Proteasome and NF-κB Inhibiting Phaeophytins from the Green Alga Cladophora fascicularis

Chemical examination of the green alga Cladophora fascicularis resulted in the isolation and characterization of a new porphyrin derivative, porphyrinolactone (1), along with five known phaeophytins 2-6 and fourteen sterols and cycloartanes. The structure of 1 was determined on the basis of spectroscopic analyses and by comparison of its NMR data with those of known phaeophytins. Compounds 1-6 displayed moderate inhibition of tumor necrosis factor alpha (TNF-alpha) induced nuclear factor-kappaB (NF-kappaB) activation, while 2 and 4 displayed potential inhibitory activity toward proteasome chymotripsin-like activation. The primary structure-activity relationship was also discussed.


Introduction
The genus Cladophora (Cladophoraceae) is a group of filamentous green algae that grows widely in eutrophic wastewater and marine intertidal zones.Previous chemical investigations indicated that Cladophora mainly containing sterol derivatives [1][2][3].Terpenes [4] from C. vagabunda in Black Sea and a brominated diphenyl ether from C. fascicularis [5] were also reported.In the course of our investigation of chemical diversity and the bioactive compounds from marine plants, the green alga C. fascicularis was collected from shallow seawater along the coastline of Qingdao, China.The EtOH extract showed moderate cytotoxicity against human tumor cell lines, including Hela and LH-1210 (inhibition rates of 92% and 85% per 20 µg/mL, respectively).Chromatography of this extract resulted in the isolation of six phaeophytin derivatives 1-6, along with eight steroids and six cycloartanes.In this paper, we report the structural elucidation of the new phaeophytin derivative porphyrinolactone (1), and the inhibitory effects of compounds 1-6 against proteasome chymotripsin-like and TNF-α-induced NF-κB activation.

Figure 3.
Effect of 1-6 on 20S proteasome ChT-L activity.20S proteasome was incubated with compounds 1-6 (50 µM of each) and fluorogenic peptide (100 µM) in 100 µL of 20 mM Tris-HCl (pH 7.8), at 37 ºC for 1 hour.The proteasome ChT-L activity was assayed after addition of specific substrate.MG132 (1.0 µM) was used as a positive control.Data were measured by means of ± SD from three independent experiments. .The ubiquitin-proteasome pathway (UPP) is a major nonlysosomal system responsible for intracellular protein degradation in eukaryotic cells [19].The key proteins modulated by the proteasome are those involving in the affection of inflammatory processes, cell cycle regulation, and gene expression.Proteasome inhibition is a potential treatment option for cancer and inflammatory conditions [20][21], NF-κB is a crucial transcription factor mediated by immune responses and cellular growth.It is regulated by interaction with an inhibitor protein named inhibitor of κB (IκB).In quiescent cells, NF-κB presents in the cytoplasm and binds to its inhibitor IκB.When a cell is stimulated by stress, growth factors, radiation, etc; IκB could be phosphorylated and degraded by the UPP, freeing NF-κB to translocate to the nucleus where it activates transcription.Proteasome inhibition could stabilize IκB-NF-κB complex making the NF-κB inactive [22].Thereby, the NF-κB signaling pathway is an important target of proteasome inhibitors for the prevention and treatment drugs against cancer and inflammation.Moreover, NF-κB constitutive activity is one of the most important reasons that leading the MRD (multidrug resistance) of tumor cells [23].Thus, the inhibitor of proteasome and NF-κB activities will be a promising candidate for new anti-inflammation or anticancer drug, especially for MRD tumour cells.

Experimental
General IR spectra were recorded on a Perkin-Elmer Nicol FT-50X spectrometer.UV spectra were measured on a UV-VIS spectrophotometer 756MC. 1 H-, 13 C-NMR and 2D NMR spectra were recorded on Bruker Avance-500 FT NMR or Varian INOVA 500 MHz spectrometers, and chemical shifts were referenced to the solvent CDCl 3 as internal standard.FABMS spectra were measured on a Bruker FTICR APEXII mass spectrometer while HRFABMS were measured on a Bruker Daltonics.Inc. APEX-II FTPICRMS.Column chromatography was carried with silica gel (200-300 mesh), and HF 254 silica gel for TLC was obtained from Qingdao Marine Chemistry Co. Ltd., Qingdao, China.Sephadex LH-20 (18-110 µm) was provided by Pharmacia Co.

