of these newly diagnosed breast tumors , 6570% will express the estrogen receptor alpha ( er ) . estrogen activation of the er is required in the development of a healthy mammary gland . however , it also can be involved in the development of both primary and secondary breast cancers due to altered er signaling [ 3 , 4 ] . activation of er by estrogen ( e ) promotes cell growth and survival of tumor cells . ae drugs can be used in the metastatic , adjuvant , and chemopreventive settings and responses are typically seen in about 70% of er+ patients selected for such treatment [ 4 , 6 ] . currently , tamoxifen ( tam ) is the most widely used ae for er+ breast cancer . tam is an example of a selective estrogen receptor modulator ( serm ) which acts as an antagonist to er in the breast , leading to a reduction in the proliferation of tumor cells . however approximately 1/3 of tumors treated with tam either possess de novo or acquire resistance to tam , leading to breast cancer recurrence . further , tam acts as an er agonist in the endometrium and in certain cases in the breast epithelium [ 4 , 8 , 9 ] . fulvestrant ( faslodex , ici 182 , 780 ; ici ) is a steroidal ae designed to have no agonist activity with the er . ici acts by degrading , and downregulating the er in the tumor cells [ 10 , 11 ] . currently , ici is used for the treatment of advanced breast cancer that is resistant to other endocrine therapies . it is effective in tumors and cell lines that are resistant to tam yet still express er . however , in the clinic the duration of response ( dor ) and time to progression ( ttp ) on ici is only 19 and 5.5 months , respectively . finding strategies to increase the sensitivity of the breast cancer cells to ici may result in increased efficacy of drug therapy . there is evidence to show that healthy changes in diet can prevent up to 40% of breast cancers . further , data is beginning to show that an increased intake of fruits and vegetables in patients recently diagnosed with breast cancer may reduce the risk of recurrent breast cancer [ 1517 ] . this preventive effect can largely be attributed to the various phytochemicals present in fruits and vegetables . these bioactive compounds have been shown to affect the development of both primary and secondary breast cancer by affecting cell proliferation , survival , and death [ 3 , 18 ] . red raspberry ( rrb ) is a readily available fruit that is part of our diets and is a rich source of phytochemicals . it is composed of compounds that inhibit the proliferation of many types of cancer cells , including breast cancer [ 1921 ] . rrb contains high levels of anthocyanins such as cyanidin-3-sophoroside , cyanidin-3-glucoside , pelargonidin-3-glucoside , and ellagic acid [ 3 , 22 ] . in vivo studies in mice further , the polyphenols present in rrb can inhibit nuclear receptors , growth factors , and kinase signaling leading to cell - cycle arrest , apoptosis , or autophagy - associated cell death . the red raspberry extract ( rre ) used in the study has been previously standardized and inhibits the growth of several cancer cell lines , including breast cancer , in a dose - dependent manner . the java plum , also called the jamun fruit , is the fruit of eugenia jambolana lam . , a tree that can be found in florida and hawaii in the united states and other various tropical zones in the world . the jamun fruit extract ( ejae ) used in this study has been previously standardized . ejae is rich in anthocyanidins including petunidin , malvidin , delphinidin , cyanidin , and peonidin [ 24 , 25 ] . ejae reduces the proliferation of mcf7-aro ( aromatase and er positive ) and mda - mb-231 ( er negative ) breast cancer cell lines . neither ejae nor rre has been previously tested in er+ , ici - resistant cell lines . clarke and coworkers have developed a series of cell lines as an in vitro model of ae resistance . the lcc series were initially derived from mcf7 cells and consists of lcc1 ( e independent , e stimulated and tam and ici sensitive ) and lcc9 ( e independent , e stimulated and tam and ici resistant ) [ 26 , 27 ] . more recently we have derived another ici - resistant variant