Effect of Extracts from Veronica peregrine on Breast Cancer Cell-induced Osteoclastic Bone Metastasis via Inhibiting Runx2 Activation / 中国实验方剂学杂志

ObjectiveTo investigate inhibitory effect of extracts from Veronica peregrina （EVP） on the osteoclastic bone metastasis induced by breast cancer cells. MethodBone metastasis model was established by injection of MDA-MB-231 cells， a human breast cancer cell line， into the left ventricle of BALB/c nude mice. The expression of human cytokeratin-19 （Ck-19） gene in mouse bone marrow was determined by nested polymerase chain reaction（PCR） to assess the bone metastasis of MDA-MB-231 cells. To assess the effects of EVP on the activation of bone marrow macrophages （BMMs）， we counted the multinuclear cells and measured the secretion of Cathepsin K. Western blot was adopted to assess the effects of EVP on receptor activator of nuclear factor-κB （RANK）， Runt-related transcription factor 2 （ Runx2 ）， phosphorylated Runx2 （p-Runx2）， and matrix metalloproteinase-9 （MMP-9） in BMMs. Gelatin zymography was employed to determine the activities of matrix metalloproteinases （MMPs）. ResultCompared with that in the blank group， Ck-19 expression was down-regulated in EVP groups （P<0.05）. The multinucleated cells increased when the BMMs were induced by soluble receptor activator of nuclear factor-κB ligand （sRANKL）， which was inhibited by EVP （P<0.05）. The level of cathepsin K in the supernatant of sRANKL group increased compared with that of the blank group， while EVP groups had lower cathepsin K levels than sRANKL group （P<0.05）. Compared with the blank group， the sRANKL group showed up-regulated RANK expression， Runx2 phosphorylation， and MMP-9 expression （P<0.05）， while the expression levels of RANK， p-Runx2， and MMP-9 were down-regulated when the cells were incubated with EVP （P<0.05）. Furthermore， exposure of BMMs to sRANKL resulted in an increase in gelatin hydrolyzation compared with the blank group （P<0.01）， which， however， was reversed in EVP groups （P<0.05）. ConclusionEVP significantly inhibits bone marrow metastasis of MDA-MB-231 cells， which may be associated with the suppression of osteoclast activation by inhibiting Runx2 phosphorylation.

Danshen Injection Inhibits SKOV3 Cell Proliferation in Vitro by Interfering with Their Interaction with Platelets / 中国实验方剂学杂志

Objective:To investigate the inhibitory effects of Danshen injection against ovarian cancer cell proliferation induced by the interaction between platelets and cancer cells. Method:The induction of platelets on SKOV3 growth <italic>in vitro</italic> and the inhibitory effect of Danshen injection at 12，24，and 48 g·L^-1 were observed by 3-（4，5-Dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide （MTT） and cell colony formation assays. The content of transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁） in the platelet-tumor cell interaction system and platelet supernatant and the effect of Danshen injection on TGF-<italic>β</italic>₁secretion were detected by enzyme-linked immunosorbent assay （ELISA）. The influences of tumor cell culture supernatant on platelet aggregation and secretion and the inhibitory effect of Danshen injection were determined by microplate assay and ELISA. The effects of Danshen injection on platelet nuclear factor kappa B （NF-<italic>κ</italic>B） signaling pathway were assayed by Western Blot. Result:Compared with the blank group， the platelet induction group exhibited significantly elevated absorbance at <italic>A</italic>₅₇₀（<italic>P</italic><0.01）， while the absorbance at <italic>A</italic>₅₇₀ in the platelet + Danshen injection group was significantly lower than that in the platelet induction group （<italic>P</italic><0.01）. The comparison with the Danshen injection group revealed that the cell proliferation inhibitory rate in the platelet + Danshen injection group at the same dose was more significant （<italic>P</italic><0.01）. The number of colonies in the platelet induction group was obviously increased in contrast to that in the blank group（<italic>P</italic><0.05）， while the number of colonies in the platelet + Danshen injection group was significantly lower than that in the platelet induction group（<italic>P</italic><0.05，<italic>P</italic><0.01）. As demonstrated by comparison with the blank group， TGF-<italic>β</italic>₁content in the supernatant of the platelet induction group rose remarkably（<italic>P</italic><0.01）， whereas that in the platelet + Danshen injection group declined（<italic>P</italic><0.01）. Compared with the Danshen injection （24 g·L^-1） group， the platelet + Danshen injection group displayed more obvious inhibition（<italic>P</italic><0.01）. Compared with the blank group， Danshen injection significantly reduced the TGF-<italic>β</italic>₁content in platelet supernatant（<italic>P</italic><0.05，<italic>P</italic><0.01）. There was no significant change in the content of TGF-<italic>β</italic>₁in SKOV3 supernatant treated with Danshen injection. The platelet aggregation， thromboxane A₂（TXB₂）， and serotonin （5-HT） secretion in the SKOV3 cell supernatant induction group were significantly increased as compared with those in the blank group （<italic>P</italic><0.01）， while such indexes in the cell supernatant induction + Danshen injection group were obviously decreased （<italic>P</italic><0.01）. Compared with the Danshen injection （24 g·L^-1） group， the cell supernatant induction + Danshen injection group displayed more obvious inhibition at the same dose（<italic>P</italic><0.01）. Compared with the blank group， the platelet induction group exhibited obviously up-regulated phosphorylated TGF-<italic>β</italic>-activated kinase-1 （TAK-1） and NF-<italic>κ</italic>B， but down-regulated phosphorylated inhibitory protein of NF-<italic>κ</italic>B （I<italic>κ</italic>B）（<italic>P</italic><0.01）， which however were significantly reversed in the platelet + Danshen injection group<bold>（</bold><italic>P</italic><0.01）. Conclusion:Danshen injection affect the proliferation of SKOV3 cells by inhibiting their interaction with platelets， which may be related to the inhibited secretion of TGF-<italic>β</italic>₁.