Inhibition of Sijunzi Decoction extract on human breast cancer cells MDA-MB-468 / 中草药

Objective: To investigate the effect of Sijunzi Decoction extract (SDE) on the growth of human triple negative breast cancer (TNBC) cell line MDA-MB-468. Methods: MDA-MB-468 cells were treated with different concentrations of SDE. The effect of SDE on the proliferation and migration of the cells were detected by CCK-8 assay and the cell wound healing assay. The colony formation assay was performed to analyze the effect on the ability of colony formation of the cells with SDE. Hoechst 33342 staining technique and flow cytometry (FCM) were used to detect apoptosis and cell cycle of the cells. Western blotting was used to detect the expression levels of STAT3, which was related with the proliferation and apoptosis of cells. Results: Compared with the control group, SDE had a certain inhibitory effect on MDA-MB-468 cells (P < 0.05), and it was dependent on the concentration and time. Cloning formation experiments showed that SDE inhibited the clonality of the cells. The cell migration experiment showed that the wound healing ability of the cells could be weakened by the extract with the medium and high dosage (P < 0.001). The results of FCM showed that the apoptosis rate of all SDE dosage increased gradually in a dose-dependent manner. And SDE with the medium and high dosage induced apoptosis of the cells significantly (P < 0.01 and 0.001). Cell cycle was affected by SDE with the obvious reduction of the cells in G2 phase (P < 0.01). The results of Western blotting showed that the expression level of STAT3 was decreased significantly. Conclusion: SDE inhibited the proliferation and clonal formation of MDA-MB-468 cells, inhibited migration, promoted apoptosis and decreased the cells of G2 phase. which may be related to the regulation of STAT3 pathway.

Mechanisms of doxorubicin resistance of breast cancer MCF-7 cells mediated by exosomes / 药学学报

This study aimed to explore the roles of exosomes in doxorubicin-resistance in breast cancer cells. Using breast cancer parental cell line (MCF-7), doxorubicin-resistant cell line (MCF-7/ADR) and sensitive cell line co-cultured with doxorubicin-resistant supernatant (MCF-7/EXO) as models, the effects of doxorubicin on proliferation or apoptosis of MCF-7, MCF-7/EXO and MCF-7/ADR cells were detected by CCK8, and light or fluorescent microscopy. Exosomes in the supernatants of cell culture were extracted by ultracentrifugation, and the quantity of exosomes was determined by transmission electron microscopy, BCA and DiI labeling assay. Expression levels of exosome-specific biomarkers CD63 and Flotillin-1 were detected by Western blot. The uptake of MCF-7/ADR cell-derived exosomes by MCF-7 cells was observed by laser confocal microscopy. Western blot was used to detect the expression levels of multidrug resistance protein ATP-binding cassette subfamily B member 1 (ABCB1) in all three cell strains. Cell proliferation assays showed that IC50 of MCF-7/EXO cells to doxorubicin was 0.83 ± 0.09 μmol·L-1, which was significantly higher than 0.15 ± 0.05 μmol·L-1 (P<0.01) of MCF-7 cells, suggesting 5.5 times of increase in drug resistance. Apoptosis of MCF-7 cells was induced after doxorubicin treatment (P<0.001), but MCF-7/EXO cells were not significantly different (P>0.05). Exosome quantification and specific marker detection showed that MCF-7/EXO cells had significantly more exosomes than MCF-7 cells (P<0.05). PKH67 tracer markers indicated that MCF-7/ADR-derived exosomes could be taken up by MCF-7 cells. Western blot showed that the expression level of ABCB1 protein in MCF-7/EXO cells was significantly higher than that in MCF-7 cells. Taken together, these results indicate that exosomes of doxorubicin-resistant breast cancer cells can transmit drug resistance to sensitive cells, and the underlying mechanism may involve ABCB1 protein transport mediated by exosomes.

Advances in understanding exosomes-mediated tumor chemoresistance / 药学学报

Chemotherapy plays an essential role in controlling tumor growth and progression. However, long-term use of chemotherapeutic drugs usually results in drug resistance in tumor cells, leading to treatment failure and disease progression. The mechanism of tumor resistance to chemotherapy and the strategy of prevention or reversal of such resistance have always been hot issues in cancer therapy research. Exosomes are small spherical vesicles secreted by cells with a diameter of 40-100 nm. They carry a variety of bioactive small molecules (including DNA, ncRNA, RNA, and proteins) and participate in regulation of cell microenvironment, thereby affecting a variety of physiological and pathological activities in the body. In recent years, studies have shown that exosomes play an important role in cancer cell resistance to chemotherapy, metastasis, and immune escape. This article reviews the role and mechanism of exosomes in the development of drug resistance in tumors, and aims to provide new ideas for the prevention or treatment of tumor resistance.

