Application Values And Mechanisms Of New LC3 Targeting Small Molecule Drug D5 In Hepatocellular Carcinoma And Other Multiple Tumors

Background and ObjectiveLiver cancer is one of China's primary malignant tumors,which has the characteristics of difficult early diagnosis,easy recurrence after treatment,and single pattern treatment choice.It is still the top priority of cancer research in the future.Autophagy is a physiological activity of various tumor cells,one of the significant reasons for tumor cells to develop drug resistance or decrease drug sensitivity during anticancer drug treatments.As an essential role in autophagy formation,LC3 is closely related to cancer patients'prognosis.This experiment focuses on exploring the inhibitory effect of the new small molecule drug D5 targeting the key protein LC3 for autophagy formation and combining it with the clinically common first-line anticancer drugs Sorafenib and Oxaliplatin to determine whether it has a synergistic effect with these anticancer drugs,to provide a preliminary research basis for D5entering clinical trials.Methods1.To explore the correlation between LC3B expression level and prognosis of HCC patients.(1)The expression of LC3B in liver cancer tissuesThe sample of liver cancer and adjacent tissues of clinical patients were collected,and the corresponding c DNA was prepared for q PCR amplification of LC3m RNA.The m RNA expression levels in cancer and para-cancerous groups were compared.(2)Relationship between LC3B expression level and patient prognosisa.Collect liver cancer and adjacent tissue samples from clinical patients,prepare corresponding c DNA to amplify LC3 m RNA by q PCR.The LC3 m RNA level of HCC patients were integrated with the patients'prognosis data,and the survival curve was drawn.b.LC3B immunohistochemical staining was performed on prefabricated tissue chips of liver cancer patients.Positive staining scores in cancer and adjacent tissues were analyzed and recorded by the software,and survival curves were drawn after integration with patients'clinical prognosis data.2.Effect of a novel LC3-targeting small molecule drug D5 on the autophagy process and hepatoma cells'malignant behavior.(1)The effect of small molecule drug D5 on autophagy of liver cancer cellsa.IC₅₀,1/2 IC₅₀,and 1/4 IC₅₀ were selected as drug administration concentrations for subsequent experiments after determining drug administration concentration by cytotoxic IC₅₀ test.b.Add the corresponding concentration of drugs to the cell culture medium and collect cell protein samples for WB analysis.c.m RFP-GFP-LC3 double-labeled adenovirus was used to infect hepatoma cells with drug treatment at the corresponding concentration for the appropriate time.A laser confocal microscope was used to take photos,and the number and proportion of autophagosomes and autophagosomes were calculated to determine the direction of autophagy flux in each group.d.The cells were treated with the drug of the corresponding concentration for the corresponding time.After collection,the cells were fixed with the electron microscope fixator,and the samples were sent to take photos.(2)The effect of small molecule drug D5 on the proliferation of liver cancer cellsCells were treated with corresponding concentration drugs,and the effect of small molecule drug D5 on the proliferation ability of HCC cells was detected by the CCK8(Cell Counting Kit-8)kit.(3)Effect of small molecule drug D5 on apoptosis of hepatoma cellsa.The cells were treated with the corresponding concentration of drugs,and the TUNEL kit stained the DNA fracture in the HCC cells and then photographed and analyzed by laser confocal microscope.b.The cells were treated with the corresponding concentration of drugs,and the cells were double-stained by Annexin V-FITC/PI through the apoptosis flow cytometry kit,and the apoptosis level was detected by flow cytometry.(4)Effect of small molecule drug D5 on migration and invasion of hepatoma cellsThe cells were treated with corresponding concentrations of drugs,and the Transwell chamber experiment compared the effects of different concentrations of drugs on cell migration and invasion ability.3.Inhibition effects of small molecule drug D5 combined with Sorafenib on liver cancer cells'malignant behaviors.