Drug-loaded Nano-micelles Of Disulfiram And Cisplatin In Inhibiting The Growth Of Of Triple-negative Breast Cancer Cell Line MDA-MB-231 In Vivo And In Vivto

Background: Triple-negative breast cancers(TNBC), characterized by absence of the estrogen receptor(ER) and progesterone receptor(PR) and lack of over expression of human epidermal growth factor receptor 2(HER-2), have no effective targeted therapy. TNBCs represent a significant treatment challenge because of the poor prognosis. Neoadjuvant chemotherapy in the clinical research results showed that cisplatin(CDDP) can achieve a higher rate of pathological complete remission, which have serious side effects. Some studies have shown that disulfiram(DSF) can inhibit the growth of the triple-negative breast cancer cell lines, and sensitized the traditional chemotherapeutics. However the study of DSF combined with CDDP in treatment of TNBC is still lacking at present. The development of nano-micelles offered a new sight in reducing the side effects of traditional chemotherapeutics. Normal tissues with dense microvascular endothelial gap and complete structure, macromolecular and nano-micelles are hard to pass through the vessel wall. However, in solid tumor tissues, the structural integrity of the newborn vascular is poor, with wider microvascular endothelial gap and obstacle lymphatic circumfluence system. Based on the enhanced permeability and retention effect,the nano-micelles can present passive targeting mechanisms and accumulated in tumor regions. The nano-micelles can significantly reduce the side effects of drugs and improve the distribution of drugs in tumor regions, have a great prospect of clinical application.Objective: The objective of this study was to evaluate the effects of CDDP combined with DSF and CDDP-loaded nano-micelles combined with DSF-loaded nano-micelles on human triple-negative breast cancer cell line MDA-MB-231 in vivo and in vitro, and to reveal the possible mechanism.Methods: 1. MTT assays were used to detect cell viability of MDA-MB-231 treated by DSF, CDDP and their nano-micelles after 72 h. When MDAMB-231 cells were exposed to free CDDP, free DSF and the combined use of free CDDP and free DSF at 24 h, flow cytometry(FACS) was applied to detect cell apoptosis.2. Exponentially growing MDA-MB-231 cells were harvested and a tumorigenic dose of 5×106 cells was injected subcutaneously into 5 weekold female BALB/c-nude mice. When tumors reached to 50 mm3, mice were divided randomly into five groups and were treated respectively with PBS, free CDDP, CDDP-loaded polymeric nano-micelles(PLGPt), DSF-loaded polymeric nano-micelles(L-DSF), or PLG-Pt&LDSF on schedule with 5 mg/kg free CDDP and 15 mg/kg free DSF. The intravenous injection was carried out at the days of 0, 2, 4, 7, 9 and 11. And the weight of mice and volume of tumor were measured before the treatment injections were given.3. Fluorescence microscope was used to observe the fluorescence intensity of intracellular reactive oxygen species(ROS) levels when MDA-MB-231 cells were exposed to CDDP with DSF or not and their nano-micelles at 3 h. And, FACS were performed to detect the intracellular ROS levels. After exposed to CDDP with DSF or not and the nano-micelles 4 h and 24 h, the intracellular platinum( Pt) concentrations were measured by Inductively Coupled Plasma-Mass Spectrometry(ICP-MS).Results: 1. In vitro, DSF combined with CDDP could inhibit MDA-MB-231 cells growth significantly on a mole ratio of 4:60. When achieve the same suppression effect, the dose of CDDP was greatly reduced, with IC50 reduced from 32.012 μM to 16.90 μM. According to the results of FACS, when MDA-MB-231 cells were exposed to CDDP alone and DSF alone, both of them could induce the cell apoptosis, with 28.2% and 21.6%, respectively. The significantly ability in inducing cell apoptosis was observed in the media which was exposed to the combination of CDDP and DSF, with the apoptosis cells increased to 52.6%(P<0.001). And the similar patterns were observed in CDDP-loaded nano-micelles combined with DSF-loaded polymeric nano-micelles or not.2. In vivo, as compared with the control group which the tumor size reached to 297.14 ± 18.85mm3, CDDP group showed a great tumor suppression effect, but serious irreversible weight loss were also observed. And the mice were all died in 5 days after the treatment was stopped. As compared with free CDDP, CDDP-loaded polymeric nano-micelles possessed improved safety and tolerance in vivo. CDDP-loaded polymeric nano-micelles showed obvious tumor suppression effect at the same dose of CDDP, with the tumor size around 115.31mm3, but the weight loss did not appear. It was noting that the combination of CDDPloaded polymeric nano-micelles and DSF-loaded polymeric nanomicelles got more obvious tumor suppression effect which the tumor size were controlled to 67.30mm3 with no weight loss compared with any other treatment group(P<0.05).3. Compared with CDDP alone, the combination of CDDP and DSF induced more ROS as demonstrated by fluorescence microscope and FACS. As compared with CDDP, when DSF were added to the media, the intracellular Pt content increased significantly.Conclusion: 1. It is found that CDDP combination with DSF can inhibit the triple-negative breast cancer cell line MDA-MB-231 which possess the characteristic of breast cancer stem cells, and induce cell apoptosis in vitro.2. In vivo, CDDP-loaded polymeric nano-micelles showed obvious tumor suppression effect at the same dose of CDDP, but the serious weight loss did not appear, and the overall survival was prolonged significantly.3. Both of CDDP-loaded polymeric nano-micelles and DSF-loaded polymeric nano-micelles can significantly inhibit the tumor growth, and the tumor suppression effect was further enhanced when combined both of them.