RESEARCH ARTICLE

Oxymatrine triggers apoptosis by regulating Bcl-2 family proteinsand activating caspase-3/caspase-9 pathway in human leukemiaHL-60 cells

Jun Liu & Yazhou Yao & Huifang Ding & Renan Chen

Received: 26 November 2013 /Accepted: 27 January 2014# International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract With the objective of identifying promising antitumoragents for human leukemia, we carried out to determine theanticancer ability of oxymatrine on the human leukemia HL-60cell line. In vitro experiments demonstrated that oxymatrinereduced the proliferation of HL-60 cells in a dose- and time-dependent manner via the induction of apoptosis and cell cyclearrest at G2/MandS phases. The proteins involved in oxymatrine-induced apoptosis in HL-60 cells were also examined usingWestern blot. The increase in apoptosis upon treatment withoxymatrine was correlated with downregulation of anti-apoptotic Bcl-2 expression and upregulation of pro-apoptoticBax expression. Furthermore, oxymatrine induced the activationof caspase-3 and caspase-9 and the cleavage of poly(ADP-ribose)polymerase (PARP) inHL-60 cells. In addition, pretreatment witha specific caspase-3 (Z-DEVD-FMK) or caspase-9 (Z-LEHD-FMK) inhibitor significantly neutralized the pro-apoptotic activityof oxymatrine in HL-60 cells, demonstrating the important role ofcaspase-3 and caspase-9 in this process. Taken together, theseresults indicated that oxymatrine-induced apoptosis may occurthrough the activation of the caspase-9/caspase-3-mediated intrin-sic pathway. Therefore, oxymatrine may be a potential candidatefor the treatment of human leukemia.

Keywords Oxymatrine . Apoptosis . HL-60 . Caspase . Bax/Bcl-2

Introduction

Leukemia is a special progressive cancer of blood-formingcells in the bone marrow, characterized by the uncontrolledaccumulation of blood cells [1, 2]. These deranged, immaturecells accumulated in the blood and organs of the body are notable to carry out the normal functions of blood cells [3].Current most widely used therapeutic options include chemo-therapy, radiotherapy, hormonal therapy, immune therapy,some supportive therapy, bone marrow transplantation, andstem cell transplantation [4, 5]. Unfortunately, despite theseimprovements, global epidemiologic studies have demonstrat-ed that the incidence and mortality of different kinds ofleukemia patients still rank high in the worldwide populationthough various treatment strategies have been developed [2,6]. Meanwhile, current treatment regimens for leukemia maylead to a wide range of side effects, such as drop in blood cellcount, complete hair loss, diarrhea, tiredness, nausea, andreduced fertility [3, 7]. There is thus an urgent need to identifynovel therapeutic agents to treat refractory and high-risk leu-kemia patients. Nowadays, growing evidences suggest thatnatural materials might be a good source to develop next-generation anticancer drugs [8]. So aiming at discovering theinnovative antitumor drug candidate from plants with moreeffective effects and low toxicity has become a very importantarea for the prevention and management of leukemia.

Recently, traditional Chinese herbal medicines are an ex-traordinary source of chemopreventive and therapeutic agentsfor the treatment of various malignant diseases, including leu-kemia, due to their antiviral, antioxidant, anti-inflammatory,and tumor apoptosis-inducing properties [9, 10]. Sophoraflavescens Ait (kushen), a traditional Chinese herb, has beenused as folk medicine for many kinds of diseases. Oxymatrine

Jun Liu and Yazhou Yao contributed equally to this work.

J. LiuDepartment of Geriatrics, Tangdu Hospital, the Fourth MilitaryMedical University, Xi’an 710038, China

Y. YaoDepartment of Haematologic and Rheumatism, Baoji CentralHospital, BaoJi 721008, China

H. DingDepartment of Haematology, Shengli Oilfield Central Hospital,Dongying 257034, China

R. Chen (*)Department of Haematology, Tangdu Hospital, the Fourth MilitaryMedical University, Xi’an 710038, Chinae-mail: chenrenantdh@126.com