(1)Effect of small molecule drug D5 combined with Sorafenib on drug sensitivity of liver cancer cellsThe cells in the 96-well plate were divided into Sorafenib group and Sorafenib+D5 group,and the cells were treated with Sorafenib with the same concentration gradient.In the Sorafenib+D5 group,small molecule drug D5 with a fixed concentration was added based on Sorafenib treatment to determine whether small molecule drug D5 could make liver cancer cells more sensitive to Sorafenib.(2)Effects of small molecule drug D5 combined with Sorafenib on the proliferation of liver cancer cellsThe cells in the 96-well plate were divided into the Control group,D5 group,Sorafenib group,and Sorafenib+D5 group and given complementary drug treatment.Each group had three duplicate wells,and the CCK8 kit was used to detect the effects of different drug combinations on the proliferation ability of liver cancer cells every day.(3)Effects of small molecule drug D5 combined with Sorafenib on apoptosis of liver cancer cellsa.The cells were divided into the Control group,D5 group,Sorafenib group,and Sorafenib+D5 group.Cell slides were made and treated with relevant drugs.b.The cells were divided into the Control group,D5 group,Sorafenib group,and Sorafenib+D5 group.The cells were treated with corresponding drugs and collected after an appropriate time,and Annexin V-FITC/PI double staining was used to detect the proportion of apoptotic cells by flow cytometry.(4)Effects of the combination of small molecule drug D5 and Sorafenib on invasion and migration of hepatoma cells.The cells were divided into the Control group,D5 group,Sorafenib group,and Sorafenib+D5 group.The cells were cultured using a Transwell chamber system,given corresponding drugs,and stained with crystal violet after treatment for the corresponding time.Photographs were taken for analysis.(5)Influence of small molecule drug D5 on Autophagy flux formation of Sorafenib induced hepatoma cellsa.m RFP-GFP-LC3 double-labeled adenovirus was used to infect hepatoma cells and the cells were divided into the Control group,D5 group,Sorafenib group,and Sorafenib+D5 group.After corresponding drug treatment,cell sliders were collected,and the number of autophagosomes and autophagolysosomal were taken by laser confocal microscope,and the proportion was calculated to determine the change direction of autophagy flux in each group.b.The cells were divided into the Control group,D5 group,Sorafenib group,and Sorafenib+D5 group.Protein samples were collected after corresponding drug treatment for Western blot analysis on an appropriate time.(6)Effects of small molecule drug D5 combined with Sorafenib on the subcutaneous tumor-forming ability of liver cancer cellsWith Huh7 cells(1 x 10⁶)grown in nude mice,tumor long diameter and short diameter were measured every day.Volume calculation starts when tumor volume of about 5 mm³,mice were divided into Control group,D5 group,Sorafenib group,and Sorafenib+D5 group,and began to give the corresponding drug by gavage,mice were euthanized at about 2 weeks later,charging tumor specimens,comparing the difference between the tumor volume and weight,and statistical analysis.4.Influence of LC3^K49R on the efficacy of small molecule drug D5(1)The effect of small molecule drug D5 on the cytotoxicity of MAP1LC3^K49Rliver cancerA lentiviral vector constructed LC3^WT/K49R overexpressing hepatocellular carcinoma cell lines.The two kinds of overexpressed cells in the 96-well plate were divided into the Sorafenib group and Sorafenib+D5 group,and Sorafenib with the same concentration gradient was used to treat the cells.In the Sorafenib+D5 group,small molecule drug D5 with a fixed concentration was added based on Sorafenib treatment to determine the difference in Sorafenib sensitivity of liver cancer cells after LC3^K49R and LC3^WToverexpression.(2)The effect of small molecule drug D5 on the proliferation of LC3^K49Rhepatoma cellsThe lentiviral vector constructed LC3^WT/K49R overexpressing liver cancer cell line,and the blank vector virus was used as the Control group.Cells in each group were evenly seeded into 96-well plates,and each overexpressed type of cells was divided into the Control group,D5 group,Sorafenib group,and Sorafenib+D5 group.3 duplicate wells were set,and the CCK8 kit detected cells'proliferation level in each group.