Inhibition Of Pro-inflammatory Cytokines In Tumor Associated Macrophages Is A Potential Anti-cancer Mechanism Of Carboxyamidotriazole And Can Be Augmented By Low Dose Dexamethasone

BackgoundCarboxyamidotriazole (CAI) is a non-cytotoxic anticancer drug in development. It inhibits proliferation and induces apoptosis of some cancer cells. A series of clinical trails have shown that the adverse effects of the drug are slight comparing with other anticancer drugs, although its effect is still in investigation. It has been suggested that CAI is a voltage-independent calcium channel inhibitor of cancer cells, but the exact anti-cancer mechanism is not quite clear yet.In our previous study, we found that CAI also possessed great anti-inflammation action in a variety of animal models of acute and chronic inflammation. In the adjuvant-induced arthritis (AA) model, the decrease of the pro-inflammatory cytokines at the site of inflammation and in serums by CAI was observed.Considering CAI has both anti-cancer and anti-inflammation activities, we then focused on its effects on macrophages, which were important both for tumor growth and inflammation processes. We wondered if CAI might also have effects on the pro-inflammatory cytokines production in tumor associated macrophages (TAMs), which might be one of the mechanisms for the anti-cancer actions of CAI.MethodsAdjuvant-Induced Arthritis (AA) Model was established and peritoneal macrophages were isolated from AA rats on day 21. Tumor necrosis factor-a (TNF-α) production in these macrophages was measured by ELISA assay. Lewis Lung Carcinoma (LLC) Model was established and the tumor tissues were obtained on day 14. TNF-αand CD31 in the tumor tissues was stained immunohistochemically and the concentration of TNF-a was measured by ELISA assay. The macrophages were isolated from the tumor tissues with collagenase and TNF-a mRNA level in these macrophages was determined by RT-PCR assay. The peritoneal macrophages from C57 mice or RAW264.7 macrophages were co-cultured with LLC cells or LLC conditioned medium. The TNF-a and Interleukin-6 (IL-6) expression in these macrophages was tested by ELISA and RT-PCR assay. In the above tests, dexamethasone (DEX) was combined with CAI as it is known to inhibit the pro-inflammatory cytokines.The proliferation of the LLC cells cultured alone or with macrophages was measured with CCK-8 agent. The migration and invasion of the LLC cells cultured alone or with macrophages was measured with crystal violet agent. The TNF-a neutralizing antibody was used to verify the action of TNF-a.A549 xenograft model in nude mice was established to see whether the great inhibition caused by the combination of CAI and DEX was a much commen effect. The blood vessel growth and TNF-a concentration in tumor tissues were also tested.ResultsCAI suppressed TNF-a production in peritoneal macrophages isolated from rats of adjuvant-induced arthritis.CAI decreased TNF-a in LLC tumor tissues and suppressed the expression of mRNA for TNF-a in tumor tissue macrophages. It also inhibited the angiogenesis in LLC tumor tissues.CAI inhibited TNF-a and IL-6 production in macrophages induced by LLC cells or its conditioned medium. Proliferation of LLC cells was promoted by peritoneal macrophages; DEX enhanced the inhibition of CAI on the proliferation of LLC cells co-cultured with peritoneal macrophages instead of LLC cells cultured alone. RAW264.7 cells promoted LLC cells invasion and the activity was inhibited if RAW264.7 cells were pre-treated with CAI. The activity was also inhibited if the TNF-a neutralizing antibody was added in the co-culture system.DEX did not affect the direct inhibition of CAI on the proliferation of LLC cells cultured alone. But the combination treatment of CAI and DEX significantly inhibited the LLC tumor growth in mice with the survival time prolonged. The addition of DEX did not influence the body weights.In the A549 xenograft model, it is also observed that combination of CAI and DEX significantly inhibited the tumor growth and blood vessels growth in the tumor tissues. The concentrations of TNF-αand TGF-βin tumor homogenates and TNF-αproduction in peritoneal macrophages of A549 tumor bearing mice were all decreased by CAI and/or DEX.ConclusionIn summary, we have found that CAI can not only act on tumor cells directly, but also on macrophages, including those in arthritis model and TAMs. It can suppress TNF-a and IL-6 production, break the "smoldering" inflammation balance in TAMs, and thus inhibit tumor growth indirectly. The findings suggest that CAI exerts anti-cancer activities may be due to its direct actions on both tumor cells and TAMs. The newly found anti-inflammation activity may make CAI one of effective tools to study the relationship between inflammation and cancer, and the combination with low dose DEX may be of great importance for clinical application of CAI.