| 1 Background and aimsHypercoagulability is common in clinical tumor patients,and its core pathological mechanism is caused by tumor thrombolus formed by "tumor-platelet"complex.In the theory of Traditional Chinese Medicine,huoxue huayu drugs are often used to treat patients with tumor blood stasis syndrome,but the anticancer application mechanism of Huoxue Huayu Chinese medicine is still unclear,and its influence on promoting and inhibiting tumor metastasis has been controversial.This topic selected representative suberect spatholobus stem,Chinese traditional medicine for activating blood circulation through the body,the experiment,in order to "tumorplatelet" interaction and tumor microenvironment blood as a breakthrough point,in order to interpret the caulis spatholobi extract(SEA)efficacy and pharmacological mechanism of tumor metastasis,cognitive development and caulis spatholobi anti-cancer mechanism clearly,and for the clinical drug research and development,In particular,it provides new hints and valuable evidence for drug development targeting tumor metastasis.2.Research strategies and methods2.1 Efficacy evaluation of ethyl acetate extract of Caulis spatholobus(SEA)for inhibiting platelet activation induced by tumor cells in vitro.① The ethyl acetate extract of subtilis spatholobi was prepared into SEA working solution(4 mg/mL)and stored at-20℃ for later use.②Blood was collected from mouse inner canthus to obtain platelet-rich plasma and prepare washing platelet suspension.③ To detect the effects of SEA on platelet LDH release and cytotoxicity of MC38 tumor cells,and determine the concentration and duration of SEA action in vitro(100μg/mL,within 24 h)on the premise of not affecting cell survival.④ Mouse MC38 cells were used to establish an in vitro induced platelet aggregation model to simulate the activation and activation effect of tumor cells on platelets in blood,and determine the optimal proportion of tumor cells to platelets in the model.⑤ The effect of SEA on platelet aggregation induced by MC38 cells was detected.⑥ The effect of SEA on the formation of "tumor-platelet" complex was determined qualitatively and quantitatively by using confocal laser and flow cytometry through CFDA-SE labeled platelets.2.2 Effects of SEA on EMT and cancer stem-like characteristics of tumor cells under "tumor-platelet" interaction.2.2.1 Effect of SEA on EMT process of tumor cells under tumor-platelet interaction.Tumor cells and platelets were co-cultured for 20 h in vitro to establish a tumor-platelet co-culture model on the basis of clear in vitro SEA blocking of platelet activation by tumor cells.① Morphological changes(length-width ratio statistics)of MC38 cells in model group and SEA administration group in co-culture system were observed and detected.② The ability of SEA to induce platelet aggregation in MC38 cells in co-culture system was detected.③ In the co-culture system,The effects of SEA on the expression of Snail-1,Vimentin,CDH-1,Slug and Twistl of EMT regulatory transcription molecules in MC38 cells were detected(qPCR experiment),and the expression of EMT-related protein molecules E-cadherin and Vimentin(WB experiment).And the effect on MC38 cell motility(wound-repair test)and invasion ability(MMP-9 test,Matrigel invasion test).2.2.2 Effect of SEA on cancer stem-like characteristics of MC38 cells under tumor-platelet interaction.On the basis of confirming that SEA can effectively reverse THE EMT transformation of MC38 cells under the "tumor-platelet" interaction model,we further detected the following effects under the interaction model:① Effects of SEA on the expression of Sox2,Oct4,Nanog and Bmil in MC38 cells(qPCR experiment).②～③Effects of SEA on protein expression of CD44,CD 133,(WB,FACS)and c-Myc in MC38 cells(WB).④The effects of SEA on MC38 cell clon-forming ability(clone proliferation experiment),colony forming ability(soft AGAR colony formation experiment)and pellet-forming ability(pellet-forming experiment),and the function of MC38 cell was evaluated.2.2.3 Effect of "tumor-platelet" interaction model on efficacy of SEA.Under the co-culture model,it was confirmed that SEA had the efficacy of reversing EMT transformation and reducing cancer stem-like characteristics of MC38 cells.In order to further determine whether SEA’s efficacy was realized based on the"tumor-platelet" interaction model,platelets were removed and tested respectively:①Effects of SEA on the expression of Sox2,Oct-4,Nanog and Bmil in MC38 cells(qPCR experiment).