Xihuang pill, an approved Chinese medicine formula (state medical permit number. Z11020073), is a commonly used adjuvant drug for cancer patients in China. Xihuang pill has a satisfactory effect in treating breast cancer in clinics, especially triple-negative breast cancer (TNBC), which is the most aggressive type of breast cancer, and finite effective therapies. However, the mechanism of Xihuang pill in treating TNBC remains unclear. The present study aims to explore the pharmacological mechanism of Xihuang pill in treating advanced TNBC. We identified the main chemical components of Xihuang pill by using HPLC-Q-TOF-MS/MS. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis shows that serum containing Xihuang pill (XS) had no obvious killing effect on any subtype of breast cancer cells, but it inhibited mammosphere colony formation of two TNBC cell lines (4T1 and HCC1806 cells) and could enhance the inhibitory effect of paclitaxel (PTX) on the proliferation of 4T1 and HCC1806 cells when combined with PTX. Seventy-six active compounds in Xihuang pill, their 300 protein targets, and 16667 TNBC stem cell–related genes were identified. The drug–herb–active compound–target gene–disease network and enrichment analyses were constructed with 190 overlapping candidate targets. Through text mining and molecular docking, the target gene NR3C2 and its active compound naringenin were selected for further validation. According to the TCGA database, we observed that a high expression of NR3C2 promoted a higher survival probability regarding overall survival (OS). In vitro experiments indicated that naringenin presented an identical effect to XS, possibly by regulating the NR3C2 expression. Overall, this study explored the effect of Xihuang pill in treating advanced TNBC cells and showed that naringenin, which is the key active compound of Xihuang pill, could lessen the stemness of TNBC cells to produce a synergistic effect on PTX by regulating the NR3C2 gene.
The present study examined the effects of brucine on the OPG/RANKL/RANK signaling pathway for exploring the mechanism of brucine suppression of bone metastasis in breast cancer. MDA-MB-231 breast cancer cells and mouse osteoblast MC3T3-E1 cells were cocultured to mimic the breast cancer bone metastasis microenvironment in vitro. qRT-PCR and Western blotting were used to detect the expressions of OPG and RANKL at the mRNA and protein levels, respectively, in brucine-treated cultures and they were compared to those in untreated cultures. We aimed to understand the effect of brucine on the entire OPG/RANKL/RANK signaling pathway after analyzing these effects. Results showed that brucine treatment significantly increased both the OPG mRNA/RANKL mRNA expression ratio and the OPG protein/RANKL protein ratio in cocultures compared to those in untreated cocultures (P < 0.01). Brucine, therefore, plays a regulatory role in the OPG/RANKL/RANK signaling pathway, suggesting that it can indirectly control osteoclasts by regulating the expression and secretion of OPG and RANKL in osteoblast cells, thereby inhibiting the differentiation and bone resorption function of osteoclasts.
Background Although the triple negative breast cancer is sensitive to chemotherapy, breast cancer stem cells (BCSCs) is the origin of tumor chemotherapy resistance, tumor recurrence and tumor invasion and metastasis. This study aims to examine the effect of tetrandrine combine with arsenic trioxide on BCSCs and potential mechanism of anti- triple negative breast cancer metastasis. Methods We cultured the triple negative breast cancer cell MDA-MB-231 and induced BCSCs sphere formation by serum-free medium for 5 days. In the MDA-MB-231 cell and MDA-MB-231 stem cell, we compared the ratio of CD44+/CD24- and sorted stem cells by flow cytometry, the expression of Oct4 and Sox2mRNA were by rt-PCR, invasion ability were by Transwell assay. We subsequently measured the effect of tetrandrine combine with arsenic trioxide on BCSCs proliferation by CCK8 method. The stem cell morphology observation was by trypan blue staining. Stem cell cycle and apoptosis were evaluated by flow cytometry. Western Blot was used to measure the protein levels of Hedgehog, Notch1 and PTEN signaling of BCSCs. Results The ratio of CD44+/CD24- in MDA-MB-231 stem cells was 95.0%, while MDA-MB-231 cell was 89.3%. The invasion number of MDA-MB-231 stem cell was significantly higher than that of MDA-MB-231 cells (p<0.01). Furthermore, we demonstrated that tetrandrine and arsenic trioxide could inhibit the BCSCs proliferation. Tetrandrine combine with arsenic trioxide could significantly promote the apoptosis (p<0.01) and increase the percentage of G0/G1 phase and decrease the G2/M phase (p<0.01) of BCSCs. Compared with the control group, arsenic trioxide, tetrandrine and the combined group could significantly reduce the expression of GLI1 and SMO and increase the expression of PTEN protein (P<0.05). Conclusions These findings revealed that tetrandrine combined with arsenic trioxide could suppress the proliferation and induce apoptosis of BCSCs by decreased Gli and SMO expression and increased PTEN expression. Targeting BCSCs treatment, this study provides potential therapeutic drugs against triple negative breast cancer metastasis.
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