| Backgroud:Lung cancer is currently the most common malignant tumor in China and even in the world.In recent years,vascular mimicry(VM)has been found to be another pathway of tumor angiogenesis,which is closely related to the poor prognosis of lung cancer.Hypoxia is an important initiating factor in the induction of VM formation,and tumor stem cells participate in VM formation through a variety of pathways.Previous studies have found that Chuanxiong and Danshen can inhibit the expression of VEGF,HIF-1α and EMT-related proteins in cancer stem-like cells(CSLCs),which are related to VM formation.Therefore,we speculate that Chuanxiong and Danshen may inhibit the formation of CSLCs VM in lung cancer,and the mechanism may be different under different oxygen environments.Objective:Firstly,the A549 CSLCs subpopulation was isolated and identified,and its stem cell characteristics were identified.Then,the effect of A549 CSLCs on VM formation was observed.Finally,the inhibitory effects of Chuanxiong and Danshen monotherapy groups and two drug compatibility group on the formation of CSLCs VM in lung cancer under different oxygen conditions were determined for observing the effects of single-drug and compatibility,which providing experimental basis for the clinical practice of Chinese medicine.Methods:(1)A549 CSLCs subgroups were sorted by serum-free spheroid culture.The cell proliferation was detected by single cell cloning assay and mouse xenograft formation experiment.The cell migration ability was detected by scratch assay and the tumor stem cell surface markers were detected by flow cytometry.These experiments were used to jointly identify the sorted spherical cells as CSLCs.(2)The ability of three-dimensional tubule formation of A549 CSLCs was detected by Matrigel tube formation assay and the endothelial cell marker proteins CD31 and vWF were detected by immunofluorescence method to observe the ability of A549 CSLCs to induce transformation into endothelial cells.These experiments were used to jointly determine the role of A549 CSLCs in promoting VM formation.(3)A549 CSLCs tumor-bearing mouse models were established and divided into normoxia group and hypoxia group.Each group was further divided into control group,Chuanxiong group,Danshen group,Chuanxiong and Danshen(Chuandan)group and Endostar group.The expression of vWF protein in each group was detected.The formation of VM in each group was detected by CD31/PAS double staining method.The effects of Chuanxiong and Danshen on A549 CSLCs VM under different oxygen conditions were observed.(4)The expression of VEGF was detected by ELISA.The mRNA expressions of VEGF and MMP-9were detected by RT-PCR.The expressions of MACC1,HGF,c-MET,TWIST,VE-Cadherin,FAK,EphA2,Integrin β1,HIF-1α were detected by Western blot.These experiments were conducted to observe the specific mechanism of the effects of Chuanxiong and Danshen on A549 CSLCs VM formation in different oxygen environments.Results:(1)The serum-free A549 cells formed regular cell spheres on the third day,and then the cell spheres gradually increased.On the fifth day,the refractive index of the cell spheres decreased.After the cells were successively passaged twice,and the spheres were more stereoregular.Single cell clone formation experiment indicated that the number of cells in a single colony formed by A549 spheroid cells were significantly more than that of the parental cells,and the data were statistically significant(P<0.05).Mouse transplanted tumor formation experiments showed that A549 spheroid cells formed tumors were earlier than primary A549 cells.At the end of the 30-day observation period,at the grade of 5×105,the tumor formation rate of A549 spheroid cells was 100%,and the tumor formation rate of primary cells was 80%.At the grade of 1×105,the tumor formation rate of A549 spheroid cells was 100%,the primary tumor formation rate was 60%.At the grade of 1×104,the tumor formation rate of A549 spheroid cells was 100%,and the tumor formation rate of primary cells was 0.These data indicated that A549 spheroid cells are more likely to form tumors than primary cells.The scratch test results showed that A549 spheroid cells had stronger cell migration ability than their primary cells at 6th,12th and 24th day,respectively(P<0.01).Flow cytometry showed that the expression of stem cell marker CD44+/CD24-/low in A549 spheroid cells was as high as 80.4%.