② Effects of SEA on protein expression of CD44,CD 133,c-Myc and Oct-4 in MC38 cells(WB experiment).③ Effect of SEA on expression of CD44 and CD 133 in MC38 cells(FACS experiment).2.3 Effect of SEA on hematogenous metastasis in tumor-bearing mice and its molecular mechanism.2.3.1 Effects of SEA on blood metastasis in tumor-bearing mice.In order to detect whether SEA has the effect of inhibiting MC38 cell metastasis in vivo and the mechanism of action,the following experiments were carried out respectively:① MC38-Luc cells were first injected into the tail vein of C57BL/6J mice(1×105/mouse)to establish the hematogenic metastasis model.SEA group was given SEA(4 mg/kg)once a day by intragastric administration.On this basis,the specific effect of SEA blocking T-P interaction to inhibit metastasis of colon cancer was confirmed by specially designing the treatment and administration regimen of tumor cells(in vitro platelet preconditioning tumor cells group and in vitro SEA administration group(100 μg/mL).Small animal imaging was performed on day 14 after inoculation.② Effect of SEA on coagulation function of normal mice and tumor-bearing mice was detected(calcium rehydration test).③ To detect the effect of SEA on the distribution and number of metastases(in vivo imaging of small animals).④ Effect of SEA on platelet count in peripheral blood of tumor-bearing mice(biochemical test).⑤ The effect of SEA on lung structure of tumor-bearing mice was detected(HE staining).⑥ The effects of SEA on the expression of CD44,N-cadherin and MMP-9 in lung metastasis tissues of tumor-bearing mice were detected(immunohistochemistry).2.3.2 Effect of SEA on "tumor-platelet" interaction mode.Mechanically,thrombin(0.5 U/mL)was used to induce platelet activation in vitro,the activated platelet fragments were separated from the platelet release,and Platelets,platelet fragments and releasate were interacting with MC38 cells for 20 h,respectively.The blank group was treated with 1640 medium for control(buffer),and respectively detected:① EMT-like morphological changes of MC38 cells.②～③Changes of protein expression of CD44 and CD133 in MC38 cells(WB and FACS).④ Further,Transwell chamber was used to isolate tumor cells from platelets and co-culture for 20 h.The expression of E-cadherin and Vimentin of EMT in MC38 cells and the effect of ⑤ on MC38 cells’ motor ability(wound repair experiment and Transwell migration experiment)in model group and SEA group were examined.2.3.3 Effects of SEA on "tumor-platelet" interaction related signaling pathways.Finally,TGF-β,NF-κB and β-catenin blockers were used to intervene and detect the expression changes of E-cadherin,a key EMT protein,so as to preliminarially study the SEA drug blocking the "tumor-platelet" interaction signaling pathway.3 Results3.1 Efficacy evaluation of SEA for inhibiting platelet activation induced by tumor cells in vitro.① LDH and CCK-8 results showed that SEA under 100μg/mL had no enhancement effect on platelet LDH release and no cytotoxicity on MC38 cells.In consideration of dose and time,SEA treatments of 25,50 and 100 μg/mL for 20 h were selected as the treatment conditions for subsequent experiments.②The results of platelet aggregation rate experiment showed that MC3 8 could induce the maximum platelet aggregation rate when the number ratio of tumor cells to platelets was 1:100.Under these conditions,SEA significantly inhibited platelet aggregation induced by MC38 cells compared with other monomer drugs(tanshinone ⅡA,salvianolic acid A,ligustrazine and β-elemene)and control drug aspirin(P<0.01).③ Confocal microscopy and flow cytometry showed that SEA significantly reduced the activation and adhesion of MC38 cells to platelets,and blocked the formation of"tumor-platelet" complex(P<0.01).3.2 Effects of SEA on EMT and cancer stem-like characteristics of tumor cells under "tumor-platelet" interaction.3.2.1 Effects of SEA on EMT process of tumor cells under "tumor-platelet"interaction.Under the "tumor-platelet" co-culture model for 20 h,the results of EMT-related detection were as follows:① Morphological observation showed that platelets in the model group induced significant EMT-like changes in MC38 cells(P<0.01),SEA could significantly reverse THE EMT-like transformation of MC38 cells(P<0.01).