Mechanism of apoptosis with the involvement of calpain and caspase cascades

in human malignant neuroblastoma SH-SY5Y cells exposed to flavonoids

Arabinda Das, Naren L. Banik and Swapan K. Ray*

Department of Neurosciences, Medical University of South Carolina, Charleston, SC

Neuroblastoma is the most common extracranial solid tumor inchildren causing death at pre-school age, as no cure has yet beendeveloped. We investigated the proteolytic mechanisms for apo-ptosis in human malignant (N-type) neuroblastoma SH-SY5Y cellsfollowing exposure to flavonoids such as apigenin (APG), (2)-epi-gallocatechin (EGC), (2)-epigallocatechin-3-gallate (EGCG) andgenistein (GST). We found decrease in viability of SH-SY5Y cellswith an increase in dose of APG, EGC, EGCG and GST. Predomi-nantly apoptosis occurred following exposure of SH-SY5Y cellsto 50 lM APG, 50 lM EGC, 50 lM EGCG and 100 lM GST for24 hr. Apoptosis was associated with increases in intracellular free[Ca

21] and Bax:Bcl-2 ratio, mitochondrial release of cytochrome c

and activation of caspase-9, calpain and caspase-3. Induction ofapoptosis with APG and GST showed activation of caspase-12 aswell. Activation of caspase-3 could cleave the inhibitor-of-caspase-activated DNase (ICAD) to release and translocate caspase-3-acti-vated DNase (CAD) to the nucleus. Activation of caspase-8 cleavedBid to truncated Bid (tBid) in cells treated with EGC and EGCG.EGC and EGCG induced apoptosis with caspase-8 activation andmitochondria-mediated pathway, whereas APG and GST causedapoptosis via an increase in intracellular free [Ca21] with calpainactivation and mitochondria-mediated pathway. Activation of dif-ferent proteases for cell death was confirmed using caspase-8 in-hibitor II, calpeptin (calpain inhibitor), caspase-9 inhibitor I andcaspase-3 inhibitor IV. Thus, plant-derived flavonoids cause celldeath with activation of proteolytic activities of calpain and cas-pases in SH-SY5Y cells, and therefore serve as potential therapeu-tic agents for controlling the growth of neuroblastoma.' 2006 Wiley-Liss, Inc.

Key words: apoptosis; calpain; caspase; flavonoids; neuroblastoma

Neuroblastoma, a childhood malignancy of the sympatheticnervous system, shows very complex biological and clinical heter-ogeneity.1 The same remarkable cure rate that has been achievedin most other childhood malignancies has not yet occurred in thistumor. The most favorable subset of this embryonic tumor may getregressed due to apoptosis after minimal therapy. However, themajority of the neuroblastomas in children over 1 year age areaggressive metastatic tumors with poor clinical outcome despiteintensive multimodal therapy, including surgery, irradiation andchemotherapy.

It has been shown repeatedly that antioxidants and their deriva-tives selectively induce apoptosis in cancer cells, including murineneuroblastoma cells, but not in normal cells in culture.2–9 Flavo-noids belong to a class of natural polyphenolic compounds, whichcontain D-a-tocopherol-like chemical structure that is responsiblefor antioxidant activities. Doses of some antioxidants and their de-rivatives, which produce apoptosis in cancer cells, can also protectnormal cells against damage produced by chemical or ionizing radi-ation. For example, a dose of D-a-tocopheryl succinate (vitamin E),which increased the levels of radiation-induced chromosomal dam-age in cancer cells, protected normal cells against such damage.10

Flavonoids also display a variety of biological activities, includinginhibition of tumor growth,11 suppression of activity of severalenzymes that regulate cell proliferation,11 prevention of cell-cycleprogression and induction of apoptosis.12 Apigenin (APG), a com-mon dietary flavonoid, is abundantly present in fruits and vegetablesand has a great potential for growth inhibition of cancer cells.13

(2)-Epigallocatechin (EGC) and (2)-epigallocatechin-3-gallate (EGCG)belong to the flavan-3-ol class of flavonoids, which are present in

green tea at low levels and capable of reducing the proliferation ofhuman breast cancer cells in culture and decreasing breast tumorgrowth in rodents.12 The anti-tumor mechanism of EGCG in cul-ture is due to modulation of the expression of key molecules incell-cycle progression,14 inhibition of nuclear factor kappa B(NFjB),14 binding to Fas,15 activation of mitogen-activated proteinkinase cascade.16 Genistein (GST), an isoflavonoid and specific in-hibitor of protein tyrosine kinase,17 is considered to be a promisingtherapeutic candidate for various cancers. GST substantially inhib-ited the growth of 5 tumor cell lines (N2A, JC, SKNSH, MSN andLan5) through induction of apoptosis and modulation of proteintyrosine kinase activity and N-myc expression.18 GST has multiplefunctions resulting in anti-tumor effects and also induces glutathi-one peroxidase in the human prostate cancer cell lines LNCaP andPC-3.19 It is also known to trigger the pathway that leads to cellulardifferentiation by stabilizing the possibility of protein-linked DNAstrand breakage in human acute myelogenous leukemia HL-60cells, chronic myelogenous leukemia K-562 cells and melanomaSK-MEL-131 cells.20

Two main pathways are known to induce apoptosis in mamma-lian cells. In receptor-mediated pathway, death factors such as Fasligand (FasL) and tumor necrosis factor-a (TNF-a) bind to their rel-evant cell-surface receptors, resulting in activation of caspase-8.21

In mitochondria-mediated pathway, cytochrome c and other apop-togenic proteins are released from the mitochondrial intermem-brane space into the cytosol.22 Once released, cytochrome c bindsto Apaf-123 and induces sequential activation of caspase-9 andcaspase-3.24 Caspase-8 activation provides a link between receptor-mediated and mitochondria-mediated pathways of apoptosis, therebyamplifying the receptor-mediated death signal.25,26 Caspase-8 activa-tion induces cleavage of Bid to truncated Bid (tBid) that triggers Baxactivation,27 resulting in change in mitochondrial permeability andthereby release of cytochrome c into the cytosol.

