they have closer resemblance to the cells growing in the in vivo tissue environment ( 2 ) . they further contain specific characteristics similar to the corresponding tissues in vivo and can stay active and functional for weeks ( 3 ) . it is important to study the cells growth , proliferation and response to chemical agents in an environment organized with cell to cell and cell to matrix interactions plus a well - defined morphological geometry . monolayer cell culture systems of cancer cell lines are commonly used to evaluate the antitumor effects of anticancer drugs ( 5 ) . most tumor cells are supported by an extracellular matrix microenvironment , with an important role in resistance against anticancer drugs ( 6 ) . three - dimensional culture systems have allowed the study of cell - to - cell interactions and tumor - mediated angiogenesis ( 3 ) . in addition , spheroid cultures that use a compound derived from basement membrane , organotypic cultures , and soft agar cultures as well as cells embedded in collagen gel have been developed to investigate the biology of cancer cell lines ( 7 ) . we have used the collagen extracted from persian gulf squid ( uroteuthis duvauceli ) as a 3d culture system to study the capacity of pectic acid to inhibit tumor cells growth in mda - mb-231 breast cancer cells . whole apple extract can inhibit mammary cancer in a dose - dependent manner in rat model ( 8) . the major component of apple is a kind of polysaccharide called pectin . dietary pectic and its degradation products ( pectic oligosaccharides ) caused decreased proliferation in ht-29 cells ( 9 ) . there are a number of studies related to pectic ( mostly citrus pectin ) and cancer . studies on the effect of pectic on different types of cancers such as blood - borne , prostate and colorectal indicated that pectic intake was beneficial to inhibit tumor growth and metastasis ( 10 , 11 ) . in addition , the effect of apple pectic on colon cancer through enhancement of apoptosis and inhibition of tumor formation has been reported both on animal and cellular models ( 12 , 13 ) . the main objective of this research was to apply the collagen extracted from persian gulf squid as a biomaterial to be used in cell culture systems in order to study the development of anticancer drugs and treatments . the skin was mechanically separated from the squid body , defatted in 10% butyl alcohol , and rinsed in acetic acid and nacl . the total collagen obtained was determined according to aoac using standard procedures ( 15 ) . ssd - page analysis was performed on 12% resolving gel and 5% stacking gel ( 16 ) . the crude extracted collagen was dissolved in phosphate buffer containing 1% sds and 0.5 m urea ( merck ) ( 17 ) to give a final concentration of 5mg / ml . the gels were stained with coomassie brilliant blue r-250 ( merck ) and detained in methanol / acetic acid . the mda - mb-231 cell line was obtained from ncbi ( national cell bank of iran ) . the cells were grown in rpmi 1640 medium ( gibco ) in tissue culture flasks at 37 c in 5% co2 until reaching confluence . the medium contained 10% fetal calf serum ( fcs ) ( gibco ) and 0.1% penicillin - streptomycin . the cells were detached with 0.25% ( w / v ) trypsin  edta ( sigma ) . cell number and viability was determined using trypan blue ( sigma ) exclusive dye and hemocytometer . the extracted collagen was prepared at 5 mg / ml concentration by suspending in sterile 0.01 m acetic acid and stirring overnight at 4 c . the gel was prepared on ice by adding dmem / ham f12 3x , 5% fbs and naoh to neutralize its acidity . to form the gel , then , mda - mb-231 cells were decanted onto the collagen in two models : collagen - coated and collagen gel ( collagel ) . for both models , mda - mb-231 cells were treated with different concentrations of pectic acid ( 0 , 0.5 , 1.5 , 3 mg / ml ) for 24 and 48 h. the effect of pectic acid on cell proliferation was measured using mtt - based assay . the cells were incubated with 10l of 5mg / ml mtt ( 3-[4 , 5-dimethylthiazole-2-yl]-2 , 5-diphenyltetrazoliumbromide ) solution for 3 h in phosphate - buffered saline ( pbs ) . the formazan crystals produced from mtt were dissolved in dmso , and their absorbance was recorded by elisa reader at 630 nm . the proliferation rate ( pr ) was calculated based on the following formula : 
 pr  percentage=[absorbance  of  drug  treatmentgroupabsorbance  of  blank / absorbance  of  controlgroupabsorbance  of  blank]100 . all the experiments were done at least 3 times , and the data were shown as mean  standard error of the mean ( sem ) . the results were analyzed for significance using anova test with tukey s post hoc test . the skin was mechanically separated from the squid body , defatted in 10% butyl alcohol , and rinsed in acetic acid and nacl . the total collagen obtained was determined according to aoac using standard procedures ( 15 ) . ssd - page analysis was performed on 12% resolving gel and 5% stacking gel ( 16 ) . the crude extracted collagen was dissolved in phosphate buffer containing 1% sds and 0.5 m urea ( merck ) ( 17 ) to give a final concentration of 5mg / ml . the gels were stained with coomassie