The Study Of New Platinum Complex That Can Inhibit Proliferation And Migration

Objective: Since cisplatin was discovered and applied clinically, people have synthesized thousands of platinum compound derivertives, among which about 30 species hve been tested in clinical trials, less than 10 were approved, while only 3 were widely accepted and used in clinical, namely carboplatin, nedaplatin and oxaliplatin. Currently, subcellular targeting of platinum complexes has become a hot research topic, to study if drug target to a specific localization can improve therapeutic efficacy. Mitochondria are essential to eukaryotic cell survival, and it plays an important role in physiology and pathology, including ATP production through oxidative phosphorylation, activation of apoptotic and necrotic pathways in vivo when the cells are damaged. Owing to mitochondria that can regulate the basic function of cells, the dysfunction of the mitochondrial has a strong correlation with the occurrence and prognosis of the tumor. In fact, mitochondria of normal cells and tumor cells are different in structure and function due to the fact that the metabolism of tumor cells is relatively strong, therefore tumor cells are more susceptible to mitochondrial injury than normal cells. As the result, mitochondrial targeted anti-cancer drugs have become a major challenge in improving therapeutic affection, which may lead to less drug resistant. Our group successfully synthesized some mitochondrial targeting platinum compounds, and they can significantly damage tumor cells than the traditional drug cisplatin, while the other biological effects have not been studied. This research aims to study on the anti-cancer mechanism and the impact on cell migration and invasion, hoping to find strong anticancer efficacy drugs, but less toxic to normal cells, even to the extent that can inhibit cell migration and invasion.Materials and methods: Mitochondria-targeted platinum complex we synthesized are different from cisplatin which are combined with nuclear DNA, they targeted to mitochondrial DNA. Due to mitochondrial DNA lacking histone and nucleotide excision repair pathway, and our platinum complex also have space steric effect, it may have a potential to reduce resistance. This research has made the following work:(1) Cell viability assay(CCK-8), comparison toxicity of several new platinum compouns of Pt-platin to cisplatin on tumor cells(A549, HeLa, HepG2, MCF7), normal cells(MRC5) and cisplatin resistant lung cancer cells(1549/DDP).(2) Tumor models in vivo were established by injecting 4T1 cells in the back flanks of male mice, When the tumors reached a size of about 100 mm3, Pt-platin were injected into the mice by intratumoral injection, equivalent PBS were served as control, the tumor size was continuous observed for 30 days.(3) Using A549 as cell model, PIP-platin as drug model, the uptake of drug at 37℃ or 4℃ was detected by using flow cytometry and inductively coupled plasma mass spectrometry(ICP-MS).(4) To further study the mechanism of uptake, cells were pretreated with four endocytic inhibitor, such as nacodazole(microtubule inhibitor), chlorpromazine(Rho GTPase inhibitor), chloroquine(inhibit endosome acidification), brefeldin(inhibition of protein transport to Golgi), then the cells treated with PIP-platin, the endocytosis was detected by ICP-MS.(5) To study whether PIP-platin enriched in mitochondria, colocalization between PIP-platin and mitochondria was observed by Laser Scanning Confocal microscope and isolated mitochondria from cells, the uptake of PIP-platin into the cells and the mitochondria was quantified by determination of the Pt content using ICP-MS, the cellular and mitochondria protein concentration of the same sample was determined by BCA Protein Assay Kit.(6) The morphological changes of mitochondria after treatment with PIP-platin was observed by Transmission Electron microscope(TEM).(7) The cell apoptosis and cell cycle rate were detected by flow cytometry.(8) ROS level, mitochondria membrane potential, ATP synthesis, cytochrome c, the ratio of Bax and Bcl-2 were detected to investigate the mitochondrial function after treated with PIP-platin.(9) In addition, 2D and 3D cell model were established, using cell adhesion assay, wound healing assay and transwell assay to study the drug effects on cell adhesion, migration and invasion.(10) Using fluorescence microscopy to observe whether adhesion releated protein ?-catenin gathered in the cell membrane.(11) The protein level of FAK and p-FAK were detected by western blot.(12) To study the affection of PIP-platin on Wnt signaling, and the protein expression of ?-catenin of cell membrane, cell cytoplasm and nucleus in the cell.Results: Cell viability assay indicates that new mitochondria-targeted platinum complex were able to inhibit cell proliferation, in a dose-dependent manner, exhibited higher cytotoxicity against all cell lines with smaller IC50 value compared to cisplatin, particularly against cisplatin-resistant, among all Pt-platin complex, PIP-platin showed relatively lower cytotoxicity(IC50 = 20.25 ?g/mL) against noncancerous cell line MRC5 than other cancer cell lines(~ 7.0-14.0 ?g/mL); In vivo antitumor activity of PIP-platin resulted in a stastically significant inhibition of tumor growth by 77%(p<0.05, n=5), while PBS control group tumor size can hardly changes; Flow cytometry and ICP-MS showed that content of platinum metal in cells increased with time; Nacodazole can inhibit endocytosis, while the other three lysosomal inhibitors did not inhibit the endocytosis. Confocal images showed that PIP-platin colocalized with mitochondria, Pearson’s correlation coefficient is more than 0.8, after mitochondria extraction, ICP-MS results revealed a significant Pt accumulation in the mitochondria at 0.99 nmol Pt/mg mitochondria protein compared to 0.35 nmol Pt/mg total cellular protein. TEM showed that 5 ?g/m L PIP-platin can injure mitochondria with leaky outer membrane and structurally obscure or vanished cristae; PIP-treated cells can produced more ROS than control group; PIP-platin can also decrease mitochondria membrane potential, decrease intracellular ATP synthesis, the release of cytochrome C from mitochondria into the cytoplasm and increase the proportion of Bax/Bcl-2 and arrest the cells in S phase. Apoptosis assay indicated that apoptosis induced by PIP-platin in a dose and time dependent manner; In 2D model, we found that PIP-platin treated cells are difficult to trypsinized and enhanced cell adhesion, wound-healing assay showed that PIP-platin can inhibit migration and invasion, transwell assay also found that PIP-treated cells were hard to migrate, further indicated that PIP-platin can inhibit migration and invasion; 3D cell model recapitulated the actual cellular microenvironment and the results showed that non-treated cells migrated from the spheroid more rapidly with larger expansion area than spheroid dosed with 5 ?g/m L PIP-platin, consistent with transwell and wound-healing assay; Fluorescence microscope images showed ?-catenin accumulated in the cell membrane; FAK protein expression did not change significantly, but phosphorylation of FAK protein p-FAK protin expression have changed, increased first and then decreased; PIP-platin inhibited Wnt signaling; PIP-platin increased cell membrane accumulation, but reduced enter to the nucleus.Conclusion:(1) PIP-platin is more toxic than the classical anticancer drug cisplatin, but less toxic to normal cells.(2) PIP-platin successfully targeted to the mitochondria, colocalized with mitochondria, disrupted mitochondria morphology, such as leaky outer membrane, vanished cristae and so on.Also mitochondria-targeted PIP-platin can induce excessive ROS, reduce mitochondria membrane potential, reduce ATP synthesis, cause cytochrome c release from mitochondria to cytoplasm, increase the ratio of Bax/Bcl-2, arrested cell cycle in S phase. Thus, it is reasonable to assume that PIP-platin can induce concurrent alterations on mitochondrial metabolism.(3) PIP-platin affected the protein level of FAK and p-FAK, resulted in increased cell adhesion and inhibited cell migration and invasion. PIP-platin promoted cell membrane translocation of ?-catenin, but reduced cell nucleus translocation, and inhibited Wnt signaling.