Novel marine phenazines as potential cancer chemopreventive and anti-inflammatory agents

Abstract

Two new (1 and 2) and one known phenazine derivative (lavanducyanin, 3) were isolated and identified from the fermentation broth of a marine-derived Streptomyces sp. (strain CNS284). In mammalian cell culture studies, compounds 1, 2 and 3 inhibited TNF-α-induced NFκB activity (IC₅₀ values of 4.1, 24.2, and 16.3 μM, respectively) and LPS-induced nitric oxide production (IC₅₀ values of >48.6, 15.1, and 8.0 μM, respectively). PGE₂ production was blocked with greater efficacy (IC₅₀ values of 7.5, 0.89, and 0.63 μM, respectively), possibly due to inhibition of cyclooxygenases in addition to the expression of COX-2. Treatment of cultured HL-60 cells led to dose-dependent accumulation in the subG1 compartment of the cell cycle, as a result of apoptosis. These data provide greater insight on the biological potential of phenazine derivatives, and some guidance on how various substituents may alter potential anti-inflammatory and anti-cancer effects.

Keywords: NFκB; apoptosis; chemoprevention; inflammation; lavanducyanin; phenazines.

Figure 1

Chemical structures of phenazines 1–3.

Figure 2

Effect of phenazines on COX-2 (black) and iNOS (gray) mRNA expression in RAW 264.7 cells. Total RNA was isolated using the TRIZOL^® Reagent method (Invitrogen) from RAW 264.7 cells (2 × 10⁵ cells/well) after treatment with samples. cDNA was synthesized using the RT² First Strand Kit (C-03, SA Biosciences) protocol. cDNA was used for quantitative real time PCRs with fluorescent Power SyBR^® Green PCR master mix (Applied Biosystems), employing GAPDH, iNOS, COX-2 specific primers, and a 7300 Real Time PCR System (Applied Biosystems). The results were derived from two independent RNA preparations employing identical triplicates in each analysis and quantitated using GAPDH as the internal control, following the manufacturer’s instructions. (A) GAPDH standard curve for quantitation of iNOS and COX-2 expression; (B) Levels of COX-2 (black) and iNOS (gray) mRNA expression. The concentration and duration of treatment had no significant effect on the viability of Raw 264.7 cells.

Figure 3

NIM-DAPI cell cycle analysis of HL-60 cells. HL-60 cells (2 × 10⁵ cells/well) were treated with different concentrations of the indicated compounds for 24 h. The media were discarded, and 4',6-diamidino-2-phenylindole solution (NIM-DAPI; Beckman Coulter) was added just before the measurement using a Cell Lab Quanta™ SC (Beckman Coulter) flow cytometer. NIM-DAPI-stained cells were analyzed after excitation. The distribution of cells in each phase of cell cycle was exhibited in a DNA histogram and percentage in subG₁ was analyzed. Results are representative of two experiments in triplicate.