Icaritin inhibits PLK1 to activate DNA damage response in NK/T cell lymphoma and increases sensitivity to GELOX regime
2022; Elsevier BV; Volume: 25; Linguagem: Inglês
10.1016/j.omto.2022.04.012
ISSN2372-7705
AutoresCan-Jing Zhang, Huiwen Xu, Xianxian Sui, Lina Chen, Bobin Chen, Haozhen Lv, Songmei Wang, Xuanyi Wang,
Tópico(s)Fungal Infections and Studies
ResumoNatural killer/T cell lymphoma (NKTCL) is a highly aggressive subtype of non-Hodgkin lymphoma. Gemcitabine, oxaliplatin, and L-asparaginase (GELOX) is one of the first-line chemotherapy regimens of NKTCL. Yet, the prognosis of NKTCL is poor. Icaritin is an herb-derived monomer from icariin with antitumor effects. We found that icaritin induced proliferation inhibition and apoptosis of NKTCL both in vitro and in vivo. Moreover, icaritin inhibited the dissemination of NKTCL in vivo. RNA sequencing revealed the Polo-like kinase 1 (PLK1) gene and DNA damage response (DDR) as the targets of icaritin. Mechanistically, icaritin inhibited PLK1 to promote checkpoint kinase 2 (Chk2) homodimerization and its T387 phosphorylation, which further activated p53, leading to the activation of the DDR pathway. Moreover, inhibiting PLK1 increased Forkhead box O3a nuclear localization, the latter of which activated ataxia telangiectasia mutated (ATM), an early sensor of DNA damage. Then ATM phosphorylated Chk2 T68 and initiated Chk2 activation. Remarkably, the combined treatment of icaritin and GELOX achieved better antitumor efficacy than single treatment in vivo. In summary, our results proved the efficacy of icaritin treating NKTCL, provided insights into its antitumor molecular mechanism, and revealed the application value of icaritin in facilitating clinical NKTCL treatment. Natural killer/T cell lymphoma (NKTCL) is a highly aggressive subtype of non-Hodgkin lymphoma. Gemcitabine, oxaliplatin, and L-asparaginase (GELOX) is one of the first-line chemotherapy regimens of NKTCL. Yet, the prognosis of NKTCL is poor. Icaritin is an herb-derived monomer from icariin with antitumor effects. We found that icaritin induced proliferation inhibition and apoptosis of NKTCL both in vitro and in vivo. Moreover, icaritin inhibited the dissemination of NKTCL in vivo. RNA sequencing revealed the Polo-like kinase 1 (PLK1) gene and DNA damage response (DDR) as the targets of icaritin. Mechanistically, icaritin inhibited PLK1 to promote checkpoint kinase 2 (Chk2) homodimerization and its T387 phosphorylation, which further activated p53, leading to the activation of the DDR pathway. Moreover, inhibiting PLK1 increased Forkhead box O3a nuclear localization, the latter of which activated ataxia telangiectasia mutated (ATM), an early sensor of DNA damage. Then ATM phosphorylated Chk2 T68 and initiated Chk2 activation. Remarkably, the combined treatment of icaritin and GELOX achieved better antitumor efficacy than single treatment in vivo. In summary, our results proved the efficacy of icaritin treating NKTCL, provided insights into its antitumor molecular mechanism, and revealed the application value of icaritin in facilitating clinical NKTCL treatment. IntroductionNatural killer/T cell lymphoma (NKTCL) is an Epstein-Barr virus-associated non-Hodgkin lymphoma subtype originated from malignant proliferating NK cell or T cell lineage.1Swerdlow S.H.C.E. Harris N.L. Jaffe E.S. Pileri S.A. Stein H. Thiele J. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Revised.Fourth edition. IARC, 2017Google Scholar NKTCL commonly occurs in the upper aerodigestive tract, with more frequent occurrence in Asia and South American.2Yamaguchi M. Suzuki R. Oguchi M. Advances in the treatment of extranodal NK/T-cell lymphoma, nasal type.Blood. 2018; 131: 2528-2540https://doi.org/10.1182/blood-2017-12-791418Crossref PubMed Scopus (110) Google Scholar Owing to the resistance to anthracycline of NKTCL, non-anthracycline-regimens containing L-asparaginase are recommended as first-line therapy.3Yamaguchi M. Miyazaki K. Current treatment approaches for NK/T-cell lymphoma.J. Clin. Exp. 