Tumor Biol.DOI 10.1007/s13277-014-1705-7

is one of the main basic quinolizidine alkaloids extracted fromthe root of this Chinese herb which displays various pharma-cological effects such as anti-hepatitis virus infection, anti-hepatic fibrosis, anti-inflammation, anti-anaphylaxis, anti-hepatic fibrosis, and other immune regulation [11–22]. It wasalso reported that oxymatrine exhibits broad activities in humanmalignant melanoma [23], gastric cancer [24], hepatoma [25],and lung cancer [26]. Recently, interests in studying the anti-cancer mechanism of oxymatrine seem to be mounting [27].The inhibition of cellular proliferation, induction of apoptosis,cell cycle arrest, and regulation of related protein expression(Bcl-2 and p53) may contribute to the anticancer mechanism ofoxymatrine [28–30]. Despite the emerging evidence of itsimportance, no studies have been reported to date to evaluatethe chemotherapeutic potential of oxymatrine in the manage-ment of leukemia. Therefore, the aim of the present study is todetermine whether treatment with oxymatrine inhibits the pro-liferation of the human leukemia HL-60 cell line and, if so, toidentify the underlying molecular mechanism. This presentstudy would provide a deeper insight into the events leadingto oxymatrine-induced apoptosis in HL-60 cells.

Materials and methods

Test product

Oxymatrine with the purity of 98 % was from Nanjing ZelangMedical Technology Co., Ltd. (Jiangsu, China). Its purity wascorroborated by measurements of melting point, IR, UV, and1H NMR spectra. The structure of the compound tested isshown in Fig. 1.

Cell culture and treatment

The human leukemia cell line HL-60 was obtained from the CellBank of Chinese Academy of Sciences (Nanjing, China) andwas maintained in RPMI-1640 medium supplemented with

10 % heat-inactivated fetal bovine serum (FBS) in a humidifiedatmosphere of 5 % CO2 and 95 % air at 37 °C. The testcompound was dissolved in dimethyl sulfoxide (DMSO) andwas added to the culture medium to give a final DMSO concen-tration of 0.1 %v/v. This concentration of DMSO had no signif-icant effect on the growth of the cell line tested (data not shown).

Cell growth inhibition assay

The cytotoxicity of oxymatrine against the HL-60 cells wasassessed via a colorimetric MTT assay [31]. HL-60 cells wereseeded at a density of 104 cells per well in a 96-well platetogether with various concentrations of drug (5, 10, 25, 50,and 100 μg/mL) and incubated for different lengths of time(24, 48, and 72 h). HL-60 cells without treatment withoxymatrine would be used for control. At the end of thecultivation, 20 μl of MTT (2 mg/mL) working solution wasadded to the wells, which were incubated for an additional 4 hat 37 °C. After discarding the MTT supernatant, 100 μlDMSO was added to dissolve the formazan crystals formedin viable cells and the plates were shaken for 10 min. Finally,the optical density of the formazan solution, as a measure ofcell viability, was read using a microplate reader (Bio-RadLaboratories, CA, USA) at 570 nm. Cell growth inhibitionrate (%) was calculated using the following equation: Inhibi-tory rate (%)=(1−Atreatment/Acontrol)×100 %. The half maxi-mal inhibitory concentration (IC50) was calculated from thecytotoxicity curves. The experiments were repeated threetimes for each cell line.

Lactate dehydrogenase (LDH) release assay

Cell death of leukemia cells was quantitatively assessed usingthe LDH kit (Roche, Indianapolis, IN, USA) according to themanufacturer’s instructions. The HL-60 cells were incubatedwith various concentrations of oxymatrine for 24, 48, and 72 h,respectively, as described above. After treatment, the mediumwas removed and combined with NADH and pyruvate solu-tions. LDH activity is proportional to the rate of pyruvate loss,which was assayed by absorbance change using a microplatereader. Relative intensity was compared to treatment with Tri-ton X-100 and was expressed with the maximum value of 100.

Flow cytometric analysis of the cell cycle

The cell cycle was analyzed with a flow cytometer afterpropidium iodide (PI) staining according to the manufac-turer’s instructions. After HL-60 cells were exposed to themedium containing different concentrations of oxymatrine (0,10, 25, and 50 μg/mL) for 48 h, HL-60 cells were harvestedby centrifugation, rinsed three times with sterile phosphate-buffered saline (PBS), and fixed in ice-cold 70 % ethanol at4 °C overnight. After centrifugation at 1,500×g for 3 min, theFig. 1 The chemical structure of oxymatrine

Tumor Biol.

cells were dyed with PI and incubated for 30 min in the dark.Distribution of cell cycle was conducted on a flow cytometer(BD FACSCalibur, BD Bioscience, USA), and the data wereanalyzed using CellQuest.

