Inhibitory Effect And Mechanism Of A Novel Tubulin Destabilizing Agent,DHPAC,on Angiogenesis And Vasculogenic Mimicry In Tumor Microenvironment

Backgrounds and objectivesThe growth and metastasis of solid tumors depends on angiogenesis,which provides tumor cells with oxygen and nutrients necessary for growth,as well as the timely removal of metabolic waste.At present,many angiogenesis inhi'bitors have been used clinically for the treatment of cancer,and a variety of new small molecule compounds are also in clinical research.However,in tumor tissues,in addition to the traditional endothelial cell-dependent angiogenesis,there are mimetic blood vessels similar to vascular networks formed by the interconnection of tumor cells themselves,that is,vasculogenic mimicry(VM).VM usually occurs in some highly invasive tumors,such as melanoma,breast cancer,ovarian cancer and lung cancer.Its appearance increases the resistance of anti-cancer therapies such as radiotherapy,chemotherapy and surgery,and the poor prognosis of cancer treatment.Non-small cell lung cancer(NSCLC)is a common cancer with high invasiveness and many kinds of NSCLC cells can form VM.Microtubule targeted agents(MTAs)exert anti-tumor effects by affecting microtubule dynamics and thus affecting the process of mitosis of tumor cells.Studies have found that MTAs have potential anti-angiogenic effects,but there are few reports on their ability to inhibit VM formation of tumor cells.Moreover,MTAs targeting microtubule colchicine binding site have better anti-angiogenic effects than those targeting paclitaxel binding site and vincristine binding site.DHPAC is designed as a compound targeting colchicine binding site by computer-assisted drug design method.This study mainly investigates the inhibitory effects of DHPAC on angiogenesis and VM formation in NSCLC in tumor microenvironment,and subsequently elucidates its mechanisms.Part 1 The anti-endothelial angiogenesis and mechanism of DHPACMethods1.The ability of DHPAC enters HUVECs and inhibitory effects of DHPAC on cell proliferation was investigated.In this thesis,human umbilical vascular endothelial cells(HUVECs)and high-invasive non-small cell lung cancer H1299 cells were selected as the research subjects.Firstly,HPLC-MS/MS was used to detect whether DHPAC could enter HUVECs.Then,the culture supernatant of H1299 cells was collected as a conditioned medium to simulate HUVECs to mimic the tumor microenvironment,and the inhibitory effects of DHPAC on the migration and invasion of endothelial cells in the tumor microenvironment were tested.Next,MTT assay was used to detect the inhibitory effect of DHPAC on the proliferation of HUVECs stimulated by normal medium and conditioned medium.2.The effect of DHPAC on the migration of HUVECs in the tumor microenvironment was examined.Wound scratch assay and Transwell migration assay were used to detect the effect of DHPAC on the migration ability of HUVECs stimulated by conditioned medium or normal medium.3.The effect of DHPAC on the angiogenic ability of HUVECs in the tumor microenvironment in vitro was examined.Matrigel was used to simulate the basement membrane matrix,and the effect of DHPAC on the angiogenesis ability of HUVECs stimulated by conditioned medium or normal medium was tested by capillary tube formation assay.4.The effect of DHPAC on tumor angiogenesis in nude mice was examined.CD31 is a specific marker of endothelial cells.CD31 immunofluorescence assay was used to stain tumor tissue slices to explore the effect of DHPAC on angiogenesis in nude mice.5.The molecular mechanism of anti-angiogenesis of DHPAC in tumor microenvironment and hypoxia was explored.Western blot and ELISA were used to detect the effect of DHPAC on the expression and secretion of VEGF in HUVECs under normoxia and hypoxia.Western blot method was then used to detect the effect of DHPAC on the expression of related proteins in the JNK/STAT3/VEGF signaling pathway in the tumor microenvironment.The effects of DHPAC on the expression of related proteins in HIF-1?/VEGF/VEGFR2 signaling pathway under hypoxic conditions were also examined.In order to further clarify the mechanism of DHPAC,JNK inhibitor SP600125 was introduced to the cell culture system to verify the inhibitory effects of DHPAC on endothelial cell angiogenesis by Transwell migration assay and tube formation assay,and the molecular mechanism of DHPAC on cellular signal pathway in tumor microenvironment and hypoxia was further confirmed.Results1.DHPAC can enter HUVECs and inhibit cell proliferation.HPLC-MS/MS results showed that DHPAC could enter HUVECs.The results of MTT assay showed that DHPAC could inhibit the proliferation of HUVECs,and more obvious inhibitory effects of DHPAC was observed under the stimulation of conditioned medium than that under normal medium.2.DHPAC significantly inhibits the migration of HUVECs in the tumor microenvironment.Wound scratch assay and Transwell migration assay results showed that DHPAC significantly inhibited the migration of HUVECs,and the inhibition was enhanced under the stimulation of conditioned medium.3.DHPAC significantly inhibits angiogenesis of HUVECs in the tumor microenvironment.Capillary tube formation assay result showed that DHPAC could inhibit the formation of blood vessels in HUVECs,and the inhibition was enhanced under the stimulation of conditioned medium.4.DHPAC inhibits tumor tissue angiogenesis in nude mice.CD31 immunofluorescence staining results show that DHPAC could inhibit tumor angiogenesis in nude mice.5.DHPAC inhibits the migration and angiogenesis of HUVECs by inhibiting the STAT3/VEGF/VEGFR2/FAK/AKT signal transduction pathway through activating JNK.Under normal culture conditions or conditioned medium-induced tumor microenvironment,DHPAC can inhibit the secretion of VEGF,inhibit the phosphorylation of STAT3,FAK and AKT proteins in HUVECs,and promote the phosphorylation of JNK and c-Jun proteins.After the introduction of JNK inhibitors,DHPAC inhibited the secretion of VEGF by HUVECs and inhibited the phosphorylation of STAT3,FAK and AKT,and the inhibition of migration and angiogenesis of HUVECs by DHPAC was also abolished.6.DHPAC inhibits HIF-1?