breast cancer is the leading cause of cancer deaths among women in developed countries [ 1 , 2 ] .
the use of natural dietary compounds to block or delay the onset of cancer is a promising chemoprevention strategy [ 37 ] .
the purpose of these chemopreventive agents is to suppress cancer cell proliferation , induce cancer cell differentiation , or initiate apoptosis in cancer cells .
studies have demonstrated that natural phytochemicals containing phenolic compounds possess anticancer properties [ 810 ] .
polyphenols have anticancer functions both in vitro and in vivo [ 1116 ] . tannic acid ( ta ) belongs to the class of hydrolysable tannins and is comprised of a pentagalloylglucose core esterified at all functional hydroxyl groups with gallic acid molecules .
apoptotic activity is increased in breast cancer and prostate cancer cells in response to exposure to tannin extracts [ 1820 ] .
type i collagen is a common tissue engineering scaffold due to its intrinsically bioactive and biodegradable qualities .
collagen is a naturally derived material and , when uncross - linked , is enzymatically degraded .
if used as a biomaterial for tissue engineering purposes the ta - crosslinked collagen type i would not only serve as an attachment scaffold for cells but also function as an extended release anticancer treatment .
ta - crosslinked collagen sheets enhance wound healing of the skin in rats   and we have previously demonstrated that ta - crosslinked collagen sheets promote adipocyte survival while inducing apoptosis in estrogen receptor - positive ( er ) breast cancer cells . since ta cross - links collagen and has antitumor properties , the combination could prove to be an effective agent to induce localized apoptosis when introduced in tumor environments . if used for tissue reconstruction , ta - crosslinked collagen could provide increased protection against localized tumor recurrence in reconstructed breasts following mastectomy or lumpectomy .
the focus of this work is twofold : ( 1 ) ta - crosslinked collagen type i can assume the form of small beads that will , in the long - term , form the basis of an injectable tissue reconstruction scaffold ; ( 2 ) it investigates the effects of ta on normal human breast epithelial cells and different types of human breast cancer cells lines in vitro .
a 1  mg / ml collagen type i solution was prepared from a stock solution of 3.1  mg / ml ( advanced biomatrix , poway , ca ) as described elsewhere .
the ta cross - linked beads were prepared using a nisco encapsulation unit var v1 electrostatic syringe pump loaded with a 60  :  40 ratio of 1  mg / ml collagen  :  1.2% alginate solution in milliq water ( sigma - aldrich , st .
louis , mo ) ; the syringe pump was programmed to pump at 10  ml / h into a 1.5%  wt / v cacl2 solution ( fisher , pittsburgh , pa ) in water .
one hour after formation , the beads were filtered out using a mesh strainer and placed in ta crosslinking solution comprising ta ( 10% , 1.0% , or 0.1%  wt / v  ta / milliq water ) , 1.5% cacl2 , 0.15  m nacl ( sigma - aldrich ) , and 1.1%  wt / v n - cyclohexyl-2-aminoethanesulfonic acid ( sigma - aldrich ) buffer overnight .
twelve hours later the ta - crosslinked beads were placed in 50  mm sodium citrate ( fisher ) for 3  h , washed with deionized ( di)h2o , and stored in phosphate buffered saline ( pbs ) at 4c .
a sample of each concentration of the ta - crosslinked beads was immersed in commercially available blue food coloring for 12  h at room temperature .
dyed beads were placed individually in dih2o and the temperature was gradually increased until the bead morphology was distorted . the normal human breast epithelial cell line mcf10a and cell lines isolated from human breast cancers , mcf7 and mda - mb-231 ( all from atcc ; manassas , va ) , were grown in dulbecco 's modified eagle medium ( dmem ) with mammary epithelial cell growth medium ( megm ) supplements ( epidermal growth factor , insulin , bovine pituitary extract , and gentamicin ; lonza ; walkersville , md ) with 10% fetal bovine serum ( atlanta biologicals ; atlanta , ga ) and 1% antibiotic / antimycotic ( life technologies , grand island , ny ) .
the cultures were maintained at 37c with 5% co2 . for studies using 0.4  m transwell inserts ( corning ; corning , ny )
, the cells were seeded into the individual wells at 30,000 cells / well of 12-well cell culture plates and grown for 24  h. collagen beads crosslinked with tannic acid at concentrations of 10% , 1.0% , or 0.1% were placed on the transwell inserts .
each concentration of ta crosslinked collagen beads was placed in three transwell inserts per plate ( 400  l / transwell insert ) .
one 12-well plate was used for each timepoint ( 24 , 48  h ) examined .
