Cuprous oxide nanoparticles inhibit the growth and metastasis of melanoma by targeting mitochondria

Abstract

Metal and its oxide nanoparticles show ideal pharmacological activity, especially in anti-tumor therapy. Our previous study demonstrated that cuprous oxide nanoparticles (CONPs) selectively induce apoptosis of tumor cells in vitro. To explore the anti-tumor properties of CONPs in vivo, we used the particles to treat mouse subcutaneous melanoma and metastatic lung tumors, based on B16-F10 mouse melanoma cells, by intratumoral and systemic injections, respectively. The results showed that CONPs significantly reduced the growth of melanoma, inhibited the metastasis of B16-F10 cells and increased the survival rate of tumor-bearing mice. Importantly, the results also indicated that CONPs were rapidly cleared from the organs and that these particles exhibited little systemic toxicity. Furthermore, we observed that CONPs targeted the mitochondria, which resulted in the release of cytochrome C from the mitochondria and the activation of caspase-3 and caspase-9 after the CONPs entered the cells. In conclusion, CONPs can induce the apoptosis of cancer cells through a mitochondrion-mediated apoptosis pathway, which raises the possibility that CONPs could be used to cure melanoma and other cancers.

Figure 1

Anti-tumor effects of CONP therapy on subcutaneous melanoma and metastatic lung tumors. (a) Representative images of stripped subcutaneous tumors. The diameters of the subcutaneous tumors of the CONP group were obviously smaller than the diameters observed in the glucose group. (b) Representative images of mice bearing subcutaneous melanoma from the same study at day 12. The tumors of the CONP group were significantly smaller than the tumors of the glucose group. (c) Plot of tumor mass versus time. Day 0 was the starting of the treatment. The mice bearing subcutaneous tumors were euthanized when exhibiting signs of illness or death. The tumor masses of the deceased mice were not included after the day of death. Each group initially contained six mice. The error bars represent±S.D. The tumors of the CONP group were significantly smaller when compared with the tumors of the control mice (*P<0.05, for day 16; **P<0.005, for days 4 and 12; and ***P<0.001, for day 8; Student's paired t-test, n=6). (d) Survival plot of mice bearing subcutaneous tumors. The survival of mice treated with CONPs was significantly longer than the mice in the glucose group (*P<0.05, log-rank test, n=6). Day 5 was the first day of CONPs injection. (e) TUNEL (terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling)-stained assay of the subcutaneous tumors of CONP-treated mice on day 8. The image shows that CONPs induced the apoptosis of the tumor cells in vivo (as indicated by pink arrow). (f) TUNEL-stained assay of the control mice in the glucose group. (g) Statistical analysis of the positive cells in the TUNEL assay showed that the CONPs induced apoptosis of the tumor cells in vivo (*P<0.05, Student's t-test). (h) Representative lung images from mice in the metastatic lung tumor experiment. The average diameter of the lung tumors of the CONP group was significantly lower than the diameter of the tumors of the glucose group. (i) Number of tumors, counted using an anatomical lens, in the lungs of mice 15 days after the initiation of treatment. Regions of lung metastasis, which were >0.1 mm in diameter, were recorded. Treatment with CONPs significantly reduced the number of metastases (*P<0.05, Student's paired t-test, n=6)

Figure 2

Assessment of toxicity and clearance of CONPs. (a) The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), and blood creatinine levels in the treatment group were the same as in the control group (n=4). (b) HE-stained images of the major organs, showing that CONPs at a total dose of 6 mg/kg 1 day did not cause significant histological changes. (c) Mice were injected via the vena caudalis at a dose of 2 mg/kg. Four hours after the injection, the concentration of copper in the tested organs increased obviously. Seven days later, the concentration of copper in the tested organs had returned to normal, which suggested that the CONPs had been effectively cleared by the mice (n=4). (d) Plot of weight changes in the mice bearing metastatic lung tumors. The weights of the CONP group were stable, whereas the weight of the mice in the glucose group decreased dramatically from day 5 to day 15 (*P<0.05, Student's paired t-test), which indicated that the CONPs inhibited tumor growth with low systemic toxicity (n=6). (e) The levels of serum ALT, AST, ALB, and blood creatinine in the mice with the metastatic lung tumors remained normal after treatment with CONPs and glucose for 15 days (all P>0.05, Student's paired t-test, n=6)

Figure 3

Determination of subcellular localization of CONPs by TEM. (a) CONPs entered the cells by endocytosis, were transported in a vesicle, and caused mitochondrion swelling. (b) CONPs broke the membrane of the mitochondrion and caused mitochondria swelling. (c) A vesicle containing CONPs touched the outer membrane of a mitochondrion. (d) CONPs entered a mitochondrion. (e and f) CONPs caused apoptosis of B16-F10 cells significantly. The cells showed obvious apoptotic phenotypes. (M, mitochondrion; CONPs in the cells were marked by the blue arrowheads)

Figure 4

Mitochondrion-mediated apoptosis signaling pathway assay. (a) Fluorescence microscopic images of the B16-F10 and HeLa cells treated with CONPs (10 μg/ml) for 5 h. The red fluorescence intensity of the cells in the CONP group was weaker than in the control group. (b) Flow cytometry showed that the mitochondrial membrane potentials of the B16-F10 and HeLa cells decreased significantly after treatment with CONPs for 5 h. (c) Western blot assay of Cyt C release. The level of Cyt C increased in the cytoplasm and decreased in the mitochondria of the B16-F10 and HeLa cells treated with CONPs (5 μg/ml) for 3 or 5 h compared with the control (0 h). This finding indicated that the CONPs caused the release of Cyt C from the mitochondria. (d) Western blot assay of caspase-3 and caspase-9 activation. Caspase-3 and caspase-9 cleavage occurred in the B16-F10 cells and HeLa cells treated with CONPs. (e) ROS in the B16-F10 and HeLa cells were upregulated substantially and in a dose-dependent manner after being treated with CONPs for 5 h

Figure 5

CONPs can significantly reduce the growth of melanoma in vivo by targeting the mitochondria and initiating the mitochondrion-mediated apoptosis signaling pathway