Recently, it has been demonstrated that a novel endoplasmicreticulum (ER)-specific apoptotic pathway is operated with cas-pase-12 activation.28,29 Caspase-12 may be activated due to itsdirect proteolytic cleavage by calpain, which is a non-caspase pro-tease. Pro-caspase-12 is localized on the cytoplasmic side of ER.Calpain, upon activation by elevated intracellular free [Ca21], isknown to translocate from the cytosol to the membrane,30 where itcan cleave pro-caspase-12 to active caspase-12. It is plausible thatmembrane association of caspase-12 is an inhibitory mechanismto prevent its activation under normal conditions. Active caspase-12 is capable of directly activating caspase-3. The final execu-tioner of cells is caspase-3 that cleaves inhibitor-of-caspase-acti-vated DNase [ICAD, i.e., DNA fragmentation factor-45 (DFF45)],resulting in release of CAD [i.e., DNA fragmentation factor-40

Grant sponsor: National Institutes of Health; Grant numbers: CA-91460,NS-31622 and NS-57811.*Correspondence to: Department of Neurosciences, Medical Univer-

sity of South Carolina (MUSC), 96 Jonathan Lucas Street, Suite 323K,P.O. Box 250606, Charleston, SC 29425, USA. Fax:11-843-792-8626.E-mail: raysk@musc.eduReceived 22 March 2006; Accepted 30 June 2006DOI 10.1002/ijc.22228Published online 20 September 2006 inWiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 119, 2575–2585 (2006)' 2006 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

(DFF40)] from the ICAD/CAD complex and translocation ofCAD to the nucleus to carry out its nuclease activity.31,32

The human malignant (N-type) neuroblastoma SH-SY5Y cellline represents a subclone of the neuroblastoma SK-N-SH cellline.33 It is useful for studying the mechanisms of neuronal deathin various diseases.34,35 We examined the mechanism of inductionof apoptosis in SH-SY5Y cells following exposure to 50 lMAPG, 50 lM EGC, 50 lM EGCG and 100 lM GST. Our currentstudies indicate that EGC and EGCG induce apoptosis by activationof caspase-8 and mitochondria-mediated pathway, whereas APGand GST induce apoptosis by activation of calpain and mitochon-dria-mediated pathway.

Material and methods

Cell culture and treatments

Human malignant (N-type) neuroblastoma SH-SY5Y cell linewas purchased from the American Type Culture Collection (ATCC,Manassas, VA, USA). Cells were grown in 75-cm2 flasks contain-ing 10 ml of RPMI 1640 (GIBCO) supplemented with 10% fetalbovine serum (FBS) and 1% penicillin and streptomycin in a fully-humidified incubator containing 5% CO2 at 37�C. Prior to drugtreatments, the cells were starved in RPMI 1640 supplemented with1% FBS for 24 hr. Dose-response studies were conducted to deter-mine the suitable doses of the drugs used for induction of apoptosisin the experiments. Finally, cells were treated with 50 lM APG,50 lM EGC, 50 lM EGCG and 100 lM GST for 24 hr for induc-tion of apoptotic death. Following treatments, apoptosis was deter-mined morphologically and biochemically. The cells were also ex-amined for alterations in expression and activity of apoptosis relatedproteins. Protease inhibitors such as caspase-8 inhibitor II, calpeptin(calpain inhibitor), caspase-9 inhibitor I and caspase-3 inhibitor IVwere obtained from Calbiochem-Novabiochem (San Diego, CA,USA) and used for pretreatment (1 hr) of the cells followed by50 lM APG, 50 lM EGC, 50 lM EGCG and 100 lM GST treat-ments for 24 hr.

Trypan blue dye exclusion test for residual cell viabilityafter flavonoid treatments

Following all treatments, the viability of attached and detachedcell populations was estimated by trypan blue dye exclusion test.36

Viable cells maintained membrane integrity and did not take uptrypan blue. Cells with compromised cell membranes took up trypanblue, and were counted as dead. At least 600 cells were counted in 4different fields and the percentage of residual cell viability was cal-culated. For calculation, we used the following formula: percentageof residual cell viability 5 [number of trypan blue negative cells/(number of trypan blue positive cells1 number of trypan blue nega-tive cells)]3 100.

Wright staining for morphological analysis of apoptosis

Cells from each treatment were detached with a cell scraper,washed twice in PBS and sedimented onto a microscopic slide usingan Eppendorf 5804R centrifuge (Brinkmann Instruments, Westbury,NY, USA) at 106g for 5 min. Cells were fixed and stained withWright stain.36 Cellular morphology was examined by optical mi-croscopy to assess apoptosis. Cells were considered apoptotic if theyshowed (i) reduction in cell volume and (ii) condensation of thechromatin or the presence of cell membrane blebbing. At least 600cells were counted in each treatment, and the percentage of apopto-tic cells was calculated.

