ble at ScienceDirect

Journal of Pharmacological Sciences 132 (2016) 122e130

Contents lists availa

Journal of Pharmacological Sciences

journal homepage: www.elsevier .com/locate/ jphs

Full paper

Calf Spleen Extractive Injection (CSEI), a small peptides enrichedextraction, induces human hepatocellular carcinoma cell apoptosis viaROS/MAPKs dependent mitochondrial pathway

Dongxu Jia a, Wenqian Lu a, Xinrui Zhang a, Guangsheng Cai a, Lirong Teng a,Xinyu Wang a, Minghai Zhang a, Yan Zeng b, Chunhua Liang c, Di Wang a, *

a School of Life Sciences, Jilin University, Changchun, 130012, Chinab JiLin AoDong Pharmaceutical Co., Ltd., Taonan, 137100, Chinac Jilin Institute for Drug Control, Changchun, 130062, China

a r t i c l e i n f o

Article history:Received 13 May 2016Received in revised form10 August 2016Accepted 22 August 2016Available online 4 September 2016

Keywords:Calf Spleen Extractive Injection (CSEI)ApoptosisHepatocellular carcinomaROS/MAPKsMitochondria

* Corresponding author. School of Life Sciences, JiliJilin Province, China.

E-mail addresses: jiadx14@mails.jlu.edu.cn (D. J(W. Lu), xrzhang15@mails.jlu.edu.cn (X. Zhang), caigtenglr543@gmail.com (L. Teng), wangxy1313@mzhangmh9913@mails.jlu.edu.cn (M. Zhang), zengyang982981710@qq.com (C. Liang), jluwangdi@outlook.co

Peer review under responsibility of Japanese Pha

http://dx.doi.org/10.1016/j.jphs.2016.08.0061347-8613/© 2016 The Authors. Production and hostinlicense (http://creativecommons.org/licenses/by-nc-n

a b s t r a c t

Calf Spleen Extractive Injection (CSEI), a small peptides enriched extraction, performs immunomodu-latory activity on cancer patients suffering from radiotherapy or chemotherapy. The present study aimsto investigate the anti-hepatocellular carcinoma effects of CSEI in cells and tumor-xenografted mousemodels. In HepG2 and SMMC-7721 cells, CSEI reduced cell viability, enhanced apoptosis rate, causedreactive oxygen species (ROS) accumulation, inhibited migration ability, and induced caspases cascadeand mitochondrial membrane potential dissipation. CSEI significantly inhibited HepG2-xenografted tu-mor growth in nude mice. In cell and animal experiments, CSEI increased the activations of pro-apoptoticproteins including caspase 8, caspase 9 and caspase 3; meanwhile, it suppressed the expressions of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and anti-oxidation proteins, such as nuclear factor-erythroid2 related factor 2 (Nrf2) and catalase (CAT). The enhanced phosphorylation of P38 and c-JunN-termi-nalkinase (JNK), and decreased phosphorylation of extra cellular signal-regulated protein kinase (ERKs)were observed in CSEI-treated cells and tumor tissues. CSEI-induced cell viability reduction was signif-icantly attenuated by N-Acetyl-L-cysteine (a ROS inhibitor) pretreatment. All data demonstrated that theupregulated oxidative stress status and the altered mitogen-activated protein kinases (MAPKs) phos-phorylation contributed to CSEI-driven mitochondrial dysfunction. Taken together, CSEI exactly inducedapoptosis in human hepatocellular carcinoma cells via ROS/MAPKs dependent mitochondrial pathway.

© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese PharmacologicalSociety. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/

licenses/by-nc-nd/4.0/).

1. Introduction

Hepatocellular carcinoma (HCC), the sixth common type ofcancer, threatens humans' life and health worldwide. Patientssuffered fromHCC are often diagnosed with severely impaired liverfunction at a late stage and die within 7e8 months after diagnosis(1). Although multiple therapeutic options have been applied in

n University, Changchun City,

ia), luwq15@mails.jlu.edu.cns15@mails.jlu.edu.cn (G. Cai),ails.jlu.edu.cn (X. Wang),_aodong@126.com (Y. Zeng),m (D. Wang).rmacological Society.

g by Elsevier B.V. on behalf of Japad/4.0/).

clinic, adverse effects including immune system disorders and liverdysfunction are noted in patients (2). Novel therapeutic agents withlower toxicity for HCC curing are urgently needed.

It has been reported that peptides, either synthetic or derivedfrom natural products, show anti-hepatocellular carcinoma activ-ities in animalmodels or clinical trials (3). A small molecular weightof polypeptide with low toxicity can readily penetrate the tumorcells without drug resistance (4). Bombesin, a 14-amino acid pep-tide originally isolated from the skin of the European fire-belliedtoad (Bombina bombina), significantly inhibits the growth on SK-Hep-1-xenografted tumor in nude mice (5). Calf Spleen ExtractiveInjection (CSEI), a small peptides enriched extraction, has beenused to ameliorate thrombocytopenia and leucopenia in cancerpatients who underwent chemotherapy and radiotherapy in China(6, 7). Additionally, efficacious therapy of CSEI combined with

nese Pharmacological Society. This is an open access article under the CC BY-NC-ND

D. Jia et al. / Journal of Pharmacological Sciences 132 (2016) 122e130 123

cyclosporine A was found in patients with chronic aplastic anemiafrom clinical observation suggested by the significantly increase ofleukocyte and platelets in blood (8). In our group, CSEI has beenproved to improve immune function in cyclophosphamide-inducedimmunosuppressed mice model via NF-kB signaling pathway (9).As a marketable drug, although occupational therapists haveclaimed that CSEI possesses anti-tumor activities, no studies sys-tematically reported these effects.

