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The sphingomyelin metabolites ceramide and sphingosine are mediators of cell death induced by c-irradiation. We studied the production of ceramide and the effects of exogenous ceramide on apoptosis in LNCaP prostate cancer cells that are highly resistant to c-irradiation-induced cell death. LNCaP cells can be sensitized to c-irradiation by tumor necrosis factor a (TNF-a) and, to a lesser degree, by the agonistic FAS antibody CH-11. TNF-a activated intrinsic and extrinsic apoptosis pathways and increased ceramide and sphingosine levels in irradiated LNCaP cells. CH-11 activated only the extrinsic apoptosis pathways and had a negligible effect on ceramide and sphingosine levels in irradiated LNCaP cells. Exogenous ceramide and bacterial sphingomyelinase sensitized LNCaP cells to radiation-induced apoptosis and had a synergistic effect on cell death after irradiation with TNF-a, but not with CH-11. Cell death effects after exposure to ceramide and irradiation were blocked by the serine protease inhibitor TLCK (Na-p-tosyl-L-lysinechloromethylketone), but not by the caspase inhibitor z-VAD (2-val-Ala-Asp(oMe)-CH 2 F). During LNCaP cell apoptosis induced by exogenous ceramide, we observed activation of caspase-9, but not caspases-8, -3, or -7. The effect of ceramide occurred largely via the intrinsic mitochondrial apoptosis pathway and enhanced TNF-a, but not CH-11 effects on irradiated cells. The data show that ceramide enhanced activation of the intrinsic apoptotic pathway and enhanced cell death induced by TNF-a with or without cirradiation. TNF-a and c-irradiation elevated levels of endogenous ceramide and activated the intrinsic cell death pathway.

LNCaP prostate cancer cells are resistant to induction of apoptosis by g-irradiation and partially sensitive to TNF-a or FAS antibody, irradiation sensitizes cells to apoptosis induced by FAS antibody or TNF-a. LNCaP cell clones stably expressing IkBa super repressor were resistant to apoptosis induced by death ligands in the presence or absence of irradiation. IkBa super repressor expression also increased clonogenic survival after exposure to TNF-a+irradiation, but had no effect on survival after irradiation alone. IkBa super repressor expression blocked the increase of whole cell and cell surface FAS expression induced by TNF-a, but did not effect induction of FAS expression and cell surface FAS expression that resulted from irradiation. In cells expressing IkBa super repressor there was diminished activation of caspases-8 and -7 and diminished production of proscaspases-8 and -7, usually required for death induction in LNCaP cells. Peptide inhibitors of caspase activation complemented the IkBa super repressor inhibition of apoptosis, but peptide inhibitors of serine proteases had no effect on LNCaP cells expressing IkBa super repressor. Moreover, cleavage of a serine protease substrate was induced by treatment of LNCaP cells with TNF-a and irradiation. The data suggest that in LNCaP cells NF-kB mediates a proapoptotic pathway that leads to activation of proapoptotic serine proteases. Cell Death and Differentiation (2002) 9 972 ± 980.

The ability of Tetra-O-methyl nordihydroguaiaretic acid (M4N) to induce rapid cell death in combination with Etoposide, Rapamycin, or UCN-01 was examined in LNCaP cells, both in cell culture and animal experiments. Mice treated with M4N drug combinations with either Etoposide or Rapamycin showed no evidence of tumor and had a 100% survival rate 100 days after tumor implantation. By comparison all other vehicles or single drug treated mice failed to survive longer than 30 days after implantation. This synergistic improvement of anticancer effect was also confirmed in more than 20 cancer cell lines. In LNCaP cells, M4N was found to reduce cellular ATP content, and suppress NDUFS1 expression while inducing hyperpolarization of mitochondrial membrane potential. M4N-treated cells lacked autophagy with reduced expression of BNIP3 and ATG5. To understand the mechanisms of this anticancer activity of M4N, the effect of this drug on three cancer cell lines (LNCaP, AsPC-1, and L428 cells) was further examined via transcriptome and metabolomics analyses. Metabolomic results showed that there were reductions of 26 metabolites essential for energy generation and/or production of cellular components in common with these three cell lines following 8 hours of M4N treatment. Deep RNA sequencing analysis demonstrated that there were sixteen genes whose expressions were found to be modulated following 6 hours of M4N treatment similarly in these three cell lines. Six out of these 16 genes were functionally related to the 26 metabolites described above. One of these up-regulated genes encodes for CHAC1, a key enzyme affecting the stress pathways through its degradation of glutathione. In fact M4N was found to suppress glutathione content and induce reactive oxygen species production. The data overall indicate that M4N has profound specific negative impacts on a wide range of cancer metabolisms supporting the use of M4N combination for cancer treatments.