brilliant blue r-250 ( merck ) and detained in methanol / acetic acid . to characterize the extracted collagen , mda - mb-231 breast cancer cell line was grown on the squid collagen . the mda - mb-231 cell line was obtained from ncbi ( national cell bank of iran ) . the cells were grown in rpmi 1640 medium ( gibco ) in tissue culture flasks at 37 c in 5% co2 until reaching confluence . the medium contained 10% fetal calf serum ( fcs ) ( gibco ) and 0.1% penicillin - streptomycin . the cells were detached with 0.25% ( w / v ) trypsin  edta ( sigma ) . cell number and viability was determined using trypan blue ( sigma ) exclusive dye and hemocytometer . the extracted collagen was prepared at 5 mg / ml concentration by suspending in sterile 0.01 m acetic acid and stirring overnight at 4 c . the gel was prepared on ice by adding dmem / ham f12 3x , 5% fbs and naoh to neutralize its acidity . to form the gel , then , mda - mb-231 cells were decanted onto the collagen in two models : collagen - coated and collagen gel ( collagel ) . for both models , mda - mb-231 cells were treated with different concentrations of pectic acid ( 0 , 0.5 , 1.5 , 3 mg / ml ) for 24 and 48 h. the effect of pectic acid on cell proliferation was measured using mtt - based assay . the cells were incubated with 10l of 5mg / ml mtt ( 3-[4 , 5-dimethylthiazole-2-yl]-2 , 5-diphenyltetrazoliumbromide ) solution for 3 h in phosphate - buffered saline ( pbs ) . the formazan crystals produced from mtt were dissolved in dmso , and their absorbance was recorded by elisa reader at 630 nm . the proliferation rate ( pr ) was calculated based on the following formula : 
 pr  percentage=[absorbance  of  drug  treatmentgroupabsorbance  of  blank / absorbance  of  controlgroupabsorbance  of  blank]100 . all the experiments were done at least 3 times , and the data were shown as mean  standard error of the mean ( sem ) . the results were analyzed for significance using anova test with tukey s post hoc test . the collagen content of the outer sheath of squid skin is high ( 18 ) . the skin collagen content of persian gulf squid was about 3% , the extracted collagen contained low amounts of fat ( 0.5% ) and approximately 98% moisture ( table 1 ) . in sds - page pattern , the extracted collagen contained two  chains , one  chain and  chain components ( 1 about 50 kda and 2 about 80 kda ) ; accordingly , it was similar to type i collagen ( fig . sds - page patterns of squid collagen showing that the collagen extracted from squid contained  and  chains physicochemical properties of collagen extracted from persian gulf squid mda - mb-231 cells displayed flat cell bodies and a sheet - like monolayer spread over the culture plates in 2d in vitro system . to investigate the extracted collagen as a scaffold for cell culture , mda - mb-231 cells were cultured in squid collagen gels for a period of four days . additionally , the cells began to proliferate and show cell - to - cell interactions , especially in collagen - coated cultures ( fig . these interactions ( cell to cell and cell to matrix ) closely resembled in vivo biological systems . mda - mb-231 cells 48 h after cell cultivation on collagen extracted from persian gulf squid . 3d population of cells can be seen in the collagen cultures ( 100x ) in order to peruse the proliferation of mdamb-231 cells on collagen , the cells were cultivated on the squid collagen , and their proliferation rate was assessed for a period of 72 h. mtt test showed that the proliferation rate of cells was increased in collagen - coated plates after 24 h ( fig . mda - mb-231 cells cultivated on the squid collagen did not show the same increase in proliferation rate as did the coated cells . the proliferation rate of the cells was significantly increased after 48 h in both the coated and squid collagen cultures , and the cells density was so high in 96 h tests ( fig . mda - mb-231 cells cultured in the collagen extracted from squid . as shown , proliferation rate is higher in the cultures containing collagen . each value shown is meansd ( n=8 ) . * significantly different from cells grown in 2d condition ( * * p<0.01 , * * * p<0.001 ) in the first step , cell proliferation assay was performed to determine the effect of pectic acid on mda - mb-231 cells . to reach this goal , mdamb-231 cells were treated with different concentrations of pectic acid ( 0 , 0.5 , 1.5 , 3 mg / ml ) for 24 h. fig . 4 indicate that pectic acid inhibited cell growth and reduced the number of attached cells after 24 h. in this condition , degenerative changes like loss of cell sheet were detected . thus , pectic acid had a cytotoxic effect on mdamb-231 cells in 2d in vitro system . subsequently , the culture of mda - mb-231 cells on the squid collagen was treated with pectic acid . even higher concentration of pectic acid ( 3 mg / ml ) could not inhibit the mda - mb-231 cells growth both on collagen - coated and collagel cultures ( fig . treatment with pectic acid for 24 h in 2d conventional condition inhibited the growth of mda - md-231 cells while pectic acid could not affect the cells in collagen - coated cultures . columns show percent viable cells ( n=8 ) ; * * indicate significant differences ( p<0.01 ) treatment with pectic acid for 24 h on the collagel culture obtained from the squid indicated no significant effect . columns show percent viable cells ( n=8 ) ; * * indicates significant differences ( p<0.01 ) to verify this result , higher concentrations of pectic acid ( 5 and 10 mg / ml ) were applied on the cells . in these circumstances , morphological analysis of the cells indicated no obvious changes in culture of mdamb-231 cells on the squid collagen ( coated and collagel ) in presence of pectic acid ( fig . 