# The Extracts of Some Marine Invertebrates and Algae Collected off the Coast Waters of Vietnam Induce the Inhibitory Effects on the Activator Protein-1 Transcriptional Activity in JB6 Cl41 Cells

Authors: S. N. Fedorov; V. B. Krasokhin; L. K. Shubina; S. A. Dyshlovoy; N. H. Nam; C. V. Minh
Journal: Journal of Chemistry
 (2013)

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## Abstract

It has previously been shown that inhibition of the transcriptional activity of the oncogenic nuclear factor AP-1 can result in cancer prevention. Marine invertebrates and alga are a rich source of natural compounds that possess various biological activities. The inhibitory effects of the extracts of Vietnamese marine organisms in relation to the AP-1 transcriptional activity were examined by the luciferase method using JB6 Cl41 cells stably expressing a luciferase reporter gene controlled by AP-1 DNA binding sequence. As was found, 71 species of marine sponges out of 148 species studied contain inhibitors of the AP-1 transcriptional activity. Therefore, marine organisms as a source of biologically active compounds have a great potential for isolation of the new cancer preventive compounds that inhibit the oncogenic AP-1 nuclear factor.


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## Body

## 1. Introduction

The activator protein-1 (AP-1) transcription factor is a heterodimeric complex that contains members of the JUN, FOS, ATF, and MAF protein families. AP-1 activity is induced by a plethora of physiological stimuli and regulates a wide range of cellular processes, including cell migration, proliferation, differentiation, inflammation, apoptosis and survival, transformation, and tumor promotion [1–5]. The upregulation of such AP-1 proteins as c-FOS, FOSB, and c-JUN was found to correlate with a positive effect on cell transformation [6–10]. The AP-1 has increased expression in different cancers including breast, ovarian, cervical, colorectal, lung, bladder, and many others [11–15]. Thus, AP-1 has been considered primarily to be an oncogene. Later, some of the AP-1 proteins, such as Jun-B and c-Fos, were shown to have tumor-suppressor activity both in vitro and in vivo [16, 17]. Activation of another AP-1 protein, c-Jun, is required for the induction of Fas L-mediated apoptosis in PC12 and human leukemia HL-60 cells [18, 19]. Activation of both AP-1 and NF-κB nuclear factors is necessary for DNA damaging agents- and ceramide-induced apoptosis in T lymphocytes and Jurkat T cells [20, 21]. The balance between AP-1 family members, c-Jun, and ATF-2 governs the choice between differentiation and apoptosis in PC12 cells [22]. The ultimate fate of the cells relies on the relative abundance of AP-1 complex, the composition of the AP-1 dimers, cell type, and cellular environment [23].AP-1 transcription factor plays a role in regulatory processes essential for the specific function of cells in the immune, endocrine, nervous, cardiovascular, and other physiological systems [24, 25], and therefore it participates not only in cancer but also in such diseases as cerebral ischaemia, stroke, seizure [26, 27], psoriasis [28], and mastitis [29].Taken together, this suggests that AP-1 is a promising target for prevention and therapy in cancer and many other diseases [30].Many works from our laboratory and other laboratoring clearly suggested that some cancer preventive and therapeutic compounds, including those of marine origin, can inhibit AP-1 activity, whereas other such compounds can induce it. For example, cancer preventive compounds resveratrol from grape skin, actinoporin RTX-A from sea anemones, steroidal glycoside LSG from starfishes are among agents that inhibit AP-1 activity [31–33]. On the other, hand anticancer drug vinblastine, cancer preventive flavonoids kaempferol and genistein, marine natural product 3-demethylubiquinone Q2 from ascidian Aplidium glabrum, and its synthetic analogs induce AP-1 activity [34–39].In this study, thein vitro effects of ethanol extracts of 148 marine invertebrate species (mainly sponges) and algae on the basal AP-1-dependent transcriptional activity in mouse epidermal JB6 Cl41 cells were evaluated.