Detection of apoptosis

The extent of apoptosis in HL-60 cells was quantified by flowcytometry using fluorescein isothiocyanate (FITC)-conjugat-ed annexin Vand PI. After treatment with oxymatrine for 48 h,HL-60 cells (5×105 cells) were collected and suspended in100 μL of binding buffer (10 mM HEPES/NaOH, 140 mMNaCl, 2.5 mMCaCl2; pH 7.4) and stained with binding bufferwith 10 μL annexin V-FITC and 5 μL PI for 30 min at roomtemperature in the dark. The binding of annexin V-FITC andPI to the cells was measured by flow cytometry(FACSCalibur, BD Biosciences) using the CellQuest software[32]. At least 10,000 cells were analyzed for each sample. Inthe annexin V/PI quadrant gating, annexin V (−)/PI (−),annexin V (−)/PI (+), annexin V (+)/PI (−), and annexin V(+)/PI (+) represented the fraction of living cells, necroticcells, early apoptotic cells, and advanced apoptotic/secondary necrotic cells, respectively.

Measurement of enzyme activity of caspase-3, caspase-8,and caspase-9

Caspase-3, caspase-8, and caspase-9 activities were deter-mined by a colorimetric assay kit (R&D Systems Inc., Min-neapolis, MN, USA) according to the manufacturer’s instruc-tions. In brief, cells were seeded in 24-well plates at a densityof 3×106 cells per well. After exposure of the cells to the testcompounds for 48 h, the cells were washed three times withPBS and then lysed in the supplied lysis buffer for 10 min onice. The lysed cells were centrifuged at 12,000×g for 10 min,and cell lysates containing 50 μg of protein were incubatedwith the supplied reaction buffer containing the colorimetrictetrapeptides, Asp-Glu-Val-Asp (DEVD)-p-nitroaniline(pNA) for caspase-3, Ile-Glu-Thr-Asp (IETD)-pNA forcaspase-8, and Leu-Glu-His-Asp (LEHD)-pNA for caspase-9, at 37 °C for 2 h. The reaction was measured by changes inabsorbance at 405 nm using a microplate reader.

Caspase inhibition assay

The caspase-9-specific inhibitor Z-LEHD-FMK (50 μM) andthe caspase-3-specific inhibitor Z-DEVD-FMK (50 μM) (allfrom R&D Systems Inc., Minneapolis, MN, USA) were dis-solved in DMSO. Cells were pretreated with either the medi-um containing 0.1%DMSO or each inhibitor for 2 h.Mediumalone or medium containing each test compound at a finalconcentration of 50 μg/mL was then added. After 48 h of

incubation, the number of apoptotic cells was determined byflow cytometry as described previously.

Western blot analysis

In order to further investigate cytoplasmic Bax, Bcl-2,procaspase-3, procaspase-8, procaspase-9, and poly(ADP-ri-bose) polymerase (PARP) protein expressions in oxymatrine-induced apoptosis, HL-60 cells (4×105 cells/mL) were cul-tured with or without different indicated concentrations ofoxymatrine (10, 25, and 50 μg/mL) for 48 h and harvested.The cells were lysed in RIPA buffer (150 mM NaCl, 1 %Triton X-100, 0.5 % sodium deoxycholate, 0.1 % sodiumdodecyl sulfate (SDS), 50 mM Tris–HCl; pH 7.4) for 30 minon ice. Cell lysates were washed by centrifugation (13,000×g,4 °C, 15 min), and protein concentrations were determinedusing the BCATM protein assay kit. Aliquots of the lysates(30 μg of protein) were resolved by 15 % SDS–polyacryl-amide gels (SDS–PAGE) and electrotransferred onto a nitro-cellulose membrane (Bio-Rad). After the nonspecific site wasblocked with 5 % (w/v) milk for 1 h on a shaker at roomtemperature, the membrane was incubated with specific pri-mary antibody (Santa Cruz Biotechnology, Santa Cruz, CA,USA.) for 1 h at room temperature. Themembranewas furtherincubated for 60 min with a peroxidase-conjugated secondaryantibody (Vector Laboratories, Burlingame, CA, USA) atroom temperature, and immunoactive proteins were visual-ized by ECL-enhanced chemiluminescence.

Statistical analysis

All data are expressed as means±SD. Significant differencesbetween the groups were determined using the unpaired Stu-dent’s t test. A value of P<0.05 was considered to be statisti-cally significant.

