| BackgroundThe close relationship between the occurrence and development of cancer and immunity has been revealed in recent years,including pro-inflammatory environment,escape from immune killing,metabolic reprogramming.Currently,the main treatments for cancer include surgery,radiotherapy,chemotherapy,targeted therapy and immunotherapy.Among them,small molecule drugs have the advantages of high bioavailability,long history of research and application,low cost and good compliance.A variety of natural products or industrial products containing disulfide or polysulfide bonds have been found to have antitumor activity and the disulfide or polysulfide bond is an indispensable group for their antitumor action.The importance of disulfide or polysulfide groups in the fields of chemistry,biology and pharmacology is well understood.The antitumor mechanisms of disulfides and poly sulfides have mostly been reported to be possibly related to reactive oxygen species,while other antitumor mechanisms still need to be further explored in depth.Compared to the risks associated with lengthy drug development cycles,high costs,and low success rates,the strategy of repurposing existing drugs,known as "drug repositioning",has been increasingly recognized due to its advantages of shorter development cycles,lower costs,and higher success rates.Disulfide compound disulfiram(DSF),a traditional medication used in alcohol abstinence therapy,have gained increasing attention for their antitumor activity.Previous studies have reported inhibition of ubiquitination process,NF-κb activity,tumor stem characteristics,as well as its ability to induce tumor cell apoptosis by enhancing reactive oxygen species(ROS)levels.However,its anti-tumor mechanism still needs to be further explored.While the multi-targeting nature of the drug suggests further exploration of other anti-tumor mechanisms of disulfiram.Another disulfide compound,SAMC,a water-soluble allicin derivative from old garlic,is obtained by natural biotransformation of allicin.With the advantages of small molecule drugs,including easy penetration of cells and biological barriers,multiple targets,controllable efficacy,relatively low price,especially fewer adverse reactions,the anti-tumor activity of SAMC has attracted more and more attention from researchers.According to previous studies,SAMC can inhibit the Wnt pathway,upregulat P53 level and inhibit the MAPK pathway.Although it has been reported in many studies,the specific molecular mechanisms of SAMC’s anti-tumour effects are still unclear,Therefore,there is an urgent need to further elucidate the anti-tumor mechanism of SAMC.In this study,we found that DSF/Cu could exert anti-tumor effects by promoting the protein degradation of MELK through an autophagy-lysosome-dependent pathway,thereby inhibiting PGK1 phosphorylation and impeding the glycolytic capacity of tumor cells,thus playing an anti-tumor role.We also found that SAMC could reduce the expression level of immune checkpoint PD-L1 to enhance the anti-tumor immune effect.Combined application of DSF/Cu and SAMC could further enhance the antitumor activity.This study not only reveals a new mechanism of the anti-tumor effects of DSF/Cu and SAMC,but also further explores the synergistic anti-tumor effects of DSF/Cu and SAMC,providing theoretical guidance for the design and development of small molecule drugs.Part Ⅰ DSF/Cu promotes MELK protein degradation and inhibits tumor cell glycolysis[Objective]First,we examined the effects of DSF on the proliferation and migration of tumor cells.Then,the DSF/Cu regulatory pathway and the key protein MELK were analyzed by means of protein spectrometry combined with bioinformatics.The anti-tumor effect of DSF/Cu and its specific molecular mechanism were detected by using molecular biology experiment methods combined with in vitro and in vivo experiments.[Methods and Results]1.DSF/Cu inhibited tumor cell proliferation,promoted tumor cell apoptosis,and suppressed tumor metastasisUtilizing multiple lung cancer cell lines,the impact of DSF/Cu on tumor cell proliferation was evaluated through MTT assays.The effect of DSF/Cu on tumor cell colony formation ability was assessed through plate clone experiments.FACS technology was employed to measure the influence of DSF/Cu on tumor cell apoptosis.Transwell and scratch assays were conducted to investigate the impact of DSF/Cu on tumor cell migration ability.The functional experimental results demonstrated that DSF/Cu inhibited tumor cell proliferation and migration,induced cell apoptosis,and suppressed tumor cell metastasis.A549 lung cancer cells were injected into nude mice subcutaneously,and the tumor volume was measured after DSF/Cu administration.The body weight of the nude mice was also recorded.After 30 days,the tumors were excised,photographed,and weighed.The results indicated that DSF/Cu could significantly inhibits tumor growth.2.DSF/Cu reduced the protein kinase