Amount of anticancer agents can successfully induce cancer cellapoptosis, which is recognized as an orderly cell autonomous deathmediated by caspases cascade and mitochondrial dysfunction(10,11). The over-generation of reactive oxygen species (ROS) canmodulate mitochondrial depolarization (12), and lead to cellularoxidative stress-induced apoptosis through c-Jun-N-terminal ki-nase (JNK), P38 and extra cellular signal-regulating kinases (ERKs)signaling pathways (13e15). Oxidation-reduction system isdestroyed in tumor cells, and high levels of oxidative stress causethe oxidative modification of amino acid residues thereby inducingDNA mutations and cell apoptosis, therefore, antioxidant agentscould interferewith carcinogenesis (16). In addition, nuclear factor-erythroid 2 related factor 2 (Nrf2) and catalase (CAT) are twoimportant factors in apoptosis resistance associated with ROS level(17, 18). Nrf2 plays a critical role in cellular redox homeostasis byattenuating oxidative-stress-associated pathological phenomena(19), and shows mutual inhibition relationship between ROS (20).

Collectively, we hypothesize that CSEI may display anti-cancereffects via oxidative stress related apoptotic pathway. To verifythis hypothesis, in the present study, the anti-hepatocellular car-cinoma activity of CSEI in HepG2 and SMMC-7721 cells is examinedsystematically, and ROS-related signalings are detected to furtherexplore its underlying mechanisms.

2. Materials and methods

2.1. CSEI information

CSEI was supplied by JiLin AoDong Pharmaceutical Co., Ltd. inChina, and is a makeable drug in China with the STATE MEDICALPERMITMENT No. H22026121. CSEI is a small peptides enrichedextraction, which is separated and purified from spleen of healthycows within 24 h after birth with the peptide concentration of2.5 ± 0.2 mg/ml. The characteristic spectrum of CSEI analyzed byhigh performance liquid chromatography combined with UV de-tector was displayed as Fig.1S. In our primary experiments, we haveconfirmed the safety of CSEI in an acute toxicity test indicating byno significant effects on body weights of Sprague Dawley rats (TabS1). CSEI showed strong cytotoxic effects on lung carcinoma cells(A549), and breast carcinoma cells (MDA and T47D) (Fig. 2S).

2.2. Cell culture

Human HCC tumor cell lines HepG2 and SMMC-7721, and hu-man embryonic kidney cell line HEK 293T obtained from ShanghaiCell Collection (Chinese Academy of Sciences, Beijing, China) werecultured in Dulbecco's Modified Eagle Media (DMEM) supple-mented with 10% fetal bovine serum (FBS), 100 units/ml penicillinand 100 g/ml streptomycin under a humidified atmosphere con-taining 5%/95% CO2/air at 37 �C. Cell culture reagents were obtainedfrom Gibco BRL (Grand Island, NY, USA).

2.3. MTT assay

HepG2, SMMC-7721 and HEK 293T were seeded into 96 wellplates at density of 1 � 104 cells/well. After incubation with0e1.5 mg/ml CSEI for 24 h, 3-(4,5-dimethylthiazol-2-yl)- 2,5-

diphenyltetrazolium bromide (MTT; SigmaeAldrich, USA) at a finalconcentration of 0.1 mg/ml was added to each well following anadditional culture at 37 �C in darkness for 4 h. In the late incubation,culture medium was removed, and 100 ml of DMSO was added todissolve formazan crystals within cells. The absorbance wasmeasured using Synergy™4 Microplate Reader (BioTek In-struments, Winooski, VT) at a wavelength of 490 nm.

In another separate test, 5 mM of N-Acetyl-L-cysteine (NAC; aROS inhibitor) was used to pretreat cells for 60 min. After co-incubation with CSEI for 24 h, cell viability was assessed.

2.4. Colony formation assays

HepG2 and SMMC-7721 cells were seeded into 6 well plate atdensity of 3 � 105 cells/well, and incubated with 0e1.5 mg/ml CSEIfor continuous 7 days. Cells were incubated with 4% para-formaldehyde for 10 min and stained with 0.05% crystal violet for60 min. The plate was washed three times with phosphate bufferedsaline (PBS), drained upside down on paper towels, and photo-graphed. The experiment was repeated for six times.

2.5. Migration assay

HepG2 and SMMC-7721 cells were plated in 6 well plates,cultured to over 90% confluence, and then scraped with a syringeneedle. After 24-h culture with CSEI (0, 0.35 and 0.70 mg/ml), thedistances of migrating cells were used to evaluate the cell migra-tory ability. The width of the wound was expressed as a percentageof 0 mg/ml CSEI-treated cells.

2.6. Hoechest 33258 staining

Hoechest 33258, a blue fluorescent dye, could penetrate the cellmembrane and specifically bind to DNA to show the form ofchromatin; therefore, it is typically used to visualize the apoptoticnuclei (21). Cells were seeded at 3 � 105 cells/well in 6 well platesand incubated overnight. After treatment with CSEI for 24 h, cellswere fixed with 4% paraformaldehyde for 10 min and then stainedwith Hoechest 33258 (1 mg/ml) in darkness for 15 min. The sampleswere examined via Nikon Eclipse TE 2000-S fluorescence micro-scope. The relative fluorescence intensity was analyzed by Image Jsoftware (National Institutes of Health, Bethesda, USA).

