Anti-Inflammatory Activity of Geldanamycin and Its Derivatives in LPS-Induced RAW 264.7 Cells

Geldanamycin (1) had been isolated as a major compound from Streptomyces zerumbet W14; an endophyte of Zingiber zerumbet (L.) Smith. Two new geldanamycin derivatives; 17-(tryptamine)-17-demethoxygeldanamycin (2) and 17-(5’-methoxytryptamine)-17-demethoxygeldanamycin (3) were synthesized and their anti-inflammatory activity was evaluated in LPS-induced macrophage RAW 264.7 cells by investigating their effects on the inhibition of production of NO, PGE2, TNF-α, IL-1β, IL-6 and IL-10. The data obtained were consistent with the modulation of TNF-α, IL-1β, IL-6, IL-10 production by these derivatives at concentration of 1 to 5 μg/ml. A similar effect was also observed when LPS-induced NO release and PGE2 production were tested. The inhibitory effects were shown in concentration-dependent manners. From the obtained results, it was concluded that two new geldanamycin derivatives possess anti-inflammatory activity on LPS-induced RAW 264.7 cells. They could be useful for the management of inflammatory diseases.

Advances in Microbiology macrocyclic ansamycin bridge. The target of geldanamycin is heat shock protein 90 (Hsp90), it deactivates Hsp90 specifically to inhibit tumor growth or virus replication and also interfere normal function of Hsp90 in normal cells [1]. The synthesized a series of new 17-and 19-modified derivatives of geldanamycin to obtain new types of Hsp90 inhibitor with weaker toxicities and higher efficiencies have been attempting [2]- [9]. In our previous study, geldanamycin had been isolated as a major compound from Streptomyces zerumbet W14; an endophyte of Zingiber zerumbet (L.) Smith. It had in vitro anti-inflammatory activity on LPS-induced RAW 264.7 cells by inhibition of mRNA expression and production of inducible NO synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) [10].
Tryptamine, a monoamine alkaloid containing an indole ring structure was derived by the decarboxylation of amino acid tryptophan. Tryptamine played important biomedical role as neurotransmitter and neuromodulator, vasoconstrictor and vasodilator, antibacterial, antifungal and antioxidant agents [11]. Its modification at different position led to many compounds of pharmacological importance. Recently, tryptamine-gallic acid hybrid molecule have been synthesized as SEGA (3a), which prevented non-steroidal anti-inflammatory druginduced mitochondrial pathology, apoptosis, and gastropathy by blocking mitochondrial oxidative stress, chelating intramitochondrial free iron, and correcting mitochondrial pathology entering into mitochondria [12]. According to this effect, the invention of tryptamine-geldanamycin hybrids has been designed and evaluated the in vitro anti-inflammatory activity. The C17 methoxyl of geldanamycin molecule could allow for the introduction of various nucleophiles, hence geldanamycin from the beginning had been apopular template for semisynthetic analogs [2]- [9].
In this study, a novel 17-substituted geldanamycin derivatives with tryptamine and 5-methoxytryptamine were synthesized and their anti-inflammatory activity was evaluated on LPS-activated macrophage RAW 264.7 cells by investigating their effects on the inhibition of production of NO, PGE 2 , TNF-α, IL-1β, IL-6 and IL-10.

Extraction and Isolation
Streptomyces zerumbet W14 was isolated from the rhizome of Zingiber zerumbet (L.) Smith by surface-sterilization technique and identified by morphological, cultural, physiological and biochemical characteristics and also 16S rDNA gene sequencing as described by Taechowisan et al. [10]. This strain was grown on ISP-2 agar at 30˚C for 14 days and then the culture medium was cut into small pieces that were extracted with ethyl acetate (3 × 500 ml). This organic solvent was pooled and then taken to dryness under rotary evaporation to give a dark brown solid. The solid was separated by column chromatography using silica gel gave a very prominent single spot of pure compound on TLC and was undertaken to investigate on NMR spectroscopy. The spectral data revealed this compound to be geldanamycin (1).

Cell Culture and Sample Treatment
RAW 264.7 murine macrophage cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). These cells were grown at 37˚C in DMEM medium supplement with 10% FBS, penicillin (100 units/ml), and streptomycin sulfate (100 µg/ml) in a humidified atmosphere of 5% CO 2 . Cells were incubated with the compound at increasing concentrations and stimulated with LPS 1 µg/ml for 24 h.

