| Background and Basis:Self-renewal, differentiation, proliferation, anti-apoptosis, a strong tumorigenic ability and maybe epithelial-mesenchymal transition (EMT) of cancer stem cells (CSCs) are the important causes to promote tumor growth and metastasis. EMT can significantly enhance the ability of cancer cells to invasion and migration, and researches showed that CSCs have the higher EMT levels than general cancer cells. So EMT could be the main pathway that breast cancer stem cells to promote breast cancer metastasis. Transforming growth factor-(3(TGF-β) is considered to be the one of main triggers inducing EMT in the breast cancer microenvironment through activating Smad pathway. This study explored the regulate roles of RHDT and peimine/peiminine against BCSCs and4T1cells, basing on the theory of reinforcing the healthy qi and dispelling toxin. It was researched that the effects of RHDT and peimine/peiminine on EMT by intervening on the microenvironment associated with breast cancer stem cells to control breast cancer invasion and metastasis. It will provide some new scientific evidences for the further studies of the biological behaviors of CSCs, cancer metastasis and its molecular mechanisms.Methods:Cultured, separated and purified breast cancer stem cells (BCSCs) basing on4T1inflammatory breast cancer cells line, and evaluated through detecting general functions and phenotype of breast cancer stem cells. The proteins levels associated with EMT in the BCSCs compared with4T1cells’. It was controlling observed that some cytokines promoting tumor metastasis in the BCSCs’and4T1cells’microenvironments, including VEGF, TGF-β, MMP-9and TF. Meanwhile, the regulate roles of RHDT and peimine/peiminine were also observed. It was researched that the inducing effects of TGF-β to enhance the extents of invasion and metastasis. We controlling studied to observe the activation of TGF-β/Smad2/3/4pathway, and the expression of key phenotype proteins with EMT in BCSCs groups and4T1cells groups. The key epithelial phenotype protein is E-cad, and the main mesenchymal phenotype proteins are including N-cad, VIM, FN, and MMP-9.Results:(1) It was showed that a small part of the4T1cells to form cancer stem cells microspheres (TumorSpheres), and further proliferation and self-renewal, While4T1cells were cultured in serum-free culture conditions for5-7days. The TumorSpheres obtained the relative purification by being passaged more than three times. The evaluation of BCSCs’ CD24-/lowCD44+phenotype showed that4T1cells groups’ CD24-/lowCD44+cells had (0.11± 0.05)%, while TumorSpheres groups had (82.56±4.13)%(P<0.01). When the3day,5day and7day, the proliferation rates of TumorSpheres were51.42%,100%and146%, respectively. The CD24-/lowCD44+/Ki-67+cells in TumorSpheres showed the ability of proliferation, and9day>6day>3day,(p<0.05). The TumorSpheres could differentiation and proliferation rapidly by serum inducing. In addition, TumorSpheres’level of apoptosis was obviously lower than4T1cells’(p<0.01). The results proved that TumorSpheres’features are mainly coinciding with the breast cancer stem cells’.(2) LC-MS results showed that RHDT had peimine8.42ug/ml and16.61ug/ml, peiminine6.26ug/ml and12.42ug/ml in the different samples, respectively.(3) The inhibition rates of4T1cells and BCSCs that be intervened by RHDT, peimine, peiminine and DDP were observed. The results showed RHDT, peimine, peiminine and DDP can significantly suppress4T1cells and BCSCs, and the inhibition rates rely on time and concentrations. The BCSCs groups’ IC50were higher than4T1cells groups’, but had no statistics significance. RHDT, peimine, peiminine and DDP were all promoting the cells apoptosis, p<0.01. It proved BCSCs had higher ability of anti-apoptosis than4T1cells.(4) EMT correlate detections including western blot and RT-PCR were showed that the key epithelial phenotype marker E-cadherin protein expression levels:4T1groups> BCSCs groups (p<0.05). On the other side, the important mesenchymal phenotype marker N-cadherin, VIM and FN proteins expression levels:BCSCs groups>4T1groups (p <0.05). These results proved that BCSCs could occur to EMT easier than general breast cancer cells.