2.7. Assessment of cell apoptosis

HepG2 and SMMC-7721 cells were seeded into 6 well plates at4 � 105 cells per well. After exposure to CSEI for 24 h, cells werewashed with PBS and stained with propidium iodide (PI) and/orAnnexin V at room temperature for 20 min. The intensity of fluo-rescence was analyzed utilizing Muse™ Cell Analyzer from Milli-pore (Billerica, MA) following manufacturer's instructions.

2.8. Assessment of caspases activities

Cells treatedwith CSEI for 24 hwere harvested using lysis buffer.After detecting the protein concentration by BCA Protein Assay Kit(Millipore, Billerica, MA), the activities of caspase 3, 8 and 9 wereexamined according to the protocols of assay kits (Nanjing Jian-cheng Bioengineering Institute, Nanjing, China).

2.9. Assessment of intracellular ROS levels

20-70-Dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-eAldrich, USA) staining was used to objectively reflect the intra-cellular ROS level. Cells treated with CSEI for 24 h were suspended

D. Jia et al. / Journal of Pharmacological Sciences 132 (2016) 122e130124

and incubated with 10 mM of DCFH-DA at 37 �C for 15 min indarkness. After washing with PBS three times, the changes ofintracellular ROS levels were examined via Nikon Eclipse TE 2000-Sfluorescence microscope. Green fluorescence intensity is on behalfof intracellular ROS accumulation.

2.10. Assessment of mitochondrial membrane potential (MMP)

5,50,6,60-tetrachloro-1,10,3,30 tetraethylbenzimidazolylcarbocya-nine iodide (JC-1; Calbiochem, USA) was used to detect MMPchanges. In normal cells, JC-1 aggregates to form a polymer in themitochondrial matrix, and the polymer emits intense red fluores-cence. In unhealthy cells, JC-1 exists only in the form of a monomerin the cytoplasm, and it emits green fluorescence (22). HepG2 andSMMC-7721 cells were seeded into 6 well plates at 3� 105 cells perwell and treated with CSEI for 24 h. After incubation with 2 mM ofJC-1 at 37 �C for 15min in darkness, the changes of fluorescent color

Fig. 1. CSEI induced cytotoxicity in hepatocellular carcinoma cells and showed little effect7721 (A), but not in HEK 293T (B) after 24-h treatment (n ¼ 6). (C) CSEI inhibited the migratiScale bar: 100 mm) (n ¼ 6). Data are expressed as the means ± S.D. *P < 0.05, **P < 0.01 andcarcinoma cells proliferation detecting via crystal violet staining after 7-day treatment (n ¼

in the mitochondria were analyzed by fluorescent microscopy viaNikon Eclipse TE 2000-S fluorescence microscope. The relative redand green fluorescence intensity was analyzed by Image J software(National Institutes of Health, Bethesda, USA).

2.11. HepG2-xenografted tumor model

5-week-old Male BALB/c nude mice (Chares River ExperimentalAnimal Technical Co.,Ltd., Beijing, China) were housed in groups oftwo in clear plastic cages and maintained on a 12 h light/dark cycleat 23 ± 1 �C with water and food available ad libitum.

The experimental animal protocol was approved by the AnimalEthics Committee of Jilin University. A total number of5� 106 cells/100 ml was subcutaneously implanted below the rightback near hind leg. 3 days after the cell injection, palpable tumorsize was measured with a caliper. Nude mice were randomlydivided into two groups (n ¼ 6 each). Mice were intraperitoneally

on HEK 293T cells. CSEI dose-dependently reduced cell viability in HepG2 and SMMC-on ability of hepatocellular carcinoma cells determined via a wound healing assay (10X;***P < 0.001 versus 0 mg/ml of CSEI-treated cells. (D) CSEI suppressed hepatocellular6).

D. Jia et al. / Journal of Pharmacological Sciences 132 (2016) 122e130 125

injected with saline solution (0 mg/kg CSEI) and 1.75mg/kg of CSEIevery other day, and the body weight and tumor size wererecorded at the same time. Tumor size was calculated using theequation length (mm) � (width (mm))2 � 0.5. After 14-daytreatment, the tumors were harvested and stored at �80 �C forwestern blot assay.

2.12. Western blot

Cells were planted into 6 well plates at 4 � 105 cells per well andtreatedwithdoses of CSEI for 24h. Cellswere harvested,washedwithcold PBS twice and lysed with RIPA buffer (SigmaeAldrich, USA)containing 1% protease inhibitor cocktail (SigmaeAldrich, USA) and

Fig. 2. CSEI induced apoptosis in hepatocellular carcinoma cells after 24-h treatment. (A) Cexamined by Hoechst 33258 staining (20X; Scale bar: 100 mm). Numerical data were demcarcinoma cell apoptosis rate analyzed via flow cytometry. Numerical data were demonstrateof caspase 3, 8 and 9 in HepG2 and SMMC-7721 cells (n ¼ 6). Data are expressed as the me

2% PMSF (Phenylmethanesulfonyl fluoride) (SigmaeAldrich, USA).Similarly, tumors were lysed in the same protocol mentioned above.The protein concentration was measured by BCA Protein Assay Kit(Millipore, Billerica, MA) for three times repeated. 40 mg of proteinswere separated using a 10%e12% SDS-PAGE gel and transferredelectrophoretically onto PVDF membranes. The transferred mem-braneswere blocked in 5% bull serum albumin (BSA) for 4 h, and thenblotted with the following primary antibodies at 4 �C overnight atdilution of 1: 1000: phosphor (P)-ERKs (ab47339), total (T)-ERKs(ab17942), P-JNK (ab192200), T-JNK (ab1798461), P-P38 (ab31828), T-P38 (ab31828), cleavedpoly (ADP-ribose) polymerase (cleaved-PARP)(ab32064), poly (ADP-ribose) polymerase (PARP) (ab32138), Bcl-2(ab32124), cleaved-caspase 3 (ab2302), cleaved-caspase 8

SEI induced nucleus apoptotic morphologic alteration in HepG2 and SMMC-7721 cellsonstrated by the average optical density (n ¼ 6). (B) CSEI enhanced hepatocellulard by the sum of early and late apoptosis rates (n ¼ 6). (C) CSEI enhanced the activationans ± S.D. *P < 0.05, **P < 0.01 and ***P < 0.001 versus 0 mg/ml of CSEI-treated cells.