Algal material
The green alga Clodophora fascicularis was collected in shallow seawater along the coastline of Qingdao, China, in November 2003.The algal species was identified by Professor Baoren Lu from the Institute of Oceanology, Chinese Academy of Science (CAS).A voucher specimen (no.20031101) is deposited at the Institute of Oceanology Chemistry Department of Marine Algae.

Detection of NF-kB activity
HCS (high-content screening) was used to detect data on multiple parameters in single cells as well as in populations of cells [24], aiming to measure the inhibitory effects of compounds on TNF-α-induced NF-κB activation, i.e. nuclear translocation of NF-κB.The HeLa cells were seeded in 96-well microplates and incubated in 24 h for fluorescence detection (Costar 3603) at 5×10 4 cells/well (100 µL/well).Exponentially growing cells were exposed to 50 µM of compounds 1-6 or PBS (untreated control) and N-carbobenzoxyl-Leu-Leu-leucinal (MG132, 5 µM, positive control), respectively, for 60 min.Afterwards, TNF-α (50 ng/mL, R&D Co.) was added and samples were kept in additional 30 min.The procedure including cell fixation, permeabilization and staining for imaging analysis was prepared according to the manufacturer's instructions for the NF-κB activation kit (HitKit K01-0001-01, Cellomics).The Cytoplasm to Nucleus Translocation BioApplication (software) is capable of measuring the activation and/or translocation of one target at a time at the single cell level.It analyzes images obtained from the HCS Readers (KineticScan™ Reader, Cellomics) and measures differences and ratios of fluorescence intensities between two sub-cellular compartments: the cytoplasm and the nucleus.In Channel 1, nuclei (DNA) stained with the fluorescent dye Hoechst 33342 defines the nuclear region called Circ.In Channel 2 (the target channel), NF-κB is stained with the second antibody conjugated with the fluorophore Alexa Fluor 488 [25].An annular region called Ring is defined in the cytoplasm beyond the nuclear (i.e., Circ) region.The size of the Circ and Ring masks in the target channel can be adjusted appropriately so that the Circ and Ring masks approximate a measure of the nuclear and cytoplasm concentration of the target respectively.The output feature MEAN_CircRingAvgIntenDiffCh2 is an indicator of activation/translocation of macromolecules between the nucleus and the cytoplasm in the target channel at the cell level.This feature reports the difference in average pixel intensity between the Circ and Ring mask regions, so it is selected to calculate the inhibitory rate of the compounds on NF-κB activity, and the rate is expressed as a percentage of negative control.All samples were performed at least duplicate and run in triplicate.

Detection for proteasome ChT-L activity
The enzymatic activity of the proteasome was assayed using fluorogenic peptides Suc-Leu-leu-Val-Tyr-AMC [Suc (succinyl) and AMC (7-amido-4-methylcoumarin) were obtained from SIGMA] for chymotryptic-like (ChT-L) activity [26].20S proteasome (1.0 µg) was purified from human erythrocytes and then incubated with compounds 1-6 (50 µM of each) and fluorogenic peptides (100 µM) in 20mM Tris-HCl (100 µL, pH 7.8) at 37ºC for 1 h, respectively.The fluorescence of released 7-amido-4-methylcoumarin (AMC) was measured by a spectrofluorimeter (Fluostar OPTIMA, BMG Germany) at excitation/emission wavelengths of 380/440 nm.MG132 (1.0 µM), a known inhibitor of proteasome ChT-L activity and 0.1% DMSO were used as positive and solvent control, respectively.By comparison with the fluorescence of solvent control, an inhibition rate for each compound was calculated.All samples were prepared at least in duplicate and run in triplicate.The bioassays mentioned above were performed under the conditions to avoid illumination.