Anticancer Efficacy Of Arsenic Trioxide In Human Nasopharyngeal Carcinoma Cell In Vitro

Arsenic compounds are natural substances that have been used medically for more than 2,400 years in Chinese and Western medicine. However it fell out of use in the mid-20th century because of the unacceptable side effects that occurred at the doses that were thought to be necessary. Since 1990s, Chinese physicians have begun to use arsenic trioxide (As2O3) in treating the patients with acute promyelocytic leukemia (APL), and the results proved to be dramatically effective. As2O3 can degrade chimeric PML-RAR proteins in APL patients and induce cancer cells apoptosis without significant myelosuppression. Further study demonstrated that AsiOs was safe and effective not only on patients with leukemia, but also on patients with many other kinds of malignancies. Recent studies have showed that As2O3 was capable of inhibiting the proliferation and angiogenesis of tumor cells, inducing apoptosis and cell partial cytodifferentiation both in vitro and in vivo. Though As2O3 has been recognized as a promising novel anticancer agentfor some solid cancers, little is known about its therapeutic efficacy in human nasopharyngeal carcinomas. In this study, we assessed the ability of As2O3 to inhibit proliferation of human nasopharyngeal carcinoma CNE-1 cell line in vitro. The apoptotic induction of As2O3 and the arsenic-mediated apoptotic pathway in nasopharyngeal carcinoma cell line CNE-1 was also investigated so as to explore the possible mechanism behind its anticancer effect.MethodsStudies of inhibition of cell proliferation:Human Nasopharyngeal Carcinoma Cell line, CNE-1, was cultured in RPMI-1640 media and exposed to different concentrations of As2O3, cisplatin (DDP) and 5-fluorouraine (5-FU) at different time course. Methyl thiazolyl tetrazolium (MTT) reduction assay was carried out to test the cytotoxicity of As2O3. The states of cell growth and morphology were observed by invert microscope and light microscope. Cell cycle phase distribution was determined by Flow Cytometry (FCM). Immunohistochemical staining method was used to measure the expression of proliferating cell nuclear antigen (PCNA) and hTRT protein.Studies of Apoptosis and related genes expression: The apoptosis phenotype of CNE-1 cells was observed by HE staining under light microscope, Hoechst33342/PI double staining under fluorescent microscopy and transmission electron microscopy. The apoptosis sub-diploid peak was detected by FCM. TUNEL method was used for in site evaluation of cell apoptosis. Apoptosis-related proteins of Bcl-2, Bax and p53 expression were examined byImmunohistochemical staining method.ResultInhibition of cell proliferation:1. Cell survival: As2O3 significantly inhibited the growth of CNE-1 cell in vitro. The proliferation inhibitory effect was superior to that of DDP or 5-FU in same concentration. The IC50 value for As2O3 at 24 h, 48 h, 72 h and 96 h were 3.12 u g/mL, 0.65 u g/mL, 0.12 u g/mL, 0.08 u g/mL, respectively.2. Cell morphology: Cells treated with As2O3 underwent growth arrest. Cells shrunk and floated in culture medium under inverted microscope examination. With HE staining, the cells in control group appeared round or oval, large nuclear-cytoplasm ratio, enlarge nucleoli and mitotic figures. Apoptotic cells were frequently seen in the As2O3 group.3. Cell cycle phase: FCM showed that G2/M phase is increasing in the As2O3.treated cells. This G2/M phase arrest effect was dose and time dependent.4. PCNA and hTRT: The average gray levels of PCNA and hTRT positive cell were decreased gradually when concentration of As2O3 was increased.Apoptosis:1. The morphological changes:(1)HE staining showed scattered apoptotic cell. Cell and nucleus shrinked, cytoplasm strongly eosinophiled, karyorrhexis and apoptotic body formed. (2)Hoechst 33342/PI double stains under fluorescencemicroscope: Using fluorescence microscope with Hoechst 33342/PI double staining, karyopyknosis, chromatin condensation and karyorrhexis we...