The Anti-cancer Activities Of Carboxyamidotriazole And Part Of Its Mechanism

BackgroundCarboxyamidotriazole (CAI) is a non-cytotoxic anti-cancer drug, which can inhibit proliferation and induce apoptosis of several tumor cell lines. Previous study shows that the mechanism of action is the inhibition of transmembrane calcium influx, but the molecular mechanism has not been fully understood. The study of pharmacological functions of CAI by our research group shows that CAI not only had favourable anti-cancer activities, but also had anti-inflammation action in a variety of animal models of acute and chronic inflammation via inhibiting the expression of several cytokines (such as tumor necrosis factor-alpha) at the site of inflammation and macrophages.The macrophages play a critical role in the relationship between cancer and inflammation. During the development of tumorgenesis, the macrophages and pro-inflammatory cytokines act as accomplices for the proliferation, migration, invasion and metastasis of neoplastic in the tumor microenvironment. There is also a consensus about the treatment of cancer through macrophages and pro-inflammatory cytokines.As mentioned above, we hypothesize that the anti-cancer and anti-inflammation effects of CAI may function via the macrophages. CAI may not only inhibit the release of pro-inflammatory cytokines of animal inflammation models, but also can inhibit the product of pro-inflammatory cytokines in the tumor microenvironment. Ifthe presumptions above are correct, we could combine a drug which can suppress pro-inflammatory cytokines with CAI in vivo and in vitro, thus increasing the usage and power of C Al.MethodsAdjuvant-Induced Arthritis (AA) Model and LPS induced RAW264.7macrophages inflammation model was established. We analyzed the effect of CAI on TNF-a production in peritoneal macrophages isolated from AArats and LPS induced RAW264.7macrophages using enzyme-linked immuno-sorbent assay (ELISA). TNF-a mRNA expression level was analyzed by RT-PCR assay. Neutral red experiment is used to examine the impact of CAI on the non-specific immune function. DNFB-induced delayed-type hypersensitivity is used to examine the impact of CAI on the cellular immunity. Lewis Lung Carcinoma xenograft Model was established and validated. The effect of CAI on anti-tumor was evaluated through measuring tumor growth, animal body weight, survival, angiogenesis level. Tumor tissues were removed for immunohistochemical experiment. The tumor homogenate was determined by ELISA assay. The macrophages extracted from tumor tissues were stained and indentified by F4/80immuno-fluorescent test. TNF-a mRNA expression level was analyzed by RT-PCR.A549xenograft model in nude mice was established and validated. The effect of CAI, DEX, and their combination on anti-tumor was evaluated using tumor growth and angiogenesis. The specimens of tumor homogenate or peritoneal macrophages were subjected to ELISA for cytokines including:TNF-α and transforming growth factor-beta (TGF-p). CAI and co-administered group (COM Group, CAI and DEX combination group) used with ELISA to detect TNF-a level in tumor conditioned medium induced murine peritoneal macrophages. Using CCK-8assay on Lewis Lung Carcinoma cells (LLC) cultured alone or a co-culture model of mouse peritoneal macrophages and LLC cells to detect how the drug affects LLC cells proliferation. CAI and co-administered group used with ELISA to detect TNF-a level in tumor conditioned medium induced RAW264.7macrophages.The TNF-a and Interleukin-6(IL-6) expression levels in RAW264.7macrophages were tested by ELISA and RT-PCR assay. The migration and invasion of the LLC cells cultured alone or with RAW264.7macrophages were measured with crystal violet agent.Results1. CAI (40umol/L) inhibited the releases of TNF-a from LPS-induced RAW264.7cells (p<0.01). The inhibition rate was35.36%(p<0.01). CAI (10,20μmol/L)suppressed the expression of mRNA for TNF-a.(p<0.01).2. CAI (20mg/kg) suppressed TNF-a production in peritoneal macrophages isolated from rats of adjuvant-induced arthritis (p<0.01)3. CAI at a dose of20mg/kg was proven to be neutral in DNFB-induced contact hypersensitivity assay. CAI (5、10、20and40μmol/L) had little influence of on phagocytosis of rat peritoneal macrophages in vitro.4. CAI (20mg/kg) in Lewis Lung Carcinoma (LLC) xenograft model inhibit the growth of mouse transplanted tumor, blood vessels growth and prolong the survival time. The anti-tumor effect of CAI combined with low dose DEX (1mg/kg) was more than that of single drug respectively. The addition of DEX did not influence the body weights.5. In20mg/kg CAI group, the TNF-a level in tumor was decreased. The inhibitory effect of CAI combined with low dose DEX on TNF-a level was more than that of single drug respectively.6. LLC tumor tissue macrophages were identified by staining F4/80. The result of realtime RT-PCR showed that, compared with PEG group, mRNA from LLC tumor tissue macrophages for TNF-a in CAI, DEX and combination group was decreased by75%,88%and91%, respectively.7. In the A549xenograft model, it is observed that combination of CAI and DEX significantly inhibited the tumor growth and blood vessels growth in the tumor tissues.8. The concentration of TNF-α and TGF-β in tumor homogenate and TNF-a production in peritoneal macrophages of A549tumor bearing mice were all decreased by CAI and/or DEX.9. CCK-8tests showed that CAI (5、10、20and40μmol/L) inhibited LLC cells proliferation in a dose and time dependent manner (p<0.01). DEX could hardly affect the proliferation of LLC cells, and the addition of DEX could not enhance the inhibition of CAI.10. CAI (30μmol/L) could suppress TNF-a, IL-6production and increase IL-10production in the macrophages cultured with LLC cells conditioned medium(LCM), and the addition of DEX (10μmol/L) could decreased TNF-α, IL-6to a dramatic low level (p<0.05)11. The proliferation of LLC cells were significantly enhanced while co-cultured with macrophages. Both CAI (30μmol/L) and DEX (10μmol/L) had the ability to inhibit the LLC cells proliferation in the co-culture system (p<0.05). The promoting action of macrophages vanished while treated with both CAI and DEX (p<0.01)12. ELISA assays showed that both CAI (20μmol/L) and DEX (10μmol/L) could decrease the level of TNF-a secreted from LCM-induced macrophages. The combination could decrease it to a lower level. 13. The result of real time RT-PCR showed that the TNF-α,IL-6and IL-1mRNA level in RAW264.7cell treated with CAI, DEX or the combination were much lower than the control group (p<0.01)14. The migration and invasion of LLC cells were all significantly enhanced while co-cultured with RAW264.7cell. Both CAI(30μmol/L) and DEX (10μmol/L) had the ability to inhibit the LLC cells migration and invasion in the co-culture system. The promoting action of macrophages vanished while treated with both CAI and DEX (p<0.05)ConclusionsCAI, not only does it have anti-tumor effects, it also has the ability to anti-inflammation via suppressing pro-inflammatory expression such as TNF-α.CAI can inhibit the production of cytokines in the tumor micro-environment.This explains how CAI indirectly inhibits tumor proliferation, migration, invasion and tumor blood vessel growth.CAI combined with low doses of dexamethasone can enhance the anti-tumor effect, and further reduce the adverse reactions.This way it can strengthen the weakness of CAI when used on its own, because as a stand alone drug it’s not very effective against tumor cells.CAI has both anti-cancer and anti-inflammatory effects and may become a tool to explore the relationship between inflammation and cancer. Through CAI mechanism, we could find the other key substances in the inflammation related cancer, which is significant for basic and clinical research.