(3)The effect of small molecule drug D5 on tumorigenesis of LC3^K49Rhepatoma cellsA lentiviral vector constructed LC3^WT/K49R overexpressing Huh7 cell,and a blank vector virus was used as the Control group.Nude mice were divided into Control group,D5 group,Sorafenib group with Sorafenib+D5 group after tumor volume gained about 5 mm³ and gave the corresponding drug by gavage,mice were euthanized at about 2 weeks later,charging Tumor specimens,comparing the difference between the Tumor volume and weight,statistical analysis.5.Inhibitory effects of small molecule drug D5 combined with Oxaliplatin on various tumor cells'malignant behaviors.(1)Effect of small molecule drug D5 combined with Oxaliplatin on drug sensitivity of different tumor cellsThe cells in the 96-well plate were divided into the Oxaliplatin group and Oxaliplatin+D5 group and treated with Oxaliplatin at the same concentration gradient.In the Oxaliplatin+D5 group,small molecule drug D5 with a fixed concentration was added based on Oxaliplatin treatment to determine whether small molecule drug D5could make HCC cells more Oxaliplatin-sensitive.(2)Effects of small molecule drug D5 combined with Oxaliplatin on the proliferation of various tumor cellsThe cells in the 96-well plate were divided into control group,D5 single treatment group,Oxaliplatin group,and Oxaliplatin+D5 group.The cells in each group were set up with 3 duplicate wells.The effects of different drug combinations on the proliferation ability of HCC cells were detected with the CCK8 kit every day.(3)Effects of small molecule drug D5 combined with Oxaliplatin on apoptosis of various tumor cellsThe cells were divided into the Control group,D5 group,Oxaliplatin treatment group,and Oxaliplatin+D5 group,and the cells were collected after an appropriate time.Annexin V-FITC/PI double staining was used to detect the proportion of apoptotic cells by flow cytometry.(4)Effect of small molecule drug D5 on migration and invasion of various tumor cells induced by OxaliplatinThe cells were divided into the Control group,D5 group,Oxaliplatin group,and Oxaliplatin+D5 group.The cells were cultured in a Transwell chamber system and given corresponding drugs.After treatment for the appropriate time,the cells were stained with crystal violet and photographed for analysis.(5)Effects of small molecule drug D5 combined with Oxaliplatin on autophagy of various tumor cellsCells were divided into the Control group,D5 group,Oxaliplatin group,and Oxaliplatin+D5 group.Protein samples were collected after corresponding drug treatment for the appropriate time,and Western blot analysis was performed.6.Statistical analysisThe data were analyzed using SPSS 23.0.For comparison between the two groups,if the variance is homogeneous,the two-tailed Student's t-test is used.If the variance is non-homogeneous,the non-parametric rank-sum test Mann-Whitney U test is used.The survival time of the two groups of data was compared using Kaplan-Meier statistical analysis.P<0.05 was considered statistically significant.Results1.LC3B expression level is closely related to the prognosis of HCC patients.(1)LC3B was highly expressed in liver cancer tissues of patientsLC3 m RNA expression level was higher in cancer tissues than in precancerous tissues.(2)LC3B expression level was negatively correlated with the prognosis of patientsThe results of clinical specimens showed that patients with higher LC3 m RNA expression levels had a poor prognosis.Patients with high LC3B expression in tissue microarray have a poor prognosis.2.Effect of a novel LC3B-targeting small molecule drug D5 on the autophagy process and hepatoma cells'malignant behaviors.(1)The small molecule drug D5 inhibits autophagy formationUsing serum starvation to induce autophagy activity in tumor cells:a.Western blot:Compared with the Control group,the LC3 level was significantly increased in the serum starvation group.With the increase of D5concentration,the LC3-I/II expression level in Hep3B and Huh7 cells gradually decreased,the expression level of SQSTM1/p62 recovered,and the c-caspase3expression level gradually increased,but the effect on ATG3,ATG5,and ATG16L1was negligible.b.Autophagy flux:Compared with the Control group,the formation of autophagosomes was significantly increased in the serum starvation group.With the increase of D5 drug concentration,the formation of autophagosomes was decreased significantly.c.Transmission electron microscopy:Compared with the serum starvation group,the application of small molecule drug D5 significantly reduced the formation of autophagosomes in Huh7 cells.