| Objective:To examine the efficacy and safety of PEM, ZA and combination with PEM with ZA for fighting against TNBC (Triple Negative Breast Cancer) both in vitro and in vivo. To clarified the expression of Mina53 genes in TNBC and its impacts upon biological behavior of TNBC. Besides, to discuss the synergistic antitumor mechanism of PEM combined with ZA on TNBC.Methods:1) Inhibitory effects of PEM and ZA on growth of MDA-MB-231-fLuc-GFP cells in TNBC were tested with CellTiter 96(?) AQueous One Solution Cell Proliferation Assay. The most appropriate concentrations were selected to verify if combined use of PEM and ZA was effective for synergistically inhibiting the growth of MDA-MB-231-fLuc-GFP cells in TNBC. Impacts of PEM, ZA and their combination upon cell cycle and apoptosis of MDA-MB-231-fLuc-GFP cells in TNBC were tested by PI staining and Annexin V-FITC/PI double staining with a flow cytometer. Effects of PEM, ZA and their combined use on mRNA expressions of CyclinDl, MMP2, VEGF, Bax and Bcl-2 genes in MDA-MB-231-fLuc-GFP cells of TNBC were tested with real-time PCR.2) A xenograft model was subcutaneously constructed for TNBC of nude mice. Changes to tumor volume were measured with a vernier caliper and changed BLI signals of cancer were acquired with a small animal imager, in order to compare the effects of PEM, ZA and their combined use for fighting against TNBC in vivo. By observing changes to nude mice’s behaviors, movements and weight, safety of such drugs was evaluated. The impacts of PEM, ZA and their combined use upon protein expressions of CD31 and CD11b in cancer tissues were observed through immunohistochemical tests.3) mRNA expressions of Mina53 genes in MDA-MB-231-fLuc-GFP, MCF-7, SK-BR-3 and MCF-10A cell lines were tested through real-time PCR. Protein expressions of Mina53 in MDA-MB-231-fLuc-GFP, MCF-7, SK-BR-3 and MCF-10A cell lines were tested by Western blot. Expressions of Mina53 genes of MDA-MB-231-fluc-GFP cells transfected by siRNA-Mina53 were inhibited with RNA interference (RNAi), and the transfection efficiency of siRNA-Mina53 was tested with real-time PCR. Meanwhile, the transfection concentration optimal for inhibiting expression of Mina53 genes in MDA-MB-231-fLuc-GFP cells of TNBC was selected for siRNA-Mina53. The transfection efficiency of siRNA-Mina53 was tested via Western blot and the optimal transfection concentration most appropriate for inhibiting expression of Mina53 protein in MDA-MB-231-fLuc-GFP cells of TNBC was determined. Impacts of Mina53 gene silencing (siRNA-Mina53) upon MDA-MB-231-fLuc-GFP cell proliferation in TNBC were experimentally tested with CellTiter 96(?) AQueous One Solution Cell Proliferation Assay. Effects of Mina53 gene silencing (siRNA-Mina53) on cell cycle and apoptosis of MDA-MB-231-fLuc-GFP cells in TNBC were tested by PI staining and Annexin V-FITC/PI double staining with a flow cytometer. Influences of Mina53 gene silencing (siRNA-Mina53) on migration and invasion ability of MDA-MB-231-fLuc-GFP cells in TNBC were assessed through Transwell assay. A cell adhesion experiment was performed to investigate impacts of Mina53 gene silencing (siRNA-Mina53) upon cell adhesion ability of MDA-MB-231-fLuc-GFP in TNBC. Impacts of Mina53 gene silencing (siRNA-Mina53) on related gene expressions in MDA-MB-231-fLuc-GFP cells were tested with real-time PCR.4) mRNA expressions in Mina53 genes in groups of PEM, ZA and their combined use were explored with real-time PCR. Mina53 protein expressions in these three groups were examined through Western blot. Mina53 protein expressions in cancer tissues of such three groups were tested by immunohistochemistry.Results:1) Both PEM and ZA were effective for inhibiting MDA-MB-231-fLuc-GFP of TNBC. Dependent upon concentration, their inhibitory effects were strengthened with the increase in concentration. In vitro experiment, PEM might best inhibit MDA-MB-231-fLuc-GFP at a concentration of lOμmol/L, and ZA would achieve the best inhibitory effect at a concentration of 100μmol/L. As PEM was combined with ZA, the cell activity was significantly weakened as compared with separate use of drugs (P<0.05), and the combination index (CI) equaled to 0.325 for combined use of PEM and ZA, which was lower than 1 and indicated that their combination was synergistically effective for proliferation of MDA-MB-231-fLuc-GFP cells in TNBC. Compared with the Control Group, PEM, ZA and combination of PEM with ZA could increase the proportion of MDA-MB-231-fLuc-GFP cell in the sub-G0/G1 phase and G0/G1 phase, whereas the proportion of cells blocked prior to G1 phase was significantly higher in the group where PEM was combined with ZA than the group where drug was separately used (P<0.001). Meanwhile, the proliferation index (PI) was 0.51889 when drugs were combined, which was significantly lower than the Control Group and the group where drug was separately used. Compared with the Control Group, significant increase was observed in total apoptosis when PEM was separately used and combined with ZA (P<0.01, P<0.001). When ZA was separately used, the total apoptosis didn’t increase significantly (P>0.05), whereas it was higher when PEM was combined with ZA than the group only using PEM (P<0.05). Expressions of CyclinDl, MMP2 and VEGF genes in MDA-MB-231-fLuc-GFP cells of TNBC were lowered no matter PEM or ZA was used or they were combined. Besides, lowered level of expressions was significantly higher in the group where drugs were combined than the group where drugs were separately used (CyclinDl, P<0.05; MMP2, P<0.01; VEGF, P<0.05). Compared with the Control Group, the ratio of Bax to Bcl-2 could be significantly increased when PEM (P<0.05) was separately used or applied in combination with ZA (P<0.001). Furthermore, increased level was significantly higher when PEM was used