Results

Cytotoxic effects of oxymatrine on HL-60 cells

To verify the effect of oxymatrine on cell proliferation, HL-60cells were treated with oxymatrine 1 (0, 5, 10, 25, 50, and100 μg/mL) for 24, 48, and 72 h and the cell viability wasassessed by MTTassay. Cells treated with 0.1 % DMSO wereused as controls. As shown in Fig. 2, oxymatrine treatmentexhibited a marked inhibition on the survival of HL-60 cells ina dose-dependent manner and reached the maximum valueafter 48 h of treatment, with IC50 value of 26.38, 14.71, and22.51 μg/mL for 24, 48, and 72 h of treatment, respectively.The data revealed that the prominent inhibitory activity ofoxymatrine on cell survival was attained after 48 h of treat-ment and the concentrations of 10, 25, and 50 μg/mL were

Tumor Biol.

appropriate as the high, moderate, and low concentrations inthe following experiment.

At the same time, the cell viability of HL-60 cells byoxymatrine was further confirmed by LDH release assay.The results showed that cell growth curves correlated wellwith the results of the MTT assay. In addition, there was nocytotoxicity effect of oxymatrine on normal cell lines (RAW264.7 cells) at the tested concentration (data not shown).

Cell cycle arrest and induction of apoptosis by oxymatrinein HL-60 cells

The change of cell cycle caused by oxymatrine in HL-60 cells is shown in Fig. 3a. We found that G0/G1

phase cells markedly decreased while G2/M and S phasecells increased obviously with the increase ofoxymatrine concentration after 48 h of incubation. The-se data indicated that oxymatrine blocked the cell cycleof HL-60 cells in G2/M and S phases.

To evaluate the effect of oxymatrine on the induction ofapoptosis, the oxymatrine-treated HL-60 cells were double-stained with annexin V-FITC and PI, followed by quantitativeflow cytometry analysis. As shown in Fig. 3b, a markedapoptosis phenomenon was observed in HL-60 cells treatedwith oxymatrine. The apoptotic rate increased from 24.5 to61.12% after the cells were treated by oxymatrine (10, 25, and50 μg/mL) for 48 h.

Effects of oxymatrine on the levels of Bcl-2 family membersin HL-60 cells

To investigate the apoptotic pathways activated byoxymatrine, we used Western blotting to measure the expres-sion of the Bcl-2 family members. As shown in Fig. 4, West-ern blot analyses revealed that the level of pro-apoptotic Baxwas not truncated and remained virtually unchanged in

response to oxymatrine treatment, whereas the level of anti-apoptotic Bcl-2 was markedly inhibited by oxymatrinetreatment in a concentration-dependent manner. Thisresult suggested the involvement of Bcl-2 family inthe apoptotic process.

0 5 10 25 50 1000

20

40

60

80

10024h

48h

72h

Oxymatrine (µg/mL)

Cel

l gro

wth

inh

ibit

ion

rat

e (%

)

Fig. 2 Cytotoxicity of oxymatrine on human leukemiaHL-60 cells. Eachvalue is expressed as means±SD of three experiments

0 10 25 50 0 10 25 50 0 10 25 500

20

40

60

80G0/G1G2/MS

Oxymatrine (µg/mL)

Cel

l cir

cle

dis

trib

uti

on

(%

)

0 10 25 500

20

40

60

80

***

***

***

Oxymatrine (µg/mL)

Ap

op

tosi

s ra

te (

%)

a

b

Fig. 3 a The change of cell cycle in human leukemia HL-60 cells afterexposure to oxymatrine for 48 h. bApoptosis analysis of human leukemiaHL-60 cells induced by different concentrations of oxymatrine for 48 h.Each value is expressed as means±SD of three experiments. ***P<0.001compared with the control group

Fig. 4 Effects of oxymatrine on the levels of the Bcl-2 family membersin human leukemia HL-60 cells

Tumor Biol.

Effects of oxymatrine on the expressions of caspase-3,caspase-8, and caspase-9 cascade, as well as cleavageof PARP in HL-60 cells

Caspase, a family of cysteine proteases, is known to formintegral parts of the apoptotic pathway [33]. To further inves-tigate the apoptotic cascades involved in the effects ofoxymatrine, we quantified the proteolytic activation of thecaspases (3, 8, and 9) using fluorogenic substrates and evalu-ated their protein expression using Western blot. As shown inFig. 5a, oxymatrine treatment markedly increased the activity ofcaspase-3 and caspase-9 in a concentration-dependent manner,but caspase-8 was not activated. Furthermore, oxymatrine treat-ment decreased the expression of procaspase-3 and procaspase-9 proteins in a concentration-dependent manner, but the expres-sion levels of procaspase-8 remained unchanged (Fig. 5b).

Poly(ADP-ribose) polymerase (PARP), an enzyme in-volved in DNA repair, is a substrate for caspase-3 [34].Subsequent Western blot analysis was performed in order to

investigate the potential involvement of PARP in oxymatrine-induced apoptosis. As shown in Fig. 5b, oxymatrine treatmentcaused the cleavage of PARP from a 116- to an 89-kDafragment, which corresponded with the activation ofcaspase-3. This suggested that apoptotic actions of oxymatrinewere closely related to the activation of caspase-3 and subse-quent cleavage of PARP.

Inhibition of oxymatrine-induced apoptosis by a caspase-3or caspase-9 inhibitor

Caspase-3 and caspase-9 are the key proteases responsible forthe cleavage of PARP and subsequent apoptosis [35]. Toconfirm whether the activation of intracellular caspase-9 andcaspase-3 is required for the induction of apoptosis byoxymatrine, HL-60 cells were pretreated with a specificcaspase-9 or caspase-3 inhibitor for 2 h, followed by treatmentwith 50 μg/mL oxymatrine for 48 h. Then the apoptosis ratewas measured by flow cytometry. As shown in Fig. 6, pre-treatment with Z-LEHD-FMK (a caspase-9 inhibitor) or Z-DEVD-FMK (a caspase-3 inhibitor) significantly blocked thecell apoptosis by oxymatrine. These suggested thatoxymatrine-induced apoptosis involved caspase-3- andcaspase-9-dependent signaling cascades.

Conclusions

Apoptosis is an important homeostatic mechanism that bal-ances cell division and cell death to maintain the appropriatecell number in the body. During this process, apoptosis ischaracterized by cell shrinkage, blebbing of the plasma mem-brane, chromatin condensation, and nuclear condensationwithout cell lysis [36, 37]. Apoptosis serves as a defensemechanism for cancer development by eliminating damagedcells which are prone to develop cancer [38]. Since

Fig. 5 a Effect of oxymatrine on activities of caspase-3, caspase-8, andcaspase-9 in human leukemia HL-60 cells. Each value is expressed asmeans±SD of three experiments. b Effect of oxymatrine on the proteinexpression of procaspase-3, procaspase-8, procaspase-9, and cleavedPARP in human leukemia HL-60 cells

0 1 2 3

0

50

100

150

200Living cells

Apoptotic cells

Oxymatrine(50 g/mL)

Z-LEHD-FMK+Oxymatrine

(50 g/mL)

Z-DEVD-FMK+Oxymatrine

(50 g/mL)

Control

Cel

l po

pu

lati

on

s (%

)

µµ

µ

Fig. 6 Effect of caspsae-3 or caspase-9 inhibitor on oxymatrine-inducedapoptosis in human leukemia HL-60 cells. Each value is expressed asmeans±SD of three experiments

Tumor Biol.

deregulation of apoptosis is the hallmark of all cancer cells[39], triggering programmed cell death in cancer cells istherefore considered as an important way for the developmentof valuable anticancer therapeutics [40, 41]. Accumulatingevidence indicates that many naturally derived componentsfrom plant species can cause tumor cell death via the inductionof apoptosis [42–44].

In this study, we investigated whether oxymatrineinduces apoptosis in human leukemia HL-60 cells andwhat mechanisms are related to the cell death. Ourresults demonstrated that oxymatrine inhibits cell prolif-eration and induces apoptosis via arresting the cell cycleat G2/M and S phases.

Members of the Bcl-2 family proteins, including Bcl-2 andBax, are critical regulators of the apoptotic pathway [45]. Wefurther examined the effect of oxymatrine on the expression ofanti-apoptotic Bcl-2 protein and pro-apoptotic Bax protein.The level of the anti-apoptotic protein, Bcl-2, declined as theconcentration of oxymatrine increased, whereas oxymatrinetreatment resulted in an increase in the level of the pro-apoptotic protein, Bax.

Activation of caspase proteases is an important biochemi-cal event of apoptosis [46, 47]. Different caspases are activat-ed at the initiation and execution phases of apoptosis.Caspase-8 and caspase-9 are upstream initiator caspases;caspase-3 is one of the downstream effectors which play thecentral role in the initiation of apoptosis [48]. Here, weshowed that oxymatrine treatment activated caspase-3 andcaspase-9 in a dose-dependent manner without activation ofcaspase-8. Subsequently, we observed that the inhibitors ofcaspase-3 and caspase-9 significantly protected HL-60 cellsfrom oxymatrine-induced apoptosis. These results suggestedthat oxymatrine induces apoptosis in HL-60 cells via caspase-9/caspase-3 activation. Caspase-3 is a major apoptosis-inducing protein that mediates the cleavage of PARP, awell-known caspase-3 substrate [49, 50]. As expected,oxymatrine treatment resulted in a cleavage of 116-kDa PARP into 85 kDa in HL-60 cells. In addition,the expression levels of death receptor-related proteinssuch as TRAIL, DR4, DR5, Fas, and FasL were rela-tively unchanged in response to oxymatrine treatment(data not shown). Taken together, our data suggestedthat the caspase-dependent intrinsic pathway was in-volved in oxymatrine-induced apoptosis in HL-60 cells.

In conclusion, this s tudy demonstrated thatoxymatrine strongly suppressed the proliferation ofHL-60 cells by induction of apoptosis through activationof the caspase-3/caspase-9-mediated intrinsic pathway.This could be evidenced by the fact that the apoptosisof HL-60 cells was induced by the activation ofcaspase-9 and caspase-3, subsequent cleavage of PARP,along with elevation of the ratio of Bax/Bcl-2. Thepresent results suggested that oxymatrine could be a

potential candidate for developing anticancer drug forthe treatment of human leukemia.

Conflicts of interest None

References

1. Wu SS, Chen LG, Lin RJ, Lin SY, Lo YE, Liang YC. Cytotoxicity of(−)-vitisin B in human leukemia cells. Drug Chem Toxicol. 2013;36:313–9.

2. Löwenberg B, Downing JR, Burnett A. Acute myeloid leukemia. NEngl J Med. 1999;341:1051–62.

3. Kang SH, Jeong SJ, Kim SH, Kim JH, Jung JH, Koh W, et al.Icariside II induces apoptosis in U937 acute myeloid leukemia cells:role of inactivation of STAT3-related signaling. PloS one. 2012;7:e28706.

4. Nau KC, Lewis WD. Multiple myeloma: diagnosis and treatment.Am Fam Physician. 2008;78:853–9.

5. Fotoohi AK, Assaraf YG, Moshfegh A, Hashemi J, Jansen G, PetersGJ, et al. Gene expression profiling of leukemia T-cells resistant tomethotrexate and 7-hydroxymethotrexate reveals alterations that pre-serve intracellular levels of folate and nucleotide biosynthesis.Biochem Pharmacol. 2009;77:1410–7.

6. Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ.Cancer statistics, 2003. CA Cancer J Clin. 2003;53:5–26.

7. Zaini RG, Brandt K, Clench MR, Le Maitre CL. Effects of bioactivecompounds from carrots (Daucus carota L.), polyacetylenes, beta-carotene and lutein on human lymphoid leukaemia cells. AnticancerAgents Med Chem. 2012;12:640–52.

8. Niu YP, Li LD, Wu LM. Beta-aescin: a potent natural inhibitor ofproliferation and inducer of apoptosis in human chronic myeloidleukemia K562 cells in vitro. Leuk Lymphoma. 2008;49:1384–91.

9. Wargovich MJ, Woods C, Hollis DM, Zander ME. Herbals, cancerprevention and health. J Nutr. 2001;131:3034S–6.

10. Boon H, Wong J. Botanical medicine and cancer: a review of thesafety and efficacy. Expert Opin Pharmacother. 2004;5:2485–501.

11. Dong Y, Xi H, Yu Y,WangQ, JiangK, Li L. Effects of oxymatrine onthe serum levels of T helper cell 1 and 2 cytokines and the expressionof the S gene in hepatitis B virus S gene transgenic mice: a study onthe anti-hepatitis B virus mechanism of oxymatrine. J GastroenterolHepatol. 2002;17:1299–306.

12. Liu J, Liu Y, Klaassen CD. The effect of Chinese hepato-protectivemedicines on experimental liver injury in mice. J Ethnopharmacol.1994;42:183–91.

13. Cao YG, Jing S, Li L, Gao JQ, Shen ZY, Liu Y, et al. Antiarrhythmiceffects and ionic mechanisms of oxymatrine from Sophoraflavescens. Phytother Res. 2010;24:1844–9.

14. Cui X, Wang Y, Kokudo N, Fang D, Tang W. Traditional Chinesemedicine and related active compounds against hepatitis B virusinfection. Biosci Trends. 2010;4:39–47.

15. Deng ZY, Li J, Jin Y, Chen XL, Lü XW. Effect of oxymatrine on thep38 mitogen-activated protein kinases signalling pathway inrats with CCl4 induced hepatic fibrosis. Chin Med J (Engl).2009;122:1449–54.

16. Zeng Z, Wang GJ, Si CW. Basic and clinical study of oxymatrine onHBV infection. J Gastroenterol Hepatol. 1999;14:295–7.

17. Chen XS, Wang GJ, Cai X, Yu HY, Hu YP. Inhibition of hepatitis Bvirus by oxymatrine in-vivo. World J Gastroenterol. 2001;7:49–52.

18. Xiang X, Wang G, Cai X, Li Y. Effect of oxymatrine on murinefulminant hepatitis and hepatocyte apoptosis. Chin Med J. 2002;115:593–6.

Tumor Biol.

19. Lu LG, Zeng MD, Mao YM, Li JQ, Wan MB, Li CZ, et al.Oxymatrine therapy for chronic hepatitis B: a randomized double-blind and placebo-controlled multi-center trial. World JGastroenterol. 2003;9:2480–3.

20. Lu LG, Zeng MD, Mao YM, Fang JY, Song YL, Shen ZH, et al.Inhibitory effect of oxymatrine on serum hepatitis B virus DNA inHBV transgenic mice. World J Gastroenterol. 2004;10:1176–9.

21. Mao YM, Zeng MD, Lu LG, Wan MB, Li CZ, Chen CW, et al.Capsule oxymatrine in treatment of hepatic fibrosis due to chronicviral hepatitis: a randomized, double blind, placebo-controlled, mul-ticenter clinical study. World J Gastroenterol. 2004;10:3269–73.

22. Fan H, Li L, Zhang X, Liu Y, Yang C, Yang Y, et al. Oxymatrinedownregulates TLR4, TLR2, MyD88, and NF-kappaB and protectsrat brains against focal ischemia. Mediat Inflamm. 2009;2009:704706. doi:10.1155/2009/704706.

23. Liu XY, Fang H, Yang ZG, Wang XY, Ruan LM, Fang DR, et al.Matrine inhibits invasiveness and metastasis of human malignantmelanoma cell line A375 in vitro. Int J Dermatol. 2008;47:448–56.

24. Luo C, ZhuY, Jiang T, LuX, ZhangW, JingQ, et al.Matrine inducedgastric cancer MKN45 cells apoptosis via increasing pro-apoptoticmolecules of Bcl-2 family. Toxicology. 2007;229:245–52.

25. Ma L, Wen S, Zhan Y, He Y, Liu X, Jiang J. Anticancer effects of theChinese medicine matrine on murine hepatocellular carcinoma cells.Planta Med. 2008;74:245–51.

26. Zhang Y, Zhang H, Yu P, Liu Q, Liu K, Duan HY, et al. Effects ofmatrine against the growth of human lung cancer and hepatoma cellsas well as lung cancer cell migration. Cytotechnology.2009;59:191–200.

27. Bao JL, Lu JJ, Chen XP,WangY. Research progress in the anti-tumoreffects andmechanisms ofmatrine and oxymatrine. Tradis ChinDrugRes Clin Pharmacol. 2012;3:369–73.

28. Song G, Luo Q, Qin J, Wang L, Shi Y, Sun C. Effects of oxymatrineon proliferation and apoptosis in human hepatoma cells. ColloidsSurf B: Biointerfaces. 2006;48:1–5.

29. Zhang MJ, Huang J. Recent research progress of anti-tumormechnism matrine. Zhongguo Zhong Yao Za Zhi. 2004;29:115–8.

30. Jin Y, Hu J, Wang Q, Li Z, Chen Y. Effects of oxymatrine on theapoptosis of human esophageal carcinoma Eca109 cell line and itsmechanism. J Huazhong Univ Sci Technol Med Sci. 2008;28:314–6.