Biochemical detection of apoptosis by ApopTag assay

Biochemical detection of apoptotic cells in situ was done withthe commercially available ApopTag kit (Intergen, Purchase,Manhattanville, NY, USA). Apoptosis was quantified by countingApopTag-stained cells on the grid of the microscope field (403objective). The cells were also counterstained with methyl green.Methyl green stained normal nuclei a pale to medium green. The

nuclei that contain DNA fragments or condensation were posi-tively stained dark brown (by the ApopTag detection procedure)and were not stained with the methyl green. Experiments wereperformed in triplicate, and the brown colored ApopTag-positivecells were counted under the light microscope to determine per-centage of apoptosis.

Determination of intracellular free [Ca21] using Fura-2 assay

The level of intracellular free [Ca21] was measured in humanneuroblastoma SH-SY5Y cells using the fluorescence Ca21 indi-cator Fura-2/AM as described previously,36 which was a modifica-tion of the original method37 for determination of intracellular free[Ca21]. The intracellular free [Ca21] was calculated spectrofluoro-metrically using the equation [Ca21]5 Kdb(R2 Rmin)/(Rmax 2 R),[where b is the ratio of maxF380 (fluorescence intensity exciting at380 nm for zero free Ca21) to minF380 (fluorescence intensity at sat-urating free Ca21), as reported previously.38 The determination offluorescence ratio (R) was done using an SLM 8000 spectrofluorom-eter (Thermospectronic, Rochester, NY, USA) at 340 and 380 nmwavelengths. The maximal (Rmax) and minimal (Rmin) ratios weredetermined using 200 ll of 250 lM digitonin (Fisher Scientific,Pittsburgh, PA) and 500 mM EGTA (Sigma Chemical Co., St.Louis, MO), respectively. The value of Kd, a cell-specific constant,was determined experimentally to be 0.451 lM for the SH-SY5Ycells using standards of the Calcium Calibration Buffer Kit withMagnesium (Molecular Probes, Eugene, OR).

Antibodies

Various primary IgG antibodies were used to probe the Westernblots. Monoclonal antibodies against Bax and Bcl-2 (Santa CruzBiotechnology, Santa Cruz, CA, USA) were used to assess the ap-optotic threshold by determining the Bax:Bcl-2 ratio. Monoclonalantibody against a-spectrin (Affiniti, Exeter, UK) was used to mea-sure the activities of calpain and caspase-3. Polyclonal antibodiesagainst caspase-8, caspase-9, caspase-12, cytochrome c and ICADwere also used (Santa Cruz Biotechnology). Polyclonal antibodyagainst Bid (Santa Cruz Biotechnology) was used to determine theBid cleavage to tBid by caspase-8 activity. Monoclonal antibodyagainst b-actin (clone AC-15, Sigma) was used to standardize pro-tein loadings on the gels. Antibody against cytochrome c oxidasesubunit 4 (COX4) (Molecular Probes, Eugene, OR, USA) was usedto standardize mitochondrial protein loadings. It should be notedthat COX4 is an inner mitochondrial membrane protein and itremains in the mitochondria regardless of activation of apoptosis.39

The secondary antibody used was goat anti-mouse IgG conju-gated with alkaline horseradish peroxidase (HRP) (ICN Biomedi-cals, Aurora, OH) except in the case of calpain, caspase-8, cas-pase-12 and a-spectrin where it was goat anti-rabbit IgG conju-gated with alkaline HRP (ICN Biomedicals).

Western blot analysis of specific proteins

Analysis of specific proteins was performed by Western blot-ting.36 Proteins were separated by (4–20%) gradient gel or 5% gelusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Following electrophoresis, resolved total proteinfrom the gel was transferred to a PVDF membrane (Millipore,Bedford, MA, USA) in an electroblotting apparatus Genie (IdeaScientific, Minneapolis, MN, USA). The membrane was thenblocked in 5% powdered non-fat milk in a Tris/Tween solution(20 mM Tris-HCl, pH 7.6, 0.1% Tween 20 in saline) for 1 hr. Theprimary antibodies were diluted (1:100 for Bax, Bcl-2, Bid, cas-pase-3, caspase-8, caspase-9, caspase-12 and CAD; 1:500 for cal-pain; 1:2,000 for a-spectrin; 1:15,000 for b-actin) in blocking solu-tion and then incubated with the membrane for 1 hr. The membranewas then covered with an alkaline HRP conjugated secondary IgGantibody (goat anti-rabbit for calpain, caspase-8, caspase-12 anda-spectrin; and goat anti-mouse for all others) at a 1:2,000 dilutionfor 1 hr. Between steps, membranes were washed 3 times in Tris/Tweensolution. Blots were incubated with ECL reagents (Amersham

2576 DAS ET AL.

Pharmacia, Buckinghamshire, UK) and exposed to X-OMAT ARfilms (Eastman Kodak, Rochester, NY, USA). The autoradiogramswere scanned on a UMAX PowerLook Scanner (UMAX Technolo-gies, Fremont, CA, USA) using Photoshop software (Adobe Sys-tems, Seattle, WA, USA). Optical density (OD) of each band wasdetermined using Quantity One software (Bio-Rad Laboratories,Hercules, CA, USA).

Preparation of mitochondrial, cytosolic and nuclear fractionsfor measurements of cytochrome c and CAD

Preparations of mitochondrial and cytoplasmic fractions40 andalso nuclear fraction41 were performed by standard procedures.Levels of cytochrome c in the mitochondrial and cytosolic frac-tions, and CAD in the nuclear fraction were analyzed by the West-ern blotting as described earlier.