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Hematol. 2013; 97: 617-623https://doi.org/10.1007/s12185-013-1317-9Crossref PubMed Scopus (49) Google Scholar In the current study, we aimed to investigate its ability to inhibit NKTCL and the underlying mechanism.Here, we found that ICT potently inhibited the proliferation and induced apoptosis of NKTCL in vitro, and inhibited its growth and dissemination in vivo. By inhibiting PLK1, ICT increased Chk2 homo-dimerization and activated p53. Inhibiting PLK1 also increased FOXO3a nuclear localization and further activated ATM, symbolizing the DDR pathway activation. Intriguingly, the combined application of ICT and the GELOX regimen achieved a better tumor suppression effect than that of the GELOX regimen alone in NKTCL xenografts.ResultsICT inhibits the growth and induces cell apoptosis of NKTCL both in vitro and in vivoSNT-8, SNK-10, and NK-92 MI cells were treated with different concentrations of ICT for 24, 48, and 72 h. A CCK-8 assay revealed lower cell viability as the ICT concentration and treatment time increased (Figure 1A ). The half maximal inhibitory concentration (IC50) values of 24 hour-ICT treatment for SNT-8, SNK-10, and NK-92 MI cells were 54.75, 44.26, and 32.92 μM, respectively (Figure 1A). The IC50 values of 48-h ICT treatment for SNT-8, SNK-10, and NK-92 MI cells were 34.26, 25.01, and 25.20 μM, respectively (Figure 1A). ICT significantly inhibited cell proliferation in a dose- and time-dependent manner (Figure 1A). Cell count data confirmed the inhibitory effect of ICT on NKTCL cells, which showed that ICT decreased the NKTCL cell number dose dependently (Figure S1). Then we examined the apoptotic rates of three NKTCL cell lines treated with DMSO or 25 or 50 μM ICT for 48 h using annexin V/PI dual staining and flow cytometry assay. The apoptosis rates of untreated SNT-8, SNK-10, and NK-92 MI cells were 9.9%, 7.9%, and 7.5%, respectively (Figure 1B). After 48 h of treatment with 25 μM ICT, the average apoptosis rates of SNT-8, SNK-10, and NK-92MI cells increased to 25.3%, 18.0%, and 15.9%, respectively (Figure 1B). After 48 h of treatment with 50 μM ICT, the apoptosis rates of SNT-8, SNK-10, and NK-92MI cells increased to 33.9%, 30.8%, and 26.8%, respectively (Figure 1B). ICT induced apoptosis in three cell lines significantly. Moreover, 50 μM ICT induced a significantly higher apoptosis rate than 25 μM ICT in SNK-10 and NK-92 MI cells (Figure 1B). ICT induced apoptosis of the SNK-10 and NK-92 MI cell lines in a dose-dependent manner (Figure 1B). In contrast, ICT did not influence the apoptosis rates in normal human cells, including primary NK cells, human embryonic kidney cell line 293T, and human liver cell line QSG-7701 (Figures S2 and S3).To investigate the in vivo efficacy of ICT treating NKTCL, a xenograft model developed using NK-92 MI cell line was used. The tumor-bearing mice were treated with dissolvent (control), 30 mg/kg, 60 mg/kg, or 120 mg/kg ICT (Figure 1C). Notably, the tumor growth was significantly suppressed by 120 mg/kg ICT, compared with those in control group, as shown by the tumor volume curve (Figure 1D). During the whole treatment period, the mice weight of the 30, 60, and 120 mg/kg ICT groups showed no difference from that of control group (Figure 1E). At the end of treatment, tumor tissues were collected and weighed (Figure 1F). The weight of the tumors in the ICT group was lighter than those in the control group (Figure 1G). To evaluate the effect of ICT on the dissemination of NKTCL, we established a mouse model using NK-92 MI cells stably transfected with luciferase gene (NK-92 MI Luc). After being treated with 120 mg/kg ICT or vehicle for 16 days, only one in five mice of the ICT group had distant dissemination of NKTCL in the whole body, compared with four in five mice in the control group, showing the profound inhibition by ICT to NKTCL xenograft dissemination (Figure 1H). Tumor tissues in the ICT groups gained significantly lower positive scores of Ki-67, which is a marker of proliferation, than in the control group (Figure 