7 ) ; how - ever , the cells treated with pectic acid in 2d cultures showed changes such as irregular cell walls and cell debris in the medium . the majority of cells were floating , became rounded , and detached from the surface ( fig . 8) . therefore , the collagen extracted from squid could mimic 3d culture conditions through increased resistance of cells to chemical agents . treatment with higher concentrations of pectic acid for 24h on the collagel culture inhibited the growth of mda - mb-231 cells . columns show percent viable cells ( n=4 ) ; * * indicates significant differences ( p<0.01 ) morphological changes of mda - mb- 231 cells treated with pectic acid on the collagen extracted from squid . there is no significant change in cell morphology compared to control cells ( 100x ) morphological changes of the mda - mb- 231 cell treated with pectic acid on 2d conventional culture system . cell debris and irregular rounded cell shapes are seen by increasing of pectic acid concentration ( 100x ) the collagen content of the outer sheath of squid skin is high ( 18 ) . the skin collagen content of persian gulf squid was about 3% , the extracted collagen contained low amounts of fat ( 0.5% ) and approximately 98% moisture ( table 1 ) . in sds - page pattern , the extracted collagen contained two  chains , one  chain and  chain components ( 1 about 50 kda and 2 about 80 kda ) ; accordingly , it was similar to type i collagen ( fig . sds - page patterns of squid collagen showing that the collagen extracted from squid contained  and  chains physicochemical properties of collagen extracted from persian gulf squid mda - mb-231 cells displayed flat cell bodies and a sheet - like monolayer spread over the culture plates in 2d in vitro system . to investigate the extracted collagen as a scaffold for cell culture , mda - mb-231 cells were cultured in squid collagen gels for a period of four days . additionally , the cells began to proliferate and show cell - to - cell interactions , especially in collagen - coated cultures ( fig . these interactions ( cell to cell and cell to matrix ) closely resembled in vivo biological systems . mda - mb-231 cells 48 h after cell cultivation on collagen extracted from persian gulf squid . in order to peruse the proliferation of mdamb-231 cells on collagen , the cells were cultivated on the squid collagen , and their proliferation rate was assessed for a period of 72 h. mtt test showed that the proliferation rate of cells was increased in collagen - coated plates after 24 h ( fig . mda - mb-231 cells cultivated on the squid collagen did not show the same increase in proliferation rate as did the coated cells . the proliferation rate of the cells was significantly increased after 48 h in both the coated and squid collagen cultures , and the cells density was so high in 96 h tests ( fig . mda - mb-231 cells cultured in the collagen extracted from squid . as shown , proliferation rate is higher in the cultures containing collagen . each value shown is meansd ( n=8 ) . * significantly different from cells grown in 2d condition ( * * p<0.01 , * * * p<0.001 ) in the first step , cell proliferation assay was performed to determine the effect of pectic acid on mda - mb-231 cells . to reach this goal , mdamb-231 cells were treated with different concentrations of pectic acid ( 0 , 0.5 , 1.5 , 3 mg / ml ) for 24 h. fig . 4 indicate that pectic acid inhibited cell growth and reduced the number of attached cells after 24 h. in this condition , degenerative changes like loss of cell sheet were detected . thus , pectic acid had a cytotoxic effect on mdamb-231 cells in 2d in vitro system . subsequently , the culture of mda - mb-231 cells on the squid collagen was treated with pectic acid . even higher concentration of pectic acid ( 3 mg / ml ) could not inhibit the mda - mb-231 cells growth both on collagen - coated and collagel cultures ( fig . treatment with pectic acid for 24 h in 2d conventional condition inhibited the growth of mda - md-231 cells while pectic acid could not affect the cells in collagen - coated cultures . columns show percent viable cells ( n=8 ) ; * * indicate significant differences ( p<0.01 ) treatment with pectic acid for 24 h on the collagel culture obtained from the squid indicated no significant effect . columns show percent viable cells ( n=8 ) ; * * indicates significant differences ( p<0.01 ) to verify this result , higher concentrations of pectic acid ( 5 and 10 mg / ml ) were applied on the cells . in these circumstances , morphological analysis of the cells indicated no obvious changes in culture of mdamb-231 cells on the squid collagen ( coated and collagel ) in presence of pectic acid ( fig . 