(2)Small molecule drug D5 inhibits the proliferation of HCC cellsCompared with the Control group,the use of small molecule drug D5 could significantly inhibit the proliferation of Hep3B and Huh7 cells,and the higher the drug concentration,the more significant the inhibition effect was.(3)Small molecule drug D5 promotes the apoptosis of HCC cellsa.TUNEL staining:Compared with the Control group,the use of small molecule drug D5 could significantly promote the apoptosis of Hep3B and Huh7 cells,and the higher the drug concentration,the more significant the effect of promoting apoptosis.b.Annexin V-FITC/PI double staining:Compared with the Control group,the administration of small molecule drug D5 significantly promoted the apoptosis of Hep3B and Huh7 cells,and the higher the drug concentration,the higher the proportion of apoptotic cells.(4)Small molecule drug D5 inhibits the migration and invasion of HCC cellsa.Migration:Compared with the Control group,the use of small molecule drug D5 could significantly inhibit the migration of Hep3B and Huh7 cells,and the higher the drug concentration,the more significant the inhibition effect.b.Invasion:Compared with the Control group,the use of small molecule drug D5 could significantly inhibit the invasion ability of Hep3B and Huh7 cells,and the higher the drug concentration,the more significant the inhibition effect was.3.Small molecule drug D5 increases the sensitivity of HCC cells to Sorafenib.(1)Effect of small molecule drug D5 combined with Sorafenib on drug sensitivity of liver cancer cellsSmall molecule drug D5 can significantly increase the Sorafenib sensitivity of Hep3B and Huh7 cells.(2)The combination of small molecule drug D5 and Sorafenib significantly inhibited the proliferation of HCC cellsBoth small molecule drug D5 and Sorafenib could inhibit the proliferation of Hep3B and Huh7 cells,but the effect of D5 was weaker than that of Sorafenib.The Sorafenib+D5 group was the strongest of the most.(3)The combination of small molecule drug D5 and Sorafenib significantly promoted the apoptosis of HCC cellsa.TUNEL staining:Compared with the Control group,small molecule drug D5and Sorafenib both promoted the apoptosis of Hep3B and Huh7 cells,but the proportion of TUNEL positive cells in the D5 group was less than that in the Sorafenib group,and the ratio of TUNEL positive cells in Sorafenib+D5 group was the highest.b.Annexin V-FITC/PI double staining:Compared with the Control group,both small molecule drug D5 and Sorafenib could promote the apoptosis of Hep3B and Huh7 cells,but the proportion of positive cells by flow apoptosis staining in the D5group was lower than that in the Sorafenib group,and the ratio of apoptosis-positive cells in the Sorafenib+D5 group was the highest.(4)The combination of small molecule drug D5 and Sorafenib significantly inhibited the migration and invasion of HCC cellsa.Migration:Both small molecule drug D5 and Sorafenib could inhibit the migration of Hep3B and Huh7 cells,but the effect of D5 was weaker than that of Sorafenib,and the combination group of Sorafenib+D5 had the strongest inhibition of cell migration.b.Invasion:Both small molecule drug D5 and Sorafenib could inhibit the invasion of Hep3B and Huh7 cells,but the effect of D5 was weaker than that of Sorafenib,and the combination group of Sorafenib+D5 had the most vital ability to inhibit cell invasion.(5)Small molecule drug D5 inhibits the formation of autophagy flux in liver cancer cells caused by Sorafeniba.Autophagy flux:compared with the control group,small molecule D5 could significantly inhibit the formation of autophagy flux,Sorafenib could dramatically promote the formation of autophagy flux,and small molecule drug D5 could dramatically inhibit the increase of autophagy caused by Sorafenib.b.Western blot:Compared with the Control group,small molecule drug D5inhibited the expression of LC3-I/II and restored the expression of p62.Sorafenib could not significantly promote the expression of LC3 but could significantly inhibit the expression of p62.Small molecule drug D5 can restore Sorafenib-induced p62expression decline and further decrease LC3-I/II expression.