## 2. Experimental

### 2.1. Marine Invertebrates and Alga

The specimens of marine invertebrates and algae were collected off the coast of Vietnam at a depth of 2–440 m by scuba diving and trawling during the 34th scientific expedition onboard the research vessel “Academic Oparin” in May-June 2007 and were immediately extracted with ethanol. The animals and algae were identified by Dr. V. B. Krasokhin. The sponges having the same names in Table1 belong to one and the same genus, but they may have different specific names, undetermined yet, and were collected at the different places of the coast of Vietnam. The voucher specimens are kept in the collection of G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia.Table 1
The effects of the extracts of the marine invertebrates and alga on AP-1-dependent transcriptional activity in JB6 Cl41 cells.
Num.*
Namex
Conc.£ mg/mL
AP-1¥
O14-34
Callyspongia sp.
0.6
103
O15-34
Oscarella sp.
0.5
94
O16-34
Dysidea sp.
0.5
100
O17-34
Mycale sp.
0.5
134
O18-34
Chondrilla sp.
0.6
110
O19-34
Monanchora sp.
0.125 (0.24)**
40
O20-34
Haliclona sp.
0.6
96
O21-34
Spirastrella sp.
0.4
111
O22-34
Ascidian unidentified
0.8
80
O23-34
Petrosia sp.
0.8
64
O24-34
Demospongiae, unidentified
0.6
88
O25-34
Spirastrella sp.
0.6
41
O26-34
Callyspongia sp.
0.5
71
O27-34
Spongia sp.
0.5
98
O28-34
Mycale sp.
0.8
106
O29-34
Callyspongia sp.
0.6
101
O30-34
Chondrilla sp.
0.6
89
O31-34
Alga unidentified
0.5
98
O33-34
Callyspongia sp.
0.5
89
O34-34
Callyspongia sp.
0.5
70
O35-34
Xestospongia sp.
0.6
29
O36-34
Spongia sp.
0.5
35
O37-34
Spirastrella sp.
0.5
122
O38-34
Demospongiae, Haplosclerida, fam. unidentified
0.5
90
O41-34
Phakellia sp.
0.7
59
O42-34
Halichondriasp.
0.5
74
O43-34
Halichondriasp.
0.4
125
O44-34
Haliclona sp.
0.6
105
O45-34
Halichondriasp.
0.6
75
O46-34
Haliclona sp.
0.6
93
O47-34
Ascidian unidentified
0.6
95
O48a-34
Ascidian unidentified
0.8
79
O48b-34
Ascidian unidentified
0.8
96
O49-34
Echinoidea unidentified
0.8
105
O50-34
Pennatulacea unidentified
0.5
91
O52-34
Alga unidentified
1.0
95
O53-34
Alga unidentified
0.6
76
O54-34
Alga unidentified
0.6
89
O55-34
Alga unidentified
0.7
93
O56-34
Alga unidentified
0.5
169
O57-34
Petrosia sp.
0.5
105
O58-34
Demospongiae unidentified
0.125 (0.27)**
19
O59-34
Haliclona sp.
0.8
74
O60-34
Pachastrella sp.
0.0625 (0.17)**
47
O61-34
Spirastrella sp.
0.5
73
O62-34
Spongia sp.
0.6
71
O63-34
Spongia sp.
0.5
102
O64-34
Stelletta sp.
0.5
62
O65-34
Spirastrella sp.
0.25 (0.35)**
52
O66-34
Dysidea sp.
0.125
77
O67-34
Spongia sp.
0.6
84
O68-34
Algae unidentified
0.5
67
O69-34
Opistobranchia unidentified
0.25
88
O70-34
Opistobranchia unidentified
0.125 (0.22)**
121
O71-34
Opistobranchia unidentified
0.8
96
O72-34
Opistobranchia unidentified
0.5
149
O73-34
Spongia sp.
0.0625 (0.1)**
137
O74-34
Myxilla sp.
0.125 (0.23)**
14
O75-34
Spirastrella sp.
0.4
108
O76-34
Xestospongia sp.
0.5
117
O77-34
Stelletta sp.
0.0625
91
O78-34
Spongia sp.
0.25 (0.54)**
38
O79-34
Callyspongia sp.
0.5
108
O81-34
Gorgonian gen unidentified
0.5
35
O82-34
Gorgonian gen unidentified