MELK levels in tumor cellsAfter treating H1299 lung cancer cells with DSF/Cu for 24 hours,the impact of DSF/Cu on protein expression in tumor cells was examined using proteomics technology.Combined with bioinformatics analysis,differentially expressed proteins regulated by DSF/Cu were analyzed,and pathway enrichment was conducted.The results revealed that DSF/Cu was involved in regulating protein kinase pathways.Through bioinformatics analysis,MELK was selected as the target for further research on the anti-tumor effects of DSF/Cu,and it was validated through Western blot and qRT-PCR experiments that DSF/Cu could reduce MELK protein expression levels.3.DSF/Cu induces MELK degradation through the autophagolysosomal pathwayH1299 and H1975 lung cancer cells were treated with the protein synthesis inhibitor CHX,followed by different durations of DSF/Cu treatment.The impact of DSF/Cu on MELK protein degradation was examined through Western blot experiments.The results demonstrate that DSF/Cu could accelerate MELK protein degradation.Using the above cell lines,the proteasome inhibitor MG 132 was utilized to inhibit proteasomes,and it did not alleviate DSF/Cu-induced MELK protein degradation.This indicated that DSF/Cu could not degrade MELK protein through the ubiquitin-proteasome pathway.Western blot experiments were conducted to examine the expression of LC3B in H1299 and H460 cell lines after DSF/Cu treatment.Immunofluorescence experiments were performed to assess the changes in the number of autophagosomes induced by DSF/Cu.The results confirm that DSF/Cu could activate autophagy.Furthermore,by employing the autophagy inhibitors chloroquine and bafilomycin A1.Western blot experiments were conducted to assess LC3B expression.RFP-GFP-LC3B adenovirus was used to infect H1299 and H460 cells,and after DSF/Cu treatment.confocal microscopy was utilized to measure the intensity of RFP and GFP fluorescence.The results indicated that DSF/Cu could increase autophagic flux.In H1299 and H1975 cell lines,the autophagy-lysosome pathway was significantly reversed by autophagy inhibitors chloroquine,bafilomycin A1,3-methyladenine and siATG5 to restore MELK protein expression levels reduced by DSF/Cu,demonstrating that DSF/Cu promoted MELK protein degradation through the autophagolysosomal pathway.4.DSF/Cu inhibited tumor progression by downregulating MELK expressionBased on the relative expression level of MELK protein in lung cancer cells,A549,H157,and H460 were selected to construct MELK overexpressing cell lines,while H1299 and H1975 were selected to construct MELK knockdown cell lines.The effects of MELK on cell proliferation and migration ability were examined using MTT assay,Transwell assay,and scratch assay in the successfully constructed MELK knockdown cell lines.The results showed that knocking down MELK effectively inhibited cell proliferation and migration.The expression levels of MELK in lung cancer tissue and adjacent tissue were detected using IHC experiment with a commercial lung cancer tissue microarray,and the results demonstrated that MELK was highly expressed in lung cancer tissue,confirming its role as an oncogene.In the successfully constructed MELK overexpressing cell lines,the effects of DSF/Cu treatment on cell proliferation ability were examined using MTT assay and colony formation assay.The apoptotic rate was measured using FACS analysis.The migration ability was evaluated using Transwell assay and scratch assay.A549 cells with high MELK expression and their control cells were subcutaneously injected into nude mice,and tumor volume and nude mouse body weight were measured after DSF/Cu treatment.Tumors were collected after 30 days,photographed,and weighed.The results showed that reconstitution of MELK effectively reversed the inhibitory effect of DSF/Cu on tumor proliferation ability,reversed the pro-apoptotic effect of DSF/Cu,reversed the inhibitory effect of DSF/Cu on tumor migration,and reversed the inhibitory effect of DSF/Cu on tumor growth.5.DSF/Cu inhibits tumor cell glycolysisFlag-MELK plasmid was transfected into 293T cells,and after 72 hours,immunoprecipitation was performed to enrich MELK protein.Mass spectrometry was used to identify the interacting proteins of MELK,and a total of 266 interacting proteins of MELK were identified.Pathway enrichment analysis showed that MELK was involved in the regulation of tumor cell glycolysis.H1299 and A549 lung cancer cell lines were treated with DSF/Cu for 24 hours,and tumor cell glycolysis was detected using Seahorse technology.The results showed that DSF/Cu significantly inhibited tumor cell glycolysis in a dose-dependent manner.Reconstitution of MELK effectively reversed the inhibitory effect of DSF/Cu on tumor glycolysis.6.DSF/Cu inhibits PGK1 phosphorylation by downregulating MELK expressionRNA-seq analysis was performed using the MELK knockdown H1299 cell line,and the results showed that MELK had minimal effect on