(5) The results of intervention roles of RHDT, peimine, peiminine and DDP in BCSCs groups showed that E-cad had different control levels:4T1negative control group> DDP group> BCSCs negative control group> RHDT group> peimine group> peiminine group, especially peiminine group mRNA expression decreased obviously, p<0.05. The main mesenchymal phenotype protein Vimentin (VIM) mRNA expression levels declined by medicinal intervention (p<0.01):BCSCs negative control group> peimine group> DDP group>4T1negative control group> RHDT group> peiminine group. The mesenchymal phenotype fibronectin (FN):RHDT group> peimine group> BCSCs negative control group> peiminine group> DDP group>4T1negative control group. Peiminine group decreased significantly, p<0.01.(6) Transwell invasion assay showed that the cells count across the membrane in4T1cells group and4T1cells stimulated by TGF-β, had a significant difference. TGF-β/4T1group>4T1group (p<0.01). On the other side, migration assay showed that the cells count across the membrane in4T1cells group and4T1cells stimulated by TGF-β, also had a significant difference. TGF-β/4T1group>4T1group(p<0.01). The results revealed that the invasion and migration levels increased obviously after4T1cells stimulated by TGF-β for24h. It is considered that TGF-β is a point trigger inducing EMT.(7) Mechanism of EMT relevant assays:The TGF-β autocrine level in4T1cells was decreased than BCSCs, BCSCs group>4T1group, p<0.01. peiminine may significantly inhibit TGF-β mRNA expression, p<0.01. RHDT, peimine and peiminine were all declined the phosphorylation level of Smad2/3, and inhibited Smad4expression. RHDT and peiminine can suppress downstream Twistl mRNA expression, p<0.01. The results revealed that RHDT, peimine and peiminine regulated EMT through down regulating TGF-β/Smad pathway, potentially.(8) The influence caused by different medicine for factors boosting tumor metastasis in the microenvironment of BCSCs and4T1cells, including VEGF, MMP-9, TF and TGF-β. The influences were significant. VEGF content: peimine group, RHDT group and DDP group were declined in4T1cells groups, p<0.05. TGF-β content in BCSCs groups and4T1groups: negative control group> peimine group> peiminine group> RHDT group> DDP group (p <0.01). Moreover, peiminine had a significant inhibitory effect on MMP-9, p<0.05.Conclusions:①It was showed that breast cancer stem cells (BCSCs) can be cultured and separation from4T1cells by phenotypic and functional verification. BCSCs can express CD24-/lowCD44+breast cancer stem cells’phenotype, and has general functions including self-renewal, differentiation, proliferation and anti-apoptotic ability.②Reinforce the Healthy Qi and Dispel Toxin Formula (RHDT), peimine and peiminine can significantly suppress BCSCs and4T1cells. In addition, liquid chromatography combined detection showed that peimine and peiminine belong to the active ingredients of RHDT.③RHDT, peimine and peiminine can induce4T1cells and BCSCs to apoptosis obviously. Meanwhile, BCSCs had higher level of anti-apoptosis than4T1cells.④Breast cancer stem cells could be more easily occurring epithelial-mesenchymal transition than the general4T1breast cancer cells.⑤RHDT and peiminine can inhibit obviously expression of key proteins that are closely related with EMT.⑥By digging out the mechanisms of EMT of BCSCs to find that TGF-β may significantly promote the occurrence of invasion and migration of4T1cells. TGF-β could be the important trigger to induce EMT. RHDT, peimine and peiminine, especially peiminine may inhibit EMT through down regulation of TGF-β/Smad pathway to reduce the main mesenchymal phenotype proteins and enhance the point epithelial phenotype protein.⑦RHDT, peimine and peiminine, especially peiminine may improve the levels of factors boosting tumor metastasis in the microenvironment of BCSCs and4T1cells in vitro, including VEGF, MMP-9, TF and TGF-β. |
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