The NF-kB inhibitors curcumin and DHMEQ exert antitumor synergy with cisplatin in hepatic cancer cells. Analysis of relationship to IL-6 production

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Both curcumin and the novel NF-kB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) exerted significant growth inhibitory and cell death inducing effects in the human hepatocellular cancer (HCC) cell lines (HepG2, HuH-6 and HA22T/VGH) expressing at different degrees constitutively activated NF-kB. Further, the agents sensitized HA22T/VGH to the antitumor effects of cisplatin; the effects of the combinations were additive in the other cell lines. Since interference with an autologous interleukin 6 (IL-6) production by the cancer cells might contribute to explain the synergistic effect, we investigated this aspect. Indeed, at difference of HepG2 or HuH-6, in ELISA HA22T/VGH cells showed to secrete substantial amounts (16.8 ng /10⁶ cells/24 h) of IL-6. Curcumin and DHMEQ strongly decreased this production, by down-regulating, through inhibition of nuclear NF-kB expression and in a caspase-independent way, IL-6 mRNA. Flow cytometry, ELISA, mRNA and Western blotting analyses were carried out to characterize the status of the IL-6 receptor in HA22T/VGH cells. They showed that the cells express the gp130 subunit at their surface, but release only traces of its soluble form. For the IL-6 receptor alpha subunit, the cells produced the mRNAs of both its membrane and soluble form. However, in immunoblotting HA22T/VGH cells exhibited only a very faint content of the same subunit, which, in addition, was neither expressed at the cell surface nor secreted. In MTT assays, incubation with a neutralizing anti-IL-6 antibody for up to 7 days did not affect the growth of HA22T/VGH cells or sensitize them to cisplatin. Also treatment with other specific anti-IL-6 approaches (siRNA or antisense oligodeoxynucleotide) failed to produce this result. Overall, inhibition of an autocrine IL-6 loop did not explain the direct antitumor effects of curcumin or DHMEQ in the HA22T/VGH model of HCC. However, since release of IL-6 by tumor cells is frequent in HCC, especially in its more advanced stages, the use of these agents in the tumor might beneficial also to contrast the adverse systemic effects (e.g. cachexia) of the cytokine.