D. Jia et al. / Journal of Pharmacological Sciences 132 (2016) 122e130126

(ab181580), cleaved-caspase 9 (ab2014), Nrf2 (ab62352), CAT(ab52477)andglyceraldehyde-3-phosphatedehydrogenase (GAPDH)(ab8245) purchased from Abcam (Cambridge, MA), followed bytreatment with horseradish peroxidase-conjugated secondary anti-bodies diluted 1:2000 (Santa Cruz, USA). Chemiluminescence wasdetectedusingECLdetectionkits (Millipore,USA)and imagingsystem(Biospectrun 600). The intensity of the bands was quantified byscanning densitometry using software Image J (National Institutes ofHealth, Bethesda, USA).

2.13. Statistical analysis

All of the experiments were carried out in sextuplicate. Allvalues were expressed as mean ± S.D. A one-way analysis of vari-ance (ANOVA) was used to detect statistical significance followedby post-hoc multiple comparisons (Dunn's test) using SPSS 16.0

Fig. 3. CSEI caused mitochondrial dysfunction in hepatocellular carcinoma cells after 24-h trbar: 100 mm). Numerical data were expressed as the ratio of red to green fluorescence intencleaved-PARP, and evidently reduced the levels of Bcl-2 in HepG2 (B) and SMMC-7721 (CGAPDH (n ¼ 6). Data are expressed as the means ± S.D. *P < 0.05, **P < 0.01 and ***P < 0.

software (IBM corporation, Armonk, USA). The value of P < 0.05 wasconsidered to be significant.

3. Results

3.1. CSEI causing cell damage in hepatocellular carcinoma cells

CSEI dose-dependently reduced the cell viability of HepG2 andSMMC-7721 cells (P < 0.001; Fig. 1A), but showed no significanteffects on HEK 293T cells after 24-h treatment (P > 0.05; Fig. 1B).The 24-h IC50 of CSEI in HepG2 and SMMC-7721 were approxi-mately 0.70 mg/ml and 0.66 mg/ml respectively. A wound healingassay was performed to observe the inhibitory effect of CSEI on themigration ability of hepatocellular carcinoma cells. Compared withnon-treated cells, whose wound areas were almost healed, themigration ability was strongly inhibited by CSEI (P < 0.01; Fig. 1C).

eatment. (A) CSEI induced the dissipation of MMP detected by JC-1 staining (20X; Scalesity (n ¼ 6). CSEI significantly enhanced the expressions of cleaved-caspase 3, 8, 9, and) cells. Quantification data of protein expressions were normalized by corresponding001 versus 0 mg/ml of CSEI-treated cells.

D. Jia et al. / Journal of Pharmacological Sciences 132 (2016) 122e130 127

The robust inhibitory on HepG2 and SMMC-7721 cell colony for-mation were apparent at 0.5 mg/ml, and from 1.0 to 1.5 mg/ml ofCSEI, the clonogenic ability was completely blocked (Fig. 1D).

3.2. CSEI inducing apoptosis in hepatocellular carcinoma cells

Hoechst 33342 staining corroborated that CSEI at doses of 0.35and 0.7 mg/ml strikingly induced nucleus apoptosis in hepatocel-lular carcinoma cells indicated by the enhanced blue fluorescenceintensity (P < 0.01; Fig. 2A). Exposure to CSEI for 24 h significantlyincreased the early and late apoptosis rate in HepG2 and SMMC-7721 cells in a dose-dependent manner, and reached the maximumof 23.97% and 15.53% respectively (P < 0.001; Fig. 2B). The activa-tion of caspase 3, 8 or 9 is considered as typical events in mito-chondrial apoptosis (23). CSEI enhanced 14.1%, 26.2% and 9.1%activities of caspase 3, 8 and 9 in HepG2 cells, respectively (P< 0.05;Fig. 2C). In SMMC-7721 cells, 32.2%, 6.3% and 22.3% enhancement ofcaspase 3, 8, 9 activities were observed in CSEI-treated cells(P < 0.01; Fig. 2C).

Fig. 4. Intracellular ROS accumulation contributes to CSEI-mediated hepatocellular carcinomand SMMC-7721 cells analyzed via DCFH-DA staining (20X; Scale bar: 100 mm). Numerical dCSEI treatment reduced the expression of Nrf2 and CAT in HepG2 and SMMC-7721 cells. Q(n ¼ 6). (C) NAC pretreatment strikingly abrogated CSEI-reduced cell viability (n ¼ 6). Data arCSEI-treated cells. ##P < 0.05 and ###P < 0.001 versus 0.35 and 0.70 mg/ml of CSEI-treat

3.3. CSEI causing apoptotic alteration on mitochondrial function

Mitochondrial function is one of the factors responsible for cellapoptosis. 24-h CSEI incubation significantly caused MMP loss inhepatocellular carcinoma cells indicated by the augment in greenfluorescence (JC-1 monomers) and the reduction in red fluores-cence (JC-1 aggregates) (P < 0.01; Fig. 3A). In CSEI-treated cells, theexpressions of cleaved-caspase 3, 8, 9, which are active formsinducing cell apoptosis, as well as cleaved-PARP, were enhancedsignificantly; conversely, the expression of Bcl-2 was suppressed inHepG2 and SMMC-7721 cells respectively (P < 0.05; Fig. 3B,C).

