Norcantharidin induces apoptosis of breast cancer cells: Involvement of activities
of mitogen activated protein kinases and signal transducers and activators
of transcription
Pei-Yu Yang
a,b, Ming-Feng Chen
b, Ying-Hsien Kao
c, Dan-Ning Hu
b,d, Fang-Rong Chang
a,
Yang-Chang Wu
a,e,f,g,⇑aGraduate Institute of Natural Products, Kaohsiung Medical University, 100, Shih-Chuan 1st Rd., Kaohsiung 80708, Taiwan, ROC b
Department of Medical Research, Show Chwan Memorial Hospital, 542, Sec. 1 Chung-Shan Rd., Changhua 500, Taiwan, ROC c
Department of Medical Research, E-DA Hospital, I-Shou University, 1, E-DA Rd., Kaohsiung 824, Taiwan, ROC d
New York Medical College, NY, USA e
Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan, ROC f
Natural Medicinal Products Research Center, China Medical University Hospital, Taichung 40402, Taiwan, ROC gCenter for Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 21 June 2010 Accepted 18 January 2011 Available online 23 January 2011
Keywords: Norcantharidin Chemosensitivity Breast cancer Estrogen receptor
Signal transducer and activator of transcription
Mitogen-activated protein kinase
a b s t r a c t
Involvement of activities of mitogen-activated protein kinases (MAPKs) and signal transducers and acti-vators of transcription (STATs) remains unsolved in norcantharidin-associated breast cancer cell apopto-sis. This study investigated the anti-cancer effect of norcantharidin and its underlying mechanism in two human breast cancer cell lines, estrogen receptor (ER) HS-578T and ER+ MCF-7 cells. Norcantharidin induced potent cytotoxicity and arrested cell growth through increasing phosphorylation of Chk1, Chk2 and total p21Waf1/Cip1and reducing cyclin B and cdc25c expression. It also induced apoptosis
through extrinsic death receptor and intrinsic mitochondrial pathways by cytochrome c release, caspase activation, oligonucleosome appearance, PARP cleavage, and aberration of Bcl-2 family protein expres-sion and phosphorylation. Although norcantharidin did not affect STAT1, STAT3, and STAT5 protein expression, it suppressed STAT3 and STAT5 phosphorylation in HS-578T cells, whereas it up-regulated STAT1 phosphorylation and down-regulated STAT5 phosphorylation in MCF-7 cells. Moreover, norcant-haridin activated MAPK family member proteins, extracellular signal-regulated kinase (ERK), p38MAPK
and c-Jun N-terminal kinase (JNK), were all phosphorylated by treatment. Pretreatment with selective kinase inhibitors significantly attenuated the norcantharidin-induced cytotoxicity in breast cancer cells. These findings suggest the potential involvement of MAPK and STAT pathways in norcantharidin-induced apoptogenesis. Norcantharidin may be an effective anti-cancer drug against breast cancer.
Ó 2011 Published by Elsevier Ltd.
1. Introduction
Breast cancer (BCa) is the second most common cancer world-wide after lung cancer and the fifth most common cause of can-cer-related death. Notably, morbidity of BCa in females is roughly 100 times higher than males, survival rates for both sexes are equal (Jemal et al., 2009). Unfortunately, severe morbidity of BCa has not been improved by surgery, radiotherapy, biological therapy, che-mo- or hormonotherapies, as BCa is highly resistant to
chemother-apy and no effective cure exists for patients with advanced disease stages (Andre et al., 2004; Orlando et al., 2007). Several biomarkers have been identified that predict response to chemotherapy to var-iable extent. Since estrogen is well known for its ability to directly modulate expression of growth factor receptor pathways and cell cycle regulatory genes (Dickson and Russo, 2000), status of estro-gen receptor (ER) in a tumor significantly correlates with the like-lihood of achieving pathologic complete response (Tewari et al., 2008). Patients who bear operable and ER-negative (ER ) tumors are more likely to achieve higher chemosensitivity than those ER+ cases (Guarneri et al., 2006; Ring et al., 2004).
Norcantharidin is a demethylated analog of cantharidin purified from the dried body of the Chinese blister beetle Mylabris, (Myla-bris phalerata Pallas), which is a traditional Chinese medicine long been used for treating malignant tumors (Wang, 1989). In compar-ison with cantharidin, the major bioactive compound in Mylabris, 0887-2333/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.tiv.2011.01.011
⇑ Corresponding author at: Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan, ROC. Tel.: +886 4 220 57153; fax: +886 4 220 60248.
E-mail addresses:[email protected](P.-Y. Yang),cmffl[email protected](M.-F. Chen), [email protected] (Y.-H. Kao), [email protected] (D.-N. Hu), [email protected](F.-R. Chang),[email protected](Y.-C. Wu).