(2)The abilities of EA.hy 926,A549 primary cells,normoxia A549 CSLCs and hypoxic A549 CSLCs to form tubules in a three-dimensional culture system were observed.The results showed that A549 CSLCs had stronger tubule formation ability compared with A549 primary cells,and the small tubes were more obvious in the hypoxic environment,which the morphology of the small tubes was closer to the small tubes formed by EA.hy 926.Immunofluorescence assay showed that the expressions of CD31 and vWF in A549 CSLCs were significantly increased under hypoxic conditions compared with normoxic environment,indicating that the ability of A549 CSLCs to transform into endothelial cells was more obvious in hypoxic environment.(3)The results of laser confocal microscopy showed that the Danshen group and Chuandan group could promote the expression of vWF in the normoxia environment compared with the control group,and there was no significant difference among the other groups.There was no significant difference in the expression of vWF between the groups in the hypoxic environment compared with the control group.Compared with the normoxic group,there was no significant difference in the expression of vWF between the groups.The results of CD31/PAS double staining showed that the formation of VM could be inhibited by Chuanxiong,Danshen,Chuandan and Endostar groups regardless of the normoxic environment or hypoxic environment.There was no significant difference between the inhibition of VM formation in each group compared with different oxygen environments.(4)The results of ELISA showed that the Chuanxiong group,Danshen group,Chuandan group and Endostar group significantly inhibited the expression of VEGF compared with the control group(P<0.01).There was no significant difference in the expression of VEGF between the groups under hypoxic conditions.Compared with the normoxic group,the expression of VEGF was significantly promoted in each group under hypoxic conditions(P<0.01),which might be related to the hypoxia itself as a signal stimulation to promote the expression of VEGF.The results of RT-PCR detection of VEGF showed that under the normoxic environment,Chuanxiong group,Danshen group,Chuandan group and Endostar group could significantly inhibit the expression of VEGF(P<0.01).There was no significant difference in the expression of VEGF between the groups under hypoxic conditions.Compared with the normoxic group,the expression of VEGF in each group was generally increased under hypoxic conditions,which might be related to the hypoxia itself as a signal stimulus to promote the expression of VEGF.The results of RT-PCR detection of MMP-9 showed that under the normoxic environment,Chuanxiong group,Danshen group,Chuandan group and Endostar group could significantly inhibit the expression of MMP-9(P<0.01).There was no significant difference in the expression of MMP-9 between the groups under hypoxic conditions.Compared with the normoxic group,the expression of MMP-9 was generally increased in hypoxic environment,which might be related to the hypoxia itself as a signal stimulus to promote the expression of MMP-9.The results of Western blot showed that under the normoxic environment,the Chuanxiong group,Chuandan group and Endostar group could inhibit the expression of MACC1.The Danshen group could inhibit the expression of HGF.The Danshen group could inhibit the expression of c-MET.The Danshen group and Endostar group could inhibit the expression of TWIST.The Danshen group and Chuandan group could inhibit the expression of VE-Cadherin.The Danshen group could inhibit the expression of FAK.The Chuanxiong group and Danshen group could inhibit the expression of EphA2,and the Danshen group could inhibit the expression of Integrin β1.In the hypoxic environment,the Chuanxiong group could inhibit the expression of MACC1.The Danshen group could inhibit the expression of HGF.The Chuanxiong group could inhibit the expression of c-MET.The Danshen group could inhibit the expression of TWIST.The Danshen group could inhibit the expression of VE-Cadherin.The Danshen group and Endostar group could inhibit the expression of FAK.The Chuanxiong group and Endostar group could inhibited the expression of EphA2,and the Chuanxiong group and Chuandan group could inhibited the expression of Integrin β1.Conclusion:(1)The spheroid cells isolated and enriched by serum-free culture have the abilites of stem cell-like cells.(2)A549 CSLCs have stronger ability to promote VM formation than A549 cells and A549 CSLCs promote VM formation in a hypoxic environment.(3)Both in the normoxic and hypoxic environment,Chuanxiong,Danshen and their compatibility can inhibit the formation of VM.However,no matter the normoxia or hypoxic environment,the compatibility of Chuanxiong and Danshen has no obvious synergistic effect on the inhibition of VM formation.(4)The inhibitory mechanism of Chuanxiong,Danshen and their compatibility on VM in different oxygen environments is not exactly the same. |
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