0.25
104
O83-34
Ascidia
0.6
91
O84-34
Haliclona sp.
0.6
74
O85-34
Spirastrella sp.
0.8
60
O86-34
Halichondriasp.
0.8
63
O87-34
Spongia sp.
0.8
60
O88-34
Xestospongia sp.
0.6
109
O89-34
Clathria sp.
0.8
105
O90-34
Ascidian unidentified
1.0
105
O92-34
Spongia sp.
0.6
93
O93-34
Spirastrella sp.
0.5
124
O94-34
Spongia sp.
0.6
77
O95-34
Spongia sp.
0.6
105
O98-34
Crinoidea
0.6
71
O99-34
Demospongiae, unidentified
0.8
93
O100-34
Demospongiae, unidentified
0.8
87
O101-34
Demospongiae, unidentified
0.25 (0.4)**
127
O102-34
Spirastrella sp.
0.5
84
O103-34
Petrosia sp.
0.5
54
O104-34
Halichondriasp.
0.5
104
O105-34
Alga unidentified
0.25
99
O106-34
Alga unidentified
0.0625 (0.11)**
169
O107-34
Alga unidentified
0.6
144
O115-34
Crinoidea
0.25 (0.42)**
73
O117-34
Nudibranchia unidentified
0.25 (0.37)**
77
O119-34
Spirastrella sp.
0.6
118
O120-34
Haliclona sp.
0.6
88
O121-34
Demospongiae unidentified
0.6
111
O122-34
Spongia sp.
0.5
38
O123-34
Callyspongia sp.
0.0625
101
O124-34
Axinella sp.
0.6
52
O125-34
Aplysina sp.
0.6
60
O126-34
Spongia sp.
0.6
57
O127-34
Spongia sp.
0.0625 (0.16)**
46
O129-34
Crinoidea unidentified
0.6
84
O130-34
Crinoidea unidentified
0.5
90
O131-34
Crinoidea unidentified
0.125
83
O132-34
Nudibranchia unidentified
0.0625
84
O133-34
Ascidian unidentified
0.25 (0.37)**
73
O134-34
Haliclona sp.
0.0625
110
O135-34
Callyspongia sp.
0.5
135
O136-34
Spongia sp.
0.5
79
O137-34
Demospongiaeunidentified
0.25
84
O140-34
Spongia sp.
0.8
41
O141-34
Clathria sp.
0.8
63
O145-34
Callyspongia sp.
0.8
43
O146-34
Axinella sp.
0.6
33
O149-34
Suberites sp.
0.8
83
O152-34
Lithistida unidentified
0.8
67
O153-34
Halichondriasp.
0.8
102
O154-34
Penaressp.
0.25 (0.34)**
37
O155-34
Petrosia sp.
0.8
67
O156-34
Pheronema raphanus
0.0625
90
O157-34
Pachastrella sp.
0.8
49
O158-34
Lithistida  unidentified
0.8
75
O159-34
Geodiasp.
0.125
98
O160-34
Demospongiae unidentified
0.8
55
O162-34
Halichondriasp.
0.8
60
O163-34
Spirastrella sp.
0.5
101
O165-34
Petrosia sp.
0.8
53
O167-34
Rhabdastrella globostellata
0.5
37
O168-34
Petrosia sp.
0.125
81
O169-34
Halichondriasp.
0.25 (0.48)**
36
O170-34
Acanthodendrilla sp.
0.0625 (0.1)**
53
O171-34
Callyspongia sp.
0.8
30
O172-34
Carteriospongia sp.
0.0625 (0.11)**
19
O173-34
Carteriospongia sp.
0.25 (0.32)**
76
O174-34
Axinella sp.
0.25 (0.41)**
33
O175-34
Halichondriasp.
0.25 (0.33)**
66
O176-34
Petrosia sp.
0.125
110
O177-34
Spongia sp.
0.125 (0.19)**
54
O178-34
Petrosia sp.
0.8
56
O179-34
Hyalonema sp.
0.25
80
O181-34
Porifera, Lithistida unidentified
0.5
43
O186-34
Porifera,  Calcarea, unidentified
0.6
55
O187-34
Callyspongia sp.
0.0625
101
O188-34
Agelas sp.
0.8
50
O189-34
Axinella sp.
0.0625 (0.16)**
67
O192-34
Agelas sp.
0.8
47
O193-34
Petrosia sp.
0.5
42
O194-34
Amphimedon sp.
0.25 (0.39)**
42
O196-34
Spongia sp.
0.125 (0.23)**
60
O197-34
Axinella sp.
0.5
60
O198-34
Plakina sp.
0.5
41
*The number of the voucher specimen.
xSystematical name of the specimen.
£Concentration of the extract dry residue in the MEM.
¥AP-1-dependent transcriptional activity, percentage of the control.
**IC50.