(6)The combination of small molecule drug D5 and Sorafenib significantly inhibited the subcutaneous tumorigenesis of hepatoma cellsCompared with the Control group,there was no significant difference in the volume of the subcutaneous tumor,and the ratio of tumor weight to weight in the small molecule drug group D5,the ratio of subcutaneous tumor volume,and ratio tumor weight to body weight in the Sorafenib group was significantly reduced.The subcutaneous tumor volume ratio to tumor weight in the Sorafenib+D5 group was significantly reduced compared with the Sorafenib group.4.LC3^K49 is a crucial target of small molecule drug D5.(1)LC3^K49R can counteract the cytotoxic effect of small molecule drug D5 on liver cancera.For small molecule drug D5,the IC₅₀ value of the LC3^K49R overexpression group was significantly higher than the LC3^WT group.b.In the case of the drug combination,there was no significant difference in IC₅₀values between the Sorafenib+D5 group and Sorafenib group in LC3^K49R.(2)LC3^K49R can counteract the inhibitory effect of small molecule drug D5 on the proliferation of HCC cellsLC3^WT group alone with p CDH group,Sorafenib+D5 combined group had more substantial tumor cell inhibition effect than Sorafenib group,while LC3^K49RSorafenib+D5 group and Sorafenib group showed no significant difference in proliferation level.(3)LC3^K49R can counteract the inhibitory effect of small molecule drug D5 on tumorigenicity of HCC cellsLC3^WT group alone with p CDH group,the tumor volume and tumor weight/body weight ratio of Sorafenib and D5 combined group were smaller than Sorafenib single group.Simultaneously,there was no significant difference in the proliferation level between LC3^K49R Sorafenib+D5 combined group and the Sorafenib group.5.Small molecule drug D5 can enhance the sensitivity of various tumor cells to Oxaliplatin.(1)Small molecule drug D5 promotes the cytotoxic effect of Oxaliplatin on tumorSmall molecule drug D5 can significantly increase the sensitivity of RKO,MKN28,and A549 cells to Oxaliplatin.(2)The combination of small molecule drug D5 and Oxaliplatin can significantly inhibit the proliferation of tumor cellsBoth small molecule drugs D5 and Oxaliplatin could inhibit the proliferation of RKO,MKN28,and A549 cells,but the effect of D5 was weaker than that of Oxaliplatin,and the combination group had the strongest inhibitory effect on proliferation.(3)The combination of small molecule drug D5 and Oxaliplatin can significantly promote the apoptosis of tumor cellsCompared with the Control group,both small molecule drugs D5 and Oxaliplatin could promote the apoptosis of RKO,MKN28,and A549 cells.Still,the proportion of positive cells by flow apoptosis staining in the D5 group was less than that in the Oxaliplatin group,and the ratio of apoptosis-positive cells in the drug combination group was the highest.(4)Small molecule drug D5 combined with Oxaliplatin can significantly inhibit the migration and invasion of tumor cellsa.Migration:Both small molecule drugs D5 and Oxaliplatin could inhibit the migration of RKO,MKN28,and A549 cells,but the effect of D5 was weaker than that of Oxaliplatin,and the combined drug group had the most vital ability to inhibit cell migration.b.Invasion:Both small molecule drugs D5 and Oxaliplatin could inhibit the invasion of RKO,MKN28,and A549 cells,but the effect of D5 was weaker than that of Oxaliplatin,and the combination group had the most substantial ability to inhibit the invasion of cells.(5)The combination of small molecule drug D5 and Oxaliplatin can significantly inhibit the autophagy of tumor cellsCompared with the Control group,the small-molecule drug D5 could inhibit the expression of LC3-I/II and restore the expression of p62 in RKO,MKN28,and A549cells.Oxaliplatin can significantly promote the expression of LC3 and inhibit the expression of p62.D5 inhibited the increase of LC3-I/II expression induced by Oxaliplatin and restored the expression of p62.Conclusion1.The expression of LC3B is negatively correlated with the prognosis of patients with liver cancer.2.Small molecule drug D5 is a novel inhibitor targeting LC3,which can inhibit autophagy flux in liver cancer cells and various tumors.3.Small molecule drug D5 can significantly increase liver cancer cells'sensitivity(or other tumor cells)to Sorafenib(or Oxaliplatin).4.LC3^K49 is a crucial target of small molecule drug D5.