Objective  This study investigates the in vitro inhibitory action of apogossypolone, a gossypol derivative (ApoG2), combined with ceramide on cell proliferation in human nasopharyngeal cancer cell line CNE-2. The possible mechanism of this tech- nique is also evaluated in this study.

  Methods  ApoG2 and ceramide of different concentrations were applied, individually or simultane- ously, to human nasopharyngeal cancer CNE-2 cells. The cell counting kit-8 (CCK-8) method was used to determine the cytotoxicity and assay the synergetic effect by calculating the value of the coefficient of drug interaction (CDI). Hoechst-33258 staining was con- ducted to observe morphological changes in the cell nucleus. Acridine-orange (AO) staining and transmission electron microscopy (TEM) were employed to observe the morphological alterations in autophagic cells. The apoptosis rate and fluorescence intensity of au- tophagy were determined by flow cytometry (FCM). The expressions of Bcl-2 and Beclinl proteins were analyzed by Western blot.

  Resuits   The CCK-8 assay showed that the inhibitory action of ApoG2 and ceramide was enhanced with increasing drug concentrations, considering the drugs were used alone. With the conjunctive use of ApoG2 and ceramide both under low concentrations, the action would be synergistic (CDI<l). Compared with the control group, Hoechst-33258 staining demonstrated the occurrence of apoptosis in the CNE-2 cells treated with ApoG2 or ceramide, or both. However, the morphological changes in the nuclear condensation and frag- mentation in CNE-2 cells treated by both drugs were most significant. AO staining revealed more bright red acidic vesicular organelles in the combination group. An increase in the number of large vacuoles and double-layered membrane structure was observed under TEM in the combination group. Compared with the other groups, the FCM assay showed increased apoptosis rate and fluorescence in- tensity of autophagy when treated with both drugs. The differences were statistically significant between the single and combined application groups (F_apoptosis=106.72, P_apoptosis=0.000; F_apoptosis=140.77, P_apoptosis=0.000). Western blot analysis showed that Bcl-2 protein expression was downregulated with statistically significant differences between the two groups (F=111.071, P < 0.001). By contrast, Beclin1 expression increased in the combined therapy group compared with the other groups. Statistically significant differences were found among the groups (F=62.271, P < 0.001).

  Conclusion  The combined application of ApoG2 and ceramide at lower concentrations promotes apoptosis and autophagy, and synergistically inhibits the proliferation of human nasopharyngeal carcinoma cells. Such effects may be related to the downregulation of Bcl-2 expression and the upregulation of Beclin1 expression.