Contents lists available atScienceDirect
Toxicology in Vitro
norcantharidin has the advantage of easy synthesis and reduced intrinsic toxicity while retaining its cancer activity. The anti-cancer mechanisms of norcantharidin have been illuminated by the evidence showing its anti-proliferation, pro-apoptotic and anti-migratory effects on many cancer cells. Norcantharidin inhib-its the growth of numerous cancer cell lines via apoptosis, includ-ing oral cancer (Kok et al., 2003), hepatoma (Chen et al., 2002), leukemia (Liao et al., 2007), colorectal adenocarcinoma (Peng et al., 2002), melanoma (An et al., 2004b), glioblastoma (Hong et al., 2000) and gallbladder carcinoma (Fan et al., 2007). Besides, animal studies demonstrated that norcantharidin reduced pul-monary metastatic capacity of colorectal adenocarcinoma cells (Chen et al., 2005, 2009b) and prolonged survival of HepG2 tu-mor-bearing nude mice (Yang et al., 1997).
The well-studied mechanism for the anti-cancer activity of nor-cantharidin is its ability to induce tumor cell apoptosis, the process of which encompasses interruption of cell cycle progression of tu-mors at mitotic phase, suppression of cell adhesion and motility, expressional modulation of Bcl-2 superfamily member proteins and of caspase activity, and induction of eventual apoptotic cell death. In fact, the apoptotic induction by norcantharidin has been evidenced in many types of tumor cells and, more recently, in hu-man breast cancer cells (Huang et al., 2009). In addition, the activ-ities of mitogen-activated protein kinase (MAPK) family members, such as extracellular signal-regulated kinase (ERK), p38MAPK, and
c-Jun N-terminal kinase (JNK), have been mechanistically implicated in the apoptogenesis of norcantharidin-treated tumor cells (An et al., 2004a; Chen et al., 2002, 2003, 2005; Hong et al., 2000; Peng et al., 2002). AlthoughHuang et al. (2009)has recently demon-strated that norcantharidin interferes with Akt phosphorylation in a highly metastatic human BCa cell line, however, not enough is known about the role that MAPKs play in the norcantharidin-elicited apoptosis of breast tumor cells. Moreover, aberrant activa-tion of signal transducers and activators of transcripactiva-tion (STATs), mainly STAT1, STAT3, and STAT5 proteins, is involved in the path-ogenesis of cancer (Bromberg, 2001). Overall, STAT1 functionally promotes apoptosis and tumor suppression, while STAT3 and STAT5 modulate cell growth and inhibit apoptosis (Battle and Frank, 2002; Bromberg, 2001). To date, the role of STATs in the apoptotic induction of BCa cells by norcantharidin remains unclear. Due to lack of knowledge for the above-mentioned viewpoints on the anti-cancer activity of norcantharidin in BCa cells, this study thus aimed at validating the norcantharidin-driven cytotoxicity in two BCa cell lines with opposite ER status (i.e., ER HS-578T and ER+ MCF-7 cells), delineating the responsive profiles of STATs and MAPKs activities to drug treatment therein, as well as elucidat-ing the pharmacological mechanism underlyelucidat-ing the drug action.
2. Materials and methods 2.1. Chemical reagents
Norcantharidin was purchased from Sigma (Sigma, St. Louis, MO, USA) and prepared by serial dilutions in culture medium. Pri-mary antibodies against Bax, Bid, Bad, Bak, Bim, Bcl-2, Bcl-xL, Mcl-1, phosphor-Bcl-2 (Ser70), c-Jun N-terminal kinase (JNK), phos-phor-JNK (Thr183/Tyr185), ERK 1/2, phosphor ERK 1/2 (Thr202/ Tyr204), p38MAPK, phosphor-p38MAPK (Thr180/Tyr182), p21Waf1/ Cip1, phosphor-p53 (Ser15), p27Kip1, phosphor-Chk1 (Ser345),
phor-Chk2 (Thr68), STAT1, phosphor-STAT1 (Tyr701), STAT3, phos-phor-STAT3 (Tyr705), STAT5, phosphor-STAT5 (Tyr694) and goat-anti-rabbit secondary antibody were obtained from Cell Signaling Technology (MA, USA). Primary antibodies against cyclin B, cdk1/ cdc2, cdc25c, and goat-anti-mouse secondary antibody were from Millipore (NY, USA). Primary antibodies against poly (ADP-ribose)
polymerase (PARP), Fas, Fas-associated death domain protein (FADD), TNFR1-associated death domain protein (TRADD), and receptor interacting protein (RIP) were from BD Biosciences (USA). Selective inhibitors, U0126 for MAPK/ERK kinase (MEK), SP600125 for JNK, SB203580 for p38MAPK, were from Sigma (St.
Louis, MO) and all dissolved in DMSO for stock preparation. 2.2. Cell culture
Both BCa cell lines, HS-578T and MCF-7, were obtained from the Cell Bank of the National Health Research Institute, Taiwan. HS-578T cells were grown in DMEM with 10
l
g/ml bovine insulin and 10% FBS. MCF-7 cells were grown in MEM-Alpha containing 0.1 mM non-essential amino acids and 10% FBS. The media for both cell lines contained 50l
g/ml gentamicin. All cultured cells were maintained in a humid chamber at 37 °C under 95% humidified air/5% CO2atmosphere.2.3. Evaluation of antitumor activity by cytotoxicity assay
Antitumor activity of norcantharidin was evaluated using a microculture tetrazolium test (MTT) as previously described ( Phil-lips et al., 1990; Yang et al., 2010). Briefly, HS-578T and MCF-7 tu-mor cells seeded into a 96-well plate (5000 cells per well) were treated with or without norcantharidin in triplicate for 48 h of con-secutive incubation at 37 °C. Then 50
l
l MTT solution (Sigma) was added to each well. Following incubation for an additional 4 h at 37 °C, supernatants were removed and 100l
l DMSO was added to dissolve the MTT-formazan product. The plate was read using a microplate reader (Labsystems, Helsinki, Finland) at 550 nm. Cell inhibition at each concentration was converted to percentage of control levels set at 100% and the IC50 value for norcantharidinwas calculated.