/VEGF/VEGFR2 signaling pathway in HUVECs by activating JNK under hypoxic conditions.Under hypoxic conditions,DHPAC can inhibit the expression of HIF-1? in HUVECs,promote the phosphorylation of JNK and c-Jun,and inhibit the phosphorylation of STAT3 and FAK.The experimental results showed that DHPAC promoted the phosphorylation of c-Jun prior to the inhibition of HIF-1? expression,suggesting that DHPAC may inhibit the expression of HIF-1? by activating c-Jun.In addition,under hypoxic conditions,after the introduction of JNK inhibitors,the inhibitory effects of DHPAC on the secretion of VEGF,the phosphorylation of STAT3 and FAK have been partly reversedIn summary,the possible molecular mechanism of DHPAC inhibiting in HUVECs;DHPAC enters endothelial cells,targets the microtubule colchicine binding site to inhibit the polymerization of tubulin,further activates JNK and promotes phosphorylation of JNK.In one aspect,activated JNK inhibits VEGF expression and secretion by inhibiting phosphorylation of STAT3.On the other hand,under hypoxic conditions,p-JNK can promote the phosphorylation of c-Jun,reducing the content of non-phosphorylated c-Jun and hindering the formation of c-Jun and HIF-1? complexes,and cannot protect HIF-1? from ubiquitination to be degraded.The decrease of HIF-1? content affects the expression and secretion of VEGF,thereby inhibiting the activation of VEGF/VEGFR2 signaling pathway and its downstream signaling pathway,and finally inhibiting angiogenesis of HUVECs.Part 2 The anti-VM of tumor cells and mechanism of DHPACMethods1.The effects of DHPAC on H1299 cells proliferation in vitro and tumor growth in vivo were investigated.MTT assay was used to observe the effect of DHPAC on the proliferation of H1299 cells in vitro.The inhibitory effect of DHPAC on the growth of H1299 cell xenografts was detected by using the nude mouse xenograft model.2.The effects of DHPAC on the invasion and migration of H1299 cells were examined.Transwell migration assay and Transwell invasion assay were used to detect the effect of DHPAC on the migration and invasion of H1299 cells.3.The effect of DHPAC on the VM formation of H1299 cells was examined.The in vitro VM formation assay was used to detect the effect of DHPAC on the VM formation of H1299 cells.4.The molecular mechanism of inhibitory effect of DHPAC on VM formation of H1299 cells was explored.Western blot and ELISA were used to detect the effect of DHPAC on the expression and secretion of VEGF in H1299 cells.Western blot was used to detect the effects of DHPAC on the protein expression of FAK,p-FAK,AKT,p-AKT,MMP2,MMP9 and Laminin 5 in vitro and in vivo.Results1.DHPAC significantly inhibited the proliferation of H1299 cells in vitro andthe growth of xenografts in nude mice.MTT results showed that DHPAC could significantly inhibit the proliferation of H1299 cells in vitro.The results of nude mice xenografts experiment showed that DHPAC could significantly inhibit tumor growth(52.24%inhibition rate of 30 mg/kg)without obvious toxic side effects on lung and heart.2.DHPAC inhibits migration and invasion of H1299 cells.Transwell migration assay and invasion assay showed that DHPAC significantly inhibit the migration and invasion of H1299 cells.3.DHPAC inhibits VM formation of H1299 cells in vitro.The results of VM formation experiments showed that DHPAC could significantly inhibit the formation of VM network in H1299 cells.4.DHPAC inhibits the expression and secretion of VEGF in H1299 cells and inhibits the intracellular FAK/AKT signal transduction pathway.DHPAC inhibits the VM formation of H1299 cells:DHPAC can inhibit the expression and secretion of VEGF after entering cells;inhibit the phosphorylation of FAK to inhibit the phosphorylation of AKT,thereby inhibiting the expression of MMP2,MMP9 and Laminin 5,and finally preventing the formation of mimetic vascular networks in tumor cells.ConclusionThe novel microtubule-targeted depolymerizing agent DHPAC can effectively inhibit endothelial cell angiogenesis,and its inhibitory effect is more obvious in the tumor microenvironment.Its anti-angiogenic effect is to inhibit STAT3/VEGF/VEGFR2 signaling pathway by activating JNK in normal medium or in tumor microenvironment.Under hypoxic conditions,DHPAC inhibits HIF-1?/VEGF/VEGFR2 signal transduction pathway via activating JNK.In in vivo and in vitro studies,DHPAC can inhibit the proliferation of H1299 cells and the formation of VM,and the mechanism is related to inhibiting the FAK/AKT signal transduction pathway.SignificanceThis study clarified the inhibitory effects and intrinsic mechanism of DHPAC,a novel microtubule-targeted depolymerizing agent,on angiogenesis of endothelial cells and VM formation of H1299 cells in the tumor microenvironment.These findings enrich the multiple mechanisms of tumor angiogenesis,reveal the anti-angiogenic mechanism of microtubule-targeted inhibitors,and propose new inhibitory effects and mechanisms of microtubule-targeted inhibitors in tumor VM formation.Therefore,DHPAC is a potential candidate compound for tumor vasculature in the treatment of NSCLC.