Colorimetric assay for the measurement of caspase-8,caspase-9 and caspase-3 activities

Measurements of caspase activities in cells were performedwith the commercially available caspase-8, caspase-9 and cas-pase-3 assay kits (Sigma). The colorimetric assays were based onthe hydrolysis of the Ac-IETD-pNA by caspase-8, Ac-LEHD-pNA by caspase-9 and Ac-DEVD-pNA by caspase-3, resulting inthe release of the p-nitroaniline (pNA) moiety. The pNA has ahigh absorbance at 405 nm (emM 5 10.5). Proteolytic reactionswere carried out in extraction buffer containing 20 lg of cytosolicprotein extract and 40 lM Ac-IETD-pNA, 40 lM Ac-LEHD-pNAor 40 lM Ac-DEVD-pNA. The reaction mixtures were incubatedat room temperature for 2 hr and the formation of pNA was mea-sured at 405 nm in a colorimeter. The concentration of the pNAreleased from the substrate was calculated from the absorbancevalues. Experiments were performed in triplicate.

Trypan blue dye exclusion assay after pretreatmentwith protease inhibitors

Loss of membrane integrity was determined by the inability ofcells to exclude the vital dye trypan blue. For the inhibitor studies,cells were cultured as described before and either left untreated orwere pretreated (1 hr) with 10 lM caspase-8 inhibitor II, 10 lMcalpeptin, 10 lM caspase-9 inhibitor I and 10 lM caspase-3 inhib-itor IV. After 24 hr, cells were removed from each treatment,diluted 1:1 with trypan blue (Sigma) and counted (at least 600cells) for calculation of percentage of residual cell viability.

Statistical analysis

All results obtained from different treatments of SH-SY5Y cells wereanalyzed using StatView (Abacus Concepts, Berkeley, CA, USA).Data were expressed as mean 6 standard error of mean (SEM) ofseparate experiments (n > 3) and compared by one-way analysis ofvariance (ANOVA) followed by Fisher’s post hoc test. The differ-ence between 2 treatments was considered significant at p � 0.05.

Results

Evaluation of residual cell viability and also morphologicaland biochemical features of apoptotic cell death

Exclusion of trypan blue dye by residual viable SH-SY5Y cellswas evaluated under a light microscope using a hemocytometer af-ter treatment with different flavonoids (Fig. 1). Also, the extent ofapoptotic cell death was determined based on morphological fea-tures after Wright staining (Fig. 2). Treatment of cells with 50 lMAPG, 50 lM EGC, 50 lM EGCG and 100 lM GST showed anincrease in the percentage of apoptotic cells, compared with con-trol (CTL) cells (Fig. 2). The results from Wright staining werefurther confirmed with biochemical detection of DNA fragmenta-tion by AopTag assay (Fig. 3). Qualitatively, CTL cells showedlittle or no brown color confirming almost absence of apoptosis.But cells treated with 50 lM APG, 50 lM EGC, 50 lM EGCG or100 lM GST demonstrated the prominent brown color in apopto-

tic cells. Thus, Wright staining and ApopTag assay clearly demon-strated the morphological and biochemical apoptotic features,respectively, in SH-SY5Y cells following exposure to the selecteddoses of the flavonoids (Figs. 1–3).

Treatment with flavonoids increased intracellular free [Ca21]

We used Fura-2 assay to determine the intracellular free [Ca21]in SH-SY5Y cells (Fig. 4). Treatment of cells with 50 lM APG,

FIGURE 1 – Dose-response to flavonoids. Dose-response (24 hr)studies were conducted using lM levels of flavonoids [APG (a), EGC(b), EGCG (c) and GST (d)] to determine the suitable doses of theseagents for use in the experiments for induction of apoptosis. Bargraphs show the percentages of residual cell viability and apoptosis ineach treatment group.

2577FLAVONOIDS INDUCE APOPTOSIS IN NEUROBLASTOMA

50 lM EGC, 50 lM EGCG and 100 lM GST for 24 hr caused sig-nificant increases in intracellular free [Ca21], compared with CTLcells. These results suggested a role for Ca21 influx in cell deathin SH-SY5Y cells.

EGC and EGCG treatments induced caspase-8 activationand proteolytic cleavage of Bid

Our results showed caspase-8 (Fig. 5a) activation in SH-SY5Ycells treated with EGC and EGCG. b-Actin expression was moni-tored to ensure that equal amounts of protein were loaded in eachlane (Fig. 5b). We found a significant increase (p 5 0.004) in cas-pase-8 active band (Fig. 5c) in SH-SY5Y cells treated with EGCand EGCG. Caspase-8 activation induced the proteolytic cleavageof Bid to tBid (Fig. 5d), which could translocate from cytosol to mi-tochondrial membrane to stimulate more efficient oligomerizationof Bax and thereby activation of the intrinsic apoptotic pathway.42

We measured the levels of tBid in mitochondrial fraction. In West-ern blotting, we used an antibody against COX4 as a mitochondrialinternal control (Fig. 5e). We found significant (p < 0.005) proteo-lytic cleavage of Bid to tBid (Fig. 5f) in SH-SY5Y cells treated withEGC and EGCG.

Apoptosis with an increase in Bax:Bcl-2 ratio

A commitment to apoptosis was measured by examining anyincrease in the ratio of Bax (pro-apoptotic protein) expression toBcl-2 (anti-apoptotic protein) expression (Fig. 6). The bax geneencodes different isoforms. The monoclonal antibody used in thisinvestigation could recognize both Baxa (21 kDa) and Baxb(24 kDa) bands (Fig. 6a). Here, we considered both bands in ourestimation of total Bax expression. We used another monoclonal anti-

FIGURE 2 – Wright staining forevaluation of morphological fea-tures of apoptosis. Five treatment(24 hr) groups: control (CTL);50 lM APG; 50 lM EGC; 50 lMEGCG and 100 lM GST. Photomi-crographs show cells from eachtreatment and the arrows indicateapoptotic cells (a). Bar graphsshow the percentage of apoptoticcells counted from each treatmentgroup (b).