1I). The terminal deoxynucleotidyl transferase (TUNEL) assay revealed a notably higher apoptotic cell rate of ICT-treated tumors, compared with the control group (Figure 1H). Taken together, ICT suppresses NKTCL growth and induces apoptosis both in vitro and in vivo.RNA-sequencing data reveals that ICT inhibits PLK1 and activates DDR and FOXO pathway in NKTCLTo decipher the underlying mechanisms of ICT inhibiting growth and inducing apoptosis of NKTCL, we conducted RNA-sequencing (RNA-seq) of three NKTCL cell lines treated with or without ICT. After standardization of the RNA sequence results, differentially expressed genes (DEGs) were identified (185 up and 180 down in SNT-8 cells, 179 up and 241 down in SNK-10 cells, and 167 up and 156 down in SNT-16 cells) (Figure 2A ). The overlap among the three DEG sets contained 110 genes (Figure 2B). Normal human cell lines 293T and QSG-7701 were used to test the safety of ICT. The RNA-seq results of 293T and QSG-7701 cells revealed only three and eight DEGs regulated by ICT, respectively (Tables S1 and S2).Figure 2Bioinformatic analysis reveals that ICT down-regulates PLK1 and activates the apoptosis, p53 and FOXO pathwayShow full caption(A) DEGs of SNT-8, SNK-10, and NK-92 MI cell lines treated with DMSO or ICT were selected with a fold change of 2 or greater and a p value of less than 0.05. Orange for up-regulated genes and blue for down-regulated genes. (B) The Venn diagram showed the overlap of 110 genes among 3 datasets. (C) The bubble plot showed the KEGG results of the 110 genes. (D) Heatmap of genes enriched in p53, FOXO and apoptosis signaling pathways. (E) The PPI network of 110 enriched genes was constructed using Cytoscape. One of the core modules of DEGs obtained by MCODE from PPI network was shown by red arrow. (F) The FPKM value of PLK1 gene in three NKTCL cell lines treated with DMSO or ICT were shown. (G) The expression of PLK1 in normal lymph nodes and NKTCL cell lines. The data were acquired from the GSE63548, GSE25297, GSE36172, and GSE19067 datasets of the GEO database. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The biological functions of the DEGs in SNT-8, SNK-10 and NK-92 MI cells were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The gene ontology (GO) analysis results showed that DEGs were significantly enriched in biological processes (BP), including cell proliferation, apoptotic process, DDR, cell cycle regulation, cell division, negative regulation of transcription from RNA polymerase II promoter, immune response, response to endoplasmic reticulum stress, and cellular response to hypoxia, and so on (Table 1). Among the DEGs involved in the top 20 BP regulated by ICT, the PLK1 gene was related to 7 of 20 processes, including the negative regulation of transcription from RNA polymerase II promoter, regulation of the cell cycle, cell proliferation, mitotic nuclear division, protein ubiquitination involved in ubiquitin-dependent protein catabolic process, anaphase-promoting complex-dependent catabolic process, and G2/M transition of the mitotic cell cycle (Table 1). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DEGs were enriched in p53, FOXO, cell cycle, cytokine-cytokine receptor interaction, and the mitogen-activated protein kinase signaling pathway (Figure 2C). The DEGs involved in the p53, FOXO, and apoptosis pathways were shown by a heatmap (Figure 2D). PLK1 was involved in the FOXO pathway (Figure 2D). To better understand the interaction between the DEGs, the PPI network of DEGs was constructed using Cytoscape, with 64 nodes and 174 edges (Figure 2E). The possible initiative genes which were closely interacting with other genes were acquired by conducting a molecular complex detection (MCODE) analysis. One core module was amplified as shown by the red arrow (Figure 2E). PLK1, as one of the hub genes, the mRNA expression was drastically reduced by ICT in three NKTCL cell lines (Figure 2F). To verify the role of PLK1 in NKTCL tumorigenesis, we collected the PLK1 mRNA expression data of normal lymph nodes and NKTCL