cultures of treated and untreated breast cells were washed once with pbs , fixed for 10  min with neutral buffered formalin , washed twice with pbs , and stained with hematoxylin and eosin . all fixation and staining were performed in wells of culture plates .
apoptotic cells were visualized using click - it tunel alexa fluor imaging kits from life technologies ( grand island , ny ) according to manufacturer instructions .
images were acquired using a zeiss camera ( thornwood , ny ) and the cells were manually counted .
at least 1500 cells per cell line and treatment ( i.e. , ta concentration ) were counted in random fields in each culture well .
the sr flica caspase 9 kit and fam caspase 3/7 kit from immunochemistry technologies , llc ( bloomington , mi ) , and the guava caspase 8 fam kit from millipore ( hayward , ca ) were used .
cultures of mcf10a , mcf7 , and mda - mb-231 cells were treated as outlined above .
protein lysates were collected using m - per mammalian protein extraction reagent ( thermo scientific ; rockford , il ) with halt phosphatase inhibitor cocktail ( thermo scientific ) added . protein concentration was determined using a pierce bca protein assay kit ( thermo scientific ) .
lysates were combined with laemmli 's sds sample buffer ( boston bioproducts ; boston , ma ) and boiled for 5  min .
proteins were separated using a 415% criterion tgx gel ( bio - rad ) , transferred to nitrocellulose , and blocked for 1  h with 1% casein in pbs ( bio - rad ) .
membranes were probed with anti - caspase 7 and anti--actin ( 1  :  1000 ; cell signaling technology ; danvers , ma ) overnight at 4c .
membranes were washed thrice with 0.1% tween-20 in pbs and incubated with hrp - conjugated secondary antibodies ( 1  :  2500 ; cell signaling technology ) for 2  h at rt .
chemiluminescence was provided by lumiglo reagent ( cell signaling technology ) and detected using a fluorchem m ( cell biosciences ; santa clara , ca ) .
each experiment was performed a minimum of three times , with at least three replicates performed within each experiment .
samples of the three concentrations of ta - cross - linking collagen type i beads underwent denaturation .
once dyed , individual beads were placed in 25  ml of dih2o and subjected to increasing temperatures until noticeable shrinkage occurred ( figure 1 ) .
the average temperature required for denaturation of the 0.1% ta beads was 58.4c    1.5c , while for 1.0% ta beads the denaturation temperature was 61.2c    1.3c and for 10% ta beads the denaturation temperature was 63.8c    1.8c ( table 1 ) .
the denaturation temperature of the 0.1% ta beads was significantly lower than that of both the 1.0% and 10% ta beads ( p < 0.05 ) .
these results are in agreement with previous reports demonstrating that uncross - linked collagen scaffolds have a denaturation temperature of 55c , while collagen scaffolds crosslinked with 1  mg / ml ta elevated the denaturation temperature to 68c .
the ta - crosslinked collagen beads are stable at human body temperature and therefore have the potential to serve as a growth scaffold for cells during tissue reconstruction . the normal human breast epithelial cell line mcf10a and the human breast cancer cell lines mcf7 and mda - mb-231 were grown in conventional 2d cultures .
mcf10a cells are immortalized normal breast epithelial cells ; mcf7 cells are er , while the mda - mb-231 cells are triple negative breast cancer cells ( er negative , progesterone receptor ( pr ) negative , and her2 negative ) [ 2729 ] .
collagen type i beads crosslinked by various concentrations ( 10 , 1.0 , and 0.1% ) of ta were added to transwell inserts placed above the growing cell lines .  
figure 2 illustrates the different effects of the various concentrations of ta on the three cell lines . in cultures of mcf10a cells treated with ta , a change in morphology of the cells treated with the two higher doses of ta ( 10% and 1.0% )
was observed after 24  h , while it was not until 48  h of treatment that a noticeable change in phenotype was observed in the cells treated with the lowest concentration of ta ( figure 2(a ) ) .
the changes in observed morphology include rounder cells with fewer protrusions , an increase in the number of detached cells , and an increase in the interstitial space between cells in the cultures .
all three concentrations of ta inhibited the growth of the mcf10a cells after 48  h of exposure ( figure 3(a ) ) .
the highest concentration of ta used to cross - link the collagen beads had a dramatic effect on the morphology of mda - mb-231 cells within 24  h of exposure ( figure 2(b ) ) .
the lower two concentrations of ta had noticeable effects on the mda - mb-231 cells after 48  h of treatment .
the changes in cellular morphology of the mda - mb-231 cells were similar to those observed in mcf10a cells , with the addition of brown precipitate seen in the cells exposed to the high concentration of ta .
