marine sponges ( porifera ) have been prolific sources of many interesting bioactive substances and are still an important resource for marine natural product chemistry . in the course of our studies on biologically active natural products of marine organisms , we have recently isolated four new melophlins p ( 1 ) , q ( 2 ) , r ( 3 ) , and s ( 4 ) together with seven known melophlins a ( 5 ) , d ( 6 ) , e ( 7 ) , g ( 8) , h ( 9 ) , i ( 10 ) , and o ( 11 ) from two marine sponges of the genus melophlus collected in palau .
melophlins are tetramic acid derivatives possessing a long alkyl chain and have been isolated from melophlus sarasinorum [ 13 ] .
compounds 111 , except 8 , showed weak to modest growth inhibitory activity against a murine leukemia cell line l1210 .
melophlins a ( 5 ) and b ( possessing the c11 alkyl chain and two methyl groups at c-5 and c-9 ) were reported to have moderate cytotoxicity against hl-60 and arrest nih3t3 fibroblasts in the g1 phase of the cell cycle .
furthermore , melophlins a and b were found to reverse the morphology of h - ras - transformed hih3t3 cells to normal and might act on the components of the ras - mediated signal transduction pathway .
therefore , we intended to investigate the influence of 11 compounds ( 111 ) on the colony formation of chinese hamster v79 cells .
we also observed the effect of 11 compounds ( 111 ) on the production of an inflammatory cytokine , il-8 , in pma - stimulated hl-60 cells since the inhibition of cell proliferation sometimes correlates with il-8 production . in this paper
, we would like to describe some structure - activity relationships of these compounds against v79 and l1210 cells .
melophlins were isolated from two marine sponges of the genus melophlus collected in palau as described previously .
dimethylsulfoxide ( dmso ) was purchased from pierce chemical co. ( rockfield , il ) , and fetal bovine serum ( fbs ) was obtained from gibco after checking the lot .
chinese hamster v79 cells were grown as a monolayer culture in eagle s mem ( nissui seiyaku co. , ltd . ,
the hl-60 cell line was obtained from the japanese cancer research resources bank ( jcrb , kamiyoga , tokyo , japan ) and maintained in tissue culture dishes in rpmi 1640 medium ( nissui seiyaku , tokyo , japan ) supplemented with 10% heat - inactivated fbs , 2 mm glutamine , 100 u / ml of penicillin g , and 100 g / ml of streptomycin . the relative plating efficiencies against v79 cells were determined as the ratio of the number of colonies in various concentrations of samples to that in the sample - free control , as described in previous papers .
two hundred cells were seeded onto a 60/15-mm plastic plate with 4 ml culture medium and incubated overnight at 37c .
after each sample in dmso ( 4 l ) was added to the culture medium , cells were further cultured for four days .
the numbers of colonies in the sample plates were counted and compared with those in the control cultures .
the il-8 concentrations of the culture supernatants under control and various test conditions were measured by elisa using a combination of monoclonal and polyclonal antibodies .
melophlins were isolated from two marine sponges of the genus melophlus collected in palau as described previously .
dimethylsulfoxide ( dmso ) was purchased from pierce chemical co. ( rockfield , il ) , and fetal bovine serum ( fbs ) was obtained from gibco after checking the lot .
chinese hamster v79 cells were grown as a monolayer culture in eagle s mem ( nissui seiyaku co. , ltd . ,
the hl-60 cell line was obtained from the japanese cancer research resources bank ( jcrb , kamiyoga , tokyo , japan ) and maintained in tissue culture dishes in rpmi 1640 medium ( nissui seiyaku , tokyo , japan ) supplemented with 10% heat - inactivated fbs , 2 mm glutamine , 100 u / ml of penicillin g , and 100 g / ml of streptomycin .
the relative plating efficiencies against v79 cells were determined as the ratio of the number of colonies in various concentrations of samples to that in the sample - free control , as described in previous papers .