MTT Assay for Cell Viability
Cytotoxicity studies were performed on a 96-well plate. RAW 264.7 cells were mechanically scraped and plated 2 × 10 5 per well on 96-well plate containing 100 µl of DMEM medium with 10% FBS and incubated overnight. The compounds were dissolved in dimethylsulfoxide (DMSO) for stock solution. The DMSO concentrations in all assays did not exceed 0.1%. Twenty-four h after seeding, 100 µl new media or test compound was added, and the plates were incubated for 24 h. Cells were washed once before adding 50 µl FBS-free medium containing 5 mg/ml MTT. After 4 h of inoculation at 37˚C, the medium was discarded and the formazan blue, which formed in the cells, was dissolved in 50 µl DMSO. The optical density was measured at 450 nm. The concentration required for reducing the absorbance by 50% (IC 50 ) compared to the control cells was determined.

Nitrite Assay
Nitrite accumulation, an indicator of NO synthesis, was measured in the culture medium by Griess reaction [13]. Briefly, 100 µl of cell culture medium was

Data Analysis
All calculations were done using the SPSS version15 statistical software package for analysis of the data. The data were presented as means ± SEM values of three independent determinations and statical analysis carried out using one-way ANOVA. Differences were considered to be of statistical significance at an error probability of less than 0.05 (p < 0.05).

Synthesis of Geldanamycin Derivatives
The mass spectral data of geldanamycin and geldanamycin derivatives were carried out by 1 H-NMR, 13 C-NMR as following.

Effects of Geldanamycin Derivatives on NO and PGE2 Production in LPS-Induced RAW 264.7 Cells
LSP caused a significant increase in NO and PGE 2 production when compared with the blank control, geldanamycin and its derivatives at concentrations of 1, 2.5 and 5 µg/ml caused a significant reduction in NO and PGE 2 production when compared with LPS-induced control group (p < 0.05). In detail, the pro- 13.27 ± 3.10 ng/ml, respectively, while the production of PGE 2 in the group treated with LPS only was 51.75 ± 6.56 ng/ml. Therefore, the inhibitory levels of geldanamycin and its derivatives on NO and PGE 2 production also showed a dose-dependent manner (Figure 4 (NO) and Figure 5 (PGE 2 )).

Effects of Geldanamycin Derivatives on Pro-Inflammatory Cytokine Production in LPS-Induced RAW 264.7 Cells
In this study, data showed that geldanamycin and its derivatives decreased pro-        µg/ml) remarkably inhibited in a LPS-induced TNF-α, IL-1β, IL-6 and IL-10 production in a dose-dependent manner.