of the zr75 - 1 cells ( zr75 - 1r ) , that was developed by culturing zr75 - 1 in sequentially increasing concentrations of ici for more than one year ( a. zwart and r clarke , unpublished data ) . these cell lines serve as in vitro models that represent some phenotypes of ici - resistant breast cancer . in this study , we tested the effects of jamun ( ejae ) and red raspberry ( rre ) extracts and the individual phenolics ellagic acid ( ea ) and delphinidin ( del ) on ici - sensitive ( lcc1 , zr75 - 1 , bt474 ) and resistant ( lcc9 , zr75 - 1r ) cells . we hypothesized that in sensitive cells , berry extracts and their compounds would have a synergistic effect with ici , increasing the inhibition of cell proliferation by a sublethal dose of ici ( < ic50 ) , and in resistant cells , the extracts would resensitize the cells to ici making them more susceptible to ici - induced growth inhibition . cell proliferation was measured in the presence of extracts / compounds with or without ici . in addition , molecular markers of apoptosis , autophagy , and er signaling were also analyzed to understand the mechanism by which ejae , rre , and their compounds reduced the growth of these cells . the lcc1 and lcc9 cells were cultured in modified imem ( without phenol - red ) containing 5% charcoal - stripped calf serum ( e level < 10  m. ) the zr75 - 1 , zr75 - 1r , and bt474 cells were cultured in modified imem containing 5% fetal bovine serum ( e levels ~10  m. ) establishment of lcc1 cells has been previously described . lcc9 and zr75 - 1r cells were derived by the long - term selection of lcc1 and zr75 - 1 cells , respectively , in sequentially increasing concentrations of ici as described . cells were then harvested at 70% confluence for use in cell proliferation and biomarker analysis . navindra p. seeram ( university of rhode island , ri ) and have been previously described . briefly , ejae was obtained by sequential extraction of freeze - dried jamun fruit in cold hexanes , ethyl acetate , and acidified methanol . ejae is the same acidified methanol extract referred to as the jamun fruit extract ( jfe ) , in a previous publication . ( irvine , ca . )   cells were plated in 48-well plates ( 5,000 cells for lcc1 and lcc9 ; 10,000 for zr75 - 1 , zr75 - 1r , and bt474 ) and incubated overnight for attachment . rre , ejae , ea , and del were dissolved in dmso and ici was dissolved in ethanol . dmso ( 0.1% ) and ethanol ( 0.01% ) were used as appropriate vehicle controls either alone or in combination . all cells were treated with 1100  g / ml rre , 0.1100  g / ml ejae , or 0.110  m ea or del with or without ici ( 1  nm and 1  m ) . for sensitive cell lines ( lcc1 , zr75 - 1 , and bt474 ) , cells were treated with 1  nm ici , which is a sublethal dose and well below the ic50 of ici for lcc1 . for resistant cell lines ( lcc9 and zr75 - 1r ) , ici was used at a concentration of 1  nm and 1  m . all groups were treated after 24  h for the first time and the treatment was repeated at 72  h for all 6  d time points . cells were stained with 0.5% crystal violet in 25% methanol , dried , and the stain dissolved in 100  mm sodium citrate in 50% ethanol . cells were plated in 6-well plates ( 200,000 cells ) and incubated overnight for attachment . the concentration of phytochemical with the most significant reduction in cell proliferation for that respective cell line was used for treatment of cells for molecular marker analysis . after 3  d , cells were suspended in radioimmunoprecipitation assay ( ripa ) buffer ( 50  mmol / l tris - hcl , ph 7.4 ; 150  mmol / l nacl ; 1% np40 ; 0.25% na - deoxycholate ; 1  mm phenylmethylsulfonyl fluoride ( pmsf ) ; 1  mm sodium orthovanadate ; 1x roche complete miniprotease inhibitor cocktail ) . twenty forty  g of protein was fractionated using an sds - page gel , transferred to a nitrocellulose membrane , and blocked with 5% milk in tbs - t . these membranes were incubated with primary ( 1  :  1000 ) followed by an appropriate hrp - conjugated secondary antibodies ( 1  :  5000 ) for 1  h at room temperature . reactive products were visualized by chemiluminescence ( thermoscientific , rockford , il ) and quantified by densitometry using the quantity one software ( bio - rad , hercules , ca ) . membranes were stripped and reprobed for -actin ( 1  :  1,000 ) as the loading control . cell proliferation data is derived from three to four independent experiments performed in triplicate for each cell line . a t - test was used to calculate the differences in mean using microsoft excel and a p value   0.05 was considered significant . the relative index ( ri ) was also calculated using the formula ri  =  s(expected)/s(observed )  =  s[a ]  s[b]/s[a + b ] , where a and b are extracts / compounds and ici , respectively . an ri  =  1 is considered additive , < 1 antagonism or absence of synergism , and > 1 presence of synergism . in this study we have used three models of endocrine resistance to test the effect of berry extracts and compounds . the lcc1 cells , originally derived from mcf7 cells , are er+ , e independent , and sensitive to both tam and ici . these cells are er+ , e independent and are tam and ici cross - resistant . previous studies have shown the effect of both rre and del in mcf7 cells [ 22 , 29 ] . we also used zr75 - 1 cells , initially derived from a tumor that was unresponsive to tam . these cells are er+ , pgr+ , tam , and ici sensitive and express a low level of p53 [ 31 , 32 ] . zr75 - 1r cells were derived from zr75 - 1 cells by a similar procedure as for lcc9 cells . they also are cross - resistant to tam and ici ( data not shown ) . the bt-474 cells belong to the luminal b molecular classification along with the zr75 - 1 cells . these cells are er+ , pgr+ and overexpress her 2 [ 32 , 33 ] . to our knowledge , this is the first study to assess the effects of berry extracts in lcc1 , lcc9 , zr75 - 1 , zr75 - 1r , and bt-474 cells . this is also the first study to evaluate the effect of berry extracts and phytochemicals on ici - resistant cell lines . all ici - sensitive cells were slightly growth inhibited by 1  nm ici ( 1030% ) . this effect was most prominent after 6  d. in lcc1 cells , 6  d ici treatment resulted in a 30% decrease in cell proliferation compared to vehicle treatment ( 0.705 ; p value =  0.0003 ) . in zr75 - 1 cells , 1  nm ici did not cause a significant reduction in cell proliferation at 3  d ( fold change  =  0.91 ) , but caused a 20% reduction at 6  d ( p = 0.02 ) . in bt474 cells , ici treatment ( 1  nm ) alone caused a 13% and 25% ( p = 0.02 ) reduction in cell proliferation after 3 and 6  d , respectively . since the growth inhibitory effect was less than 50% ( < ic50 ) , we considered 1  nm ici to be sublethal dose in sensitive cells . on the other hand , resistant cells were not growth inhibited by ici concentrations of up to 1  m . thus , we tested the effects of extracts and compounds to resensitize lcc9 and zr75 - 1r at two different concentrations of ici ( 1  nm and 1  m ) . we found that all berry extracts and compounds had an estrogenic effect on lcc1 cells indicated by the moderate to significant , dose - dependent , increase in cell proliferation after 6  d treatment ( figures 1(a)1(d ) ) . further , this effect was found to be er - mediated since cotreatment of lcc1 cells with ici abrogated the effects of extracts / compounds ( figures 1(a)1(d ) ) . these effects were more prominent after 6  d rather than a 3  d treatment . of note , del has a significant proliferative effect on lcc1 at 0.1  m , 1  m , and 10  m with a fold change of 1.26 ( p = 0.046 ) , 1.33 ( p = 0.035 ) , and 2.04 ( p = 0.008 ) , respectively ( compared to vehicle value of 1.00 ) ( figure 1(c ) ) . after 6  d treatment , concentrations of 0.1  m and 1  m of ea showed a nonsignificant proliferative effect , but 10  m of ea showed no change in cell proliferation ( figure 1(d ) ) . this shows that there is a specific range of concentrations at which ea may have estrogenic properties . while berry extracts and compounds had significant growth promoting effects on e - independent lcc1 cells , they showed a dose - dependent but moderate growth inhibitory effects of zr75 - 1 and bt474 cells grown in e - sufficient medium ( figures 1(e)1(l ) ) . ejae alone , dose dependently inhibited the proliferation