### 2.2. Cell Culture

The mouse epidermal JB6 Cl41 AP-1 cells were cultured in monolayers at 37C and 5% CO2 in MEM containing 5% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin [40].


### 2.3. MTS Cytotoxicity Assay

Each ethanol extract of a marine invertebrate or alga was evaporated to dryness. Then, the solutions of the dry extract residue in 5% FBS/MEM with the residue concentrations of 1.0; 0.9; 0.8; 0.7; 0.6; 0.5; 0.4; 0.25; 0.125; and 0.0625 mg/mL were prepared. The effect of these solutions  on JB6 Cl41 AP-1 cells viability was evaluated using MTS reduction into its formazan product [41] as described in [42]. In brief, the JB6 Cl41 AP-1 cells were cultured for 12 h in 96-well plates (6,000 per well in 0.1 mL of 5% FBS/MEM). The media was then replaced with fresh 5% FBS/MEM containing the studied substances at various concentrations in a total volume of 0.1 mL and the cells were incubated with obtained solutions  for 22 h. Then, 20 μL of the MTS reagent was added into each well and MTS reduction was measured 2 h later spectrophotometrically at 492 and 690 nm as background using the Multiskan MS microplate reader (Labsystems, Finland). The solution with the first nontoxic concentration (more than 80% of living cells in the corresponding experiment) of an extract residue (see Table 1) was used in the luciferase assay (see Section 2.4).


### 2.4. Luciferase Assay for AP-1-Dependent Transcriptional Activity

The effect of the studied substances on AP-1 transcriptional activation was investigated in JB6 Cl41 cells stably expressing a luciferase reporter gene controlled by an AP-1 DNA binding sequence as described earlier [43]. In brief, viable JB6 Cl41 AP-1cells (8×103) suspended in 100 μL of 5% FBS/MEM were added into each well of a 96-well plate. Plates were incubated overnight and the media was then replaced with fresh 5% FBS/MEM containing the extract residue at subtoxic concentration (see Section 2.3) in a total volume of 0.1 mL and the cells were incubated with the extract residue for 24 h. Then, the cells were disrupted for 1 h at room temperature with lysis buffer (0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT, 2 mM EDTA) and 30 μL of lysate from each well were transferred into a plate for luminescent analysis and luciferase activity was measured. Results are expressed in Table 1 as an AP-1-dependent transcriptional activity in percentage relative to untreated control cells (100%).