two hundred cells were seeded onto a 60/15-mm plastic plate with 4 ml culture medium and incubated overnight at 37c .
after each sample in dmso ( 4 l ) was added to the culture medium , cells were further cultured for four days .
the numbers of colonies in the sample plates were counted and compared with those in the control cultures .
the il-8 concentrations of the culture supernatants under control and various test conditions were measured by elisa using a combination of monoclonal and polyclonal antibodies .
melophlin a ( 5 ) has been reported to inhibit the cell proliferation of hl-60 at the ic50 value of 0.2 g / ml ( 0.57 m ) .
this compound arrested nih3t3 fibroblast cells in the g1 phase of the cell cycle at 1 g / ml ( 2.85 m ) and reversed the morphology of h - ras transformed nih3t3 cells to normal at 5g / ml ( 14.2 m ) . on the other hand , melophlins c , e ( 7 ) , g ( 8) , h ( 9 ) , i ( 10 ) , m , n , and o ( 11 ) were not cytotoxic to hl-60 , hela , or tf-1 cells .
in our experiment , compounds 17 and 911 showed weak to modest inhibitory activity against l1210 cells ( table 1 ) ; therefore , we examined the effect of compounds 111 on the rate of colony formation using v79 cells .
since the production of an inflammatory cytokine , il-8 , is , in some cases , correlated with the inhibition of cell proliferation , we also observed the influence of 11 compounds on the il-8 production system mediated by pma - stimulated hl-60 cells .
although the inhibitory activities of the 11 compounds on the colony formation of v79 were not remarkable , melophlins h ( 9 ) and o ( 11 ) showed activity at ec50 values of 8.5 and 9.6 m , respectively ( table 1 ) ; however , compounds 111 did not affect the production of il-8 .
linear - chain - type compounds ( 1 , 5 , 6 , and 8) were least active among the four chain types ( table 1 ) .
the methyl group at c-5 reduced the activity of linear - chain - type and iso - type compounds ( 2 , 7 , and 9 ) , while the activity of anteiso - type compounds ( 3 , 10 , and 11 ) were increased by c-5 methylation .
activity was decreased as the chain length increased for linear - chain - type compounds . on the other hand ,
activity was increased as the chain length decreased for iso - type and anteiso - type compounds .
these structure - activity relationships were detected against both v79 and l1210 cells except for the iso - type compound with the c-5 methyl group ( compound 2 ) , which showed stronger activity against l1210 cells than compound 9 .
the activity of the 10-methyl compound ( 4 ) against v79 and l1210 cells was stronger than that of the iso - type compound ( 2 ) and weaker than that of the anteiso - type compound ( 3 ) .
therefore , it will be interesting to examine the in vitro growth inhibitory activity of compounds possessing iso - type or anteiso - type chains with a chain - length of less than c12 against human solid tumor cell lines .
 we have recently isolated four new melophlins p ( 1 ) , q ( 2 ) , r ( 3 ) , and s ( 4 ) together with seven known melophlins a ( 5 ) , d ( 6 ) , e ( 7 ) , g ( 8) , h ( 9 ) , i ( 10 ) , and o ( 11 ) from two marine sponges of the genus melophlus collected in palau . in this study , the influence of these compounds on the colony formation of chinese hamster v79 cells and the production of il-8 in pma - stimulated hl-60 cells were examined . 
 these 11 compounds did not show any effect on il-8 production . 
 the ec50 values of compounds 2 , 3 , 4 , 5 , 7 , 9 , 10 , and 11 against v79 cells were 44.0 , 13.3 , 16.7 , 27.2 , 19.8 , 8.5 , 23.1 , and 9.6 m , respectively . 
 the linear - chain - type compounds ( 1 , 6 , and 8) were not active against v79 cells at 50 m . 
 although the growth inhibitory activity of these melophlins was not remarkable , some structure - activity relationships of these compounds against v79 and murine leukemia l1210 cells were observed . 