of zr75 - 1 cells after 6  d treatment ( figure 1(e ) ) . this effect was enhanced by cotreatment with ici but significant only at 100  g / ml of ejae ( p = 0.05 ; ri  =  0.90 ) . in bt474 cells , there were no synergistic or additive effects seen after addition of 1  nm ici ( figure 1(i ) ) . however , it must be noted that the lower dose had a greater effect when combined with ici suggesting that doses in physiologically achievable ranges may achieve an additive effect with drug therapy . a lower dose of rre ( 1  g / ml ; 22% ) was more effective than the higher dose ( 100  g / ml ; 13% ) in zr75 - 1 cells after 3  d. however , at 6  d , only the 100  g / ml rre showed a 20% reduction in cell proliferation ( figure 1(f ) ) . this effect was slightly modified to 26% by the addition of 1  nm ici . in bt474 cells , both 1 and 100  g / ml rre showed  15% reduction in cell proliferation without ici ( p  0.05 ) after 3  d treatment . however , after 6  d , rre alone showed a dose - dependent 1528% reduction ( p  0.05 at 10  g / ml ) ( figure 1(j ) ) . however , an ri of 0.86 in zr75 - 1 and of 0.89 in bt474 suggests that at higher doses ( 100  g / ml ) rre and ici may trend toward antagonizing the action of each other . in zr75 - 1 cells , we observed only 28% and 20% antiproliferative effect at the highest dose of del ( 10  m ) at 3 and 6  d , respectively ( ns ; figure 1(g ) ) . however , a linear dose response and a synergistic effect were observed when ici was added ( ri    1.0 ) ( ns ; figure 1(g ) ) . in bt474 cells , after 3  d , the lowest dose of del was the most effective in curbing proliferation ( 15% reduction ; p = 0.02 for 0.1  m versus 5% for 1 and 10  m ) . an additive effect was also evident with 1  nm ici ( ri  =  1.0 ; 22% reduction for 0.1  m del+ ici versus 13% for ici alone or other doses of del+ici ) . after 6  d , all doses of del ( 0.1  m10  m ) achieved a  30% reduction in the presence of ici showing an ri . =  1.0 , suggesting that the effects were additive ( figure 1(k ) ) . ea treatment did not significantly alter the proliferation of either cell line at doses tested with or without ici after 6  d treatment ( figures 1(h ) and 1(m ) ) . in lcc9 cells , ejae alone ( 1  g / ml ) caused a modest , but significant , reduction in cell proliferation after 3  d ( 19% ; p = 0.028 ) . however this effect was not present at 6  d ( figure 2(a ) ) . ejae did not significantly resensitize lcc9 cells to either 1  nm ( figure 2(a ) ) or 1  m ici at any concentration tested ( data not shown ) . at 6  d , rre ( 10  g / ml ) increased the inhibitory response of lcc9 cells to 1  nm ici ( 33% ; ns . ; ri  =  1.34 ) ( figure 2(b ) ) . however , we did not observe this synergistic effect at 1  m ici ( data not shown ) , suggesting that specific cellular responses are elicited at specific doses of the drug ( ici ) and the extract ( rre ) . del alone had no effect on cell proliferation at 3 or 6  d. in the presence of 1  nm ici and 0.1  m del , there was a 35% induction of cell proliferation ( p = 0.03 ) at 3  d. however , this effect was not seen at 6  d ( figure 2(c ) ) . rre alone ( 100  g / ml ) caused a 28% and 18% nonsignificant reduction at 3 and 6  d , respectively . this effect was not altered by ici ( 1  nm ) after 3  d ( 27% ) or 6  d ( 11% ) . ea neither had a significant growth inhibiting effect on lcc9 cells after 3  d ( data not shown ) or 6  d treatment , nor showed an ability to resensitize lcc9 cells to 1  nm ici ( figure 2(d ) ) . zr75 - 1r cells were overall more susceptible to the effects of both ejae and rre than lcc9 cells . after 3  d , ejae alone at 100  g / ml and 0.01  g / ml showed a reduction in cell proliferation by 20% and 35% , respectively . however , after 6  d treatment , the reduction was sustained only at the highest dose ( figure 2(e ) ) . in the presence of 1  nm ici , ejae ( 100  g / ml ) showed a reduction in cell proliferation ( 35% ; ri  =  1.23 ) . this effect is slightly greater than that seen for ejae alone ( figure 2(e ) ) . however , in the presence of 1  m ici , we observed a linear dose response with 1 , 10 , and 100  g / ml of ejae reducing cell proliferation by 15% , 15% , and 40% , respectively ( ri    1.2 for each treatment ) ( figure 3(a ) ) . an interesting rre dose response with an inverted - u curve was observed in zr75 - 1r cells . at 6  d , lower doses ( 1  g / ml and 10  g / ml ) showed a proliferative effect , whereas higher dose ( 100  g / ml ) showed a 22% reduction in cell proliferation ( figure 2(f ) ) . addition of 1  nm ici does not change this effect ( figure 2(f ) ) . however , in the presence of 1  m ici , rre significantly and dose - dependently inhibited zr75 - 1r growth by 2550% ( p = 0.05 at 100  g / ml ; ri    1.4 ) ( figure 3(b ) ) . both 1  g / ml and 10  g / ml rre had a highly synergistic effect in the presence of 1  m ici with ri values of 1.98 and 1.71 , respectively ( figure 3(b ) ) . del treatment of zr75 - 1r did not show any significant change in cell proliferation after 3 or 6  d treatment with or without 1  nm ici ( figure 2(g ) ) . ea treatment of zr75 - 1r did not show a change in cell proliferation ( figure 2(h ) ) . however , ea treatment with 1  nm ici showed a synergistic dose response after 3  d as 1  m and 10  m displayed a 15% ( ri . this shows that ea could resensitize the cells to ici at 3  d. however , this response was not seen after 6  d ea treatment ( figure 2(h ) ) .    in order to understand the molecular mechanisms by which berry extracts and its constituents cause the observed effects in lcc1 and lcc9 cells , we assessed the levels of various molecular markers in these cells after treatment with berry extract / compound alone or in the presence of ici . since the antiproliferative effects of the compounds were evident at 3  d , we chose this time point to study molecular mechanisms . also , we specifically chose those concentrations of extracts / compounds that produced the greatest reduction in cell proliferation at 6  d ( figures 1(a)1(h ) ) . as expected in lcc1 cells , ejae and del alone showed an estrogenic response with an observed increase of 3- and 2.5-fold , respectively , in progesterone receptor ( pgr ) expression ( figure 4(a ) ) . interestingly , ea and del alone also raised er levels ( figure 4(b ) ) . we also assessed molecular markers of cell death ( parp cleavage ) , autophagy ( p62 , beclin 1 , light chain-3 ( lc3 ) ) , and the levels of the antiapoptotic b - cell lymphoma ( bcl ) family members ( bcl2 , bclw , and bclxl ) . it has been previously shown that combined knockdown of bcl2 and bclw leads to resensitization of lcc9 cells to tam and ici . although we observed a clear increase in parp expression by ea alone and a reduction in levels after ici treatment across all extracts , we did not see cleavage of parp in any of the treatments ( figure 4(c ) ) . ejae , rre , and del caused a 2-fold induction in lc3ii levels , which was reversed by the addition of ici , except for ejae ( figure 4(f ) ) . ejae  +  ici caused a 3.3-fold induction of lc3ii in lcc1 cells ( figure 4(f ) ) . on the other hand , ejae , del , and ea increased the levels of p62 to various extents ( 1.4 to 2.5-fold ; figure 4(d ) ) . as expected , ici treatment reduced p62 levels by 60% , suggesting active autophagy . ejae did not alter this response ; however , rre , ea , and del all reversed this reduction . rre and ea treatments significantly increased the levels on beclin-1 ( figure 4(e ) ) . ejae induced beclin-1 levels by 5-fold in the presence of ici ( figure 4(e ) ) . the effect of extracts / compounds on the bcl2-family members was neither uniform nor significant ( figures 4(g)4(j ) ) . ea increased the expression of bcl-2 > 1.5-fold without or with ici ( figure 4(g ) ) . bclw and bclxl expression was induced by ea and del alone and ejae  +  ici ( figures 4(h ) and 4(j ) ) .   in lcc9 cells , ejae  +  ici treatment increased lc3ii levels by 13.7-fold over vehicle or ici - only treatment , suggesting that ejae may increase autophagosome formation in lcc9 cells . however , we failed to see a baseline increase in lc3ii with ici - only treatment . on the other hand , rre  +  ici , del alone , and del  +  ici showed an across - the - board increase in the expression of all bcl2-family proteins ( bcl2 , bclw , and bclxl ) , as well as beclin-1 . rre  +  ici treatment and del increased p62 expression 22.5 fold suggesting an inhibition of autophagy . in this study , we tested whether berry extracts / compounds synergize with a sublethal dose of ici and increase drug response in ici - sensitive cell lines . we also tested whether cotreatment of berry extracts reverses the resistant phenotype in ici - resistant cells , thereby leading to an increased cell death in the presence of ici . we used multiple cell lines with different molecular characteristics that are representative of the er+ tumors commonly seen in the patient population . the ici - sensitive cells used can be divided into luminal a ( er+ , pgr+ , her2- ; lcc1 ) and b ( er+ , pgr+ , her2 + ; zr75 - 1 and bt-474 ) subtypes   and the resistant cells derived from the same subtypes ( lcc9 from lcc1 and zr75 - 1r from zr75 - 1 ) . further , these cells were also cultured in different media with lcc1 and lcc9 in e - deficient medium ( < 10  m  e ) and all other cell lines in an e - sufficient medium ( ~10 m  e ) . in addition to this , we tested two berry extracts ( ejae and rre ) and their representative compounds ( del and ea ) . this complex design was purposely selected to mimic the complex nature of breast cancer as it presents in the clinic . in the clinic , we are likely to see patients , both pre- ( e sufficient ) and postmenopausal ( e deficient ) , with er+ tumors of either subtypes ( luminal a and b ) that will be treated with ici . furthermore , dietary recommendations will involve whole fruits and not pure components . finally , we included individual components from each berry extract to determine the contribution of the food matrix . the results from the cell proliferation studies are straightforward and answer many of these questions . both berry extracts and compounds synergize with a sublethal dose of ici ( 1  nm ) to cause an inhibition of cell proliferation in ici - sensitive cell lines . the berry extracts / compounds are most effective in bt-474 cells and least effective in lcc1 cells . the effects of the extract / compounds alone vary greatly with the doses used and the type of cell - line . we observed that both extracts and compounds had an estrogenic effect on lcc1 cells grown in an e - deficient medium ( figures 1(a)1(d ) ) . this effect was significant and dose dependent for ejae and del ( figures 1(a ) and 1(c ) ) . however , the same extracts / compounds showed a dose - dependent inhibition of cell proliferation in zr75 - 1 and bt-474 cells grown in e - sufficient medium ( figures 1(e)1(l ) ) . this effect is similar to that observed with tam , which stimulates mcf-7 cell growth under e - deficient , and inhibits proliferation under e - sufficient conditions ( aiyer , unpublished results ) . the serm activity of tam reported in literature also suggests such effects [ 6 , 36 ] . many polyphenolic compounds including those present in berries have been reported to show serm - like effects . further , they also show a dose - dependent selective recruitment of coactivators and repressors to the er , which dictates whether these compounds will act as estrogens or antiestrogens [ 3 , 37 ] . for example , rre shows a slight estrogenic effect ( increased cell growth ) at 1  g / ml , whereas inhibits growth at 10 and 100  g / ml ( figure 1(g ) ) . regardless of their effects alone , all extracts / compounds tested show varying degrees of either additive or synergistic response with ici in sensitive cells . both bt-474 and zr-75 cells are her2 + , whereas lcc1 cells are not [ 6 , 33 ] . although all cell lines possess er , the constitutive levels of this receptor vary greatly among these cell lines at baseline ( aiyer , unpublished results ) . thus , the differential effect of berry extracts / compounds seen among various cell lines could be partially explained by these differences . many berry compounds have been shown to deactivate tyrosine kinase signaling and can inhibit her2-mediated effects on cell proliferation ( reviewed in ) . there is an extract - induced increase in pgr levels ( figure 4(a ) ) , which suggests a classic genomic response due to er activation . additionally , treatment with extracts seems to stabilize er levels and antagonize the ici - mediated degradation of er in lcc1 cells ( figure 4(b ) ) . the implications of such effects on long - term ici - treatment and resistance development must be explored further .   