Study On The Inhibition Of Tumor Treated With Metallfullerene And Cisplatin Combination

Water-soluble metallofullerene [Gd@C₈₂(OH)₂₂]_n is a novel nanomaterial, It has attracted considerable attention globally for its low toxicity and high performance for inhibiting tumor. Now it plays an important role in anti-cancer research due to its unique biological properties. Studies focused on anti-cancer mechanisms and toxicity would provide crucial information both theoretically and practically, thus it will be benefit for the further clinical cancer treatment. In our study we characterized the physical properties of the metallofullerene [Gd@C₈₂(OH)₂₂]_n , discussed its anti-cancer activity, toxicity, and ability to circumvent multidrug resistance when combined with cisplatin on the normal mice , H22 ascites tumor bearing mice and H22 solid tumor bearing mice models. Electron microscopy, Dynamic light scattering, Flow cytometry, Mass spectrometry, Histochemistry and Molecular technology were used in this study. Our results demonstrated that:1. [Gd@C₈₂(OH)₂₂]_n have an stable structure in biological systems,providing the feasibility to study the effect of inhibiting tumor.The fine cage-structure of Gd@C82(OH)22 is soluble in aqueous solution, and it can form soft aggregates of up to 50nm. The value of Zeta potential was recorded as -8.52±1.65 mV, and the microcrystalline structure was stable. It also was stable and well dispersed in saline solution and 1640 media, These are preconditions for its inhibiting tumor properties.2. In combination with cisplatin, [Gd@C₈₂(OH)₂₂]_n can improve the inhibitive rate of hepatoma solid tumor of mice comparing with cisplatin alone.These particles can effectively inhibit H22 solid tumor (50% inhibition), but fail to inhibit hepatoma ascites tumor due to highly malignancy of tumor and indirect cytotoxicity to tumor cells via intraperitoneally injection. In addition, the inhibiting effect on H22 solid tumor by [Gd@C₈₂(OH)₂₂]_n in normal mice was better than in the nude mice. It indicates that [Gd@C₈₂(OH)₂₂]_n could inhibit the solid tumor through activating the cellular immunity. In vivo tests show a significant increase in H22 solid tumor inhibition (up to 17%) when cisplatin is combined with [Gd@C₈₂(OH)₂₂]_n, comparing with cisplatin alone.3. For the first time to study the relationship between the iron metabolism and spleen injury initiated by cisplatin. The specificity injury initiated by cisplatin in mice spleen can be related with iron deposited in the spleen macrophages.[Gd@C₈₂(OH)₂₂]_n were not toxic to organism by histochemistry analysis. Low dose cisplatin has no distinguish injury to organs of mice except spleen and could lead to increased deposition of hemosiderin in spleen. The mechanism was found to be that: Cisplatin treatment can provoke erythrocytes damage, then the damaged erythrocytes damaged are endocyted by macrophages in spleen, which increases the iron concentration in macrophages. High iron concentration induced the expression of FPN1, Ferritin and Hepcidin. Ferritin will compete with FPN1 to bind iron thus limiting the excretion of iron, whereas hepcidin joined to FPN1 initiates FPN1 internalization, also resulting in iron reduced excretion. Finally, these two pathways lead to iron deposition in spleen macrophages, which cause damage to spleen. In this study, we established a model for rapid iron depositon in spleen and this fact supports the study of iron metabolism in animal. In addition, the result in which low doses of cisplatin show no distinguish toxicity in organs of mice except to spleen, can be taked in account as a reference in clinic cancer therapy. 4. Study the immunostimulation effects of [Gd@C₈₂(OH)₂₂]_n combined with ciaplatin can provide important data for clinical research.[Gd@C₈₂(OH)₂₂]_n significantly increased the ratio of CD4+ T cell through the detection of flow cytometry and ELISA. In a parallel treatment with normal mice and tumor-bearing mice, comparing with control, the ratio of CD4+ T cells increased 21.9% and 62.6%, respectively. [Gd@C₈₂(OH)₂₂]_n stimulated an increasing secretion of IL-2 and IFN-α, and they promoted solid tumor immunoinhibition through proliferation increasing of Th1 subtype CD4+ T cells. IL-2 can promote CD8+ T cells proliferation thus enhance the cytoimmunotoxicity to tumor cell. Meanwhile [Gd@C₈₂(OH)₂₂]_n stimulate the proliferation of macrophages, increase TNF-αsecretion, then kill the tumor cells. Cisplatin alone can not enhance immunoresponse and strongly inhibit those effects. But when the cisplatin was combined with [Gd@C₈₂(OH)₂₂]_n , immune system was notably activated, which indicated that [Gd@C₈₂(OH)₂₂]_n could utilize as immunostimulator combined with chemotherapy agents in clinical treatments.5. [Gd@C₈₂(OH)₂₂]_n can effectively circumvent multidrug resistance in cancer combined with cisplatin, relating with intracellular cisplatin concentration increase through endocytosis reinforced in resistant tumor cells.Cisplatin combined with [Gd@C₈₂(OH)₂₂]_n can effectively kill cisplatin resistant tumor cells. The endocytosis inhibition tests proved that, [Gd@C₈₂(OH)₂₂]_n circumvented multidrug resistant tumor cells through enhancing endocytosis of the tumor cell, increasing intracellular cisplatin concentration, thus promoting cisplatin cytotoxicity in resistant cells. Conclusion: [Gd@C₈₂(OH)₂₂]_n can significantly inhibit hepatoma solid tumor in mice, but fail to treat hepatoma ascites tumor. Cytotoxicity tests confirmed that the nanoparticles have no direct toxicity to H22 tumor cell. The following mechanism study revealed that [Gd@C₈₂(OH)₂₂]_n can stimulate immune system through promoting the Th1 CD4+ T cell proliferation and they can increase the secretion of IFN-γ, IL-2 and TNF-α, leading to tumor inhibition. These nanoparticles in combination with cisplatin can notably enhance the effectiveness of the treament, activating immune system and reducing systematic toxicity. In vitro tests with cisplatin resistant cell lines indicated that combination therapy of cisplatin and [Gd@C₈₂(OH)₂₂]_n can effectively circumvent drug resistance in PC-3 cisplatin resistance cell lines probably through enhancing endocytosis to increase intracellular cisplatin cancentration, which leads to enhanced cytotoxicity. Low dose of cisplatin generate severe spleen damage, although it may not be that toxic to the other organs. The main phenomenons of severe spleen damage were dramatically shrinking, increasing hemociderin deposition and splenic iron accumulation, significant up-regulation of FPN1 and Ferritin. These results indicat that cisplatin interference in spleen iron metabolism may be of great importance to spleen damage.