the proliferation of the mda - mb-231 cultures was inhibited for 24  h after initial exposure to the ta ; the cell numbers increased after 48  h but not to that of untreated levels ( figure 3(b ) ) .
the effects of ta on mcf7 cells were similar to those seen on mcf10a cells .
there was a change in cell morphology , from spindle shaped to more round cells ( figure 2(c ) ) . at the highest concentration of ta , brown precipitate
if ta reduces cell numbers and alters cell morphology of breast cells by inducing apoptosis , ta - treated cultures of breast cell lines were subjected to tunel assays .
previous studies have demonstrated that mcf7 cells are sensitive to the effects of ta [ 24 , 30 ] .
only the highest concentration of ta , 10% , significantly induced apoptosis in the normal mcf10a cells after 24  h of exposure ( figure 4(a ) ) .
two concentrations of ta induced significant levels of apoptosis in mda - mb-231 cells compared to untreated mda - mb-231 cells ( figure 4(b ) ) .
mcf7 cells were more sensitive to ta exposure , as all three concentrations induced significant apoptosis within 24  h of exposure and the percentage of apoptotic cells remained significantly higher after 48  h in response to the two higher concentrations of ta , unlike in the other two cell lines ( figure 4(c ) ) .
the addition of the highest dose of ta initiated apoptosis in all three cell lines within 24  h of treatment , with the breast cancer cells having a greater number of apoptotic cells when exposed to the lower levels of ta compared to the normal mcf10a cells .
the levels of activated caspase 9 found in mcf7 cells were significantly higher than the levels observed in mcf10a and mda - mb-231 cells , for the two highest ta concentrations used after 24  h of exposure ( figure 5(a ) ) .
levels of activated caspase 9 in mcf10a and mda - mb-231 cells were only significantly elevated in response to the highest concentration of ta .
all ta - induced elevated levels of caspase 9 remained elevated after 48  h of exposure .
levels of activated caspase 3/7 were elevated in response to ta exposure in all three cell lines .
mcf7 cells are most sensitive to the proapoptotic effects of ta , as significant elevated levels of caspase 3/7 were found in response to all concentrations of ta ( figure 5(b ) ) .
the levels of activated caspase 3/7 were also elevated in mda - mb-231 cells and in mcf10a but not to the extent observed in mcf7 cells .
the triple negative mda - mb-231 cells had significantly higher levels of activated caspase 3/7 in response to the highest dose of ta compared to the mcf10a cells . the flow cytometry - based assay does not discriminate between caspases 3 and 7 .
caspases 3 and 7 share 57% sequence homology and have similar substrate preference [ 31 , 32 ] .
the results of the flow cytometry - based caspase activity assays were confirmed using western blotting .
we found levels of cleaved caspase 7 elevated in a concentration - dependent manner in mcf10a cells after 24  h of ta exposure ( figure 6(a ) ) .
the levels of cleaved caspase 7 remained elevated after 48  h of ta treatment .
a similar pattern of elevated cleaved caspase 7 was also observed in cell lysates from mda - mb-231 cells ( figure 6(b ) ) .
additional forms of cleaved caspase 7 were found in mcf7 cell lysates following ta exposure .
the smaller cleaved caspase 7 isoforms were only found in er mcf7 cell lysates , not in lysates from the normal mcf10a cells or the triple negative mda - mb-231 cells . in accordance with the flow cytometry results , the mcf7 cells demonstrated the highest levels of caspase 7 activity in response to ta exposure . according to the protein determination assay , equal amounts of protein were loaded in all lanes .
however the -actin loading control demonstrates that this did occur in the lanes labeled ta 10 , the highest concentration of ta used .
we theorize that the high levels of ta crosslinked additional proteins thereby interfered with the protein concentration determination and protein separation .
this did not interfere with flow cytometry as intact cells were analyzed in that assay .
the er mcf7 cells were more sensitive to the effects of ta , based on activated caspases 9 and 3/7 , as lower concentrations of ta were able to induce elevated levels of the caspases compared to the normal mcf10a cells and the triple negative mda - mb-231 breast cancer cells . in all conditions analyzed ,
the number of cells expressing caspase 8 was less than 1.5% , equal to untreated cells , indicating that caspase 8 is not playing a major role in ta - induced apoptosis ( data not shown ) .
ta - crosslinked collagen type i beads are stable at human body temperature and reduce er breast cancer cell numbers at a higher rate than normal breast epithelial cells and triple negative breast cancer cells .
the reduction in cell number is via induction of apoptosis through activation of caspases 3/7 and 9 .
in this study the effects of ta on normal breast epithelial and breast cancer cells were investigated .
previous studies have investigated the effects of ta on one type of breast cancer cell and colon cancer cells .