2.4. Apoptotic cell death determination
Apoptosis-associated DNA fragmentation was determined by measuring the cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) using a Cell Death Detection ELISA Plus kit (Roche, Mannheim, Germany) as previ-ously described (Yang et al., 2010). Briefly, HS-578T and MCF-7 cells seeded into 96-well plates were treated with or without nor-cantharidin for 24 h. Then cell lysates were collected and trans-ferred to a streptavidin-coated plate supplied by the manufacturer. A mixture of anti-histone–biotine and anti-DNA-POD were added to cell lysates and incubated for 2 h. The complex was then conjugated to form an immune complex on the plate, which then was read for optical density at 405 nm by a microplate reader (Labsystems). The contents of mono-oligonucleosomes in lysates were calculated as absorbance of norcantharidin-treated cells/absorbance of untreated controls.
2.5. Cytochrome c assay
The cytochrome c ELISA kit (Assay Designs, Ann Arbor, USA) was used to determine the cytochrome c release from mitochondria in norcantharidin-treated cells, as previously described (Yang et al., 2010). Briefly, after treatment with norcantharidin, cells were col-lected and washed with PBS, resuspended in Digitonin cell perme-abilization buffer, and incubated for 5 min on ice. After centrifugation, supernatants were collected for the determination of cytochrome c in the cytosol. All samples were diluted and added into the cytochrome c 96-well plate. After incubation for 1 h at room temperature, the cytochrome c conjugate was added into each well and incubated for another 30 min. After the substrate solution was added to each well, the plate was incubated for
45 min at room temperature. The cytochrome c level was read at 405 nm by a microplate reader (Labsystems) after adding the stop solution.
2.6. Caspase activity assay
Caspase activities in the HS-578T and MCF-7 cells treated with or without norcantharidin were determined according to the man-ufacturer’s protocol (R&D systems, Minneapolis, MN, USA). Briefly, cell lysates (100
l
g total protein) were added to the reaction mix-tures (final volume of 50l
l) containing colorimetric substrate pep-tides specific for caspase-3 (DEVD-pNA), caspase-8 (IETD-pNA), or caspase-9 (LEHD-pNA). The reaction was performed at 37 °C for 2 h. Absorbance at 405 nm was determined with a microplate reader (Labsystems).2.7. Western blot analysis
The HS-578T and MCF-7 cells were cultivated with norcanthari-din for 24 h and then lysed in RIPA protein lysis buffer. Protein con-centration was determined using the BCA Protein Assay kit (Novagen, USA). Western blotting was performed as previously de-scribed (Yang et al., 2010). Equal amounts of sample lysate were resolved by 10% sodium dodecyl polyacrylamide gel electrophore-sis (SDS–PAGE), and transferred onto a nitrocellulose membrane. After blocking the membrane with 5% non-fat milk in TBST buffer (50 mM Tris–base, pH 7.4; 150 mM NaCl, 0.05% Tween-20) for 1 h at room temperature, the blots were incubated overnight at 4 °C with the diluted primary antibodies. After washing, the membrane was incubated with the appropriate secondary antibody (horserad-ish peroxidase conjugated goat-anti-mouse or goat-anti-rabbit IgG). Antibody binding was detected using an enhanced chemilu-minescence detection kit (Perkin-Elmer, USA). b-Actin antibody was used as an internal control.
2.8. Statistical analysis
All statistical analyses were performed using the Student’s t-test and ANOVA, with P < 0.05 considered as statistically signifi-cant. All P values were determined using two-tailed tests. 3. Results
3.1. Cytotoxicity of norcantharidin in BCa cells
To examine the cytotoxic effects of norcantharidin on estab-lished BCa cell lines, ER HS-578T and ER+ MCF-7 BCa tumor cells were selected for 48 h of treatment with norcantharidin. The dose– responsive survival curves were determined by MTT cytotoxicity assay (Fig. 1). The results indicate that norcantharidin significantly reduced viability of both lines of BCa tumor cells in a dose-depen-dent manner. The calculated IC50values for HS-578T and MCF-7
cells exposed to norcantharidin were 13.06 ± 1.27 and 18.27 ± 0.77
l
g/ml, respectively. The distinction between the IC50values of these two cells clearly indicates that ER HS-578T cells possess higher chemosensitivity to norcantharidin treatment than ER+ MCF-7 cells.
