A Novel Phenothiazine Derivatives Anti-Proliferative Action on the Colon Cancer Cell Line and Its Immunomodulatory Activity on LPS Activated Mammalian Macrophages

Abstract

Background: In recent years, agents with anti-cancer, immunomodulatory and immunostimulatory potential have gained great importance. In this study, the cytotoxic and anti-prolifative activity of a new 10-[4-(4-chlorobutoxy)butyl]-10H-phenothiazine (OZF2) phenothiazine derivative against a colon cancer cell line as well as its antiinflammatory effects on lipopolysaccharide (LPS)-activated macrophage cell line (RAW 264.7) were examined. Methodology: An N-substituted phenothiazine derivative named 10-[4-(4-chlorobutoxy)butyl]-10H-phenothiazine (OZF2) was synthesized and its molecular structure was confirmed by ¹H Nuclear Magnetic Resonance (NMR) and ¹³C NMR. The molecule contains electronegative oxygen and chlorine atoms in the long alkyl chain attached to the nitrogen atom in the phenothiazine ring. The anti-cancer activity of the OZF2 phenothiazine derivative was evaluated with the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) test on the colon cancer cell line. The anti-inflammatory activity of OZF2 was tested by measuring the levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) released from macrophage cells in the presence and absence of LPS and/or OFZ2 using enzyme-linked immunosorbent assay (ELISA). Quantitative real time polymerase chain reaction (q-RT-PCR) analysis was performed to evaluate expression levels of these cytokines also in presence and absence of LPS and/or OFZ2. Results: OFZ2 had anti-proliferative activity on the colon cancer cell line and anti-inflammatory activity on the mammalian macrophages at subtoxic concentrations. It did not stimulate IL-6 and TNF-α cytokines in the absence of LPS in ELISA. But when stimulated with LPS, a significant decrease in the production level of the IL-6 cytokines was observed, while the TNF-α production was not affected. No change in the gene expression of TNF-α was observed, whereas IL-6 gene expression significantly decreased in the presence of OZF2. Conclusions: The anti-proliferative activity of OZF2 against colon cancer cells makes of it a potential drug candidate for colon cancer research and treatment. It also has the potential to be an anti-inflammatory drug agent that can suppress the production of IL-6-based autoimmune and inflammatory diseases.