FIGURE 3 – ApopTag assay fordetecting DNA fragmentation in ap-optotic cells. Five treatment (24 hr)groups: control (CTL); 50 lM APG;50 lM EGC; 50 lM EGCG and100 lM GST. Photomicrographsshow cells from each treatment group,and the arrows indicate apoptotic cells(a). Bar graphs show the percentageof apoptotic cells counted from eachtreatment group (b).

FIGURE 4 – Determination of percent increase in intracellular free[Ca21] using Fura-2 assay. The data were from SHSY5Y cells grownin phenol-red free medium, treated with flavonoids and then exposedto Fura-2. Five treatment (24 hr) groups: control (CTL); 50 lM APG;50 lM EGC; 50 lM EGCG and 100 lMGST. Percent change in intra-cellular free [Ca21] is shown. Significant difference from CTL valuewas indicated by * (p < 0.01) or ** (p < 0.005).

2578 DAS ET AL.

body to monitor the expression of 26 kDa Bcl-2 protein (Fig. 6b).b-Actin expression was monitored to ensure that equal amountsof cytosolic protein samples were loaded in all lanes (Fig. 6c).The Bax:Bcl-2 ratio was measured in all 5 treatment groups usingWestern blotting (Fig. 6d). Cells treated with 50 lM APG, 50 lMEGC, 50 lM EGCG and 100 lMGST showed a significant increase(p 5 0.004) in the Bax:Bcl-2 ratio (Fig. 6d), compared with CTLcells. The rise in Bax:Bcl-2 ratio in cells exposed to flavonoids ver-sus CTL was influenced more by a change in Bax than by a changein Bcl-2.

Treatments with flavonoids induced mitochondrial cytochrome crelease and activation of caspase-9

To analyze the involvement of mitochondrial release of cyto-chrome c for apoptosis in SH-SY5Y cells, proteins from both cyto-solic and mitochondrial fractions were prepared and analyzed for thelevels of cytochrome c by Western blotting. We examined COX4as an internal control for the mitochondrial fractions (Fig. 7a).

The treatment of cells with flavonoids promoted disappearance of15 kDa cytochrome c from the mitochondrial fraction (Fig. 7b) ofthe cells treated with flavonoids and caused an appearance of15 kDa cytochrome c in the cytosolic fraction (Fig. 7c). Thus, themechanism of cell death involved the mitochondrial release ofcytochrome c, which subsequently could cause activation of cas-pase-9. b-Actin expression was monitored to ensure that equalamounts of cytosolic protein samples were loaded in all lanes(Fig. 7d). We found a significant difference in cytochrome crelease from mitochondria (Fig. 7e). Our results also showed anincrease in active 37 kDa caspase-9 fragment in SH-SY5Y cellsfollowing treatment with flavonoids (Fig. 7f). These results sug-gested that caspase-9 activation might be a consequence of cyto-chrome c release from the mitochondria. There was a significantdifference in caspase-9 activation (Fig. 7g) between CTL cells andcells treated with flavonoids.

Degradation of a-spectrin to specific fragments by calpainand caspase-3 activities

We determined calpain and caspase-3 activities by measuring thecalpain-specific 145 kDa SBDP43 and caspase-3-specific 120 kDaSBDP,44 respectively, on the Western blots (Fig. 8). Cells treatedwith APG, EGC, EGCG and GST showed significant increases ingeneration of 145 kDa SBDP and 120 kDa SBDP (Fig. 8a). Almostuniform levels of b-actin in all lanes indicated that equal amountsof cytosolic protein samples were loaded in all lanes (Fig. 8b). Thelevels of 145 kDa SBDP (Fig. 8c) and 120 kDa SBDP (Fig. 8d) incells treated with APG, EGC, EGCG and GST were higher thanCTL cells, indicating that both calpain and caspase-3 activities wereincreased after treatment with flavonoids.

Caspase-3 activation as determined from generationof 20 kDa caspase-3 active band

Caspase-3 activation was measured by Western blot analysis of20 kDa caspase-3 active band (Fig. 9a). Again, almost uniform ex-pression of b-actin in all treatments served as an internal control andindicated equal amounts of protein loadings in all lanes (Fig. 9b). Theintensity of the 20 kDa caspase-3 active band in cells treated withflavonoids was almost twice the intensity seen in CTL cells (Fig. 9c).

FIGURE 5 – Measurement of caspase-8 activation and also Bid cleav-age to tBid using Western blotting. Five treatment (24 hr) groups: con-trol (CTL); 50 lMAPG; 50 lM EGC; 50 lM EGCG and 100 lMGST.The representative Western blots show levels of caspase-8 bands (a)and b-actin (b). Densitometric analysis show percent change in the18 kDa caspase-8 active band in treatment over CTL (c). The other rep-resentative Western blots show mitochondrial tBid (d) and COX4 (e).Densitometric analysis show percent change in the 15 kDa tBid bandover CTL cells (f). Significant difference from CTL value was indicatedby * (p< 0.01) or ** (p< 0.005).

FIGURE 6 – The Bax:Bcl-2 ratio measured by Western blot analysis.Five treatment (24 hr) groups: control (CTL); 50 lM APG; 50 lMEGC; 50 lM EGCG and 100 lM GST. The representative Westernblots show levels of Bax (a), Bcl-2 (b) and b-actin (c). Densitometricanalysis shows the Bax:Bcl-2 ratio in all treatment groups (d). Sig-nificant difference from CTL value was indicated by * (p < 0.01) or** (p < 0.005).