3.2. Biomodulatory effects of norcantharidin on cell cycle regulators in BCa cells
Although norcantharidin is known to induce cytostaticity in MDA-MB-231 BCa cells by G2/M arrest (Huang et al., 2009), not en-ough is known about the molecular mechanism by which it elicits G2/M growth arrest in BCa tumor cells. Since our preliminary data
had confirmed that norcantharidin also induced G2/M arrest of both HS-578T and MCF-7 BCa cells (data not shown), this study continued to delineate the expression profiles of cell cycle regula-tory proteins in both cell lines exposed to norcantharidin (Fig. 2). Western blotting was used to measure intracellular contents of cell cycle regulation proteins (cyclin B, cdc2 and cdc25c), phosphoryla-tion status of cell cycle checkpoint proteins (p53, Chk1 and Chk2) as well as levels of cyclin-dependent kinase inhibitors (p21Waf1/ Cip1
and p27Kip1). In HS-578T cells, norcantharidin treatment for 24 h increased phosphorylated levels of p53, Chk1, and Chk2 as well as total protein level of p21Waf1/Cip1, accompanied with
down-regulation of cyclin B, cdc2, and cdc25c; no marked change was noted in p27Kip1protein expression. In MCF-7 cells,
norcantha-ridin up-regulated phosphorylation of both Chk1 and Chk2, ele-vated p21Waf1/Cip1 protein contents, whereas it down-regulated
cyclin B and cdc25c levels. Besides, p53 phosphorylation as well as cdc2 and p27Kip1protein expression remained constant. These
0 20 40 60 80 100 120 0 20 40 60 80 100 120 Norcantharidin (µg/ml) Viability (%) HS-578T MCF-7
Fig. 1. Induction of cytotoxicity in BCa cell lines by norcantharidin. HS-578T and MCF-7 BCa cells were treated with norcantharidin at indicated concentrations for 48 h. The drug effect on cell viability was measured using an MTT assay. Data are representative results in three independent experiments.
p21Waf1/Cip1 cdc25c Cyclin B cdc2 p27Kip1 p-Chk2 p-Chk1 p-p53 ββ -Actin Norcantharidin (μ g/ml) Norcantharidin (μ g/ml) 0 3 10 30 0 3 10 30 HS-578T MCF-7
Fig. 2. Expression of cell cycle regulatory molecules in norcantharidin-treated BCa cell lines. HS-578T and MCF-7 BCa cells were cultured with norcantharidin at indicated concentrations for 24 h. Cellular protein extracts were resolved by Western blot analysis and probed with antibodies specific for either cell cycle checkpoint proteins (phosphor-p53, phosphor-Chk1, and phosphor-Chk2), cyclin-dependent kinase inhibitors (p27Kip1
and p21Cip1/Waf1
), or cell cycle regulatory proteins (cyclin B, cdc2, and cdc25c). Meanwhile, b-Actin was used as an internal loading control. Representative images from three independent experiments are shown.
findings strongly suggest that cyclin B and cdc25c play a pivotal role in norcantharidin-induced BCa cell cycle arrest.
3.3. Apoptotic induction in BCa cells by norcantharidin treatment Norcantharidin-induced apoptosis has been evidenced in many types of tumor cells and, more recently, in MDA-MB-231 BCa cells (Huang et al., 2009). To better characterize the mechanism under-lying the norcantharidin-induced apoptogenesis, HS-578T and MCF-7 BCa cells were treated with norcantharidin for 24 h and the lysates were used for detection of various apoptosis-associated parameters. Fragmentation of chromosomal DNA into oligonucleo-somes, which is one of characteristics of apoptosis, was dose-dependently induced by norcantharidin in both BCa cell lines (Fig. 3A). The normalized data indicated that 30
l
g/ml of norcant-haridin increased oligonucleosomal formation up to 14.73- and 3.63-folds in HS-578T and MCF-7 cells, respectively. Exposure of HS-578T and MCF-7 cells to various norcantharidin concentrations resulted in appearance of proteolytic cleavage of PARP, as revealed by Western blotting detection (Fig. 3B). Again, caspase activity as-say clearly indicated that norcantharidin resulted in dose-depen-dent increases in the activities of pro-apoptotic caspases, such as caspase-3, -8, and -9 in HS-578T (Fig. 3C) as well as caspase-8 and -9 in MCF-7 cells (Fig. 3D). Intriguingly, the undetectable cas-pase-3 activity in MCF-7 cells is consistent to previous report, mainly due to an exon 3 deletion in CASP-3 gene transcript and its functional deficiency (Jänicke et al., 1998). All evidence supports that norcantharidin potently induces apoptosis in BCa cells.3.4. Involvement of both extrinsic and intrinsic pathways in norcantharidin-induced BCa cell apoptosis
To clarify the pharmacologic action of norcantharidin on the apoptogenesis of BCa cells, we first explored the role of the extrin-sic membrane death receptor-mediated pathway in norcanthari-din-induced BCa cell apoptosis. Western blotting results indicated that norcantharidin treatment for 24 h up-regulated Fas in HS-578T cells, although expression of FADD, RIP and TRADD is slightly reduced by 30
l