## 2.1. Marine Invertebrates and Alga

The specimens of marine invertebrates and algae were collected off the coast of Vietnam at a depth of 2–440 m by scuba diving and trawling during the 34th scientific expedition onboard the research vessel “Academic Oparin” in May-June 2007 and were immediately extracted with ethanol. The animals and algae were identified by Dr. V. B. Krasokhin. The sponges having the same names in Table1 belong to one and the same genus, but they may have different specific names, undetermined yet, and were collected at the different places of the coast of Vietnam. The voucher specimens are kept in the collection of G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia.Table 1
The effects of the extracts of the marine invertebrates and alga on AP-1-dependent transcriptional activity in JB6 Cl41 cells.
Num.*
Namex
Conc.£ mg/mL
AP-1¥
O14-34
Callyspongia sp.
0.6
103
O15-34
Oscarella sp.
0.5
94
O16-34
Dysidea sp.
0.5
100
O17-34
Mycale sp.
0.5
134
O18-34
Chondrilla sp.
0.6
110
O19-34
Monanchora sp.
0.125 (0.24)**
40
O20-34
Haliclona sp.
0.6
96
O21-34
Spirastrella sp.
0.4
111
O22-34
Ascidian unidentified
0.8
80
O23-34
Petrosia sp.
0.8
64
O24-34
Demospongiae, unidentified
0.6
88
O25-34
Spirastrella sp.
0.6
41
O26-34
Callyspongia sp.
0.5
71
O27-34
Spongia sp.
0.5
98
O28-34
Mycale sp.
0.8
106
O29-34
Callyspongia sp.
0.6
101
O30-34
Chondrilla sp.
0.6
89
O31-34
Alga unidentified
0.5
98
O33-34
Callyspongia sp.
0.5
89
O34-34
Callyspongia sp.
0.5
70
O35-34
Xestospongia sp.
0.6
29
O36-34
Spongia sp.
0.5
35
O37-34
Spirastrella sp.
0.5
122
O38-34
Demospongiae, Haplosclerida, fam. unidentified
0.5
90
O41-34
Phakellia sp.
0.7
59
O42-34
Halichondriasp.
0.5
74
O43-34
Halichondriasp.
0.4
125
O44-34
Haliclona sp.
0.6
105
O45-34
Halichondriasp.
0.6
75
O46-34
Haliclona sp.
0.6
93
O47-34
Ascidian unidentified
0.6
95
O48a-34
Ascidian unidentified
0.8
79
O48b-34
Ascidian unidentified
0.8
96
O49-34
Echinoidea unidentified
0.8
105
O50-34
Pennatulacea unidentified
0.5
91
O52-34
Alga unidentified
1.0
95
O53-34
Alga unidentified
0.6
76
O54-34
Alga unidentified
0.6
89
O55-34
Alga unidentified
0.7
93
O56-34
Alga unidentified
0.5
169
O57-34
Petrosia sp.
0.5
105
O58-34
Demospongiae unidentified
0.125 (0.27)**
19
O59-34
Haliclona sp.
0.8
74
O60-34
Pachastrella sp.
0.0625 (0.17)**
47
O61-34
Spirastrella sp.
0.5
73
O62-34
Spongia sp.
0.6
71
O63-34
Spongia sp.
0.5
102
O64-34
Stelletta sp.
0.5
62
O65-34
Spirastrella sp.
0.25 (0.35)**
52
O66-34
Dysidea sp.
0.125
77
O67-34
Spongia sp.
0.6
84
O68-34
Algae unidentified
0.5
67
O69-34
Opistobranchia unidentified
0.25
88
O70-34
Opistobranchia unidentified
0.125 (0.22)**
121
O71-34
Opistobranchia unidentified
0.8
96
O72-34
Opistobranchia unidentified
0.5
149
O73-34
Spongia sp.
0.0625 (0.1)**
137
O74-34
Myxilla sp.
0.125 (0.23)**
14
O75-34
Spirastrella sp.
0.4
108
O76-34
Xestospongia sp.
0.5
117
O77-34
Stelletta sp.
0.0625
91
O78-34
Spongia sp.
0.25 (0.54)**
38
O79-34
Callyspongia sp.
0.5
108
O81-34
Gorgonian gen unidentified
0.5
35
O82-34
Gorgonian gen unidentified
0.25
104
