Cucurbitacin B Inhibits The Migration And Invasion Of Breast Cancer Cells By Altering The Biomechanical Properties Of Cells

Objective:Breast cancer is the most common cancer occurring in women worldwide,however,the main cause of death in of breast cancer more than 90%patients is tumor metastasis.Presently,research on the mechanism of tumor metastasis has mainly focused on processes such as integrin-mediated cell adhesion,epithelial-mesenchymal transition and tumor angiogenesis,lymphangiogenesis,and hypoxic microenvironment.However,few studies have focused on the effects of mechanical forces generated during tumor metastasis on cell invasion of the extracellular matrix and migration in the matrix.Altering the biomechanical properties of cells can affect cell migration and invasion.Cucurbitacin B(CuB)is a tetracyclic triterpenoid compound widely present in cucurbitaceous plants and has various anticancer effects.Many studies have shown that cucurbitacin inhibits proliferation,induces apoptosis,G2/M phase arrest,induces autophagy,inhibits cell migration and invasion,and it plays a role in different tumor cells through unique molecular mechanisms.However,few studies have studied the mechanical mechanism of cucurbitacin in inhibiting breast cancer metastasisCombined with relevant research background,we will explore the effect of CuB on breast cancer metastasis from the perspective of biomechanics.This paper aims to investigate whether CuB can inhibit the migration and invasion of breast in vivo and in vitro by changing the biomechanical properties of breast cancer cells,and to explore the mechanisms for CuB to change the biomechanical properties of breast cancer cells in vivo and in vitroMethods:1.Using MTS assay to detect the effects of different concentrations of CuB on the proliferation of breast cancer SKBR-3 and MDA-MB-231 cell lines:SKBR-3 and MDA-MB-231 cells were cultured with 0.04,0.2,1,5,25,125 μM CuB,respectively The proliferation of SKBR-3 and MDA-MB-231 cells were detected at 24 h and 48 h 2.The prepared fibronectin and Matrigel were coated in 96-well plates.The control group and the low,medium and high dose groups were set up.CuB concentrations of 10,20,30 nmol/L were added to the drug-added group,and the cells were cultured for 2 hours in a 37℃ incubator.After 2 hours,the unadhered cells were washed away by PBS,observed under a microscope and photographed,and the adhesion inhibition rate of fibronectin and Matrigel was measured by MTS.3.Microtubule aspiration was used to detect the changes of mechanical properties such as deformability and viscoelasticity of SKBR-3 and MDA-MB-231 cells:a photo was taken every 92 ms to record the length of cells in the tube,recorded for 6-10s.The length of breast cancer cells transiently inhaled into the microtubules was measured using LAS.X measurement software.The experimental results were fitted using a standard solid viscoelastic model:σ+(μ/K2)(?)σ/(?)t=K1ε+μ[1+(K1/K2)](?)/(?)t,where,σ and ε are stress and strain;(?)σ/(?)t and(?)ε/(?)t are partial derivatives of stress and strain as a function of time,respectively;K1 and K2 are elastic elements,and μ is a viscous element.4.The effects of CuB at 10,20,30 nmol/L on the migration and invasion of SKBR-3 and MDA-MB-231 cell lines were detected by Wound healing assay and Transwell assay.5.0.1 mL SKBR-3 cells suspension were injected into the female BALB/c mice by tail vein to establish a lung metastasis model.Mice were randomly divided into three groups:control group,CuB group and vincristine group.The body weight of each group was measured every three days.The size and number of metastases in the liver and lung of each group of mice were visually observed.The tissues were fixed in 4%paraformaldehyde,and paraffin sections were prepared,stained,and observed following hematoxylin and eosin(H&E)staining.6.Immunofluorescence was used to detect the expression and distribution of vimentin,F-actin,FAK and Vinculin in breast cancer cells treated by 10,20,30 nmol/L CuB and to explore the effect of CuB on the cytoskeleton of breast cancer cells.7.Using Western Blot to investigate the expression levels of integrin β1,Rac1,CDC42 and WAVE 2/3 and the effect of Arp 2/3 protein expression level and RhoA/ROCK1 expression level in the Racl/CDC42 pathway in SKBR-3 and MDA-MB-231 cells treated by 10,20,30 nmol/L CuB to explore the mechanism by which CuB alters the biomechanical properties of breast cancer cells8.Establish a mouse xenograft model,remove the tumor for immunohistochemical staining,compare the CuB treatment group with the control group and vincristine treatment,and explore whether CuB changes the expression of Rho family protein in vivo and inhibits breast cancer metastasisResults:1.CuB effect on adhesion of SKBR-3 and MDA-MB-231 cellsCuB inhibits the proliferation of SKBR-3 and MDA-MB-231 cells in a time and dose dependent manner.10,20,30 nM of CuB has little effect on the proliferation of SKBR-3 and MDA-MB-231 cells but can significantly inhibitin SKBR-3 and MDA-MB-231 cells adhesion to