Pyxinol Proline Derivative Induced Cell Cycle Arrest And Apoptosis On HCC Via DDIT3-CDK2 Pathway

BackgroundHepatocellular carcinoma(HCC)is the most common primary liver cancer,accounting for the sixth most common type of cancer and the second leading cause of death among all cancers.Treatment in the early stages of HCC includes surgical resection,radiofrequency ablation,transarterial chemoembolization,or liver transplantation.However,when HCC is diagnosed at an advanced stage,tyrosine kinase inhibitors(TKIs)can only be selected as systemic therapy,but they can only benefit a small proportion of patients,have more adverse reactions,and cause resistance within6 months.It is imperative to develop new drugs for the treatment of HCC.Because natural drugs have the characteristics of high efficiency and low toxicity,they can provide better efficacy for patients.Pyxinol is the liver metabolite of 20(S)-Protopanaxadiol(PPD),which is the main intestinal metabolite of ginsenosides.Pyxinol has a variety of pharmacological effects,such as inhibiting myocardial infarction and inhibiting inflammatory diseases,but its application and development are greatly limited due to its low solubility and bioavailability in vitro and in vivo.In a previous study by our group,we tested multiple amino acid derivatives of pyxinol and found that pyxinol proline derivative(PFY)inhibited hepatoma cell growth in a dose-dependent manner.However,the specific target and detailed mechanism still need to be clarified.Transcriptome analysis suggests that DDIT3(DNA-damage inducible transcript 3)is a novel target of PFY against HCC and it has previously been reported to interact with CDK2(cyclin-dependent kinase 2)in the nucleus.PFY induced cell cycle arrest in G1-S phase,increased DDIT3 expression and decreased CDK2 expression in BEL-7404 hepatoma cell line and H22 tumor bearing mice.Furthermore,using an in vivo xenograft model,we demonstrated that PFY treatment significantly inhibited the growth of hepatoma cells.Pharmacodynamic studies by UPLC-MS showed higher PFY content in H22 tumors than in serum.In conclusion,PFY can induce cell cycle arrest and apoptosis in HCC through DDIT3-CDK2 pathway.PurposesIn this study,transcriptome sequencing analysis was used as a research clue,combined with in vitro and in vivo experiments to verify and explore the main targets and mechanisms of PFY against HCC.The aim is to provide theoretical basis and experimental basis for the treatment of patients with liver cancer.Contents and results(1)PFY(Pyxinol proline derivative)inhibits cell proliferation and promotes apoptosisSensitivity of a series of hepatoma cell lines(BEL-7404,H22,SMMC-7721),cancer cell lines(A2780,MDA-MB-231,4T1),and non-cancer cell lines(HUVEC,H9C2)to PFY and its components pyxinol and proline was examined by MTT assay.The results showed that PFY had better selectivity for the growth of cancer cells,especially HCC cells.Flow cytometric analysis of Annexin V/PI staining showed a significant increase in apoptosis in BEL-7404 cells treated with PFY-40 μM,whereas pyxinol had a minor effect on apoptosis in BEL-7404 cells at the same concentration.In cell cycle experiments,PFY was shown to arrest the BEL-7404 cell cycle at the G0/G1 phase.(2)Transcriptomic analysis and validation of antitumor effect of pyxinol proline derivativesTo further clarify the effects of PFY on HCC cells,we performed transcriptome analysis.A threshold of ∣log 2(FC)∣>0 and P < 0.05 was used to filter differentially expressed genes.Results showed 2568 genes were upregulated and 2146 genes were downregulated.Hierarchical clustering of DEGs indicated that cells with or without PFY treatment could be effectively distinguished.GO analysis of cellular fractions(CC)indicated that DEGs affected by PFY were significantly enriched in nucleoplasm,cytosol,and nucleus.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed that the DEGs regulated by PFY were significantly enriched in protein processing in endoplasmic reticulum pathway,and DDIT3 had the most fold change in this pathway.DDIT3 is associated with cell cycle arrest.Differential gene analysis of the "cell cycle" pathway showed CDKN1 A as the most significant differential gene.CDK2 is a key gene affecting cycle inhibition,and it interacts with DDIT3.Western Blot showed that DDIT3/CHOP expression increased in BEL-7404 cells treated with PFY for 24 h in a dose-dependent manner,after knockdown of DDIT3,the cell viability of the knockdown group was higher than that of the DDIT3-negative control group.Expression of CDKN1A/p21 was slightly upregulated in PFY10 μM and 20 μM groups,but slightly decreased in PFY 30 μM group.CDK2 expression showed a similar trend to CDKN1A/p21.(3)PFY has inhibitory effect on H22 subcutaneous transplanted tumor mouse tumorMale ICR mice were used to establish a subcutaneous tumor xenograft model of H22 cells.We treated tumor-bearing mice with indicated doses of PFY and pyxinol for10 days.Results showed PFY dose-dependently reduced HCC tumor growth at 100mg/kg and 150 mg/kg.Whereas pyxinol 150 mg/kg slightly reduced tumor size.Hematoxylin–Eosin staining showed vacuolar degeneration,loss of cellular structure,increase in apoptotic necrotic cells of tumor sections treated with PFY and pyxinol.Moreover,PFY at the same dose had a better antitumor effect than pyxinol.Immunohistochemical staining showed that the expression of DDIT3/CHOP in tumor tissues of the treatment group was increased.Total protein was extracted from tumor tissue,and western blot showed that PFY could up-regulate DDIT3/CHOP and downregulate CDK2 expression.These results indicated that PFY inhibits the development of HCC by acting on DDIT3-CDK2 pathway in vivo.(4)PFY can prolong the life extension rate of mice with ascites tumorMale ICR mice were used to establish the H22 ascites tumor mouse model,and mice with ascites tumors were treated with PFY and pyxinol at the indicated doses,and it was found that mice in the model group began to die on day 17 and more than half of the deaths occurred on day 18,while 83% of mice in the PFY high-dose(100 mg/kg)group survived on day 18,and the number of survivors was higher than that in the pyxinol group at the same dose.These results indicate that the life extension rate of mice with ascites tumor treated with PFY is significantly increased.(5)PFY is the main active ingredient in anti-tumorUPLC-MS detected the amount of PFY in serum and tumor/ascites at different time points after a single dose.The results showed higher concentrations of PFY in tumors than in serum at 4 h after PFY administration,suggesting that PFY may play a role in tumors in its original form.The content of PFY in serum of mice with ascites tumor gradually approached that in ascites with the increase of time,indicating that PFY entered ascites and played a role.ConclusionIn summary,we screened Pyxinol proline derivatives(PFY),a new anticancer compound.The results showed that PFY could inhibit the growth of HCC cells in a dose-dependent manner in vitro and in vivo,and the mechanism was through DDIT3-CDK2 pathway to induce cell cycle arrest and apoptosis in HCC cells.