the transcription of glycolysis genes.According to the aforementioned mass spectrometry results,DSF/Cu treatment did not affect the expression of glycolysis proteins in tumor cells.Further results showed that DSF/Cu treatment,MELK overexpression,and MELK knockdown had no significant effect on the protein expression of PGK1,LDHA,and PFKP.Immunoprecipitation experiments were performed using antibodies against PGK1,LDHA,and PFKP to investigate the interaction of endogenous proteins in tumor cells after DSF/Cu treatment.The results showed that DSF/Cu treatment significantly reduced the binding of PGK1,LDHA,and PFKP to MELK,which was also confirmed in the MELK overexpressing cell line.Immunoprecipitation was performed to enrich PGK1,LDHA,and PFKP,and phosphorylation modification of these proteins was detected using a pan-phosphorylation antibody.The results showed that DSF/Cu reduced the phosphorylation levels of PGK1,PFKP,and LDHA,and knocking down MELK reduced the phosphorylation of PGK1 but had no significant effect on the phosphorylation of LDHA and PFKP.Treatment of MELK overexpressing A549 cell line with DSF/Cu showed that high MELK expression increased PGK1 phosphorylation and effectively reversed the DSF/Cu-induced decrease in PGK1 phosphorylation.[Conclusion]1.DSF/Cu promotes protein kinase MELK degradation through the autophagolysosomal pathway.2.DSF/Cu inhibits tumor progression by reducing MELK protein levels.3.DSF/Cu inhibits the glycolytic ability of tumor cells by reducing the expression of MELK protein.4.DSF/Cu inhibits the phosphorylation of PGK1,a key glycolytic enzyme,by reducing the level of MELK protein.Part Ⅱ SAMC enhances anti-tumor immunity by suppressing PD-L1 expression[Objective]The aim of this study was to investigate the anti-tumor effect and mechanism of another disulfide compound,SAMC,by modulating the immune system through in vitro and in vivo experiments,combined with biochemical and molecular biology techniques.[Methods and Results]1.In vitro and in vivo analysis of the anti-tumor activity of SAMCUsing various human lung cancer and gastric cancer cell lines,as well as multiple mouse tumor cell lines,the direct cytotoxicity of SAMC on tumor cells was evaluated using the MTT assay.The results showed that SAMC had low in vitro direct anti-tumor activity.Using the mouse colorectal cancer CT26 cell line,subcutaneous tumor models were established in normal mice and nude mice.SAMC was administered orally,and tumor volume and mouse weight were measured.After 20 days,tumors were removed,photographed,and weighed.Tail vein metastasis models were established in normal mice and nude mice using CT26 cells.After oral administration of SAMC,mouse weight was measured,the date of mouse death was recorded,and lung tissues were collected for HE staining.The results showed that low-dose SAMC had no significant inhibitory effect on subcutaneous tumor growth in nude mice but significantly inhibited tumor growth in normal mice.Low-dose SAMC had no significant effect on lung metastasis in nude mice but significantly inhibited lung metastasis in normal mice,suggesting that low-dose SAMC might exert anti-tumor activity by modulating the immune system.2.SAMC could regulate anti-tumor immunityTumor-infiltrating lymphocytes were extracted from subcutaneous tumors in normal mice and analyzed by flow cytometry.The results showed that the proportion of CD8+T cells and NK cells among tumor-infiltrating lymphocytes significantly increased after low-dose SAMC treatment,while the proportion of Treg cells significantly decreased.Immune cells were extracted from lung tissues in the metastasis model in normal mice and analyzed by flow cytometry.The results showed that the proportion of CD8+T/CD4+ T cells and NK cells in lung tissues significantly increased after SAMC treatment,while the proportion of Treg cells significantly decreased.Lymphocytes from tumor-draining lymph nodes and spleen cells in the subcutaneous tumor model in normal mice were analyzed by flow cytometry.The results showed that the proportion of CD8+ T cells and NK cells among tumor-infiltrating lymphocytes significantly increased after low-dose SAMC treatment,while the proportion of Treg cells significantly decreased.Serum samples from the above two tumor models were collected,and ELISA experiments showed that SAMC significantly upregulated IFN-y levels in the serum.3.In vitro lymphocyte culture system validated the regulation of SAMC on immune function in inhibiting tumor growth and metastasisNormal mouse bone marrow cells were extracted and induced to differentiate into bone marrow-derived dendritic cells(BMDCs)using GM-CSF.CT26 tumor cell lysate was prepared through repeated freeze-thaw cycles,and BMDCs were matured by protein induction with the lysate.The matured BMDCs were co-cultured with spleen cells to activate immune cells.Subsequently,the lymphocytes were co-cultured with CT26 cells and treated with SAMC.Flow