Induction of non-apoptotic cell death by oxidative product of opioids

Objectives: Opioids have been used to alleviate the pain of cancer patients, but the mechanism of anticancer action of opioids is unclear. We investigated here which type of cell death is induced by codeinone and morphinone, oxdative metabolites of codeine and morphine, respectively and the cytotoxicity induced by them against human tumor cell lines.

Methods:Cytotoxicity against normal oral human cells (gingival fibroblast HGF, pulp cells HPC, periodontal ligament fibroblast HPLF) and human tumor cell lines (oral squamous cell carcinoma (OSCC) HSC-2, HSC-3, HSC-4, NA, Ca9-22, promyelocytic leukemia HL-60, cervical carcinoma HeLa) was assayed by the MTT method. DNA fragmentation was monitored by agarose gel electrophoresis. Activation of caspase-3, -8, and -9 was monitored by the extent of cleavage of each substrate. Induction of the formation of acidic organelle against HSC-2 and HSC-4 cells was monitored by acridine orange. Changes in fine cell structure were observed under transmission electron microscope (TEM).

Results:Codeinone and morphinone showed higher cytotoxic activity against human tumor cell lines (five OSCC cell lines, one promyelocytic leukemia and one cervical carcinoma) than against normal oral human cells (HGF, HPC and HPLF). Codeinone and morphinone induced internucleosomal DNA fragmentation in HL-60 cells, and activated marginally caspase-3 without activation of caspase-8 and -9 in both HL-60 and HSC-2 cells. TEM demonstrated that morphinone induced mitochondrial shrinkage and production of autophagosomes engulfing the broken organelles in the HL-60 cells. In addition, We observed the accumulation of acidic organelles distributed in the cytoplasm as granular dots, detected by acridine orange staining.

Conclusions: Since both codeinone and morphinone are classified as a,b-unsaturated ketones, the observed phenomena may be specific to a,b-unsaturated ketone family. The present study further substantiates our hypothesis that a,b-unsaturated ketones induce non-apoptotic cell death.