3.4. ROS accumulation being responsible for CSEI-inducedhepatocellular carcinoma cell apoptosis

CSEI caused significant intracellular ROS accumulation indicatedby the enhanced green fluorescence in HepG2 and SMMC-7721 cells (P < 0.05; Fig. 4A). Nrf2 and CAT, two oxidative stressmarkers, which could reduce the production of ROS, were selected

a cell apoptosis. (A) CSEI dose-dependently increased intracellular ROS levels in HepG2ata were demonstrated by the average of green fluorescence density (n ¼ 6). (B) 24-huantification data of protein expressions were normalized by corresponding GAPDHe expressed as the means ± S.D. *P < 0.05, **P < 0.01 and ***P < 0.001 versus 0 mg/ml ofed cells.

Fig. 5. MAPKs activation is involved in CSEI-mediated apoptosis in hepatocellularcarcinoma cells. After 24-h CSEI treatment, the reduced P-ERKs expression and theenhanced P-JNK and P-P38 expressions were observed in HepG2 and SMMC-7721 cells.Quantification data of P-ERKs, P-JNK and P-P38 expressions were normalized by cor-responding total protein expressions. Data are expressed as mean ± S.D. (n ¼ 6) andanalyzed using a one-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 versus 0 mg/ml of CSEI-treated cells.

D. Jia et al. / Journal of Pharmacological Sciences 132 (2016) 122e130128

to characterize the effects of CSEI on oxidative status of hepato-cellular carcinoma cells. CSEI significantly reduced the expressionsof Nrf2 and CAT in hepatocellular carcinoma cells (Fig. 4B). Furtherdata showed that the reduced cell viability caused by CSEI wasstrongly abolished via pre-treatment with 1 mM of NAC in HepG2and SMMC-7721 cells (P < 0.01; Fig. 4C) for 60 min. Data indicatedthat CSEI-mediated hepatocellular carcinoma cell apoptosis may beassociated with intracellular ROS accumulation.

3.5. CSEI regulating MAPKs activation in hepatocellular carcinomacells

The activation of mitogen-activated protein kinases (MAPKs) isinvolved in cell proliferation, differentiation, inflammation andapoptosis (24). CSEI dose-dependently suppressed the phosphory-lation of ERKs, and enhanced the activities of JNK and P38 in HepG2and SMMC-7721 cells after 24-h treatment (P < 0.05; Fig. 5).

3.6. CSEI inhibiting HepG2-xenografted tumor growth in nude mice

14-day CSEI treatment at 1.75 mg/kg strikingly suppressed theHepG2-xenografted tumor growth in nude mice without influ-encing their bodyweights, suggesting that CSEI is an effective agentwithout aggressive side-effects (Fig. 6A, B and D). Compared withnon-treated mice, 29.6% tumor size was decreased in CSEI-treatedmice at the end of the treatment (P < 0.01; Fig. 6C). CSEIenhanced the expressions of cleaved caspase 3 and cleaved-PARP,conversely, suppressed the levels of Nrf2 and CAT (P < 0.05;Fig. 6E). Furthermore, in tumor tissues, CSEI resulted in a 30%reduction of P-ERKs level, and 30% and 90% enhancement of P-JNKand P-P38 expression indicating that MAPKs may play importantroles during CSEI-mediated anti-hepatocellular carcinoma(P < 0.01; Fig. 6F).

4. Discussion

Recently, the incidence and mortality of cancer continue to rise,especially for the rapid development of hepatocellular carcinoma inAsia and Africa (25). Due tomulti-target effects and low toxicity, themulti-component drug has become a research focus. CSEI, widelyused as an adjuvant therapy agent, alleviates chemotherapy-induced adverse reactions, especially improves immune ability(26,27). In cyclophosphamide-induced immunosuppressed micemodel, we have successfully confirmed the immunomodulatoryactivity of CSEI. Encouragingly, in our present study, the anti-hepatocellular carcinoma activities of CSEI in human HCC tumorcells and HepG2-xenografted tumor mouse model were confirmed,and the roles of ROS-dependent mitochondrial pathway during thiseffect were further explored.

CSEI enhanced the dissipation of MMP in hepatocellular carci-noma cells. Bcl-2, locating in outer mitochondrial membrane, reg-ulates nucleic mitochondrial membrane permeability and wasreduced by CSEI (28). Bcl-2 expression is known as a hallmark todetect whether the cell damage related withmitochondria function(29). Following with MMP reducing, mitochondrial permeabilitytransition pores (PTP) are opened, which further leads to pro-apoptotic molecules release; consequently, caspase 3 and PARPare activated (30). As an apoptotic biomarker, cleaved-PARPexpression was enhanced by CSEI, which may be regulated bycaspase 3 (31). Mitochondria and caspases engage in a self-amplifying pathway of mutual activation (32). As reported,cleaved-caspase 8 induces cleavage and activation of Bid acting onthe intrinsic pathway (33). Cytochrome c released from mito-chondria activates caspase 9 via binding to apoptotic proteaseactivating factor 1 (Apaf-1) (34,35). Through proteolytic cleavage,

caspase 3 is activated by caspase 8 and caspase 9, and then itcleaves vital cellular proteins or other caspases causing dissectionof DNA molecules and leading to apoptosis (36). Corn peptideswhich triggered the activation of cleaved-caspase 3 showed a pro-apoptotic effect in HepG2 cells (37). The activation of caspase 3, 8and 9 has been noted in CSEI-induced cell apoptosis suggesting theroles of mitochondria-dependent intrinsic pathway.

On the other hand, it has demonstrated in amount of studiesthat oxidative stress induces apoptosis through DNA damage andmitochondrial dysfunction (38,39). ROS accumulation contributesto abnormal changes in mitochondrial membrane permeability(40). As a positive feedback loop between intracellular ROS andmitochondria, during mitochondria-dependent apoptosis, a largeamount of ROS is released into cytoplasm (41). NAC, a common ROSinhibitor, strongly abolished CSEI-reduced cell viability, whichconfirmed the central role of ROS. ROS regulates CATand Nrf2 levelswhich are usually over-expressed in tumor cells, and promotes Nrf2to transfer into the nucleus (42). It has been proved that thereduction of Nrf2 and CAT promotes apoptosis induced by oxidativestress (43). Therefore, the increased ROS levels and decreased Nrf2and CAT expression in hepatic carcinoma cells may be responsiblefor CSEI-caused apoptosis.

Moreover, ROS accumulation contributes to MAPKs activation(44,45). ROS inhibits JNK phosphatase and inactivates other po-tential inhibitory factors to promote tumor cell apoptosis (46). ERKsand JNK regulate antioxidant response element (ARE) to influenceantioxidases expressions. Nrf2 could be phosphorylated by P38inducing strengthen binding with Kelch-like ECH-associated

Fig. 6. CSEI inhibited HepG2-xenografted tumor growth in nude mice. BALB/c athymic nude mice inoculated with HepG2 cells were treated with 0 and 1.75 mg/kg of CSEI for 14days. Tumor-possessing nude mice (A) and tumor tissues (B) were separated from each mouse. (C) Growth curves of HepG2-xenografted tumors in nude mice. Tumor volumes weremeasured every other day. (D) Mean (±S.D.) body weight of 0 and 1.75 mg/kg of CSEI-treated mice. (E and F) CSEI significantly enhanced the expressions of cleaved-caspase 3,cleaved-PARP, P-JNK and P-P38, and evidently reduced the expressions of Nrf2, CAT and P-ERKs in tumor tissues. Quantification data of protein expressions were normalized bycorresponding GAPDH and/or total protein expressions, respectively. Data are expressed as mean ± S.D. (n ¼ 6) and analyzed using a one-way ANOVA. *P < 0.05, **P < 0.01, and***P < 0.001 versus 0 mg/ml of CSEI-treated mice.

D. Jia et al. / Journal of Pharmacological Sciences 132 (2016) 122e130 129

protein1 (Keap1), which leads to activity inhibition (47,48). Anti-oxidants like diallyl sulfide or apo-80-lycopenal cause nucleartranslocation of Nrf2 via activation of ERK/P38 (49,50). Arsenicenhances ROS levels to induce neuronal cell apoptosis via MAPKs-mediated mitochondrial pathway with a sharp reduction of Nrf2(47). Since JNK and P38 are reported to influence mitochondrialfunction (51,52), ROSmay serve as a link betweenMAPKs activationand mitochondrial apoptotic pathway during CSEI-induced hepa-tocellular carcinoma cell damage.

In conclusion, the anti-hepatocellular carcinoma effect of CSEI inin vitro and in vivo experiments was confirmed in our study. CSEIinduces ROS accumulation, migration inhibition, caspase cascade,MMP dissipation and abnormal expression of apoptosis relatedproteins. Incremental ROS generation serves as a link betweenmitochondrial dysfunction and MAPKs activation. Valuable phar-macological evidences for CSEI as a potential chemotherapeuticagent of hepatocellular carcinoma and other primary tumors wereprovided in the present study.

Conflict of interest

The authors indicated no potential conflicts of interest.

Ethical approval

The experimental animal protocol was approved by the AnimalEthics Committee of Jilin University.

Authors contribution

DWand LT conceived and designed the experiments. DJ, WL, XZ,XW and MZ performed the experiments. CL, GC and YZ analyzedthe data. DJ wrote the manuscript. LT and DW revised the paper.

Acknowledgements

This work was supported by the significant transformation ofscientific achievements of “Double Tenth Project” in Jilin Province(Grant No. 20140301003YY) in China and the Natural Sciencefoundation of P.R.China (Grant No. 81402955).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jphs.2016.08.006.

References

(1) Varela M, Sala M, Llovet JM, Bruix J. Treatment of hepatocellular carcinoma: isthere an optimal strategy? Cancer Treat Rev. 2003;29:99e104.

(2) Arii S, Yamaoka Y, Futagawa S, Inoue K, Kobayashi K, Kojiro M, et al. Results ofsurgical and nonsurgical treatment for small-sized hepatocellular carcinomas:a retrospective and nationwide survey in Japan. The Liver Cancer Study Groupof Japan. Hepatology. 2000;32:1224e1229.

(3) Tsang FH, Lee NP, Luk JM. The use of small peptides in the diagnosis andtreatment of hepatocellular carcinoma. Protein Pept Lett. 2009;16:530e538.

(4) Bidwell 3rd GL, Davis AN, Fokt I, Priebe W, Raucher D. A thermally targetedelastin-like polypeptide-doxorubicin conjugate overcomes drug resistance.Invest New Drugs. 2007;25:313e326.

(5) Szepeshazi K, Schally AV, Treszl A, Seitz S, Halmos G. Therapy of experimentalhepatic cancers with cytotoxic peptide analogs targeted to receptors forluteinizing hormone-releasing hormone, somatostatin or bombesin. Anti-cancer Drugs. 2008;19:349e358.

(6) Zhao F, Wang Z, Zheng R. Efficacy of calf spleen extract injection combinedwith chemotherapy in the treatment of advanced gastric cancer. J BengbuMed Coll 2010:779e781.

(7) Liu Z, Zhang X, Ma J, Wang L, Yang Y. Calf spleen extract injection combinedwith docetaxel Saijiakapei gemcitabine treatment of patients with advancedbreast cancer program in clinical research. Cancer Res Prev 2014:1116e1119.

(8) Xia L, Sun S. 30 Cases of calf spleen extractive injection applied in the therapyof chronic aplastic anemia. Tumor 2009:602.

(9) Jia D, Lu W, Wang C, Sun S, Cai G, Li Y, et al. Investigation on immunomod-ulatory activity of calf spleen extractive injection in cyclophosphamide-induced immunosuppressed mice and underlying mechanisms. Scand JImmunol. 2016;84:20e27.

D. Jia et al. / Journal of Pharmacological Sciences 132 (2016) 122e130130

(10) Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death.Am J Pathol. 1995;146:3e15.

(11) Yu XH, Perdue TD, Heimer YM, Jones AM. Mitochondrial involvement intracheary element programmed cell death. Cell Death Differ. 2002;9:189e198.

(12) Zhang C, Jia X, Bao J, Chen S, Wang K, Zhang Y, et al. Polyphyllin VII inducesapoptosis in HepG2 cells through ROS-mediated mitochondrial dysfunctionand MAPK pathways. BMC Complement Altern Med. 2016;16:58.

(13) Repicky A, Jantova S, Cipak L. Apoptosis induced by 2-acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile in human leukemia cells in-volves ROS-mitochondrial mediated death signaling and activation of p38MAPK. Cancer Lett. 2009;277:55e63.

(14) Chen KC, Chang LS. Arachidonic acid-induced apoptosis of human neuro-blastoma SK-N-SH cells is mediated through mitochondrial alteration elicitedby ROS and Ca(2þ)-evoked activation of p38alpha MAPK and JNK1. Toxi-cology. 2009;262:199e206.

(15) Singh AP, Lange TS, Kim KK, Brard L, Horan T, Moore RG, et al. Purifiedcranberry proanthocyanidines (PAC-1A) cause pro-apoptotic signaling, ROSgeneration, cyclophosphamide retention and cytotoxicity in high-risk neu-roblastoma cells. Int J Oncol. 2012;40:99e108.

(16) Mileo AM, Miccadei S. Polyphenols as modulator of oxidative stress in cancerdisease: new therapeutic strategies. Oxid Med Cell Longev. 2016;2016:6475624.

(17) Son YO, Pratheeshkumar P, Roy RV, Hitron JA, Wang L, Zhang Z, et al. Nrf2/p62signaling in apoptosis resistance and its role in cadmium-induced carcino-genesis. J Biol Chem. 2014;289:28660e28675.

(18) Chelikani P, Fita I, Loewen PC. Diversity of structures and properties amongcatalases. Cell Mol Life Sci. 2004;61:192e208.

(19) Jayachandran M, Chandrasekaran B, Namasivayam N. Geraniol attenuatesoxidative stress by Nrf2 activation in diet-induced experimental atheroscle-rosis. J Basic Clin Physiol Pharmacol. 2015;26:335e346.

(20) Liu WJ, Wang T, Wang B, Liu XT, He XW, Liu YJ, et al. CYP2C8-derivedepoxyeicosatrienoic acids decrease oxidative stress-induced endothelialapoptosis in development of atherosclerosis: role of Nrf2 activation.J Huazhong Univ Sci Technol Med Sci. 2015;35:640e645.

(21) Matsuyama M, Yoshimura R. Peroxisome proliferator-activated receptor-gamma is a potent target for prevention and treatment in human prostate andtesticular cancer. PPAR Res. 2008;2008:249849.

(22) Prathapan A, Vineetha VP, Raghu KG. Protective effect of Boerhaavia diffusa L.against mitochondrial dysfunction in angiotensin II induced hypertrophy inH9c2 cardiomyoblast cells. PLoS One. 2014;9:e96220.

(23) Rosado JA, Lopez JJ, Gomez-Arteta E, Redondo PC, Salido GM, Pariente JA. Earlycaspase-3 activation independent of apoptosis is required for cellular func-tion. J Cell Physiol. 2006;209:142e152.

(24) Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, et al.Mitogen-activated protein (MAP) kinase pathways: regulation and physio-logical functions. Endocr Rev. 2001;22:153e183.

(25) Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin.2012;62:10e29.

(26) Wang Y, Liu L, Du X, Sun X. Treatment of calf spleen extractive injectionameliorates radiotherapy and chemotherapy-induced thrombocytopenia andclinical observation in improvement of symptoms. Tumor 2004:494e496.

(27) Olontseva OI, Drozhennikov VA, Liashenko VA, Perevezentseva OS, Orlova EB.Biological activity of calf spleen extracts containing a DNAase I inhibitorduring irradiation. Radiobiologiia. 1983;23:259e263.

(28) Tsujimoto Y. Role of Bcl-2 family proteins in apoptosis: apoptosomes ormitochondria? Genes Cells. 1998;3:697e707.

(29) Raisova M, Hossini AM, Eberle J, Riebeling C, Wieder T, Sturm I, et al. The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J Invest Dermatol. 2001;117:333e340.

(30) Bensassi F, Gallerne C, el Dein OS, Hajlaoui MR, Bacha H, Lemaire C. Mecha-nism of alternariol monomethyl ether-induced mitochondrial apoptosis inhuman colon carcinoma cells. Toxicology. 2011;290:230e240.