g/ml of norcantharidin (Fig. 4). In compar-ison with the expression patterns of death receptor-associated pro-teins in HS-578T cells, norcantharidin did not alter Fas expression, slightly suppressed FADD content, whereas up-regulated RIP and TRADD levels in MCF-7 cells. These findings suggest that the death-receptor signaling may participate in norcantharidin-in-duced apoptogenesis, or at least be affected by drug treatment.Next, to determine whether intrinsic signaling activity is in-volved in norcantharidin-induced BCa cell apoptosis, the cytosolic level of cytochrome c, a key initiator of the intrinsic apoptotic path-way, was assessed. The results indicated that norcantharidin sig-nificantly increased the cytosolic cytochrome c level in both HS-578T and MCF-7 cells in a dose-dependent manner (Table 1). Since the intrinsic pathway hinges on the balance of activities between pro- and anti-apoptotic members of the Bcl-2 superfamily proteins, Western blotting analysis was utilized to acquire the expression profiles of Bcl-2 superfamily members in norcantharidin-treated cells (Fig. 5). The results showed that 24 h of incubation with nor-cantharidin up-regulated the expression of Bax, Bak, and Bad pro-teins in HS-578T cells, while it only increased that of Bax, Bad, and
MCF-7 0 0.5 1 1.5 2 2.5 0 3 10 30 Norcantharidin (µg/ml) Caspase activity (fold of control)
caspase 8 caspase 9
*
*
*
HS-578T 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 3 10 30 Norcantharidin (µg/ml) Caspase activity (fold of control)caspase 3 caspase 8 caspase 9
*
*
*
*
0 2 4 6 8 10 12 14 16 18 0 3 10 30 Norcantharidin (µg/ml) Enrichment of oligonucleosomes (fold of control) HS-578T MCF-7*
*
*
*
Norcantharidin (
μμ g/ml)
0 3 10 30
HS-578T MCF-7 PARP (116 kD) CleavedPARP (89 kD) PARP (116 kD) Cleaved PARP (89 kD)A
B
C
D
Fig. 3. Induction of apoptosis of BCa cell lines by norcantharidin. HS-578T and MCF-7 BCa cell lines were treated with norcantharidin at indicated concentrations for 24 h. Cellular lysates were collected for measuring apoptosis-associated parameters. (A) Dose-dependent induction of apoptotic oligonucleosome formation by norcantharidin was evidenced by using a commercially available ELISA kit. (B) Western blotting detection demonstrated that the cleavage of poly(ADP-ribose) polymerase (PARP) emerged in norcantharidin-treated HS-578T and MCF-7 cells. (C) and (D) Elevation of intracellular levels of caspases (caspase-3, -8, and -9) by norcantharidin was seen in HS-578T and MCF-7 cells, except undetectable caspase-3 activity in MCF-7. Data are presented as means ± SD from three independent experiments. indicates P < 0.05 compared with corresponding control levels.
p-Bcl-2 in MCF-7 cells (Fig. 5A). Conversely, norcantharidin in-duced down-regulation of Bcl-xL and Mcl-1 expressions in HS-578T cells as well as that of only Bcl-xL in MCF-7 cells (Fig. 5B). Of note, Bcl-2 expression in HS-578T was constitutively much low-er than that in MCF-7 cells, which was intriguingly up-regulated by norcantharidin.
3.5. Biomodulatory effect of norcantharidin on STATs in BCa cells To determine the dose-responsive effect of norcantharidin on STAT-mediated signaling activity, both total and phosphorylated protein levels of STATs, including STAT1, STAT3, and STAT5, in HS-578T and MCF-7 BCa cells with 24 h of treatment were exam-ined by Western blot (Fig. 6). The results clearly showed that nor-cantharidin did not affect total STAT1, STAT3, and STAT5 levels in both HS-578T and MCF-7 cells. Moreover, STAT1 phosphorylation level was constitutively low in HS-578T cells, whereas the consti-tutive STAT3 and STAT5 phosphorylation were prominently down-regulated by norcantharidin. By contrast, MCF-7 cells pos-sessed higher constitutive levels of phosphorylated STAT5 pro-teins, which could be further suppressed by norcantharidin. Conversely, the low constitutive level of STAT1 phosphorylation in MCF-7 cells was abruptly up-regulated by norcantharidin treatment.
3.6. Involvement of MAPK activities in norcantharidin-induced BCa cytotoxicity
Since the activities of MAPKs, including p38MAPK, ERK1/2, and
JNK, are known to mediate the norcantharidin-elicited apoptogen-esis in cancer cells (An et al., 2004a, 2005; Chen et al., 2003, 2008), their roles in the drug-induced apoptosis of breast tumor cells
re-main unclear. Therefore, the expression of total and phosphory-lated protein levels of MAPK family member proteins was monitored in both cell lines under exposure to norcantharidin. Norcantharidin at concentrations higher than 10
l
g/ml elevated phosphorylation levels of p38MAPKand JNK in both HS-578T andMCF-7 cells, while ERK1/2 phosphorylation was more sensitive and inducible at 3
l
g/ml therein (Fig. 7A and B). In addition, nor-cantharidin slightly reduced total JNK and p38MAPKprotein contentin MCF-7 cells. These results suggest that ERK1/2, JNK, and p38MAPK
may participate in norcantharidin-induced BCa cell apoptosis. To further determine whether phosphorylation of MAPK family members is involved in the norcantharidin-induced BCa cytotoxic-ity, a blockade of kinase activity kinase-specific inhibitors prior to drug treatment was performed to scrutinize the respective contri-bution of MAPK kinase to the norcantharidin-elicited BCa cytotox-icity. The data showed that pretreatment with U0126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor) as well as SB203580 (p38MAPK inhibitor), although partially but significantly, attenuated the nor-cantharidin-induced cell death of HS-578T and MCF-7 cells (Fig. 8). This finding confirms and strongly suggests that the activity of MAPK signaling pathway, indeed, plays an important role in gov-erning the drug-induced apoptogenesis in BCa cells.