② The results of platelet aggregation rate experiment showed that compared with MC38 cells treated with platelet for 20 h,the ability of platelet aggregation induced by untreated MC38 cells was significantly increased(P<0.01),SEA still significantly inhibited platelet aggregation induced by MC38 cells compared with other compounds(P<0.01).③ qPCR showed that SEA significantly reversed the expression of EMT-related transcription molecules snail-1,Vimentin,CDH-1 and Slug in the model group(P<0.01).④ WB results showed that SEA significantly reversed the protein expression of E-cadherin and Vimentin in the model group(P<0.01);⑤ The results of scratch repair experiment showed that,under the premise of consistent initial scratch width,the scratch width of MC38 cells in SEA treatment group was significantly larger than that in model group after 20 h(P<0.01);(6)Compared with the model group,the MMP-9 activity and the number of transmembrane cells in SEA treatment group were significantly decreased(P<0.01);3.2.2 Effect of SEA on cancer stem-like characteristics of MC38 cells under tumor-platelet interaction.Under the "tumor-platelet" co-culture model for 20 h,the results as follows:①qPCR results showed that SEA significantly reduced the transcription levels of Sox2,Oct4 and Nanog(P<0.01).②WB and FACS results showed that the expression of CD44,CD 133 and c-Myc protein in colon cancer was significantly decreased after SEA intervention(P<0.01).In the function experiment,the results showed that:③Compared with the model grope,the number of MC38 cell clones in SEA treatment group was significantly decreased(P<0.01);④Compared with model group,unanchored colony formation ability of MC38 cells in SEA treatment group was significantly reduced(P<0.01);⑤Compared with model group,the pellet-forming ability of MC38 cells in SEA treatment group was significantly decreased(P<0.01).3.2.3 Effect of "tumor-platelet" interaction model on efficacy of SEA.① qPCR results showed that SEA alone had no significant effect on the expression of Sox2,Oct4,Nanog and Bmi1 in MC38 cells(P>0.05).② WB and FACS results showed that SEA alone had no significant effect on CD44,CD133,c-Myc and Oct-4 of MC38 cells(P>0.05).3.3 Study on the effect of SEA on hematogenous metastasis in tumor-bearing mice and its molecular mechanism3.3.1 Effects of SEA on blood metastasis in tumor-bearing miceThe blood metastasis model of colon cancer in mice was established by tail vein injection of MC38-Luc cells,and the specific efficacy of SEA in blocking MC38 cell metastasis in vivo was verified by special treatment and administration of MC38 cells in vitro.The experimental results were as follows:① Calcium rehydration test showed that compared with normal mice,there was no significant change in calcium rehydration time in mice without tumor after SEA drug administration(P>0.05),but the calcium recovery time of tumor-bearing mice was significantly lower than that of non-tumor-bearing mice(P<0.01);Compared with tumor-bearing mice,plasma recalcification time was significantly increased after daily administration of SEA drug(P<0.01)and return to normal level.②In vivo imaging results of small animals showed that the fluorescence signal of lung was obvious in the negative control group,and the metastases were concentrated in the lung.After daily administration of SEA drug in mice,the signal of lung metastasis was significantly reduced(P<0.01);In addition,compared with the negative control group,the fluorescence signal of lung metastases in vivo was significantly enhanced after MC38 cells were pretreated with platelets in vitro(P<0.01),indicating that platelets enhanced the metastasis ability of MC38 cells.On this basis,the number of lung metastases in the in vitro SEA administration group was significantly lower than that in the control group(P<0.01).These results suggest that SEA inhibits MC38 cell metastasis in vivo by blocking tumor-platelet interaction.③ The platelet count of tumor-bearing mice was significantly lower than that of normal mice(P<0.01),and SEA therapy in vivo or in vitro significantly improved platelet count reduction in the corresponding model group(P<0.01).The results suggest that SEA regulation of tumor-platelet interaction is independent of platelet count.