(31) Qvarnstrom OF, Simonsson M, Eriksson V, Turesson I, Carlsson J. GammaH2AXand cleaved PARP-1 as apoptotic markers in irradiated breast cancer BT474cellular spheroids. Int J Oncol. 2009;35:41e47.

(32) Green D, Kroemer G. The central executioners of apoptosis: caspases ormitochondria? Trends Cell Biol. 1998;8:267e271.

(33) Tang D, Lahti JM, Kidd VJ. Caspase-8 activation and bid cleavage contribute toMCF7 cellular execution in a caspase-3-dependent manner duringstaurosporine-mediated apoptosis. J Biol Chem. 2000;275:9303e9307.

(34) Reubold TF, Eschenburg S. A molecular view on signal transduction by theapoptosome. Cell Signal. 2012;24:1420e1425.

(35) Allan LA, Clarke PR. Apoptosis and autophagy: regulation of caspase-9 byphosphorylation. FEBS J. 2009;276:6063e6073.

(36) Boucher D, Blais V, Drag M, Denault JB. Molecular determinants involved inactivation of caspase 7. Biosci Rep. 2011;31:283e294.

(37) Li JT, Zhang JL, He H, Ma ZL, Nie ZK, Wang ZZ, et al. Apoptosis in humanhepatoma HepG2 cells induced by corn peptides and its anti-tumor efficacy inH22 tumor bearing mice. Food Chem Toxicol. 2013;51:297e305.

(38) Wochna A, Niemczyk E, Kurono C, Masaoka M, Kedzior J, Slominska E, et al.A possible role of oxidative stress in the switch mechanism of the cell deathmode from apoptosis to necrosisestudies on rho0 cells. Mitochondrion.2007;7:119e124.

(39) Denning TL, Takaishi H, Crowe SE, Boldogh I, Jevnikar A, Ernst PB. Oxidativestress induces the expression of Fas and Fas ligand and apoptosis in murineintestinal epithelial cells. Free Radic Biol Med. 2002;33:1641e1650.

(40) Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, andapoptosis. Free Radic Biol Med. 2010;48:749e762.

(41) Degli Esposti M, McLennan H. Mitochondria and cells produce reactive oxygenspecies in virtual anaerobiosis: relevance to ceramide-induced apoptosis.FEBS Lett. 1998;430:338e342.

(42) Mukhopadhyay D, Srivastava R, Chattopadhyay A. Sodium fluoride generatesROS and alters transcription of genes for xenobiotic metabolizing enzymes inadult zebrafish (Danio rerio) liver: expression pattern of Nrf2/Keap1 (INrf2).Toxicol Mech Methods. 2015;25:364e373.

(43) Kalayarasan S, Sriram N, Sureshkumar A, Sudhandiran G. Chromium (VI)-induced oxidative stress and apoptosis is reduced by garlic and its derivativeS-allylcysteine through the activation of Nrf2 in the hepatocytes of Wistarrats. J Appl Toxicol. 2008;28:908e919.

(44) Park GB, Choi Y, Kim YS, Lee HK, Kim D, Hur DY. ROS-mediated JNK/p38-MAPK activation regulates Bax translocation in Sorafenib-induced apoptosisof EBV-transformed B cells. Int J Oncol. 2014;44:977e985.

(45) Hseu YC, Lee MS, Wu CR, Cho HJ, Lin KY, Lai GH, et al. The chalcone fla-vokawain B induces G2/M cell-cycle arrest and apoptosis in human oralcarcinoma HSC-3 cells through the intracellular ROS generation and down-regulation of the Akt/p38 MAPK signaling pathway. J Agric Food Chem.2012;60:2385e2397.

(46) Lee NK, Choi YG, Baik JY, Han SY, Jeong DW, Bae YS, et al. A crucial role forreactive oxygen species in RANKL-induced osteoclast differentiation. Blood.2005;106:852e859.

(47) Lu TH, Tseng TJ, Su CC, Tang FC, Yen CC, Liu YY, et al. Arsenic induces reactiveoxygen species-caused neuronal cell apoptosis through JNK/ERK-mediatedmitochondria-dependent and GRP 78/CHOP-regulated pathways. ToxicolLett. 2014;224:130e140.

(48) Tian H, Zhang D, Gao Z, Li H, Zhang B, Zhang Q, et al. MDA-7/IL-24 inhibitsNrf2-mediated antioxidant response through activation of p38 pathway andinhibition of ERK pathway involved in cancer cell apoptosis. Cancer GeneTher. 2014;21:416e426.

(49) Ho CY, Cheng YT, Chau CF, Yen GC. Effect of diallyl sulfide on in vitro andin vivo Nrf2-mediated pulmonic antioxidant enzyme expression via activationERK/p38 signaling pathway. J Agric Food Chem. 2012;60:100e107.

(50) Yang CM, Huang SM, Liu CL, Hu ML. Apo-80-lycopenal induces expression ofHO-1 and NQO-1 via the ERK/p38-Nrf2-ARE pathway in human HepG2 cells.J Agric Food Chem. 2012;60:1576e1585.

(51) Garcia-Fernandez LF, Losada A, Alcaide V, Alvarez AM, Cuadrado A, Gonzalez L,et al. Aplidin induces the mitochondrial apoptotic pathway via oxidativestress-mediated JNK and p38 activation and protein kinase C delta. Oncogene.2002;21:7533e7544.

(52) Zhang Z, Teruya K, Eto H, Shirahata S. Fucoidan extract induces apoptosis inMCF-7 cells via a mechanism involving the ROS-dependent JNK activation andmitochondria-mediated pathways. PLoS One. 2011;6:e27441.