4. Discussion
As current chemotherapeutic agents, such as anti-estrogens, taxanes and aromatase inhibitors, have limited effectiveness and many side effects in BCa patients (Marsh and McLeod, 2007),
iden-RIP FAAD TRADD Fas ββ -Actin Norcantharidin (μg/ml) Norcantharidin (μ g/ml) 0 3 10 30 0 3 10 30 HS-578T MCF-7
Fig. 4. Biomodulatory effect of norcantharidin on expression of death receptor-mediated extrinsic apoptotic pathway. HS-578T and MCF-7 BCa cells were treated with norcantharidin at indicated concentrations for 24 h. Cellular protein extracts were resolved by Western blot analysis and probed with antibodies specific for Fas, Fas-associated death domain (FADD), TNFR1-associated death domain (TRADD) and receptor-interacting protein (RIP). b-Actin was used as an internal loading control. Representative images from three independent experiments are shown.
Table 1
The norcantharidin-induced dose-dependent alteration in cytosolic cytochrome c (Cyt c) levels in HS-578T and MCF-7 cells.
Dose (lg/ ml) HS-578T MCF-7 Cyt c (lg/ 106cells) Ratios to control Cyt c (lg/ 106cells) Ratios to control 0 27.51 1 34.23 1 3 53.27 1.94 33.46 0.98 10 116.93 4.25 114.85 3.35 30 395.80 14.39 125.00 3.65
Cyt c in cytosolic fraction was determined in both cell lines treated with norcant-haridin for 24 h. Bim Bax Bak Bid Bad ββ -Actin
A
Norcantharidin (μg/ml) Norcantharidin (μg/ml) 0 3 10 30 0 3 10 30 HS-578T MCF-7 Bcl-2 Mcl-1 Bcl-xL β -ActinB
Norcantharidin (μg/ml) Norcantharidin (μg/ml) 0 3 10 30 0 3 10 30 HS-578T MCF-7 p-Bcl-2Fig. 5. Expression profiles of Bcl-2 superfamily members in norcantharidin-treated BCa cell lines. HS-578T and MCF-7 BCa cells were treated with norcantharidin at indicated concentrations for 24 h. Cellular protein extracts were subjected to Western blot analysis and probed with antibodies specific for pro-apoptosis member proteins (Bax, Bak, Bad, Bid, and Bim) (A) as well as with those against anti-apoptosis member proteins (Bcl-2, p-Bcl-2, Bcl-xL, and Mcl-1) (B). b-Actin was used as an internal loading control. Representative images from three independent experiments are shown.
tifying new agents effective for treating BCa with both ER status is important. This study evaluated the in vitro chemosensitivity of two immortalized BCa cell lines with opposing ER status after treatment with an anti-cancer drug, norcantharidin. The signifi-cantly different IC50values between ER+ and ER tumor cells in
re-sponse to norcantharidin treatment confirmed that norcantharidin is more potent in inhibiting ER than that ER+ BCa cells. In fact, other line of evidence derived from primarily isolated BCa cells also supports that the ex vivo chemosensitivity of ER– breast tumor cells was apparently higher than those ER+ cells (unpublished data), supporting again the crucial contribution of ER pathway to the resistance of cancer cells to chemotherapy.
The biomodulatory effect of norcantharidin on cell cycle pro-gression in BCa cells has recently been reported (Huang et al., 2009). Norcantharidin was demonstrated to arrest BCa cell growth at G2/M phase in a dose-dependent manner. This growth-arresting effect may be associated with the upstream regulator, ataxia-telan-giectasia mutated (ATM), in human leukemia Jurkat cells (Liao et al., 2007). Despite no alteration in Cdk1 and p27Kip1expressions
in human hepatoma cell lines, the enhancement of cdc25c and p21Waf1/Cip1phosphorylation, the reduction of cyclin B level and
ki-nase activity, as well as the decrease in p53 protein expression were all involved in norcantharidin-regulated M phase arrest (Chen et al., 2002). In human gallbladder carcinoma cells, norcant-haridin retarded cell growth through down-regulation of prolifera-tion-related genes, PCNA, Ki-67, and cyclin-D1, and through
up-regulation of p27Kip1(Fan et al., 2007). In this study, we
addition-ally found that norcantharidin decreased the expressions of cyclin B and cdc25c, increased the phosphorylated contents of Chk1, Chk2 and total p21Waf1/Cip, whereas did not affect p27Kip1expression in
norcantharidin-treated MCF-7 and HS-578T cells (Fig. 2). Notably, norcantharidin decreased cdc2 abundance in ER HS-578T, but not in ER+ MCF-7 cells. Since cdc2 is one of key kinase mediators for G2/M checkpoint (Taylor and Stark, 2001), the differential responsiveness of cdc2 down-regulation between ER and ER+ BCa cells may explain the higher chemosensitivity in ER cells. Ta-ken together, norcantharidin pharmacologically may be an effec-tive agent for inducing G2/M growth arrest in BCa cells.