7 ) ; how - ever , the cells treated with pectic acid in 2d cultures showed changes such as irregular cell walls and cell debris in the medium . the majority of cells were floating , became rounded , and detached from the surface ( fig . 8) . therefore , the collagen extracted from squid could mimic 3d culture conditions through increased resistance of cells to chemical agents . treatment with higher concentrations of pectic acid for 24h on the collagel culture inhibited the growth of mda - mb-231 cells . columns show percent viable cells ( n=4 ) ; * * indicates significant differences ( p<0.01 ) morphological changes of mda - mb- 231 cells treated with pectic acid on the collagen extracted from squid . there is no significant change in cell morphology compared to control cells ( 100x ) morphological changes of the mda - mb- 231 cell treated with pectic acid on 2d conventional culture system . cell debris and irregular rounded cell shapes are seen by increasing of pectic acid concentration ( 100x ) collagen is involved in many cellular processes , including regulation of cell motion , cell proliferation and apoptosis ( 20 ) . for example , type i collagen has been used as coating for culture dishes or as scaffold for microbiological adherence and invasion test systems . a 3d - culture matrix of collagen can act as a support for cells and mimic the real extracellular matrix ( ecm ) ( 21 ) . alternative sources of collagen have been developed since the use of main sources of collagen ( land - based animals such as bovine or porcine ) involves hygienic , social and cultural concerns . marine creatures might offer a good collagen source since high obtainability , lack of disease transmission risk and religious barriers as well as extraction capacity of high - yield collagen ( 22 , 14 ) . moreover , fish waste , including bones , skins , scales as well as squid collagenous membranes detached during mechanical processing may be a new source for collagen ( 23 ) . the collagen extracted from persian gulf squid ( uroteuthis duvauceli ) contained type i collagen , and could be formed as collagel . finally , the mda - mb-231 breast cancer cells grown in 3-d culture of squid collagel are more resistant to pectic agents than the cells in 2-d culture . the cells cultured within three - dimensional ( 3d ) conditions exhibited phenotypes and responses to stimuli analogous to in vivo biological systems . this characteristic can be used in tissue engineering and in vitro tumor models ( 24 ) . morphological and protrusion analyses of mda - mb-23 cells indicated that cell to cell and cell to matrix interactions occurred during incubation of cells within the collagen extracted from persian gulf squid . in addition , the collagen extracted from persian gulf achieved biocompatibility to support cellular proliferation . these results were observed in both the collagen - coated and collagel cultures , which are consistent with a report ( 25 ) . in this condition , the cell s response to drugs was significantly changed . the cells cultured in 3d culture systems ; we expect to obtain an in vivo phenotype ( 26 ) . co - culture of mda - mb-231 cells within squid collagen caused higher concentrations of pectic acid to inhibit proliferation of cells . pectic acid could inhibit mda - mb-231 breast cancer cell growth , correlated with cell death and apoptosis induction ( 27 ) . the data achieved in this study indicated that the half - maximal inhibitory concentration of pectic acid was about 1 mg / ml when the mda - mb-231 cells were cultured in a 2d traditional system . this concentration showed a 5-fold increase in the 3d culture system using the collagen extracted from persian gulf squid . squid collagen contains a porous structure sufficient to promote cellular adhesion , aggregation , morphogenesis and growth . as sem analysis showed , a fibrous network and porous structure was observed including interconnected joints in an irregular and wavy shape for the extracted collagen ( data not shown ) . these data imply that the collagen extracted from persian gulf squid is capable of generating a 3d culture system , which closely mimics in vivo conditions . our data are in agreement with the report that showed 3d type i collagen system maintained the growth of mdamb-231 and mcf-7 breast cancer cells and decreased the cytotoxicity of apoptotic drugs such as adriamycin in this system ( 2829 ) . in addition , the mda - mb-231 cells cultured in 3d hydrogel of collagen scaffolds demonstrated greater resistance to paclitaxel as a cytotoxic chemotherapeutic agent ( 30 ) . in all such these studies , collagen i extracted from rat - tail was used as cell 3d scaffold . the collagen extracted from persian gulf squid conventional cell culture models are useful to investigate certain biological functions ; however , physiologically relevant 3d cell culture models , including the one used in this study , have the potential to yield more effective and cost - efficient results for development of anticancer drugs and treatments . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .