BIOMEDICAL REPORTS 5: 579-584, 2016 Effects of blueberries on migrtion, invsion, prolifertion, the cell cycle nd poptosis in heptocellulr crcinom cells WEI ZHAN 1*, XIN LIAO 2*, LEI YU 3, TIAN TIAN 3, XING LIU 3, JING LIU 2*, LI-JUN CAI 4, XUAN XIAO 3*, RU-JIA XIE 3 nd QIN YANG 3 Deprtments of 1 Anus nd Colorectl Surgery, 2 Rdiology, 3 Pthology nd 4 Neurology, The Affilited Hospitl of Guizhou Medicl University, Guiyng, Guizhou 550004, P.R. Chin Received April 25, 2016; Accepted July 11, 2016 DOI: 10.3892/br.2016.774 Abstrct. The im of the present study ws to investigte the effects of blueberry consumption on the migrtion, invsion, prolifertion, cell cycle nd poptosis in humn heptocellulr crcinom (HCC) cells, in order to provide clinicl tretment nd prevention strtegies for liver cncer using nticncer therpeutic gents. Rbbiteye blueberry ws prepred s fresh juice nd fed to rts t low, moderte nd high dosges (25, 50 nd 100%, respectively) by dily gstric gvge. Seven dys lter, the rts were scrificed nd the blood serum ws obtined for co-culture with HEPG2 cells. The MTT ssy ws used for detecting cell prolifertion, Trnswell ssy ws performed for migrtion nd invsion evlution, nd cell cycle nd poptosis were ssessed by flow cytometry. After co-culturing with the blood serum of rts tht were fed different dosges of blueberry juice, the inhibition rte of HEPG2 cells in the three groups ws significntly lower thn tht in the control group t 48 nd 72 h (P<0.05). The number of migrted nd trnsmembrne HEPG2 cells in the three groups ws significntly lower thn tht in the control group t 48 nd 72 h (P<0.05). The number of migrted HEPG2 cells in the high dosge group ws significntly lower thn tht in the low dosge group t 48 h, nd the numbers of migrted HEPG2 cells in the high nd moderte dosge groups were significntly lower thn tht in the low dosge group t 72 h (P<0.05). The number of trnsmembrne HEPG2 cells in the high dosge group ws significntly lower thn tht in the low dosge group t 48 h (P<0.05). The numbers of HEPG2 cells t the G 2 /M stge in the three groups were significntly Correspondence to: Dr Ru-Ji Xie or Dr Qin Yng, Deprtment of Pthology, The Affilited Hospitl of Guizhou Medicl University, 4 Beijing Rod, Guiyng, Guizhou 550004, P.R. Chin E-mil: xieruji_06@163.com E-mil: yngqin_06@163.com * Contributed eqully Key words: blueberry, HEPG2, prolifertion, invsion, histone cetyltion lower thn tht in the control group, nd the number of HEPG2 cells in the high dosge group ws significntly lower thn tht in the low dosge group, t 48 nd 72 h (P<0.05). The poptosis rte in the three groups ws significntly higher thn tht in the control group, nd the poptosis rte in the high dosge group ws significntly higher thn tht in the low dosge group t 48 nd 72 h (P<0.05). Thus, blueberries my fcilitte the clinicl tretment of HCC, providing novel therpeutic nd prevention strtegy for HCC s n nticncer therpeutic gent. Introduction Liver cncer ws the fourth most common cuse of mortlity worldwide, nd Chin ccounted for ~53% of ll liver cncer ssocited mortlities (1). The incidence of liver cncer grdully incresed in developing (2) nd developed (3) countries (4,5). In Chin, >90% of ptients with primry liver cncer presented with heptocellulr crcinom (HCC), which ws the second-leding cuse of cncer-ssocited mortlity influencing individuls of ll ges (6,7). Although tretment nd prevention strtegies hve been cliniclly developed, the globl, overll survivl of HCC ptients remined prticulrly poor (8,9). The mjority of the poor prognoses were ssocited with recurrence nd metstsis following tretment, including curtive resection (10,11). Therefore, the mechnisms of liver cncer recurrence nd intervention strtegies for liver cncer recurrence nd metstsis re required, thus future investigtions with lrger smple sizes re required. Blueberry is member of the Vccinicee fmily (genus, Vccinium). In the Koren Peninsul nd the Northest of Chin, blueberries re grown widely nd re commonly dministered s trditionl Chinese therpeutic gent for treting inflmmtory diseses. Blueberry nthocynins (BAs) were min medicinl ctive ingredient. Previous studies indicted tht BA inhibited tumor growth nd induced poptosis of tumor cells in brest (12), lung (13), nd colorectl (14) cncer, mongst others. In ddition, BA ws reported to be involved in the control of obesity (15) nd dibetes mellitus (16), prevention of crdiovsculr disese (17), vision ugmenttion (18) nd cerebrl function (19). In the present study, different concentrtions of blueberry juice were dministered to rts by gstric gvge nd, fter
580 ZHAN et l: EFFECTS OF BLUEBERRIES ON HCC MIGRATION 7 dys, blood serum ws obtined for co-culture with HEPG2 cells. Prolifertion, migrtion, invsion, cell cycle nd poptosis were detected in HEPG2 cells to investigte the effects of blueberry on the prolifertion, poptosis nd histone cetyltion in HEPG2 cells. The im of the present study ws to estblish n nticncer therpeutic gent for the clinicl tretment nd prevention of liver cncer. Mterils nd methods Fresh blueberry juice. Rbbiteye blueberries were obtined from the Blueberry Production Field (M-Jing, Chin) of the Guizhou Acdemy of Sciences (Guiyng, Chin) nd stored t -20 C. The fresh juice ws prepred from the crude blueberries by homogeniztion, nd diluted in physiologicl sline to finl volume of 1 ml [originl blueberry juice (100%) contined 100 g blueberry, which ws further diluted to 25 nd 50% in physiologicl sline]. Animls. Ethicl pprovl ws obtined for the niml experiments from Guizhou Medicl University (Guiyng, Chin), nd niml tretment ws in ccordnce with the Guidelines for Animl Cre nd Use. Twelve, mle, specific pthogen-free (SPF) Wistr rts (weight, 200±20 g) were obtined from the niml center of Guizhou Medicl University (Guiyng, Chin) nd mintined in n SPF room, t 25 C with 12-h of light/drk cycle. The rts hd free ccess to food nd wter, nd were housed seprtely. The 12 rts were rndomly divided into four equl groups s follows: Low-dose group, fed 25% blueberry juice, which ws from the originl blueberry juice (100%) diluted in sline; moderte-dose group, fed 50% blueberry juice; high-dose group, fed 100% blueberry juice; the control group, fed 1 ml physiologicl sline. Gstric perfusion ws performed twice per dy t 9:00.m. nd 9:00 p.m. for 7 dys in totl. One dditionl gstric perfusion ws performed 2 h fter the finl gstric perfusion, in order to prevent vomiting nd mintin the blueberry juice concentrtion. One hour fter the repeted gstric perfusion, 10-ml blood smples were collected from the femorl rtery of the rts nd centrifuged t 1,500 x g for 15 min t 4 C. The superntnts obtined from blood smples of rts in the sme group were combined. The complement in the blood serum ws inctivted t 56 C for 30 min using wter bth nd 0.22-µm filter (Merck Millipore, Drmstdt, Germny) ws used for removing bcteri. The smples of blood serum from rts tht were fed different dosges of blueberry juice were stored t -80 C. The rts were scrificed in sturted CO 2. Serum preprtion. Serum from the four groups ws diluted with 10% Dulbecco's modified Egle's medium (DMEM) contining Gibco 10% fetl bovine serum (FBS; Thermo Fisher Scientific, Inc., Shnghi, Chin) for co-culture with the HEPG2 cells. HEPG2 cell culture. HEPG2 cells were obtined from Americn Type Culture Collection (ATCC) nd cultured in DMEM contining 10% FBS in 37 C, 5% CO 2 nd sturted humidity. When the cell confluence reched 90%, the HEPG2 cells were digested with 0.25% (w/v) trypsin contining 0.53 mm EDTA (Thermo Fisher Scientific, Inc.) nd subcultured s described bove. MTT detection of HEPG2 cell prolifertion. HEPG2 cells in the logrithmic growth phse were digested with trypsin s single cell suspension (density, 1x10 5 /ml) nd resuspended with DMEM contining 10% FBS. Cell suspension (100 µl) ws dded to ech well of 96-well plte for 24 h until dhesion. The medium ws removed from the 96-well plte, nd serum ws dded to ech well, with 8 prllel wells/group. The cells were then cultured for 48 nd 72 h, nd the cultured superntnt ws collected nd stored t -20 C. The cells were wshed once with 3 ml DMEM nd centrifuged to discrd the DMEM. Subsequently, 100 µl DMEM nd 20 µl MTT (5 mg/ml; Sigm-Aldrich Chin, Inc., Shnghi, Chin) were dded nd incubted t 37 C for 4 h. Dimethyl sulphoxide (150 µl; Sigm-Aldrich Chin, Inc.) ws dded nd gitted for 15 sec in the drk for dissolution. An enzyme lbeling instrument ws used to detect the bsorption (A) vlues t 492 nm. The MTT verge vlue of ech group ws obtined nd the prolifertion rte (%) ws clculted s follows: Control group - tretment group/control group x 100% Trnswell ssy detection of migrtion nd invsion in HEPG2 cells. For detecting invsion, 4 µl Mtrigel (BD Biosciences, Frnklin Lkes, NJ, USA) ws plced in Trnswell chmber (Corning Life Sciences, Shnghi, Chin) in ech well. HEPG2 cells were co-cultured t 37 C with DMEM contining 10% FBS nd 10% serum from the four groups. After 48 nd 72 h, the medium ws replced with DMEM without FBS or serum, nd the HEPG2 cells were cultured for further 6 h t 37 C. The HEPG2 cells in the logrithmic growth phse were digested with trypsin nd the cell density ws djusted to 1x10 5 /ml by resuspension with DMEM without FBS. The cell suspension (0.4 ml) ws dded to the upper prt of the chmber, nd 0.6 ml DMEM contining 20% FBS (Thermo Fisher Scientific, Inc.) ws dded to the lower prt of the chmber in 24-well plte, with three prllel holes per group. Subsequently (24 h), the Mtrigel nd the non-migrting cells in the chmbers were crefully clened. Phosphte-buffered sline (PBS; Wuhn Boster Biologicl Technology, Ltd., Wuhn, Chin; 1 ml) ws dded to the chmbers for wshing nd ws discrded. The cells were fixed with 4% prformldehyde (Beijing Solrbio Science & Technology Co., Ltd., Beijing, Chin) for 15 min, then stined with 0.1% crystl violet (Beijing Solrbio Science & Technology Co., Ltd.) solution t 25 C for 20-45 min. The cells were subsequently wshed twice with PBS nd the stined cells were observed under microscope (BX51/BX51M; Olympus Corportion, Tokyo, Jpn). For detecting cell migrtion, the steps were the sme s for the detection of invsion, however, Mtrigel ws not used. Flow cytometry detection of the HEPG2 cell cycle nd poptosis. Following co-culture with serum from the four groups for 48 nd 72 h, the HEPG2 cells were digested with trypsin nd centrifuged t 1,000 x g for 5 min t 25 C. The superntnt ws discrded nd the cells were wshed twice with PBS. A further centrifugtion ws performed (t 1,000 x g for 5 min
BIOMEDICAL REPORTS 5: 579-584, 2016 581 Tble I. A-vlue nd inhibition rte of HEPG2 cells following 48- nd 72-h co-culturing with serum from rts fed with different concentrtions of blueberry juice. Dt re presented s mens ± stndrd devitions. Co-culturing --------------------------------------------------------------------------------------------------------------------------------------- Vrible time (h) Control group Low-dosge, 25% Moderte-dosge, 50% High-dosge, 100% A-vlue 48 0.58±0.19 0.46±0.07 0.41±0.12 0.39±0.05 72 0.63±1.77 0.43±0.10 0.40±0.22 0.38±0.20 Inhibition rte (%) 48 100.00±0.00 21.97±8.54 21.33±10.60 20.05±12.85 72 100.00±0.00 20.58±9.67 22.03±8.14 21.90±10.03 P<0.05 vs. the control group t the corresponding time. A, bsorbnce. Tble II. Number of migrted or trnsmembrne HEPG2 cells following 48- nd 72-h co-culturing with serum from rts fed with different concentrtions of blueberry juice. Dt re presented s mens ± stndrd devitions. Co-culturing --------------------------------------------------------------------------------------------------------------------------------------------- Process time (h) Control group Low-dosge, 25% Moderte-dosge, 50% High-dosge, 100% Migrtion 48 65.38±13.94 50.42±20.90 44.67±19.58 40.03±21.36,b 72 70.93±16.03 46.89±22.64 40.91±26.50,b 38.05±23.36,b Invsion 48 110.82±25.54 91.44±31.26 88.47±28.95 83.39±34.16,b 72 116.85±30.62 86.59±36.80 82.54±29.47 80.10±31.34 P<0.05 vs. the control group; b P<0.05 vs. the low-dosge group. Results Figure 1. Inhibition rte of HEPG2 cells following 48- nd 72-h co-culturing with serum from rts fed with different concentrtions of blueberry juice * P<0.05 vs. control group. t 25 C) to remove the PBS. Precooled ethnol (0.6 ml) ws dded nd the HEPG2 cells were plced on ice for 30 min. The cells were then centrifuged t 1,000 x g for 5 min t 25 C. Subsequently, 0.05 ml RNse A (10 mg/ml; Sigm-Aldrich Chin, Inc.) ws dded nd mintined t 25 C for 1 h. PBS (0.15 ml) nd 0.2 ml propidium iodide (Sigm Aldrich Chin, Inc.) were then dded to stin the cells. The cell cycle nd poptosis were then nlyzed by flow cytometry (BD FACSClibur; BD Biosciences). Sttisticl nlysis. Dt re presented s the men ± stndrd devition nd were nlyzed by SPSS 11.0 softwre (SPSS, Inc., Chicgo, IL, USA). Comprisons between groups were nlyzed by Student's t-test nd P<0.05 ws considered to indicte sttisticlly significnt difference. HEPG2 prolifertion. Tble I nd Fig. 1 demonstrte the A-vlue nd inhibition rte of HEPG2 cells fter co-culturing with the serums from the different tretment groups for 48 nd 72 h. Compred with the control group t 48 h, the inhibition rtes of HEPG2 cells in the low- (21.97±8.54%), moderte- (20.33±10.60%) nd high- (20.05±12.85%) dosge groups were significntly lower (P<0.05). However, there were no significnt differences in HEPG2 cell prolifertion between the three blueberry tretment groups t 48 h. Furthermore, the inhibition rte decresed s the concentrtion of blueberry juice intke incresed. Compred with the control group t 72 h, the inhibition rtes of HEPG2 cells in the low- (20.58±9.67%), moderte- (21.03±8.14%) nd high- (21.90±10.3%) dosge groups were significntly lower (P<0.05). However, there were no significnt differences in HEPG2 cell prolifertion between the three blueberry tretment groups t 72 h. Furthermore, the inhibition rte decresed s the concentrtion of blueberry juice intke incresed. In ddition, the inhibition rtes between the different groups t 48 nd 72 h were not sttisticlly different. Migrtion nd invsion in HEPG2 cells. Tble II demonstrtes the number of migrted or trnsmembrne HEPG2 cells following co-culturing with serums for 48 nd 72 h, which were obtined from rts tht were fed different concentrtions of blueberry juice.
582 ZHAN et l: EFFECTS OF BLUEBERRIES ON HCC MIGRATION Tble III. HEPG2 cell cycle nlysis following 48- nd 72-h co-culturing with serum from rts fed with different concentrtions of blueberry juice. Dt re presented s mens ± stndrd devitions. Co-culturing -------------------------------------------------------------------------------------------------------------------------------------------- Stge time (h) Control group Low-dosge, 25% Moderte-dosge, 50% High-dosge, 100% G 0 /G 1 (%) 48 65.58±2.68 61.40±2.80 73.02±1.48 76.21±2.53 72 59.37±3.90 68.14±2.30 74.95±3.13 69.62±2.98 S (%) 48 20.96±2.05 31.09±2.46 23.17±1.55 22.89±2.60 72 25.24±3.07 18.79±2.33 17.20±1.98 23.95±2.65 G 2 /M (%) 48 13.96±2.73 7.53±1.12 4.60±1.08 1.52±0.97,b 72 17.53±3.54 13.82±2.04 7.48±1.40 6.30±1.52,b P<0.05 vs. the control group; b P<0.05 vs. the low-dosge group. Figure 2. Number of migrted HEPG2 cells following 48- nd 72-h coculturing with serum from rts fed with different concentrtions of blueberry juice. * P<0.05 vs. the control group; # P<0.05 vs. the low-dosge group. Figure 4. Number of HEPG2 cells t the G 2 /M stge following 48- nd 72-h co-culturing with serum from rts fed with different concentrtions of blueberry juice. * P<0.05 vs. the control group; # P<0.05 vs. the low-dosge group. Figure 3. Number of trnsmembrne HEPG2 cells following 48- nd 72-h co culturing with serum from rts fed with different concentrtions of blueberry juice. * P<0.05 vs. the control group; # P<0.05 vs. the low-dosge group. Compred with the control group (65.38±13.94) t 48 h, the numbers of migrted HEPG2 cells in the low- (50.42±20.90), moderte- (44.67±19.58) nd high- (40.03±21.36) dosge groups were significntly lower (P<0.05). The number of metsttic HEPG2 cells in the high-dosge group ws significntly lower thn tht in the low-dosge group (P<0.05). Furthermore, the number of metsttic HEPG2 cells decresed s the concentrtion of blueberry juice intke incresed (Tble II nd Fig. 2). Compred with the control group (70.93±16.03) t 72 h, the numbers of migrted HEPG2 cells in the low- (46.89±22.64), moderte- (40.91±26.50) nd high- (38.05±23.36) dosge groups were significntly lower (P<0.05). The numbers of metsttic HEPG2 cells in the moderte- nd high-dosge groups were significntly lower thn in the control group (P<0.05). The number of metsttic HEPG2 cells decresed s the concentrtion of blueberry juice intke incresed (Tble II nd Fig. 2). Compred with the control group (110.82±25.54) t 48 h, the numbers of trnsmembrne HEPG2 cells in the low- (91.44±31.26), moderte- (88.47±28.95) nd high- (83.39±34.16) dosge groups were significntly lower (P<0.05). The number of trnsmembrne HEPG2 cells in the high-dosge group ws significntly lower thn in the control group (P<0.05). The trnsmembrne HEPG2 cell number tended to decrese s the concentrtion of blueberry juice intke incresed (Tble II nd Fig. 3). Compred with the control group (116.85±30.62) t 72 h, the numbers of trnsmembrne HEPG2 cells in the low- (86.59±36.80), moderte- (82.54±29.47) nd high- (80.10±31.34) dosge groups were significntly lower (P<0.05). However, no significnt differences were identified in trnsmembrne HEPG2 cell numbers mong the three groups tht were dministered blueberry juice. The trnsmembrne HEPG2 cell number tended to decrese s the concentrtion of blueberry juice intke incresed (Tble II nd Fig. 3). Flow cytometric detection of the HEPG2 cell cycle nd poptosis. Tble III nd Fig. 4 demonstrte the HEPG2 cell cycle (in G 2 /M) subsequent to co-culturing for 48 nd 72 h with serum from rts tht were dministered different concentrtions of blueberry juice.
BIOMEDICAL REPORTS 5: 579-584, 2016 583 Tble IV. Apoptosis rte of HEPG2 cell following 48- nd 72-h co-culturing with serum from rts fed with different concentrtions of blueberry juice. Dt re presented s mens ± stndrd devitions. Co-culturing ------------------------------------------------------------------------------------------------------------------------------------------------------------ time (h) Control group Low-dosge, 25% Moderte-dosge, 50% High-dosge, 100% 48 4.25±0.78 7.66±1.14 10.96±1.38 30.42±3.95,b 72 5.32±1.79 13.57±1.97 20.42±2.06,b 67.64±1.85,b P<0.05 vs. the control group; b P<0.05 vs. the low-dosge group. low-dosge group. The poptosis rte incresed s the concentrtion of blueberry juice intke incresed. Discussion Figure 5. Apoptosis rte of HEPGH2 cells following 48- nd 72-h co-culturing with serum from rts fed with different concentrtions of blueberry juice. * P<0.05 vs. the control group; # P<0.05 vs. the low-dosge group. Compred with the control group (13.96±2.73%) t 48 h, the numbers of HEPG2 cells t the G 2 /M stge in the low- (7.53±1.12%), moderte- (4.60±1.08)% nd high- (1.52±0.97%) dosge groups were significntly lower (P<0.05). The number of HEPG2 cells t the G 2 /M stge in the high-dosge group ws significntly lower thn in the low-dosge group (P<0.05). The number of HEPG2 cells t the G 2 /M stge decresed s the concentrtion of blueberry juice intke incresed. Compred with the control group (17.53±3.54%) t 72 h, the numbers of HEPG2 cells t the G 2 /M stge in the low- (13.82±2.04%), moderte- (7.48±1.40%) nd high- (6.30±1.52%) dosge groups were significntly lower (P<0.05). The number of HEPG2 cells t the G 2 /M stge in the high-dosge group ws significntly lower thn in the low-dosge group (P<0.05). The number of HEPG2 cells t the G 2 /M stge decresed s the concentrtion of blueberry juice intke incresed. Tble IV nd Fig. 5 demonstrte the poptosis rte of HEPG2 cells subsequent to co-culturing for 48 nd 72 h with serum from rts tht were dministered different concentrtions of blueberry juice. Compred with the control group (4.25±0.78%) t 48 h, the poptosis rtes in the low- (7.66±1.14%), moderte- (10.96±1.38%) nd high- (30.42±3.95%) dosge groups were significntly higher (P<0.05). The poptosis rte in the high dosge group ws significntly greter thn in the low-dosge group. The poptosis rte incresed s the concentrtion of blueberry juice intke incresed. Compred with the control group (5.32±1.79%) t 72 h, the poptosis rtes in the low- (13.57±1.97%), moderte- (20.42±2.06%) nd high- (67.64±1.85%) dosge groups were significntly higher (P<0.05). The poptosis rte in the high dosge group ws significntly higher thn in the Blueberries contin numerous components, some of which hve been reported to fcilitte with the prevention of different diseses. For exmple, nthocynin is min component of blueberry, nd the high nthocynin content of blueberries ws found to be chemopreventive nd exerted therpeutic effects ginst brest cncer (19). Pterostilbene, primrily found in blueberries, is n ntioxidnt tht cts s n effective nticncer gent in vrious common mlignncies, including brest nd colon cncer (20). In ddition, ellgic cid hs been ssocited with the prevention of oxidtive DNA dmge nd modultion of DNA repir gene expression (21). These components interct nd influence biologicl processes in the humn body, resulting in the potentilly protective nd preventive ctions of blueberries. In the present study, the blueberry components were not detected nd the importnt components were not extrcted from the fresh blueberries. However, fresh blueberry juice ws fed to the rts nd the serum ws collected. The serum from the blueberry-fed rts ws used for co-culturing with HEPG2 cells, nd the prolifertion, invsion, migrtion, cell cycle nd poptosis in HEPG2 cells ws detected following culture with serums tken from rts fed with different concentrtions of blueberry juice. The results indicted tht the blueberry juice exerted significnt ntitumor nd therpeutic effects on HEPG2 cells. The different concentrtions of blueberry juice nd vrying tretment times resulted in distinct influences on the invsion, migrtion, prolifertion, cell cycle nd poptosis in the HEPG2 cells. Following co-culture with serum obtined from blueberry fed rts, the inhibition rtes of HEPG2 cells in the low-, moderte- nd high-dosge groups were significntly lower thn in the control group, t 48 nd 72 h (P<0.05). The migrted nd trnsmembrne HEPG2 cells in the three tretment groups were significntly lower thn in the control group, t 48 nd 72 h (P<0.05). The number of migrted HEPG2 cells in the high-dosge group ws lower thn in the low-dosge group t 48 h, nd tht of the high- nd moderte-dosge groups were significntly lower thn in the low-dosge groups t 72 h (P<0.05). The number of trnsmembrne HEPG2 cells in the high-dosge group ws significntly lower thn in the low-dosge group t 48 h (P<0.05). Furthermore, the numbers
584 ZHAN et l: EFFECTS OF BLUEBERRIES ON HCC MIGRATION of HEPG2 cells t the G 2 /M stge in the tretment groups were significntly lower thn in the control group, nd the number of HEPG2 cells t the G 2 /M stge in the high-dosge group ws significntly lower thn in the low-dosge group t 48 nd 72 h (P<0.05). The poptosis rtes of the tretment groups were significntly higher thn those in the control group, nd the poptosis rte in the high-dosge group ws significntly higher thn tht in the low-dosge group t 48 nd 72 h (P<0.05). In the study by Fri et l (12) the nthocynins nd nthocynin-pyruvic cid dducts were extrcted from blueberries, nd demonstrted nticncer properties in brest cncer cell lines. Similr results were observed by Li et l (22) nd Lu et l (23). The mjority of previous studies were bsed on the extrction of key components of blueberries; however, there re fewer studies with the comprehensive detection of their (blueberry components or the originl blueberry juice) influence on HEPG2 cells. There were, however, certin limittions of the present study s follows: No specific components were extrcted from the blueberries. The blueberry juice ws diluted to different concentrtions to feed the rts; however, it ws the serum of the rts, nd not the blueberry juice directly, tht ws co-cultured with the HEPG2 cells. Furthermore, the tretment times were short nd, therefore, did not provide dt on the long-term therpeutic nd protective effects of blueberries on HCC or HEPG2 cells. Thus, the detiled effects of blueberry on HCC or other types of cncer require further clinicl investigtion. 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