O83-34
Ascidia
0.6
91
O84-34
Haliclona sp.
0.6
74
O85-34
Spirastrella sp.
0.8
60
O86-34
Halichondriasp.
0.8
63
O87-34
Spongia sp.
0.8
60
O88-34
Xestospongia sp.
0.6
109
O89-34
Clathria sp.
0.8
105
O90-34
Ascidian unidentified
1.0
105
O92-34
Spongia sp.
0.6
93
O93-34
Spirastrella sp.
0.5
124
O94-34
Spongia sp.
0.6
77
O95-34
Spongia sp.
0.6
105
O98-34
Crinoidea
0.6
71
O99-34
Demospongiae, unidentified
0.8
93
O100-34
Demospongiae, unidentified
0.8
87
O101-34
Demospongiae, unidentified
0.25 (0.4)**
127
O102-34
Spirastrella sp.
0.5
84
O103-34
Petrosia sp.
0.5
54
O104-34
Halichondriasp.
0.5
104
O105-34
Alga unidentified
0.25
99
O106-34
Alga unidentified
0.0625 (0.11)**
169
O107-34
Alga unidentified
0.6
144
O115-34
Crinoidea
0.25 (0.42)**
73
O117-34
Nudibranchia unidentified
0.25 (0.37)**
77
O119-34
Spirastrella sp.
0.6
118
O120-34
Haliclona sp.
0.6
88
O121-34
Demospongiae unidentified
0.6
111
O122-34
Spongia sp.
0.5
38
O123-34
Callyspongia sp.
0.0625
101
O124-34
Axinella sp.
0.6
52
O125-34
Aplysina sp.
0.6
60
O126-34
Spongia sp.
0.6
57
O127-34
Spongia sp.
0.0625 (0.16)**
46
O129-34
Crinoidea unidentified
0.6
84
O130-34
Crinoidea unidentified
0.5
90
O131-34
Crinoidea unidentified
0.125
83
O132-34
Nudibranchia unidentified
0.0625
84
O133-34
Ascidian unidentified
0.25 (0.37)**
73
O134-34
Haliclona sp.
0.0625
110
O135-34
Callyspongia sp.
0.5
135
O136-34
Spongia sp.
0.5
79
O137-34
Demospongiaeunidentified
0.25
84
O140-34
Spongia sp.
0.8
41
O141-34
Clathria sp.
0.8
63
O145-34
Callyspongia sp.
0.8
43
O146-34
Axinella sp.
0.6
33
O149-34
Suberites sp.
0.8
83
O152-34
Lithistida unidentified
0.8
67
O153-34
Halichondriasp.
0.8
102
O154-34
Penaressp.
0.25 (0.34)**
37
O155-34
Petrosia sp.
0.8
67
O156-34
Pheronema raphanus
0.0625
90
O157-34
Pachastrella sp.
0.8
49
O158-34
Lithistida  unidentified
0.8
75
O159-34
Geodiasp.
0.125
98
O160-34
Demospongiae unidentified
0.8
55
O162-34
Halichondriasp.
0.8
60
O163-34
Spirastrella sp.
0.5
101
O165-34
Petrosia sp.
0.8
53
O167-34
Rhabdastrella globostellata
0.5
37
O168-34
Petrosia sp.
0.125
81
O169-34
Halichondriasp.
0.25 (0.48)**
36
O170-34
Acanthodendrilla sp.
0.0625 (0.1)**
53
O171-34
Callyspongia sp.
0.8
30
O172-34
Carteriospongia sp.
0.0625 (0.11)**
19
O173-34
Carteriospongia sp.
0.25 (0.32)**
76
O174-34
Axinella sp.
0.25 (0.41)**
33
O175-34
Halichondriasp.
0.25 (0.33)**
66
O176-34
Petrosia sp.
0.125
110
O177-34
Spongia sp.
0.125 (0.19)**
54
O178-34
Petrosia sp.
0.8
56
O179-34
Hyalonema sp.
0.25
80
O181-34
Porifera, Lithistida unidentified
0.5
43
O186-34
Porifera,  Calcarea, unidentified
0.6
55
O187-34
Callyspongia sp.
0.0625
101
O188-34
Agelas sp.
0.8
50
O189-34
Axinella sp.
0.0625 (0.16)**
67
O192-34
Agelas sp.
0.8
47
O193-34
Petrosia sp.
0.5
42
O194-34
Amphimedon sp.
0.25 (0.39)**
42
O196-34
Spongia sp.
0.125 (0.23)**
60
O197-34
Axinella sp.
0.5
60
O198-34
Plakina sp.
0.5
41
*The number of the voucher specimen.
xSystematical name of the specimen.
£Concentration of the extract dry residue in the MEM.
¥AP-1-dependent transcriptional activity, percentage of the control.
**IC50.