2579FLAVONOIDS INDUCE APOPTOSIS IN NEUROBLASTOMA

Caspase-8, caspase-9 and caspase-3 activities as measuredcolorimetrically

Proteolytic activities of caspase-8, caspase-9 and caspase-3 werefurther determined in all treatment groups using colorimetric assays(Fig. 10). There was no significant difference (p 5 0.981) betweencaspase-8 activity (Fig. 10a) in CTL cells and that in cells treatedwith APG and GST, whereas cells treated with EGC and EGCGshowed significant increase (p 5 0.002) in caspase-8 activity whencompared with CTL cells. Cells treated with APG, EGC, EGCGand GST showed a significant increase (p 5 0.001) in activation ofcaspase-9 (Fig. 10b) and caspase-3 (Fig. 10c). Notably, GST causedthe highest increase in caspase-3 activity. These results further sup-ported that increases in 37 kDa caspase-9 and 20 kDa caspase-3active fragments were due to treatment with APG, EGC, EGCG andGST.

FIGURE 7 – Western blot analysis for determining cytochrome crelease from mitochondria and caspase-9 activation. Five treatment(24 hr) groups: control (CTL); 50 lM APG; 50 lM EGC; 50 lMEGCG; and 100 lM GST. The Western representative blots showlevels of mitochondrial COX4 (a), mitochondrial cytochrome c (b),cytosolic cytochrome c (c) and cytosolic b-actin (d). Densitometricanalysis show percent change in the cytochrome c levels from mito-chondrial fractions (e). The other representative western blot showslevel of active caspase-9 band (f). Densitometric analysis show per-cent change in the active caspase-9 band (g). Significant differencefrom CTL value was indicated by * (p < 0.01) or ** (p < 0.005).

FIGURE 8 – Determination of calpain and caspase-3 activities in a-spectrin cleavage using Western blot analysis. Five treatment (24 hr)groups: control (CTL); 50 lM APG; 50 lM EGC; 50 lM EGCG and100 lM GST. The representative Western blots show levels of 145 kDaSBDP and 120 kDa SBDP generated from a-spectrin (a) and b-actin(b). Densitometric analysis showing percent change in the calpain-specific 145 kDa SBDP over CTL (c). Densitometric analysis showingpercent change in the caspase-3-specific 120 kDa SBDP over CTL (d).Significant difference from CTL value was indicated by * (p < 0.01) or** (p< 0.005).

FIGURE 9 – Determination of caspase-3 activation using Western blotanalysis of 20 kDa active caspase-3 band. Five treatment (24 hr) groups:control (CTL); 50 lM APG; 50 lM EGC; 50 lM EGCG and 100 lMGST. The representative Western blots show levels of caspase-3 activa-tion (a) and b-actin (b). Densitometric analysis show percent change inthe 20 kDa caspase-3 active band over CTL (c). Significant differencefrom CTL value was indicated by * (p < 0.01) or ** (p< 0.005).

2580 DAS ET AL.

APG and GST induced caspase-12 activation

We also monitored activation of caspase-12 (Fig. 11). The gener-ation of 40 kDa caspase-12 active band was examined by Westernblotting (Fig. 11a). Again, almost uniform expression of b-actin inall treatments served as an internal control and indicated equalamounts of protein loadings in all lanes (Fig. 11b). The levels of40 kDa caspase-12 active fragment were increased in cells treatedwith APG and GST (Fig. 11c). There was a significant difference(p 5 0.004) in intensities of the 40 kDa caspase-12 active fragmentbetween the cells treated with APG or GST and the CTL cells. Wefound non-significant change (p 5 0.921) in caspase-12 activationin cells treated with EGC and EGCG.

Flavonoids induced CAD activation

For analyzing nuclear fractions, we ran 2 sets of SDS-PAGEgels at the same time. After SDS-PAGE, 1 set of gels with theresolved proteins was stained with Coomassie Blue to confirmloading of equal amounts of nuclear protein in all lanes (data notshown); and the other set of gels was used for analyzing levels of

CAD by Western blotting (Fig. 12). Our results showed an appear-ance of 40 kDa CAD in the nuclear fraction of SH-SY5Y cellstreated with APG, EGC, EGCG and GST (Fig. 12a). There was asharp difference in CAD levels between the CTL cells and cellstreated with APG, EGC, EGCG and GST (Fig. 12b).

Prevention of cell death by pretreatment with differentprotease inhibitors

Pretreatment of SH-SY5Y cells for 1 hr with a caspase-3 inhibi-tor IV inhibited cell death in SH-SY5Y cells following APG,EGC, EGCG and GST treatments (Fig. 13). In contrast, caspase-9inhibitor I showed partial inhibitory effect. Pretreatment of cellswith calpeptin (calpain inhibitor) blocked cell death in APG andGST treatments, whereas caspase-8 inhibitor II blocked cell deathin EGC and EGCG treatments. But, blocking of more than 60%cell death occurred by calpeptin in cells treated with EGC and

FIGURE 10 – Determination of activities of caspase-8, caspase-9 andcaspase-3 using colorimetric assays. Five treatment (24 hr) groups: con-trol (CTL); 50 lM APG; 50 lM EGC; 50 lM EGCG and 100 lM GST.Activities of caspase-8 (a), caspase-9 (b) and caspase-3 (c) were deter-mined colorimetrically using specific peptide substrates. Significant dif-ference from CTL value was indicated by * (p< 0.01) or ** (p< 0.005).

FIGURE 11 – Determination of caspase-12 activation using Westernblot analysis. Five treatment (24 hr) groups: control (CTL); 50 lMAPG; 50 lM EGC; 50 lM EGCG and 100 lM GST. The representa-tive Western blots show levels of caspase-12 activation (a) and b-actin (b). Densitometric analysis show percent change in the 40 kDacaspase-12 active band over CTL (c). Significant difference from CTLvalue was indicated by * (p < 0.01) or ** (p < 0.005).

