and CAT levels in LLC cells:Colorimetry showed that the concentrations of Fe²⁺（F=353.507,P<0.001）,LPO（F=380.356,P<0.001）,and MDA（F=230.938,P<0.001）increased significantly in LLC cells treated by AIL for 24h in a concentration-dependent manner.In addition,AIL treatment decreased the contents of GSH（F=67.112,P<0.001）,T-SOD（F=74.961,P<0.001）,and CAT（F=88.188,P<0.001）in a concentration-dependent manner.8.AIL down-regulated x CT protein and up-regulated GPX4,FTH,and TFRC protein expression:Compared with the control group,the expressions of x CT（F=2055.887,P<0.001）,GPX4（F=160.192,P<0.001）,and FTH（F=402.311,P<0.001）in lung cancer cells were significantly decreased after different doses of AIL treatment.TFRC（F=484.205,P<0.001）protein expression was significantly increased in a dose-dependent manner.Conclusion:AIL inhibited the viability of lung cancer LLC cells and induced autophagy and ferroptosis.Part 2 Antitumor effect of ailanthone in vivoObjective:To investigate the effects of AIL on tumor proliferation,autophagy and ferroptosis in two mouse models of lung cancer in vivo.Methods:1.A lung cancer mouse model of Lewis subcutaneous transplantation tumor was established:LLC cells in the logarithmic growth phase were collected to make a single-cell suspension,and the cell concentration was adjusted to 1×106 cells per mouse.Each C57BL/6J mouse was subcutaneously injected with 200μl of the cell suspension to the right hind limb of the femur,which was identified as the first-generation Lewis lung cancer subcutaneous transplantation mouse model.When the subcutaneous tumor volume reached 800mm3（about 2 weeks）,the mice were sacrificed and the tumor was aseptically dissected and inoculated into the deep groin of the right hind limb of the anesthetized mice,which was recorded as the second-generation Lewis lung cancer subcutaneous transplantation mouse model.The state of the mice was observed every day.When the subcutaneous tumor volume of the second-generation Lewis lung cancer mice reached 800mm3（about 2 weeks）,the tumor tissue inoculation procedure was repeated to construct the third-generation Lewis lung cancer subcutaneous transplantation tumor mouse model.2.Lewis lung cancer subcutaneous transplantation mice were divided into groups and treated:Thirty-two C57BL/6J mice with third-generation Lewis lung cancer subcutaneous transplantation tumor were randomly divided into four groups: model group（Control）,AIL 1mg/kg group,AIL 5mg/kg group,and cisplatin group（DDP 5mg/kg）.Treatment was performed the next day.The control group was given normal saline every day,and the mice in the experimental group were treated with AIL daily,according to 1mg/kg or 5mg/kg concentration.Positive control group mice were treated with 5mg/kg DDP once a week.All Routes of administration were via intraperitoneal injection.3.Physiological characteristics of Lewis lung cancer subcutaneous transplantation tumor model mice were observed:During the experiment,mice were weighed every three days.Tumor volume was measured with vernier calipers and calculated,and the overall state of mice was monitored.4.Sample collection:Serum was collected,and tumors and major organs of mice were stripped and weighed.The collected tumor tissues were stored in liquid nitrogen and 4% paraformaldehyde for fixation,and then dehydrated to make paraffin sections for the later experiment.5.Urethane-induced lung cancer mice were grouped and drug delivery:Sixty BALB/c mice were randomly divided into control group（Negative Control,NC）,Model group（Control）,AIL 1mg/kg group,AIL 5mg/kg group,and cisplatin group（DDP 4mg/kg）.Mice in each group（except the NC group）were intraperitoneally injected with urethane solution（800mg/kg）once a week for 10 weeks.Since the modeling date,mice in NC group and Control group were given equal volume normal saline at the same time.Urethane was administered the next day after inoculation.The cisplatin group was given 4mg/kg DDP once a week for 4 weeks.The AIL groups were intraperitoneally injected with 1mg/kg and 5mg/kg AIL,respectively,once every three days for 20 weeks,and the experiment ended in the 21 st week.6.The physiological signs of urethane-induced lung cancer mice were monitored:During the experiment,the mice were weighed every 2 weeks,and the overall condition of the mice was monitored.7.Sample collection:The main organs of the mice were dissected,and the number of lung tumor nodules was calculated.The lung tissues with tumors were stored in liquid nitrogen and 4% paraformaldehyde until dehydrated and made into paraffin sections for the later experiment.8.HE staining was performed on each major organ of the mice.9.Immunofluorescence assay was used to detect the protein expression levels of autophagy-related genes P62,LC3 B,ATG5,and Beclin1 in tumor tissues of mice.10.Colorimetric assay was used to detect the expression levels of Fe2+,LPO,MDA,GSH,T-SOD,and CAT in the serum of mice.Results:1.Effects of AIL on the general state of Lewis lung cancer xenograft mice:After the subcutaneous tumor transplantation experiment in C57BL/6J mice,the cisplatin group showed a decrease in activity.The mental state of mice in other groups was acceptable.Mice in the different treatment groups had similar body weights at the beginning of the experiment,and there was no difference in body weight among the groups during the course of the experiment.2.AIL inhibits tumor growth in Lewis lung cancer