Two major pathways that lead to apoptosis have been identi-fied, namely, the extrinsic death receptor pathway leading to cas-apse-8 activation and the intrinsic pathway that involves the release of cytochrome c from mitochondria and activation of cas-pase-9 (Hengartner, 2000). From the viewpoint of extrinsic path-way, we observed that norcantharidin up-regulated Fas/APO-1 expression in HS-578T cells and increased TRADD and RIP in MCF-7 cells (Fig. 4). Supportively, norcantharidin induces human colorectal cancer cell apoptosis by activating the CD95 receptor system and caspase-8 (Peng et al., 2002). In the light of intrinsic pathway, elevation of caspase-3 and -9 activities has been identi-fied in norcantharidin-treated melanoma and hepatoma cells (An et al., 2004a; Chen et al., 2002, 2003). The activities of all three caspases were similarly up-regulated in norcantharidin-treated
B
HS-578T 0 0.2 0.4 0.6 0.8 1 1.2 1.4 p-STAT3/STAT3 p-STAT5/STAT5 Ra ti o 0 µg/ml 3 µg/ml 10 µg/ml 30 µg/ml*
*
MCF-7 0 2 4 6 8 10 12 14 16 18 p-STAT1/STAT1 p-STAT5/STAT5 Ra ti o 0 µg/ml 3 µg/ml 10 µg/ml 30 µg/ml*
**
*
*
**
STAT3 p-STAT1 STAT1 p-STAT3 STAT5 p-STAT5 ββ -Actin Norcantharidin (μg/ml) Norcantharidin (μg/ml) 0 3 10 30 0 3 10 30 HS-578T MCF-7A
Fig. 6. Regulation of STATs in norcantharidin-treated BCa cell lines. HS-578T and MCF-7 BCa cells were cultured with norcantharidin at indicated concentrations for 24 h. Cellular protein extracts were resolved by Western blot analysis and probed with antibodies raised against either total or phosphorylated type of STAT1, STAT3, and STAT5. b-Actin was used as an internal loading control. Representative images (A) and densitometrical analysis data (B) from three independent experiments are shown.
HeLa cells (An et al., 2004a). In this study, induction of BCa cell apoptosis by norcantharidin was evidenced with dose-responsive cytochrome c release (Table 1) and up-regulation of caspase activ-ities in both HS-578T and MCF-7 cells (Fig. 3C and D), thereby lead-ing to proteolytic cleavage of downstream target PARP (Fig. 3B). The results strongly suggest that both extrinsic and intrinsic path-ways are involved in the norcantharidin-induce apoptosis.
With regard to the regulatory role of Bcl-2 superfamily mem-bers in norcantharidin-driven apoptogenesis, phosphorylation of Bcl-2 and Bcl-xL could be increased, while Bax or Bad expression were not affected in Hep-G2 cells (Chen et al., 2002). Similar to our results, the enhancement of Bax expression and the down-reg-ulation of Bcl-xL by norcantharidin have been noted in SAS cells (Kok et al., 2005). The pro-apoptogenic propensity of norcanthari-din in terms of decreased ratios of Bcl-2/Bax in A375-S2 (An et al., 2004b) and MDA-MB-231 cells (Huang et al., 2009) has also been documented. To support this, our data addressed further evidence showing that norcantharidin induced up-regulation of pro-apopto-tic Bad, Bak, or Bax proteins, but down-regulation of anti-apoptopro-apopto-tic Bcl-xL and Mcl-1 expressions in HS-578T and MCF-7 cells (Fig. 5). Consistent with previous findings (An et al., 2004b; Kok et al., 2005; Huang et al., 2009), although norcantharidin up-regulated
Bcl-2 expression in MCF-7 cells, it decreased the ratio of anti-apop-totic to pro-apopanti-apop-totic Bcl-2 family protein in HS-578T and MCF-7 cells. Interestingly, the lower constitutive level of Bcl-2 in ER-neg-ative HS-578T cells may account for the higher chemosensitivity of tumor cells to norcantharidin treatment, as Bcl-2-targeted therapy increases the apoptotic response to chemotherapy (Emi et al., 2005; Yin et al., 2004). Conversely, ER+ MCF-7 cells possess not only higher constitutive level of Bcl-2, but also an integral respon-siveness to the insult from anti-cancer drugs. The regulatory role of ER-mediated pathway in the constitutive Bcl-2 expression and rel-evant chemosensitivity in BCa cells is suggested by various reports (Tabuchi et al., 2009; LaPennsee and Ben-Jonathan, 2010). Estro-gens affected both increasing cell growth and reducing cell death through activation of PI3K/Akt survival signals and Bcl-2 protein in ER+ BCa cells (Rodrik et al., 2005). Moreover, ER affected resis-tance to chemotherapeutic drugs by regulating Bcl-2 expression. In addition, post-translational modification on Bcl-2, such as mul-ti-site phosphorylation, has been known to alter cellular chemo-sensitivity and regulates apoptosis. Since the Bcl-2 phosphorylation at serine 70 has been demonstrated to result in a loss of the binding ability of Bcl-2 to Bax and subsequent cell death (Shitashige et al., 2001), it may, at least in part,
mechanisti-B
MCF-7 0 10 20 30 40 50 60 70 80 p-JNK/JNK(p54) p-JNK/JNK(p46) p-p38/p38 p-ERK/ERK Rat io 0 µg/ml 3 µg/ml 10 µg/ml 30 µg/ml * * ** * ** ** * ** * * ** HS-578T 0 20 40 60 80 100 120 140 p-JNK/JNK(p54) p-JNK/JNK(p46) p-p38/p38 p-ERK/ERK Ra ti o 0 µg/ml 3 µg/ml 10 µg/ml 30 µg/ml * * * ** ** ** * * * * p-JNK p-p38 p-ERK JNK p38 ERK ββ -Actin Norcantharidin (μg/ml) Norcantharidin (μg/ml) 0 3 10 30 0 3 10 30 HS-578T MCF-7A
Fig. 7. Involvement of MAPK activation in norcantharidin-induced BCa cytotoxicity. HS-578T and MCF-7 cells were incubated with norcantharidin at indicated concentrations for 24 h. Cellular protein extracts were resolved by Western blot analysis and probed with antibodies raised against either total or phosphorylated type of JNK, p38MAPK
, and ERKs. b-Actin was used as an internal loading control. Representative images (A) and densitometrical analysis data (B) from three independent experiments are shown.