## 2.2. Cell Culture

The mouse epidermal JB6 Cl41 AP-1 cells were cultured in monolayers at 37C and 5% CO2 in MEM containing 5% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin [40].


## 2.3. MTS Cytotoxicity Assay

Each ethanol extract of a marine invertebrate or alga was evaporated to dryness. Then, the solutions of the dry extract residue in 5% FBS/MEM with the residue concentrations of 1.0; 0.9; 0.8; 0.7; 0.6; 0.5; 0.4; 0.25; 0.125; and 0.0625 mg/mL were prepared. The effect of these solutions  on JB6 Cl41 AP-1 cells viability was evaluated using MTS reduction into its formazan product [41] as described in [42]. In brief, the JB6 Cl41 AP-1 cells were cultured for 12 h in 96-well plates (6,000 per well in 0.1 mL of 5% FBS/MEM). The media was then replaced with fresh 5% FBS/MEM containing the studied substances at various concentrations in a total volume of 0.1 mL and the cells were incubated with obtained solutions  for 22 h. Then, 20 μL of the MTS reagent was added into each well and MTS reduction was measured 2 h later spectrophotometrically at 492 and 690 nm as background using the Multiskan MS microplate reader (Labsystems, Finland). The solution with the first nontoxic concentration (more than 80% of living cells in the corresponding experiment) of an extract residue (see Table 1) was used in the luciferase assay (see Section 2.4).


## 2.4. Luciferase Assay for AP-1-Dependent Transcriptional Activity

The effect of the studied substances on AP-1 transcriptional activation was investigated in JB6 Cl41 cells stably expressing a luciferase reporter gene controlled by an AP-1 DNA binding sequence as described earlier [43]. In brief, viable JB6 Cl41 AP-1cells (8×103) suspended in 100 μL of 5% FBS/MEM were added into each well of a 96-well plate. Plates were incubated overnight and the media was then replaced with fresh 5% FBS/MEM containing the extract residue at subtoxic concentration (see Section 2.3) in a total volume of 0.1 mL and the cells were incubated with the extract residue for 24 h. Then, the cells were disrupted for 1 h at room temperature with lysis buffer (0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTT, 2 mM EDTA) and 30 μL of lysate from each well were transferred into a plate for luminescent analysis and luciferase activity was measured. Results are expressed in Table 1 as an AP-1-dependent transcriptional activity in percentage relative to untreated control cells (100%).


## 3. Results and Discussion

In continuation of our search for cancer preventive compounds from marine organisms [32, 33, 38, 43, 44], we studied the influence of the extracts from 148 species of marine invertebrates and algae on the AP-1-dependent transcriptional activity in mouse epidermal JB6 Cl41 cells. The JB6 Cl41 cells undergo the malignant transformation under the treatment of EGF as a promoter. The transformation involves the activation of AP-1 nuclear factor which regulates the transcription of various genes related to inflammation, proliferation and metastasis. Thus, it can be said that the extracts which show the inhibition of the AP-1-dependent transcriptional activity in JB6 Cl41 cells may contain some cancer preventive compounds. To study the effects of the ethanol soluble substances from the studied species on the AP-1 transcriptional activity, we used the luciferase assay and JB6 Cl41 AP-1 cells stably expressing a luciferase reporter gene controlled by an AP-1 DNA binding sequence. The results of the study are presented in Table 1.We studied 148 species of marine invertebrates and algae, and there are 116 sponges, 7 ascidians, 10 species of algae, 1 species of Echinoidea, 4 species of Opistobranchia, 2 species of Nudibranchia, 2 species of Gorgonacea, 5 species of Crinoidea, 1 species of Pennatulacea. The extracts of 71 species studied showed inhibitory effects (less than 80% of activity in comparison to control) on AP-1-dependent transcriptional activity at noncytotoxic concentrations, whereas extracts of 12 species demonstrated activation (more than 120% of activity in comparison to control) of the AP-1 nuclear factor in JB6 Cl41 cells. As shown in Table1, sponges, belonging to genera Spongia, Petrosia, Halichondria, Callyspongia, Spirastrella, and Axinella are very promising sources of the AP-1 nuclear factor inhibitors, whereas 2 species of Opistobranchia and 3 species of Algae contain activators of the AP-1 activity. We hope that our study will facilitate the further search and isolation of the AP-1 nuclear factor inhibitors and activators from marine sources.


## 4. Conclusion

In conclusion, the extracts of 71 species of marine organisms out of 148 species studied showed inhibitory effects on the AP-1 transcriptional activity in JB6 Cl41 AP-1 cells, and only 12 extracts demonstrated activation of the AP-1 nuclear factor. The search for natural compounds that inhibit or induce AP-1 activity may lead to the development of the new promising preventive or therapeutic drugs against various diseases including cancer.



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*Source: 896709-2013-01-10.xml*