cytometric analysis of the collected lymphocytes showed that SAMC could increase the proportion of CD8+T cells and NK cells while decreasing the proportion of Treg cells.Clone formation and scratch assays demonstrated that lymphocytes treated with SAMC significantly inhibited tumor cell proliferation and migration.4.SAMC downregulated the expression of PD-L1 on tumor cell surfaceWestern blot experiments were conducted to examine the effect of SAMC on the expression of various immune checkpoint molecules in H460 cells.The impact of SAMC on PD-L1 protein expression was evaluated in H460 and H1299 cells.Western blot and FACS experiments were performed to assess the effect of SAMC on PD-L1 protein expression in CT26 and MFC cells.The results indicated that SAMC could inhibit the surface expression of PD-L1 on tumor cells,which was further confirmed in mouse tumors.5.SAMC suppressed T cell activity by downregulating PD-L1 expressionH460 cells,which had a relatively high baseline expression of PD-L1,were cocultured with Jurkat cells activated by CD3/CD28 stimulation.The expression level of IFN-γ was used as an indicator of T cell activation.The results showed that SAMC significantly promoted T cell activation after acting on tumor cells.siPD-L1 was used to knock down PD-L1 in H460 cells,which were then co-cultured with activated Jurkat cells.The results showed that SAMC had no significant effect on the expression level of IFN-γ in PD-L1-knockdown cells.Immunohistochemical staining of tumor tissues revealed that SAMC significantly promoted the infiltration of CD3+ T cells and CD8+ T cells into tumors.In addition,the mRNA levels of various markers of T cell activation,including TNF-α and IFN-y,were significantly increased in tumor tissues.6.SAMC inhibited PD-L1 transcriptional expression by suppressing the STAT3 signaling pathwayH460 and CT26 cells were treated with SAMC,and qRT-PCR experiments were performed to measure the mRNA expression levels of PD-L1.The results showed that SAMC could inhibit the transcriptional expression of PD-L1.Western blot and immunofluorescence experiments were conducted to examine the phosphorylation and nuclear localization of STAT3.Nuclear and cytoplasmic proteins were extracted separately,and Western blot experiments were performed to measure STAT3 phosphorylation levels.The results showed that SAMC inhibited STAT3 phosphorylation.H460 and CT26 cells were treated with the STAT3 inhibitor C188-9 to inhibit STAT3 phosphorylation,and at this time SAMC did not further reduce PDL1 expression.H460 cells were transfected with a dual luciferase reporter gene plasmid system,and the results showed that SAMC could significantly reduce the transcriptional activity of STAT3 in the PD-L1 promoter region.However,SAMC had no effect on transcriptional activity in the absence of the STAT3 binding site in the reporter system.[Conclusion]1.SAMC can inhibit the growth and metastasis of tumors in immune-functioning mice.2.SAMC activates both local and systemic immune systems in tumors.3.SAMC suppresses tumor growth and metastasis by reducing PD-L1 activation of the immune system.4.SAMC downregulates PD-L1 transcriptional expression by inhibiting STAT3 phosphorylation levels.Part Ⅲ The combined application of two organic sulfides,DSF and SAMC,exhibits a more significant anti-tumor effect[Objective]Through the first and second parts,the molecular mechanisms by which DSF/Cu inhibits lung cancer progression by affecting glycolysis and SAMC impedes tumor progression by activating anti-tumor immunity have been elucidated.Based on previous reports,simultaneous inhibition of glycolysis and activation of the immune system have shown more significant anti-tumor effects.Therefore,this section aims to preliminarily explore the synergistic effects of DSF and SAMC in combination therapy for enhancing anti-tumor efficacy.[Methods and Results]A CT26 subcutaneous tumor model was established in normal mice,and after treatment with DSF/Cu and SAMC in combination,the tumor volume was measured,and the mouse body weight was recorded.After 30 days,the tumor tissues were collected and the tumor weight was measured.Western blot experiments were performed on the tumor tissues from the aforementioned mouse model to detect the protein expression of MELK and PD-L1.The heart,liver,spleen,lung,and kidney tissues from the aforementioned mouse model were collected for HE staining to assess the tissue toxicity of DSF and SAMC in combination.The results showed that DSF/Cu and SAMC had a systemic anti-tumor effect.DSF/Cu reduced the expression of MELK without affecting the expression of PD-Ll,while SAMC reduced the expression of PDL1 without affecting the expression of MELK.Furthermore,the combined application showed no significant tissue toxicity.[Conclusion]1.DSF/Cu and SAMC demonstrate synergistic anti-tumor effects.2.The combined application of DSF/Cu and SAMC shows no significant tissue toxicity. |