FIGURE 12 – Western blot analysis of amounts of CAD in nuclearfraction. Five treatment groups: control (CTL); 50 lM APG; 50 lMEGC; 50 lM EGCG; 100 lM GST for 24 hr. The representative West-ern blot shows levels of CAD in nuclear fraction (a). Densitometric anal-ysis show percent change in the nuclear CAD over CTL (b). Significantdifference from CTL value was indicated by * (p<0.01) or ** (p< 0.005).

2581FLAVONOIDS INDUCE APOPTOSIS IN NEUROBLASTOMA

EGCG, and caspase-8 inhibitor II in cells treated with APG andGST. Results indicated roles of calpain and caspases in apoptosis.

Discussion

Plant-derived agents have recently received extensive attentionbecause of their pharmacological properties such as anti-bacterial,anti-mutagenic and anti-tumor effects.13,14 The most extensivelystudied property has been the anti-tumor effect in which severalcharacteristic phenomena of apoptosis were observed: cell-cyclearrest,45 DNA damage46 and activation of caspases.47 In this investiga-tion, we have demonstrated that APG, EGC, EGCG and GST are effec-tive in inducing apoptosis via activation of proteolytic pathways inhuman malignant neuroblastoma SH-SY5Y cells. This cell modelwas chosen on the basis of the aggressive behavior and high meta-static potential of this human malignant neuroblastoma SH-SY5Ycell line. Our investigation showed that exposure of SH-SY5Y cellsto APG, EGC, EGCG and GST produced morphological (Figs. 1and 2) and biochemical (Fig. 3) features of apoptosis with anincrease in Ca21 influx (Fig. 4). Our results indicate that EGC andEGCG activate caspase-8 followed by Bid cleavage to tBid (Fig. 5),increase in Bax:Bcl-2 ratio (Fig. 6), release of cytochrome c frommitochondria (Fig. 7), activation of caspase-9 (Fig. 7) and site-spe-cific degradation of a-spectrin, indicating activation of proteotyticactivities of both calpain (Fig. 8) and caspase-3 (Figs. 8–10).Increases in proteolytic activities of caspase-8, caspase-9 and cas-pase-3 were further confirmed in all treatment groups using colori-metric assays (Fig. 10). Our results also suggested activation of cas-pase-12 (Fig. 11) by APG and GST. Activation of caspase-9 (Fig. 7)prompted activation of caspase-3 and CAD (Fig. 12). Use of prote-ase inhibitors confirmed that cell death in SH-SY5Y cells followingexposure to APG, EGC, EGCG and GST required involvement ofmultiple proteolytic pathways (Fig. 13).

A role for calpain in both neuronal48 and non-neuronal49 celldeath has been documented. In this investigation, we explored themechanism by which calpain caused cell death in neuroblastomaSH-SY5Y cells following treatment with flavonoids. Calpain canplay a dual role, mediation of Ca21 influx and proteolysis subse-quent to Ca21 influx, during cell death.50,51 Our findings support a

direct relationship between an increase in intracellular free [Ca21](Fig. 4) and cell death mediated by elevation of calpain activity(Fig. 8) following exposure to APG, EGC, EGCG and GST (Fig. 4).Increase in intracellular free [Ca21] causes calpain activation anddegradation of cytoskeletal proteins,52 destabilizing the cellular in-tegrity and leading to cell death.53 Our results indicate that calpainactivation occurs in SH-SY5Y cells following exposure to APG,EGC, EGCG and GST.

Recently, it has been reported that a novel Ca21-mediated,calpain/caspase-12-dependent apoptotic pathway exists in breastcancer cells and that regulation of intracellular free [Ca21] innormal and cancer mammary epithelial cells is different.54–56

Our findings provide evidence for a sustained increase in intra-cellular free [Ca21] for activation of the Ca21-dependent calpainand the Ca21/calpain-dependent caspase-12 for the apoptoticmechanism of APG and GST in SH-SY5Y cells (Fig. 11).

It has previously been reported that green tea polyphenol indu-ces death receptor-mediated caspase-8 activation followed by cas-pase-3 activation.57 We found that EGC and EGCG activated cas-pase-8 in SH-SY5Y cells (Fig. 5), indicating that the receptor-mediated pathway was involved in apoptosis of cells treated withEGC and EGCG. It was also reported previously that green teapolyphenols induced apoptosis in human prostate carcinomacells58 and monocytic leukemia U937 cells15 via caspase-8 activa-tion. We found that caspase-8 activation caused Bid cleavage totBid in SH-SY5Y cells treated with EGC and EGCG (Fig. 5). TheC-terminal of tBid binds to mitochondria and transduces apoptoticsignal, suggesting that caspase-8 potentiates the mitochondria-mediated pathway of apoptosis.59 In this study, we also found thatEGC and EGCG induced apoptosis via caspase-9 activation, indi-cating involvement of mitochondria-mediated caspase pathway aswell (Fig. 7). Thus, our results suggest that EGC and EGCG acti-vate the receptor-mediated and mitochondria-mediated caspasepathways, which work together to induce apoptosis (Fig. 14).