two breast cancer cell lines , mcf7 and mda - mb-231 , were used in order to determine if the ta affected different forms of breast cancer differently .
the two breast cancer cell lines were compared to the mcf10a cell line , representing normal breast epithelial cells .
the mcf7 cell line is er , while the mda - mb-231 cell line is triple negative ( erprher2 ) .
our results indicate that the er mcf7 cells were more susceptible to the apoptotic - inducing effects of the ta .
type i collagen is used in a variety of tissue engineering methods , including breast reconstruction following trauma or mastectomy , due to its intrinsic bioactivity , its biodegradability , and its availability .
uncross - linked collagen type i is unstable over a period of months , but when a crosslinking agent is added the altered mechanics result in decreased enzymatic and thermal degradability .
a leading factor in breast - cancer - related death is the recurrence of a tumor following mastectomy [ 35 , 36 ] .
if an anticancer treatment could be incorporated into a tissue - engineered product , the recurrence of cancer might be reduced .
the ta - crosslinked beads used in this study have the potential to be such a tool for breast tissue engineering .
the ta - crosslinked beads are stable at body temperature ( table 1 ) and the released ta possesses anticancer properties .
ta - crosslinked collagen sheets are biocompatible and biodurable and have demonstrated a capacity to decrease wound healing time .
taken together , these properties make ta - crosslinked collagen type i , either as sheets or small injectable beads , a biomaterial with great potential in the fight against breast cancer recurrence .
multiple epidemiological studies have indicated that diets rich in vegetables and fruits possess anticancer properties .
chemicals present in fruits and vegetables trigger apoptosis in cancer cells and have the potential to be effective in cancer prevention and treatment [ 312 ] . in most cases ,
the caspase ( cysteinyl - directed aspartate - specific proteases ) family of cysteine proteases are central regulators of apoptosis [ 37 , 38 ] .
apoptosis can be initiated by numerous methods , including activation of cell surface receptors , such as tnf receptor and the fas receptor , or by extracellular stress that induces dna damage or mitochondrial damage [ 39 , 40 ] .
each of these pro - apoptotic pathways uses different members of the caspase family . caspases 8 and 9
are initiator caspases , while caspases 3 and 7 are involved in cellular breakdown . in this study ,
caspase 8 is usually regulated by cell surface receptors such as tnfr and the fas receptor / cd95 ; as such , this indicates that ta is not activating a cell surface death receptor .
activation of caspases 3 , 7 , and 9 can occur due to extracellular stress .
caspase 9 is an initiator caspase that , once activated , cleaves and activates downstream caspases such as 3 and 7 .
the results show that ta exposure facilitated increased caspase 3/7 and caspase 9 but did not increase caspase 8 . one possible explanation for the increased sensitivity of the er mcf7 cells to the effects of the ta may be due to the lack of expression of caspase 3 by mcf7 cells .
both caspase 3 and caspase 7 are downstream of caspase 9   and caspase 3 can act as a feedback regulator of caspase 9 . the lack of caspase 3 within the mcf7 cells may allow unchecked activation of the executioner caspase 7 by the initiator caspase 9 .
we found levels of both activated caspase 9 and caspase 7 elevated in response to ta . in conclusion , our results demonstrate a previously undocumented mechanism where ta induces apoptosis in breast epithelial cells via activation of caspases 9 and 7 .
we demonstrated that the er cell line mcf7 was more susceptible to the effects of ta exposure compared to normal mcf10a cells and triple negative breast cancer cells mda - mb-231 .
ta crosslinks collagen and collagen is a biomaterial used in breast reconstruction surgeries following mastectomies ; thus ta - crosslinked collagen represents a new type of biomaterials that possess anticancer properties .
from a clinical perspective this represents a new avenue in the fight against er breast cancer recurrence in patients .
 research efforts investigating the potential of natural compounds in the fight against cancer are growing . tannic acid ( ta ) belongs to the class of hydrolysable tannins and is found in numerous plants and foods . 
 ta is a potent collagen cross - linking agent ; the purpose of this study was to generate ta - cross - linked beads and assess the effects on breast cancer cell growth . 
 collagen beads were stable at body temperature following crosslinking . 
 exposure to collagen beads with higher levels of ta inhibited proliferation and induced apoptosis in normal and cancer cells . 
 ta - induced apoptosis involved activation of caspase 3/7 and caspase 9 but not caspase 8 . 
 breast cancer cells expressing the estrogen receptor were more susceptible to the effects of ta . 
 taken together the results suggest that ta has the potential to become an anti - er+ breast cancer treatment or preventative agent . 
1. Introduction
2. Materials and Methods
3. Results
4. Conclusions
5. Discussion