Study On Effect And Mechanism Of Inhibiting Hepatocellular Carcinoma Cells With Hydroxyapatite Nanoparticles

In recent years, there has been a growing interest in nanoparticles due to theirdistinct effect of nanobiology for the prevention and treatment of tumors.Hydroxyapatite (HAP) nanoparticles have good biocompatibility again. Therefore,they have aroused much attention in the research of anti-hepatocellular carcinoma.In this study, three kinds of HAP nanoparticles with different sizes wereprepared by means of homogeneous precipitation. The main measured particle sizes:HAP₁ was between 44.6nm and 86.8nm, HAP₂ was between 301.6nm and 1339.3nm,and HAP₃ was between 843.2nm and 2443.0nm. They were used for the treatment ofhepatocellular carcinoma. The inhibition effects were determined for the 0.14mmol/L,0.28 mmol/L,0.56 mmol/L concentration of HAP₁ nanoparticles and the0.56 mmol/L concentration of HAP₂ and HAP₃ particles by MTT assays, growthcurves and colony formation rate measurements, and morphology observations. Theresults showed that HAP₁ nanoparticles effectively inhibited the proliferation ofhepatocellular carcinoma cells with dose and time dependent, and 0.56 mmol/Lconcentration of HAP₁ nanoparticles was most effective. However, there was nodistinct effect for HAP₂ and HAP₃ particles at 0.56 mmol/L concentration. Theseindicate that the inhibition effect of HAP₁ nanoparticles on hepatoma carcinoma cellsis related to diameter and concentration of particle as well as exposure time, whichprovides evidence for anti-hepatocarcinoma research in vivo and in vitro.Through morphological observation and analysis, it is clear that HAP₁nanoparticles can be easily combined with hepatoma carcinoma cell membrane, and agreat number of HAP₁ nanoparticles used caveolae-mediated endocytosis to enterhepatoma carcinoma cells. However, for HAP₂ and HAP₃ particles, a few of thementered hepatoma carcinoma cells in same way because it is not easy for them to becombined with hepatoma carcinoma cell membrane. The hepatoma carcinoma cellstreated with HAP₁ nanoparticles were observed with fluorescence microscope afterFluo-3 fluorescent probe is used to mark the intracellular free Ca²⁺. The resultsshowed fluorescence was enhanced, which denoted that endochylema promoted degradation of HAP₁ nanoparticles.Having being treated with HAP₁nanoparticles, the hepatoma carcinoma cellswere stained by means of the Feulgen stain, AgNOR stain and immunocytochemistrystain for PCNA, then observed under a hight microscope and quantified with imageanalysis techniques. The results showed that HAP₁ nanoparticles lowered the averageDNA content, AgNOR quantity and PCNA expression in hepatocellular carcinomacells, which from molecular biology angle, further confumed the inhibition effect ofHAP₁ nanoparticles on hepatoma carcinoma cells, and revealed the inhibitionmechanism. Through inhibiting DNA synthesis, mainly inhibiting the PCNA activityof DNA polymerase that was essential in the DNA synthesis process, throughinhibiting rDNA transcription to inhibit rRNA synthesis, further to inhibit proteinsynthesis, and through inhibiting PCNA activity to prolong cell generation cycle,HAP₁ nanoparticles finally inhibited the proliferation of hepatocellular carcinomacells. The flow cytometry detected that hepatoma carcinoma cell were arrested at G1phase of cell cycle.Through these experiments, it can be found that HAP₁ nanoparticles were first asa drug inhibiting proliferation before they induced hepatocellular carcinoma cells todie rather than kill them quickly. Because the degradation product and nanoparticlesthemselves affect the functions of hepatocellular carcinoma cells gradually, theirultrastructure were changed form the partial to the whole. Finally, they were inducedto oncosis.The transplantation tumor models of human hepatoma in nude mice wereestablished and they possessed the morphology characteristics of human hepatoma.By intra-tumor injection of HAP₁ nanoparticles sol, the growth of transplanted tumorswas obviously inhibited. The inhibition rate of tumor growth under HAP₁nanoparticles treatment for 7days and 14days was 77ï¼…and 51ï¼…respectively. Themean survival time for the nude mice bearing cancer was obviously prolonged. Theparaffin sections from transplanted tumor were made and stained by the Feulgen stain,AgNOR stain and immunocytochemistry stain for PCNA methods. The samples wereobserved under the light microscope and The DNA, PCNA and AgNOR werequantified through image analysis techniques. It was found that there was significant decrease of the DNA content, the number of AgNOR granules and the PCNAexpression in hepatoma carcinoma cells, which from molecular biology angle, furtherconfirmed the inhibition effect of HAP₁ nanoparticles on hepatoma carcinoma cells intransplantation tumor, and revealed the inhibition mechanism in vivo.There were HAP₁ nanoparticles to be found in hepatoma carcinoma cellsthrough observing the TEM sample from transplantation tumor. Similar uptakeprocesses are possible for the hepatoma carcinoma cells in vivo to the hepatomacarcinoma cells in vitro, similar degradation of HAP₁ nanoparticles was conducted inthem, and similar effect on function of them by degradation product and nanoparticlesthemselves. The hepatoma carcinoma cells in vivo were finally induced to die. Therewas a different density distribution for HAP₁ nanoparticles in the solid tumors. As aresult, cells were usually caused to die in the apoptosis way or in the autoschizis way.In short, the anti-hepatocarcinoma mechanism of HAP₁ nanoparticles in vivo can bedescribed as follows: HAP₁ nanoparticles inhibited the proliferation of hepatomacarcinoma cells, through inhibiting DNA synthesis, mainly through inhibiting thePCNA activity of DNA polymerase that was essential in the DNA synthesis process,through inhibiting rDNA transcription to inhibit rRNA synthesis, to further inhibitprotein synthesis, and through inhibiting PCNA activity to prolong cell generationcycle. HAP₁ nanoparticles are also likely to induce the differentiation ofhepatocellular carcinoma cells. Futhermore, they can induced these cells' death in theapoptosis way or in the autoschizis way.