Small interfering RNA targeting STAT3 enhances the drug susceptibility of adriamycin in breast cancer cells / 中国药学杂志

OBJECTIVE: To further define the modulation effect of signal transducers and activators of transcription 3 (STAT3) in adriamycin-resistant breast cancer and to promote the clinical application of the inhibitors of STAT3 in reversing multidrug resistance in cancer. METHODS: Firstly, the levels of STAT3 and phosphorylated STAT3 (pSTAT3) expression in clinical breast cancer tissue samples were determined by Western blotting. The expression of STAT3 and pSTAT3 in adriamycin-sensitive and adriamycin-resistant breast cancer cell lines was evaluated by RT-PCR and Western blotting. Secondly, the expression of STAT3 was detected by Western blotting after blocking the STAT3 signal pathway with small interfering RNA targeting STAT3 (STAT3-siRNA). Finally, after the expression of STAT3 was blocked by STAT3-siRNA, immunofluorescence was performed to study the proliferation activity of breast cancer cells and MTT was used to determine the IC50 of the cells in order to observe whether STAT3-siRNA had any inhibitory effects on the growth of breast cancer cells and whether it could promote the efficacy of adriamycin in breast cancer cells. RESULTS: STAT3 and pSTAT3 were highly expressed both in clinical breast cancer tissue samples and in adriamycin-sensitive-and-resistant breast cancer cells. The expression of pSTAT3 in adriamycin-resistant breast cancer cells was significantly higher than in adriamycin-sensitive breast cancer cells. STAT3-siRNA conspicuously decreased the expression of STAT3 protein and inhibited the growth of adriamycin-sensitive breast cancer cells. Compared with the single application of adriamycin, IC50 of adriamycin-resistant breast cancer cells decreased by 4 fold when adriamycin was used in combinaiton with STAT3-siRNA. Meanwhile, an inhibition of the expression of the anti-apoptotic protein mediated by STAT3 was observed in adriamycin-resistant breast cancer cells. CONCLUSION: This study reveals that the development of breast cancer is related to the activation of STAT3. The activation of STAT3 in adriamycin-resistant breast cancer cells was more notable than in adriamycin-sensitive cells. The inhibition of the STAT3 pathway could improve the adriamycin sensitivity of adriamycin-resistant breast cancer cells and lead to their apoptosis. The RESULTS of this study explores the feasibility of the reversal of drug resistance in cancer by blocking STAT3 pathway and establishes the experimental basis for promoting the clinical use of the inhibitors of STAT3 pathway and the chemotherapeutics to overcome the multidrug resistance in cancer.

Construction of a eukaryotic expression vector expressing human IFN-gamma and its inhibitory effect on HBV replication in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the inhibitory effect of a eukaryotic expression vector expressing human IFN-gamma (pcDNA3.1- IFN-gamma) on HBV replication in hepG2.2.15 cells.</p><p><b>METHODS</b>The eukaryotic expression vector expressing human IFN-gamma was constructed using PCR and gene recombination technique. hepG2.2.15 cells were transfected with pcDNA3.1-IFN-gamma and the culture supernatant was collected to determine the expression of IFN-gamma protein by ELISA. The HBV DNA copies and the concentration of HBeAg and HBsAg were measured by fluorescence real-time PCR and ELISA kit, respectively.</p><p><b>RESULTS</b>Compared with that of negative control and blank 2.2.15 cells, the concentration of HBeAg in the supernatant of 2.2.15 cells transfected with pcDNA3.1- IFN-gamma were decreased by 49%, and HBsAg concentration was lowered by 35% and 33%, respectively. A significant decrease of HBV DNA copies was observed in pcDNA3.1- IFN-gamma-transfected cells in comparison with the two control cells. No significant differences were noted in all the results between the two control groups.</p><p><b>CONCLUSION</b>We have successfully constructed the eukaryotic expression vector expressing human IFN-gamma, which provides a basis for anti-HBV gene therapy using human IFN-gamma.</p>