cell lines from the GEO databases GSE63548, GSE25297, GSE36172, and GSE19067. The PLK1 expression in NKTCL cell lines was significantly higher than that in normal lymph nodes (Figure 2G). Collectively, the RNA-seq results indicated that ICT regulated the p53 and FOXO pathway, and DDR in NKTCL cells. Among all the DEGs regulated by ICT, PLK1 was a core gene involved in the proliferation and apoptosis processes and the FOXO pathway.Table 1The top 20 BP regulated by ICTTermCountp ValueGenesGO:0000122∼negative regulation of transcription from RNA polymerase II promoter132.34E-04PLK1, FASLG, KLF16, SFPQ, MYC, DDIT3, MDM2, CREBRF, TXNIP, TRIB3, MXD1, ATF3, HIST1H1CGO:0008284∼positive regulation of cell proliferation105.27E-04CDC20, CCPG1, MYC, CXCR3, TNFSF4, NOP2, OSM, MDM2, FASLG, ATF3GO:0045944∼positive regulation of transcription from RNA polymerase II promoter100.056048KDM3A, CCPG1, MYC, CXCR3, DDIT3, ZNF292, OSM, E2F5, ATF3, NFE2L1GO:0051726∼regulation of cell cycle73.65E-05HSPA8, CCNB1, GADD45A, CCNG2, PLK1, CCNG1, E2F5GO:0008283∼cell proliferation70.010058MYC, PLK1, OSM, POLR3G, MXD1, DLGAP5, BYSLGO:0006955∼immune response70.018886RGS1, TNFSF4, OSM, CTLA4, CCL2, FASLG, LTBGO:0032436∼positive regulation of proteasomal ubiquitin-dependent protein catabolic process61.68E-05PLK1, MDM2, RNF19B, TRIB3, TRIB1, AURKAGO:0007050∼cell cycle arrest67.05E-04PPP1R15A, GADD45A, MYC, DDIT3, JMY, HBP1GO:0042787∼protein ubiquitination involved in ubiquitin-dependent protein catabolic process60.001018CDC20, CCNB1, PLK1, MDM2, RNF19B, AURKAGO:0043065∼positive regulation of apoptotic process60.017274BNIP3L, GADD45A, JMY, TXNIP, CTLA4, FASLGGO:0051301∼cell division60.030981CDC20, CCNB1, PSRC1, CCNG2, CCNG1, AURKAGO:0034976∼response to endoplasmic reticulum stress55.41E-04PPP1R15A, DDIT3, CREBRF, TRIB3, BBC3GO:0071456∼cellular response to hypoxia50.001365BNIP3L, CCNB1, MDM2, NDRG1, BBC3GO:0006974∼cellular response to DNA damage stimulus50.020447PPP1R15A, MYC, DDIT3, CTLA4, BBC3GO:0042981∼regulation of apoptotic process50.022086BNIP3L, SDF2L1, NDRG1, INHBE, BBC3GO:0007067∼mitotic nuclear division50.035793CDC20, CCNG2, PLK1, CCNG1, AURKAGO:0070059∼intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress45.91E-04PPP1R15A, DDIT3, TRIB3, BBC3GO:0006977∼DDR, signal transduction by p53 class mediator resulting in cell cycle arrest40.003692CCNB1, GADD45A, MDM2, AURKAGO:0031145∼anaphase-promoting complex-dependent catabolic process40.007265CDC20, CCNB1, PLK1, AURKAGO:0000086∼G2/M transition of mitotic cell cycle40.031355CCNB1, PLK1, HMMR, AURKA Open table in a new tab ICT induces NKTCL cell apoptosis by decreasing PLK1 expressionBecause the RNA-seq results predicted that PLK1 was a possible core gene in the antitumor mechanism of ICT, we detected the mRNA and protein levels of PLK1 in NKTCL cell lines treated with ICT. In accordance with the RNA-seq results, the mRNA level of PLK1 was decreased by ICT in three NKTCL cell lines (Figure 3A ). Further, the total and phosphorylation levels of PLK1 protein were also decreased by ICT (Figure 3B). Quantitative analyses of the protein levels of p-PLK1 and PLK1 are shown (Figures 3C and 3D). To determine the role of PLK1 inhibition in ICT-induced proliferation inhibition and apoptosis, we constructed the tetracycline-inducible (tet on) PLK1 overexpression and the empty vector (EV) NK-92 MI cells. To test the PLK1 overexpression efficiency, doxycycline (DOX) was used to treat the tet on PLK1 cells or EV cells. The mRNA expression of PLK1 drastically increased after 12 h or 24 h treatment of 2 μg/mL or 4 μg/mL DOX (Figure 3E). The protein level of PLK1 also improved after 2 μg/mL DOX treatment for 24 h (Figure 3F). These results indicated the successful induction of PLK1 expression by DOX. To know whether overexpressing PLK1 in ICT-treated NKTCL cells could rescue ICT-induced cell apoptosis, we examined the apoptosis rate of tet on PLK1 and EV cells after being treated by ICT for 24 h followed with or without DOX treatment. The DOX tre
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