together with ZA than the group where PEM was separately used (P<0.001). Nevertheless, the in vivo experiment proved that compared with the Control Group, PEM and ZA didn’t have any significant impacts on behaviors, movements and weight of nude tumor-bearing mice by the end of observation. The growth of TNBC could be inhibited by PEM, ZA or their combined use. For the group where PEM was used in combination with ZA, 86.942±3.695% of tumor volume was inhibited at maximum, which was significantly higher than the group where PEM or ZA was separately used (53.238±5.513%, 31.506±6.359%). The differences between any two of them were statistically significant (P<0.001, P<0.001). Additionally, the inhibitory rate was higher in the group where PEM was combined with ZA than the sum of inhibitory rate when PEM or ZA was separately used. By the end of observation, the strength of BLI signals was the highest in the Control Group than other groups whenever PEM or ZA was used separately or combined (P<0.05, P<0.05, P<0.01). It was lower in the group where PEM was combined with ZA than the group where drugs were applied separately (P<0.05). In the experiment, all immunohistochemical results of all groups’tumor tissues suggested that both PEM and ZA could reduce expressions of CD31 protein in tissues, while the lowered level was more significant when both drugs were used in combination. ZA was apparently effective for inhibiting CD11b protein expressions in tumor tissues, whereas PEM didn’t play a significant inhibitory role.3) Compared with normal mammary epithelial cells, the expression of Mina53 gene was significantly increased in the breast cancer cell line (P<0.001), and it was significantly higher in MDA-MB-231-fLuc-GFP cells of subtype breast cancer cell line in TNBC than that in other subtypes (P< 0.001). siRNA-Mina53 could be effectively transfected to MDA-MB-231-fLuc-GFP cells to inhibit expressions of Mina53 genes. There were no significant differences in expressions of Mina53 genes between the Blank Control Group (normal MDA-MB-231 cells) and the negative Control Group (P<0.001). It was verified that the concentration would be most appropriate for transfection when siRNA was mixed with Mina53 at a ratio of 1/2. Compared with the Control Group, the cell proliferation rate began to significantly decline since the 48th hour as the target cell MDA-MB-231-fLuc-GFP was transfected by siRNA-Mina53 to express silenced Mina53 gene (P<0.001). The proportion of cells blocked in sub-GO/G1 phase to those in G0/G1 phase significantly increased (P<0.001) and total apoptosis significantly increased (P<0.001). Compared with the Control Group, the mRNA expressions of CyclinDl, MMP2 and VEGF were significantly lowered after target cell MDA-MB-231-fLuc-GFP was transected by siRNA-Mina53 to express silenced Mina53 genes (P<0.0001). Nonetheless, a significant increase was observed in the proportion of mRNA expressions between Bax and Bcl-2 (namely the expression level of genes Bax/Bcl-2 related to apoptosis) (P<0.001).4) Expressions of Mina53 genes in MDA-MB-231-fLuc-GFP cells were significantly lower when they were treated by PEM or combination of PEM and ZA than the Control Group (P<0.001, P<0.001). The lowered level of expressions wasn’t significant in the group where ZA was used (P>0.05), and mRNA expressions of Mina53 were significantly lower in the group where PEM was used in combination with ZA than the group where only PEM was used (P<0.001). As to cells and rumor tissues subsequently treated by different drugs, identical results were obtained in detecting the level of Mina53 protein expressions.Conclusions:1. All in vitro and in vivo experiments demonstrated that combined use of PEM and ZA had synergistic effects for fighting against TNBC. They suggested that their combination was safe within certain range of concentration. Besides, nude tumor-bearing mice were considered to be able to well tolerate combined use of PEM and ZA without any serious side effects.2. High-level Mina53 expressions were observed in breast cancer cells as compared with normal mammary epithelial cells. Besides, Mina53 was found to have higher-level expressions in subtypes of breast cancer cells of TNBC than other pertinent subtypes. Silenced target gene Mina53 could significantly reduce and weaken proliferation, migration, invasion and adhesion abilities of MDA-MB-231-fLuc-GFP cells in TNBC while imposing impacts on expressions of CyclinD1, MMP2, VEGF and Bax/Bcl-2 in cells. Mina53 was suggested to be potential for becoming a target gene for treating TNBC.3. The synergistic mechanism of the combined use of PEM and ZA for fighting against TNBC was possibly as follows:PEM could inhibit expression of Mina53 in TNBC, while decreased level of Mina53 expressions would lead to lower-level expressions of CyclinDl, MMP2 and VEGF, etc in TNBC as well as increased proportion of mRNA expressions between genes Bax and Bcl-2, namely two genes related to apoptosis, in order to play their roles in fighting against TNBC by reducing and weakening proliferation, migration, invasion and cell adhesion abilities of cancer cells. ZA decreased the expression level of factors such as MMP2 and VEGF, secreted and released by M2 macrophages which were positively expressed by CD11b under micro-environment in cells of TNBC by reducing the infiltration of these cells, so as to impact the cell proliferation directly or indirectly. As PEM was combined with ZA, they would be synergistically effective for fighting against cancer by different acting mechanisms.4. ZA was preliminarily presumed to possibly make tissues of TNBC more sensitive to PEM, in order to enhance its anti-tumor effects, whereas the specific acting mechanism shall be further defined by experimental research. |
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