Study On The Radiosensitizing Effect Of Tirapazamine-gold Nanoparticle Compounds On Human Hepatoma HepG2 Cells

Aims:Gold Nanoparticles(GNPs) have increasingly been applied to the area of cancer diagnosis and therapy, showing a great potential and efficacy in recent years. However,mounting evidence exhibits GNPs alone are easily eliminated by the reticuloendothelial system in vivo. In addition, it is necessary to combine GNPs with special drugs so as to achieve a curative effect under some special tumor physiological conditions such as for hypoxic tumor cells. This study was aimed at conjugating Tirapazamine(TPZ), a drug specific to hypoxic tumor cells, with polyethylene-glycolcoated GNP(PEG-GNP) to form new compounds TPZs-PEG-GNP. Subsequently the radiosensitizing effect of TPZs-PEG-GNP exposed to low linear energy transfer(LET)X-rays and high-LET carbon ions was investigated. Moreover, the intracellular mechanism underlying the radiosensitizing effect was explored in order to provide basis data for the clinical application of the combination of TPZ and GNP in radiotherapy, especially in heavy ion cancer therapy.Materials and Methods:Human Hepatoma Hep G2 cells cultured in 2D in vitro were used in this study.First, Tirapazamine-gold nanoparticle compounds(TPZs-PEG-GNP) were synthesized and their UV-vis absorption spectrum at wavelengths from 200 nm to 800 nm were measured with a microplate reader and the TPZs-PEG-GNP uptake kinetics by Hep G2 cells were determined using inductively coupled plasma mass spectrometry(ICP-MS).The influence of TPZs-PEG-GNP on Hep G2 cell viability was subsequently examined by means of the MTT method. In addition, the clonogenic survival assay was used to evaluate the radiosensitizing effect of TPZs-PEG-GNP on Hep G2 cells irradiated with X-rays and carbon ions. The immunofluorescence assay was adopted to analyse the damage effect of TPZs-PEG-GNP on the nuclear DNA of Hep G2 cells exposed to the X-rays. The influence of TPZs-PEG-GNP on cell cycle was detected with a flow cytometer. Furthermore, the intracellular yield of reactive oxygen species(ROS) in Hep G2 cells with TPZs-PEG-GNP exposed to X-rays was determined using DCFH-DA probes. The radiation enhancement effect of hydroxide radical production in ultra-pure water with TPZs-PEG-GNP exposed to X-rays and carbon ions was detected using coumarin-3-carboxylic acid(3-CCA) probes.Results:TPZs-PEG-GNP was almost nontoxic to HepG2 cells. An obvious radiosensitizing effect of TPZs-PEG-GNP on Hep G2 cells was observed with some damages to nuclear DNA under aerobic conditions. The sensitization enhancement ratios of TPZs-PEG-GNP on Hep G2 cells exposed to the X-rays and carbon ions were1.23 and 1.47 at 10% survival level, respectively. G1/S phase arrest was induced by TPZs-PEG-GNP in Hep G2 cells. The yield of intracellular ROS in Hep G2 cells with TPZs-PEG-GNP exposed to the X-rays increased obviously as well as hydroxide radical production in ultra-pure water with TPZs-PEG-GNP exposed to X-rays and carbon ions.Conclusion:TPZs-PEG-GNP, which had hardly toxicity to Hep G2 cells, could elicit an obvious radiosensitizing effect on Hep G2 cells. Moreover, its radiation enhancement effect was greater in Hep G2 cells when exposed to the carbon ions than to the X-rays.The radiosensitizing effect of TPZs-PEG-GNP on Hep G2 cells was achieved via enhancing the yeild of intracellular ROS.