xenograft mice:Compared with the untreated control group,AIL with 1mg/kg group,AIL 5mg/kg group,and cisplatin group mice inhibited the tumor growth.AIL inhibited tumor volume（F=35.099,P<0.001）and weight（F=25.639,P<0.001）in a dose-dependent manner in AIL 1mg/kg and 5mg/kg groups.3.AIL induces tumor autophagy in Lewis lung cancer xenograft mice:Immunohistochemical staining showed that autophagy-associated proteins P62（F=951.235,P<0.001）,Belcin1（F=97.252,P<0.001）,ATG5（F=1282.965,P<0.001）,LC3B（F=88.430,P<0.001）were significantly different among the control group,AIL 1mg/kg group,AIL 5mg/kg group,and cisplatin group.Compared with the control group,the expression of P62（P<0.001）protein in the tumor tissue of mice treated with AIL was decreased,while the expression of Beclin1（P<0.001）,ATG5（P<0.001）,and LC3B（P<0.001）were gradually increased in a dose-dependent manner.4.AIL induced ferroptosis in Lewis lung cancer xenograft mice:Colorimetry results showed that the levels of serum iron ion and lipid peroxide in mice,including Fe2+（F=181.330,P<0.001）,LPO（F=58.238,P<0.001）,and MDA（F=145.838,P<0.001）were different among the model group,AIL 1mg/kg,5mg/kg groups and cisplatin group.Moreover,the expression levels of GSH（F=48.670,P<0.001）,T-SOD（F=52.604,P<0.001）,and CAT（F=120.710,P<0.001）were significantly different in groups.Compared with the model group,the serum Fe2+（P<0.001）,LPO（P<0.05）,and MDA（P<0.001）significantly increased in AIL-treated mice.The contents of GSH（P<0.05）,T-SOD（P<0.05）,and CAT（P<0.05）decreased in a dose-dependent manner.5.Effects of AIL on main organs of Lewis lung cancer xenograft mice: The main organs of tumor-bearing mice were removed,weighed,and stained with HE.Compared with the control group,the cisplatin group had a significant increase in the organ coefficient of the liver（F=17.224,P=0.001）.There were no statistical differences among the other groups.No macroscopic lesions were observed under HE staining.6.Effects of AIL on the general state of urethane-induced lung cancer mice:The mental state and activity of the mice in the AIL groups were acceptable,while the mice in the control and cisplatin groups showed reduced activity,decreased desire for food,lethargy,and poor response to external stimuli.The mental state of the mice in the cisplatin group was particularly poor.Compared with the beginning of the experiment,the body weight of the control group increased steadily in the first 10 weeks（P=0.003）and tended to be stable in the later period（P=0.158）.The mice in the model group lost weight in the first 5 weeks（P=0.002）,and regained weight after drug withdrawal（P=0.223）.In the cisplatin group,the body weight of mice decreased significantly during the first 10 weeks of drug treatment（P=0.007）and gradually returned to normal after the cessation of urethane and cisplatin injection（P=0.057）.The overall body weight of AIL1mg/kg and 5mg/kg groups tended to be stable from 1 to 10 weeks（P>0.05）,and the body weight of the AIL 1mg/kg group（P=0.022）and AIL 5mg/kg group（P<0.001）showed an upward trend.7.AIL reduced the incidence of lung cancer and the number of lung tumor nodules in urethane-induced lung cancer mice:The incidence of lung cancer and the number of lung tumor nodules in the AIL 1mg/kg group,AIL 5mg/kg group and cisplatin groups were lower than those in the control group（P<0.05）.Compared with the 1mg/kg AIL group,the incidence of lung cancer（P<0.001）and the number of lung tumor nodules（P=0.031）were reduced in the AIL 5mg/kg group,and the differences were statistically significant.8.AIL induced autophagy in lung tumors of urethane-induced lung cancer mice:Immunohistochemical staining showed that the expression levels of autophagy-related proteins P62（F=96.019,P<0.001）,LC3B（F=284.211,P<0.001）,ATG5（F=669.711,P<0.001）,and Belcin1（F=815.193,P<0.001）are different among the NC group,control group,AIL 1mg/kg group,AIL 5mg/kg group,and cisplatin group.Compared with the control group,the expression of P62 protein decreased in the tumor tissues of the AIL 1 mg/kg group（P=0.016）and AIL 5 mg/kg group（P<0.001）,while the expression of LC3B（P<0.001）,ATG5（P<0.001）,and Beclin1（P<0.001）increased gradually in a dose-dependent manner.9.AIL induced ferroptosis in urethane-induced lung cancer mice:The levels of serum Fe2+ and lipid peroxide in mice were determined by colorimetry,and results showed that Fe2+（F=43.677,P<0.001）,LPO（F=53.223,P<0.001）,MDA（F=286.015,P<0.001）,and antioxidant enzymes GSH（F=690.885,P<0.001）,T-SOD（F=1211.322,P<0.001）,and CAT（F=16.227,P<0.001）were significantly different among the NC group,control group,AIL 1mg/kg group,AIL 5mg/kg group,and DDP group.Compared with the control group,the concentrations of Fe2+（P<0.001）,LPO（P<0.05）,and MDA（P<0.001）in serum were significantly increased in AIL-treated mice.The contents of GSH（P<0.05）,T-SOD（P<0.001）,and CAT（P<0.05）were decreased in a dose-dependent manner.10.Effects of AIL on major organs in urethane-induced lung cancer mice:The main organs of the mice were weighed and stained with HE.Compared with the control group,the liver organ coefficient increased significantly in the cisplatin group（F=3.642,P=0.044）.There were no statistical differences among the other groups.No obvious lesions were observed under HE staining.Conclusion:AIL effectively inhibited tumor growth and induced autophagy and ferroptosis in both Lewis lung cancer subcutaneous tumor grafts and urethane-induced lung cancer mice.