The mechanism of cell death involving calpain activation alsorequires mitochondrial co-operation.53 In course of apoptosis, Baxis translocaled to mitochondria with an involvement of calpain inthis process.60 The relative levels of Bax (pro-apoptotic) and Bcl-2(anti-apoptotic) in mitochondria determine the life-or-death fate of

FIGURE 13 – Pretreatment with protease inhibitors prevented flavonoid-induced cell death. Cells were pretreated with a protease inhibitor for1 hr. Twenty five treatment (24 hr) groups:1, control (CTL); 2, 10 lM caspase-8 inhibitor II; 3, 10 lM calpeptin; 4, 10 lM caspase-9 inhibitor I;5, 10 lM caspase-3 inhibitor IV; 6, 50 lM APG; 7, 10 lM caspase-8 inhibitor II 1 50 lM APG; 8, 10 lM calpeptin 1 50 lM APG; 9, 10 lMcaspase-9 inhibitor I 1 50 lM APG; 10, 10 lM caspase-3 inhibitor IV1 50 lM APG; 11, 50 lM EGC; 12, 10 lM caspase-8 inhibitor II 150 lM EGC; 13, 10 lM calpeptin 1 50 lM EGC; 14, 10 lM caspase-9 inhibitor I 1 50 lM EGC; 15, 10 lM caspase-3 inhibitor IV 1 50 lMEGC; 16, 50 lM EGCG; 17, 10 lM caspase-8 inhibitor II 1 50 lM EGCG; 18, 10 lM calpeptin 1 50 lM EGCG; 19, 10 lM caspase-9 inhibi-tor I 1 50 lM EGCG; 20, 10 lM caspase-3 inhibitor IV 1 50 lM EGCG; 21, 100 lM GST for 24 hr; 22, 10 lM caspase-8 inhibitor II 1100 lM GST; 23, 10 lM calpeptin 1 100 lM GST; 24, 10 lM caspase-9 inhibitor I 1 100 lM GST; 25, 10 lM caspase-3 inhibitor IV 1100 lM GST. Significant difference from CTL value was indicated by * (p <0.01) or ** (p < 0.005).

2582 DAS ET AL.

the cells,61 as heterodimerization of Bax with Bcl-2 prevents apo-ptosis while Bax homodimerization triggers apoptotic process withthe release of cytochrome c from mitochondria.62 Increase inexpression of calpain has previously been suggested to coincidewith elevation in expression of Bax relative to Bcl-2, suggesting thatalterations in expression of these Bcl-2 family members play an im-portant role in cell death.63 Therefore, we examined the relative lev-els of Bax and Bcl-2 in SH-SY5Y cells following treatment with theflavonoids (Fig. 6). Our results showed alterations in levels ofexpression of Bax and Bcl-2 proteins, resulting in an increase inBax:Bcl-2 ratio in SH-SY5Y cells treated with APG, EGC, EGCGand GST. An increased level of Bax promotes the opening of thevoltage-dependent anion channels in the outer mitochondrial mem-brane to release cytochrome c for induction of apoptosis.64

Cytochrome c release from mitochondria is central to apopto-sis,62 but the events leading to it are not yet clear. A number ofpro- and anti-apoptotic members of the Bcl-2 protein family regu-late the release of cytochrome c from the mitochondrial intermem-brane space into the cytosol.65 Our results demonstrated an in-crease in cytosolic cytochrome c level due to its release frommitochondria in SH-SY5Y cells treated with APG, EGC, EGCGand GST (Fig. 7). Once cytochrome c is released from mitochon-dria, subsequent events occur in the cytosol for activation of cas-pases, leading to apoptosis.66 We observed activation of caspase-9(Fig. 7) after cytochrome c release from mitochondria. Takingthese results together, we propose that the release of cytochrome cfrom mitochondria and subsequent activation of caspase-9 playkey roles in apoptosis of SH-SY5Y cells treated with flavonoids.

Further, our results demonstrated that APG, EGC, EGCG andGST induced activation of caspase-3 for apoptosis of SH-SY5Ycells (Figs. 8–10). CAD is a DNase with high specific activity and

it exists as an inactive enzyme when complexed with ICAD in liv-ing cells.67–69 In course of apoptosis in SH-SY5Y cells, caspase-3cleaved ICAD and thus released CAD for its translocation to thenucleus (Fig. 12) for degradation of genomic DNA.

Our studies using different protease inhibitors showed that SH-SY5Y cells treated with APG, EGC, EGCG and GST committedapoptosis by using activities of multiple proteases (Fig. 13). Whilewe suggest that APG, EGC, EGCG and GST cause activation ofmitochondria-mediated pathway of apoptosis because of mito-chondrial release of cytochrome c into cytosol, activation of cas-pase-9 and caspase-3, and DNA fragmentation, we also show apossibility of activation of the receptor-mediated pathway of apo-ptosis due to activation of caspase-8 following EGC and EGCGtreatments. The role of EGCG in activation of receptor-mediatedpathway of apoptosis has already been demonstrated by anothergroup of investigators.70 Nevertheless, our investigation in SH-SY5Y cells described herein is novel and has not previously beenelucidated. Activation of caspase-8 by EGC and EGCG maydirectly activate caspase-3 or cleave Bid to tBid that subsequentlyinduces mitochondrial release of cytochrome c for activation ofcaspase-9 and caspase-3, as we depict schematically (Fig. 14). Also,our results suggest that APG and GST induce apoptosis in SH-SY5Y cells mainly via mitochondria-mediated pathway due to asustained increase in intracellular free [Ca21] and activation of cal-pain, caspase-12, caspase-9 and caspase-3 (Fig. 15).

In conclusion, our results demonstrated that APG, EGC, EGCGand GST could induce activation of calpain and caspase cascadesfor mediation of apoptosis in human neuroblastoma SH-SY5Ycells. Therefore, plant-derived flavonoids should be further exploredin animal models to determine their potential as therapeutic agentsfor treating neuroblastoma.

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