title: Differential cytotoxicity and particle action of hydroxyapatite nanoparticles in human cancer cells.
abstract: OBJECTIVE
  While hydroxyapatite nanoparticles (HAPNs) have been reported to exhibit anticancer effects on several types of human cancer cells, no investigation has been performed to compare their cytotoxicity with different types of cancer cells. The objective of the present study is to investigate the cytotoxic action of HAPNs in different types of human cancer cell and to explore the possible mechanisms involved.
  
  
  METHODS
  Rod-shaped HAPNs were prepared by the aqueous precipitation method and then labeled with fluorescein isothiocyanate to visualize the cellular uptake and distribution. Their cytotoxicity to three human carcinoma cell lines (gastric cancer cells [MGC80-3], cervical adenocarcinoma epithelial cells [HeLa] and hepatoma cells [HepG2], as well as to normal human hepatocyte cells [L-02]) was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was characterized by the changes in nuclear morphology with 4',6-diamidino-2-phenylindole staining and by flow cytometric analysis with Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Furthermore, the activity of apoptotic proteins (caspase-3, -8 and -9), intracellular reactive oxygen species and glutathione levels were analyzed in HAPN-treated cells. The cellular uptake of HAPNs was studied using flow cytometry analysis, and changes in intracellular calcium levels were investigated using the Ca(2+)-sensitive fluorescent dye, fluo-3 AM.
  
  
  RESULTS
  HAPNs significantly inhibited cell proliferation and induced apoptosis of cancer cells with an order of MGC80-3 > HepG2 > HeLa, but had no impact on normal hepatic cells (L-02). The increase in apoptosis was accompanied by the activation of caspase-3 and -9, but not activation of caspase-8. Moreover, HAPN treatment led to reactive oxygen species generation and decreased intracellular glutathione in cancer cells, with the most remarkable reactive oxygen species burst in HeLa cells. The degree of cytotoxicity did not correlate with the cellular uptake efficiency of HAPNs. However, more HAPNs were found inside the nucleus of MGC80-3 cells, and an increase in the intracellular calcium level was observed in all cancer cells, with the highest level also detected in MGC80-3.
  
  
  CONCLUSIONS
  Varying cytotoxicity of HAPNs was observed in different cancer cell types. Our results suggest that possible mechanisms of cytotoxicity in various types of cancer cells could be different. The elevated calcium concentration and nuclear localization of the particles might be the main mechanism of growth inhibition by HAPNs in cancer cells.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
authors.forename: Yuan
authors.initials: Y
authors.lastname: Yuan
authors.forename: Changsheng
authors.initials: C
authors.lastname: Liu
authors.forename: Yuequn
authors.initials: Y
authors.lastname: Wu
authors.forename: Xun
authors.initials: X
authors.lastname: Lu
authors.forename: Jiangchao
authors.initials: J
authors.lastname: Qian
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D017886:Durapatite / Q000737:chemistry / Q000627:therapeutic use*; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry / Q000627:therapeutic use* / Q000648:ultrastructure; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D017382:Reactive Oxygen Species; D017886:Durapatite; D053148:Caspase 3; D002118:Calcium