④ HE staining showed that there were some tumor cells infiltrating in the lung tissue of NC group,and the alveolar structure was damaged.In the Platelets group,the degree of tumor cell infiltration was more serious,the lung tissue structure was severely damaged,and the degree of consolidation was more obvious.In both the in vivo and in vitro SEA administration groups,the lung tissue morphology was significantly improved,the lung structure was basically normal,the degree of lung tissue consolidation was significantly reduced,and there was basically no tumor cell infiltration.Our results suggest that the pharmacodynamic activity of SEA against lung metastasis of colon cancer is mediated by blocking the"tumor-platelet" interaction.⑤Immunohistochemical results showed that CD44,N-cadherin and MMP-9 were significantly expressed in NC group,and the expression of CD44,N-cadherin and MMP-9 was further increased in Platelets group with a large number of metastases.In both the in vivo and in vitro treatment groups,the positive signal was significantly reduced,and no tumor metastasis was observed.3.3.2 Effect of SEA on "tumor-platelet" interaction model.On the basis of confirming the efficacy of SEA in inhibiting MC3 8 cell hematogenesis in vivo by blocking the "tumor-platelet" interaction,an in-depth study was conducted on the "tumor-platelet" interaction mode and the intervention of SEA,and the results showed that:① Morphological observation showed that both intact platelets and platelet fragments could induce EMT-like changes in MC38 cells,while platelet release could not induce EMT-like changes.SEA could block emT-like changes induced by platelets and platelet fragments.②On the basis of the morphological observation above,the expression of CD44 and CD133 in each group was detected by WB and FACS.The results showed that the expression of CD44 and CD 133 was significantly increased after MC38 cells were incubated with intact platelets or platelet fragments for 20 h(P<0.01),SEA administration group could significantly inhibit the expression in model group(P<0.01);Platelet release group had no significant effect on the expression of CD44 and CD 133(P>0.05).We further isolated MC38 cells from platelets in the tumor-platelet interaction model using Transwell cells and treated them with SEA,and added thrombin to induce platelet activation on the basis of the model group.Results:(3)WB results showed that the expression of EMT-related molecules E-cadherin and Vimentin in MC38 cells was not changed in model group,SEA group and thrombin treatment group(P>0.05).④～⑤ Scratch test and migration test showed that there were no significant changes in the motility and migration of MC38 cells in model group,SEA group and thrombin treated group(P>0.05).The experimental results suggest that the pharmacodynamic mechanism of SEA is to block the direct contact between tumor cells and platelets.3.3.3 Effects of SEA on "tumor-platelet" interaction related signaling pathways.TGF-β,NF-κB and β-catenin blockers were used for intervention.The results showed that:①TGF-β blocker decreased the expression of E-cadherin protein in SEA group(P<0.01).The results suggest that SEA does not work by blocking TGF-β.②β-catenin blocker did not change the expression level of E-cadherin in SEA group(P>0.05).The experimental results showed that the efficacy of SEA was the same asβ-catenin in blocking EMT.③ The addition of NF-κB blocker could further restore the expression of E-cadherin on SEA(P<0.01),indicating that blocking NF-κB can synergistically exert the efficacy of SEA,indicating that NF-κB signaling pathway is not the mechanism of SEA.These experiments suggest that the mechanism of SEA blocking tumor-platelet interaction depends on blocking β-catenin signaling pathway.4 ConclusionSEA can specifically inhibit platelet aggregation induced by MC38 tumor cells,block the direct contact between tumor cells and platelets,and reduce the malignant degree of tumor cells by inhibiting the β-catenin signaling pathway,thus playing a role in blocking the metastasis of colon cancer and inhibiting the generation of metastasis. |
PDF Full Text Request