31. Zhang Y, Wang Q,Wang T, Zhang H, Tian Y, Luo H, et al. Inhibitionof human gastric carcinoma cell growth in vitro by a polysaccharidefrom Aster tataricus. Int J Biol Macromol. 2012;51:509–13.

32. Zheng L, He M, Long M, Blomgran R, Stendahl O. Pathogen-induced apoptotic neutrophils express heat shock proteins and elicitactivation of human macrophages. J Immunol. 2004;173:6319–26.

33. Tayarani-Najaran Z, Mousavi SH, Vahdati-Mashhadian N, EmamiSA, Parsaee H. Scutellaria litwinowii induces apoptosis through bothextrinsic and intrinsic apoptotic pathways in human promyelocyticleukemia cells. Nutr Cancer. 2012;64(1):80–8.

34. Choi BH, Kim W, Wang QC, Kim DC, Tan SN, Yong JW, et al.Kinetin riboside preferentially induces apoptosis by modulating Bcl-2 family proteins and caspase-3 in cancer cells. Cancer Lett.2008;261:37–45.

35. Lik F, Kumar A, Bhushan S, Khan S, Bhatia A, Suri KA, et al.Reactive oxygen species generation and mitochondrial dysfunctionin the apoptotic cell death of human myeloid leukemia HL-60 cellsby a dietary compound withaferin Awith concomitant protection byN-acetyl cysteine. Apoptosis. 2007;12:2115–33.

36. Buendia B, Santa-Maria A, Courvalin JC. Caspase-dependent prote-olysis of integral and peripheral proteins of nuclear membranes andnuclear pore complex proteins during apoptosis. J Cell Sci. 1999;112:1743–53.

37. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phe-nomenon with wide-ranging implications in tissue kinetics. Br JCancer. 1972;26:239–57.

38. Hengartner MO. The biochemistry of apoptosis. Nature. 2000;407:770–6.

39. Kim JH, Choi YW, Park C, Jin CY, Lee YJ, da Park J, et al. Apoptosisinduction of human leukemia U937 cells by gomisin N, adibenzocyclooctadiene lignan, isolated from Schizandra chinensisBaill. Food Chem Toxicol. 2010;48:807–13.

40. KimYS, Jin SH, Lee YH,Kim SI, Park JD. Ginsenoside Rh2 inducesapoptosis independently of Bcl-2, Bcl-xL, or Bax in C6Bu-1 cells.Arch Pharm Res. 1999;22:448–53.

41. Ahmad N, Feyes DK, Nieminen AL, Agarwal R, Mukhtar H. Greentea constituent epigallocatechin-3-gallate and induction of apoptosisand cell cycle arrest in human carcinoma cells. J Natl Cancer Inst.1997;89:1881–6.

42. Bhalla K, Ibrado AM, Tourkina E, Tang C, Mahoney ME, Huang Y.Taxol induces internucleosomal DNA fragmentation associated withprogrammed cell death in human myeloid leukemia cells. Leukemia.1993;7:563–8.

43. Sharma RA, Gescher AJ, Steward WP. Curcumin: the story so far.Eur J Cancer. 2005;41:1955–68.

44. Su YT, Chang HL, Shyue SK, Hsu SL. Emodin induces apoptosis inhuman lung adenocarcinoma cells through a reactive oxygen speciesdependent mitochondrial signaling pathway. Biochem Pharmacol.2005;70:229–41.

45. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ.Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell.1993;75:241–51.

46. Fadeel B, Orrneius S. Apoptosis: a basic biological phenomenonwithwide-ranging implications in human disease. J InternMed. 2005;258:479–517.

47. Kekre N, Griffin C, McNulty J, Pandey S. Pancratistatin causes earlyactivation of caspase-3 and the flipping of phosphatidylserine follow-ed by rapid apoptosis specifically in human lymphoma cells. CancerChemother Pharmacol. 2005;56:29–38.

48. Salvesen GS, Dixit VM. Caspase activation: the induced-proximitymodel. Proc Natl Acad Sci U S A. 1999;96:10964–7.

49. Chang JT, Chen YL, Yang HT, Chen CY, Cheng AJ. Differentialregulation of telomerase activity by six telomerase subunits. Eur JBiochem. 2002;269:3442–50.

50. Allen RT, Hunter 3rdWJ, Agrawal DK.Morphological and biochem-ical characterization and analysis of apoptosis. J Pharmacol ToxicolMethods. 1997;37:215–28.

Tumor Biol.