in ici - resistant cells , neither extracts nor pure compounds resensitized the cells to lower dose of ici ( 1  nm ) ( figures 2(a)2(h ) ) . the resistant cells continue to grow in the presence of ici up to 1  m and hence may be resistant to many chemopreventive agents tested at lower doses . however , both ejae and rre at all concentrations tested synergize with 1  m ici to cause a significant reduction in resistant ( zr75 - 1r ) cell growth ( figure 3 ) . a limitation of this study is the lack of clear mechanistic detail regarding how berry extracts and compounds reduce cell growth . we studied the induction of autophagy using p62 , lc3ii , and beclin 1 as surrogate markers . many compounds present in berries have been shown to induce autophagy - associated cell death [ 3 , 3941 ] . however , in terms of endocrine resistance , autophagy is possibly a survival mechanism and inhibition of autophagy alone , or in combination with bcl2+bclw coinhibition , resensitizes the resistant cells to aes . there is some evidence to suggest that rre , del , and ea may interfere with autophagy in lcc1 cells . they reverse the ici - induced degradation of p62 to various extent ( figure 4(d ) ) . it is clear that ejae    ici consistently increases autophagy in both lcc1 and lcc9 cells . since we have not directly counted lc3 punctae formation , the data is inconclusive as to whether berry extracts / compounds inhibit autophagy and the exact mechanisms by which they do so . however , we only analyzed these markers in lcc1 and lcc9 cells , where the effects of the extracts / compounds were minimal . the assessment of the markers in other cell lines is currently underway and should provide a clearer understanding of the various mechanisms by which berry extracts / compounds increase cell - line responsiveness to ici .   other research into berry extracts and compounds previous studies show that the ic50 of rre in mcf7 cells grown in e - sufficient medium is 190  g / ml at 48  h . this is comparable to the effect seen in the zr75 - 1 and bt474 cells , grown under similar conditions , in this study . ejae has been tested in breast cancer cell lines such as mcf7-aro and mda - mb-231 with an ic50 of 27 and 40  g / ml , respectively , at 72  h . have shown that hydrolyzed extract of the jamun fruit pulp was effective in reducing the growth of the non - small cell lung cancer cell - line a549 with an ic50 of 59  g / ml at 72  h. such studies have typically reported the cytotoxic effects of extracts / compounds within 72  h. by contrast , our data presents the effect at both 72  h ( 3  d ) and at 6  d. it is seen that many of the effects at 3  d are not carried over at 6  d. this could be due to the clonogenic expansion of the cells that were initially unresponsive to the chemopreventive agent . since effects of dietary bioactive compounds on cancer cells are a chronic process , 2 or 3  d cell proliferation studies , as is typically performed for a majority of these agents , may lead to the overrepresentation of the effectiveness of such compounds .    in conclusion , using a clonogenic assay , we have shown that berry extracts and compounds can increase the cell - death response of ici - sensitive breast cancer lines to a sublethal dose of ici ( 1  nm , < ic50 dose ) . additionally , berry extracts resensitize ici - resistant cells to ici treatment showing a synergistic response , especially at the higher dose ( 1  m ) . finally , we found that extracts were more effective at lower , physiologically relevant doses than at higher experimental doses in some cell lines . these results indicate that berry extracts and compounds can potentially interact with ici in breast epithelial cells to alter drug - response . further in vivo research is warranted regarding the implications of such food - drug interactions in response to ici treatment and the development of drug resistance .