Discussion
LPS was a typical inflammatory stimulus, which could trigger inflammation and activate the cellular signal transduction, and caused the intracelluar cascades [16]. LPS activates macrophages and detects the characteristics of the cells, which is a common means to evaluate the anti-inflammatory activity of many drugs. Therefore, in this study, LPS was utilized to induce macrophage RAW TNF-α, IL-1β and IL-6 mRNA [10]. In addition, the experimental results also showed that the doses of geldanamycin and its derivatives (1, 2.5 and 5 µg/ml) had no effect on the viability of RAW 264.7 cells experimentally. These results further demonstrated the pro-inflammatory effect of LPS on macrophages, and provided a reliable inflammatory model as well as a proper concentration range of geldanamycin and its derivatives for the follow-up experiments. Inflammatory is a very complex process, including the accumulation of inflammatory cells, proliferation and production of inflammatory mediators and cytokines [17]. Macrophages function by releasing pro-inflammatory mediators and cytokines, such as COX-2, iNOS, MMP-2, MMP-9, NO, PGE 2 , ROS, TNF-α, IL-1β, IL-6, IL-10 and chemokines (chemokine C-C motif ligand 2; CCL2) [18] [19] [20] [21] [22]. These mediators and cytokines play a principal role in inflammatory diseases and processes [23] [24] [25]. Hence, it is an effective means to prevent the occurrence and development of inflammatory response by blocking the excessive production of these inflammatory mediators and cytokines [26] [27] [28]. TNF-α, which can cause pro-inflammatory effects to many types of cells, is produced by macrophages, mononuclear cells and T cells. IL-1β, IL-6 and IL-10 are regarded as an endogenous mediator of LPS-induced fever [29] [30]. This study found that, under certain conditions, geldanamycin and its derivatives can significantly reduce the level of these cytokine productions in LPS-induced RAW 264.7 cells. These results suggested that geldanamycin and its derivatives could play an anti-inflammatory role by inhibiting TNF-α, IL-1β, IL-6 and IL-10 productions. NO and PGE 2 are also produced by inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) respectively [31] [32] [33]. These mediators drive the recruitment and initiation of macrophages and other immune cells to complete a full cycle of inflammation and interfere with metabolic functions [34]. In this study found that LPS can increase the expression of NO and PGE 2 obviously, but if co-incubated with geldanamycin and its derivatives simultaneously, the expression of NO and PGE 2 was significantly decreased and the decrease is dose-dependent. The data demonstrated that geldanamycin and its derivatives can suppress inflammatory response by inhibiting the production of NO and PGE 2 in LPS-stimulated RAW 264.7 cells. In the literatures, geldanamycin has been reported as a potent anti-inflammatory compound that target heat-shock protein 90 and glucose-related protein 96 [35] [36] [37], which act as intracellular chaperones that maintain the structural integrity of cytoplasmic and endoplasmic reticulum-associated proteins, respectively. It binds to, and inactivates the function of, an Hsp90-Hsp70 multichaperone machine [38]. This multisubunit complex maintains the conformation and activity of regulatory kinases (e.g. c-src, cyclin-dependent protein kinase 4 [CDK4], Raf-1, and p38) [39] [40] [41], and nitric oxide synthase (NOS) [42] in eukaryotic cells. Because several Hsp90 substrates promote cellular activation and cell growth by maintanining the structural integrity of kinases such as c-Src, Raf-1, CDK4, extracellular signal-regulated kinase 1, JNK, p38 MAPK, lymphocyte-specific these proteins have potent antiproliferative effects both in vitro and in vivo [53].
Other Hsp90 substrates (e.g. NOS) promote inflammation. Because of this, geldanamycin has potent anti-inflammatory effects [37] [54] by binding to the amino-terminal ATPase domain of Hsp90 and inactivates its function [35] [55]. Inhibition of the function of Hsp90 substrates with the use of geldanamycin has antiproliferative and anti-inflammatory effects [36] [56] [57] [58] [59]. However, geldanamycin was toxic to normal cells, it exhibited hepatotoxicity at its effective concentrations [60], thereby limiting dose regimens should be considered for patient safety with medication administration [61] [62]. As previous studies have verrified that derivatization of geldanamycin by substituting the C17-position of geldanamycin with an amino group has greatly decreased the toxicity while retaining the anti-tumor activity. [63] [64] [65] [66]. According to this effect, numerous geldanamycin derivatives with reduced hepatotoxicity have been designed, and several of them have entered clinical trials to treat patients [67] [68] [69]. In this study, synthetic geldanamycin derivatives devoid of C17 methoxy group by tryptamine and 5'methoxytryptamine substitution could potentially provide new agents with improved toxicity profiles over geldanamycin by increasing the percentage of cell viability with retaining the inhibitory effect of pro-inflammatory cytokines and mediators in LPS-induced RAW 264.7 cells. However, it should be noted that geldanamycin and its derivatives showed to be toxic at the concentration of 10 to 40 µg/ml in LPS-induced RAW 264.7 cells. The reason for this phenomenon is due the benzoquinon moiety which has an affinity for Hsp90 [70]. In this study, the inhibitory effects of geldanamycin derivatives on the productions of proinflammatory cytokines and mediators in LPS-stimulated RAW 264.7 cells were more potent than that of geldanamycin. A literaure review revealed only one study of the anti-inflammotry effects of tryptamine hybrid compounds [71]. The reason for this phenomenon is due to tryptamine group could synergize with geldanamycin in the inhibitory effects on production of inflammatory mediators and proinflammatory cytokines in LPS-stimulated RAW 264.7 cells. In addition, cytotoxic effect of geldanamycin and its derivatives was evaluated in the absence or presence of LPS. When treated alone, these compounds did not affect the cell viability at all concentrations used (1 to 5 µg/ml). The potencies of the inhibitory activities of geldanamycin and its derivatives on RAW 264.7 macrophage cells are expected that these compounds would be useful for the treatment of inflammatory diseases that show the increased expression of proinflammatory cytokines and mediators.
The results obtained here demonstrate that the effects of geldanamycin deriv- results suggest that tryptamine hybrid geldanamycins can inhibit inflammatory response and may be a potential therapeutic candidate for the treatment of chronic inflammation and autoimmune inflammatory diseases. They could be useful for future drug development.