Mechanisms On Proliferation Inhibition Of Hepatocellular Carcinoma HepG2Cells By Three-dimensional Nanographene

Hepatocellular carcinoma (HCC) is the fifth most frequent malignant tumors, and the third leading cause of cancer-related mortality in the world. HCC patients are usually diagnosed when the tumor is in an advanced stage which makes patients lose the opportunity for curative surgery. The systemic chemotherapy often fail due to the toxic side effects and drug resistance in tumor. Nano drug delivery systems have been developed to improve the selective toxicity of anticancer drugs by utilizing the enhanced EPR effect and tumor-specific targeting. The three-dimensional nanographene based on a triptycene structure bearing three hexa-peri-hexabenzocoronene (HBC) moieties was synthesized by National Engineering Research Center for Nanomedicine, Wuhan, China. This novel nanomaterial exhibited unique intrinsic fluorescence and supramolecular assembly abilities due to its large π-systems. In this thesis, we compared the biological effect of NG on hepatocellular carcinoma HepG2cells and normal hepatocellular HL7702cells. The main results are as follows:To direct the biological application of NG, NG nanoparticles (NPs) were fabricated by evaporation-induced self-assembly method using THF as solvent. DLS analysis displayed that the hydrodynamic size of these nanoparticles was169nm with polydispersity index (PDI)0.019. Transmission electron micrograph (TEM) showed that NG NPs had a spherical shape with a size around50nm. NG NPs had a strong exciting wavelength range from360nm to420nm with emission peak wavelength at470,500, and556nm when excited at363nm.To explore the potential biological activities, the cytotoxicity induced by NG NPs on HepG2cells and HL7702cells was evaluated by MTT assay. The results showed that the NG NPs could selectively kill HepG2cells against HL7702cells. NG NPs significantly suppressed the colony formation of HepG2cells.Flow cytometric evaluation by Annexin V-FITC/PI staining revealed that apoptosis was the predominant form of cell death induced by NG NPs. Western blot analysis implied that NG NPs induced PARP cleavage in HepG2cells more than in HL7702cells. The raise of y-H2AX level in HepG2cells treated with NG NPs suggested that the apoptosis of HepG2cells was triggered by DNA damage caused by NG NPs.To clarify the mechanism on the selective killing of HepG2cells, the uptake and intracellular translocation of NG NPs was monitored by confocal microscopy and the intracellular fluorescence intensity was quantified by fluorescence spectrophotometery. HepG2cells showed preferential uptake of NG NPs compared with HL7702cells. Furthermore the endocytic parthways between two cell lines were compared by using endocytosis inhibitors. The results indicated that clathrin-mediated endocytosis played a predominant role in the cellular uptake of NG NPs in HepG2cells. Both clathrin-and caveolae-mediated pathways were involved in the uptake of NG NPs in HL7702cells. In order to examine the intracellular localization of NG NPs, specific fluorescent dyes and plasmids for expressing fluorescent protein targeting to organelles were used. The results showed that NG NPs were almost completely colocalized with lysosomes and largely colocalized with Golgi apparatus in HL7702cells. NG NPs in HepG2cells were majorly entrapped in the lysosomes or Golgi apparatus and partially colocalized with mitochondria.The underlying mechanism of selective cytotoxicity of NG NPs was further explored. Oxidative stress is one commonly proposed toxicological mechanism of nanomaterials, so we compared the cellular ROS levels and GSH/GSSG levels in HepG2cells and HL7702cells. Treatment with NG NPs for3h caused a marked increase in ROS levels and the depletion of GSH in HepG2cells. In contrast, very slight increase of ROS levels in HL77021cells was induced. N-acetyl-L-cysteine (NAC) could effectively reverse ROS generation and apoptosis induced by NG NPs in HepG2cells. Furthermore we determined the mitochondrial membrane potential (MMP) in HepG2cells after treatment with NG NPs by using JC-1dye. HepG2cells incubated with10μg/ml NG NPs for3h showed a significant loss of MMP. Co-treatment with5mM NAC attenuated the loss of MMP caused by NG NPs. From these results we assumed that NG NPs transported to mitochondria disrupted the mitochondrial membrane and caused an accumulation of ROS in HepG2cells.ROS stress may induce adaptive stress responses including activation of redox-sensitive transcription factors. The transcriptional activities of nuclear factor κB (NF-κB), activator protein-1(AP-1) and p53were determined by Dual Luciferase Reporter Gene Assay when HepG2cells were treated with NG NPs. The results showed that the transcriptional activities of these transcription factors were activated by NG NPs in HepG2cells and NAC treatment can abolish the activation induced by NG NPs. Furthermore, we determined the role of NF-kB in NG NPs-induced apoptosis in HepG2cells. Wild type IKKα and IKKβ increased the NG NPs-induced activation of NF-κB activity while dominant-negative mutants of IKKa and IKKβ (IKKα K44A and IKKβ K44A) abolished the NG NPs-induced activation of NF-κB activity. Furthermore blocking NF-κB pathway using parthenolide inhibitor or transfection with IKKα K44A and IKKβ K44A sensitizes HepG2cells to NG NPs treatment. These results implicated that IKK/NF-κB signaling pathway was involved in NG NPs-induced apoptosis in HepG2cells and NF-κB played an anti-apoptotic role in this process.In this study, we evaluated the potential of a novel3D NG NPs based on triptycene for liver cancer therapy. The NG NPs showed selective induction of apoptosis of HepG2cells against HL7702cells. The different sensitivity to the NG NPs in the two cell lines was related to the internalization pathways and inherent vulnerability to oxidative stress. Our study provides essential information for further application of NG NPs for cancer therapy and a basis for designing nanomaterials to preferentially kill cancer cells. The assembling mechanism and in vivo behaviour of NG NPs should be further investigated.