fibronectin and Matrigel(P<0.05)2.CuB can effect on the biomechanical properties of SKBR-3 and MDA-MB-231 cellsThe elastic modulus K1 of MDA-MB-231 and SKBR-3 cells treated with CuB was increased compared with the control group(P<0.05).The elastic coefficient K2 of MDA-MB-231 cells after CuB treatment was not significantly different from that of the control group(P>0.05),while the elastic coefficient K2 of SKBR-3 cells was significantly decreased(P<0.05).The calculated results of the equilibrium Young’s modulus of elasticity(E∞)showed that the E∞ values of MDA-MB-231 and SKBR-3 cells after CuB treatment were significantly increased(P<0.05).The viscosity coefficient(μ)of MDA-MB-231 and SKBR-3 cells was significantly reduced after CuB treatment(P<0.05).However,the instantaneous Young’s modulus(E0)of the CuB-treated groups at each concentration was not significantly different from the control group(P>0.05)3.CuB can inhibit the migration and invasion of SKBR-3 and MDA-MB-231 cells in vitroIn this study,cells were treated with 10,20,30 nM CuB for 12 h and 24 h.The inhibition of SKBR-3 and MDA-MB-231 cells migration and invasion were examined by wound healing assay and transwell assay.The results of the scratch test showed that CuB significantly inhibited the migration of breast cancer cells(P<0.05).The most pronounced inhibition occurred after treatment of breast cancer cells at a concentration of 30 nM for 12 h and 24 h.The inhibitory effect of CuB on migration of MDA-MB-231 and SKBR-3 cells were similar,and at 24 h,the scratches of the control group of the two cells nearly healed.In the transwell migration and invasion assay,the CuB group showed a significant reduction in the number of migrated and invaded cells compared to controls(P<0.05).CuB showed a significant dose-dependent inhibition of MDA-MB-231 and SKBR-3 cell migration and invasion.4.CuB inhibits the metastasis of breast cancer in miceThe mouse lung metastasis model was established.Mice were sacrificed after 21 days and liver and lung tissues were observed.The lung and liver metastasis of the CuB and vincristine groups were lower than those of the control group(P<0.05),and pulmonary inflammation in the vincristine group was more severe than in the CuB group(P<0.05).In addition,we found that the weight loss of mice after injection of vincristine was greater than that of the control group and the CuB group.These results suggest that CuB significantly inhibits breast cancer invasion of lung and liver tissue and reduces lung tissue inflammation5.CuB induced changes in cytoskeleton of breast cancer cellThe expression of Vimentin/F-actin/Vinculin/FAK in the cells was detected by immunofluorescence.The results indicate that CuB causes a decrease in Vimentin expression in MDA-MB-231 and SKBR-3 cells(P<0.05),thereby inhibiting cell migration.Furthermore,after CuB treatment,as the concentration increased,F-actin aggregated around the nucleus,and expression in the cytoplasm decreased as the concentration of CuB increased(P<0.05).Vinculin and FAK were expressed in MDA-MB-231 and SKBR-3 cells,and their expression gradually decreased as the concentration of CuB increased(P<0.05)6.Mechanism of CuB-induced changes in mechanical properties of breast cancer cells in vitro and in vivoAfter treatment of MDA-MB-231 and SKBR-3 cells with 10,20,30 nM CuB for 24 hours,the expression levels of integrin β1 as well as Racl,CDC42,WAVE 2/3,and Arp 2/3 in the Racl/CDC42 pathway were dose-dependently decreased.Furthermore,we detected another important force-chemical signaling pathway RhoA/ROCK1 downstream of Integrin,which affects cell migration and invasion.Similarly,the expression levels of RhoA and ROCK1 proteins dose-dependently decreased after CuB(10,20,and 30 nM)treatment.We immunohistochemically examined the expression of mechanically relevant proteins in the tumors.The results showed that the expression of ROCK,CDC42,and Rac1 proteins in the CuB and Vincristine was lower than that in the normal saline group(P<0.05).These results indicate that CuB downregulated the expression of the Rho family proteins,regulated the reorganization of the cytoskeleton,changed the mechanical properties of the cells,and reduced cell migration.Conclusion:1.CuB can effectively inhibit the adhesion of MDA-MB-231 and SKBR-3 cells2.CuB can inhibit the migration and invasion ofMDA-MB-231 and SKBR-3 cells by changing the biomechanical properties of breast cancer cells in vitro3.CuB can inhibit the metastasis of breast cancer in the liver and lung of mice,and can reduce the degree of inflammation in the lungs4.CuB can change the distribution and composition of breast cancer cytoskeleton,inhibit the expression ofvimentin/F-actin/FAK/Vinculin in MDA-MB-231 and SKBR-3,and change the biomechanical properties of cells to inhibit cell migration and adhesion.5.CuB can inhibit breast cancer metastasis by inhibiting the signal transduction of the important force-chemical signaling pathway,Rho family GTPases Racl/CDC42/RhoA,to reduce breast cancer cell deformation and adhesion.