cally explain the norcantharidin-triggered up-regulation of Bcl-2 protein expression and its phosphorylation at serine 70. Taken to-gether, our findings support that norcantharidin induces apoptosis mainly through modulating the expression and the interaction among Bcl-2-related proteins in BCa cells.
The STAT proteins are a family of transcription factors that reg-ulate cell proliferation, differentiation, survival, and apoptosis ( Bat-tle and Frank, 2002; Bromberg, 2001). Accumulating clinical evidence has pointed out the aberrant activities of STAT1, STAT3, and STAT5 in a diversity of tumors, including breast cancers. Among the STAT family members, STAT1 is generally thought to promote apoptosis and tumor suppression (Kim and Lee, 2007), while STAT3 and STAT5 modulate cell growth and inhibit apoptosis (Silva, 2004). Accordingly, STAT3 and STAT5 play important and distinct roles in mammary development and are activated in breast cancer in a manner of reciprocal regulation. Analysis of the activa-tion state of both STAT3 and STAT5 has been thought to provide important diagnostic and prognostic information in breast cancer (Walker et al., 2009). Conversely, targeting constitutive STAT3 sig-naling is thus an attractive therapeutic approach for cancers ( Kun-igal et al., 2009). In terms of the role that STAT1 plays in norcantharidin-associated apoptogenesis, it was found that STAT1 protein directly interacts with TNFR1 and TRADD in HeLa cells (Wang et al., 2000) and that norcantharidin induces STAT1 nuclear translocation, thereby initiating Jurkat and CT26 cell apoptosis (Chen et al., 2009a; Liao et al., 2007). The norcantharidin-elicited STAT1 phosphorylation (Fig. 6) and TRADD protein up-regulation (Fig. 4) seen in MCF-7 cells may fortify the concept that norcantha-ridin possesses anti-cancer potency through activating extrinsic apoptotic pathway in tumor cells. Furthermore, Kunigal et al. (2009)demonstrated that siRNA-mediated STAT3 knockdown re-duced expression of Bcl-xL and survivin in MDA-MB-231 cells, and induced the Fas-mediated extrinsic apoptotic pathway by acti-vating caspases-3, -8, and -9 and PARP1 cleavage. The norcanthari-din increased Fas protein level (Fig. 4) and the attenuated STAT3 phosphorylation (Fig. 6) found in HS-578T cells, again,
demon-strated the involvement of extrinsic pathway. However, the mech-anisms through which STATs mediate BCa cell apoptosis remain further elucidation.
The importance of MAPK-associated signaling activity in nor-cantharidin-induced apoptosis of various cancer cells has been widely addressed (An et al., 2004a, 2005; Chen et al., 2003, 2008). Although inhibition of Akt and NF-
j
B signaling has recently been implicated in the norcantharidin-induced apoptosis of human BCa cells (Huang et al., 2009), to our knowledge this is the first evi-dence showing that activities of ERK, JNK and p38MAPKcontributeto norcantharidin-induced death of HS-578T and MCF-7 cells (Figs. 7 and 8). Given that the STAT1 phosphorylation has been suggested to be mediated by MAPKs (Kim and Lee, 2007; Wen et al., 1995), the coincidental dose-responsive profiles of MAPKs and STATs raised the possibility that the cross-talking effect be-tween MAPK and STAT signaling pathways may be responsible for the norcantharidin-induced BCa cell apoptosis. Nevertheless, it remains to be determined whether p38MAPKphosphorylation
in-duced by norcantharidin treatment contributes to STAT1 phos-phorylation and/or STAT1-mediated pro-apoptotic effect in BCa cells.
In conclusion, norcantharidin may disturb cell cycle distribution of BCa cells through p53- and Chk-related pathways. Alternatively, it may induce BCa cell apoptosis via both extrinsic and intrinsic pathways. The MAPK- and STAT-mediated signaling pathways are mechanistically involved in the apoptogenesis of BCa cells trig-gered by norcantharidin. Therefore, norcantharidin is suggested as a chemotherapeutic agent for treating BCa.
5. Conflict of interest statement None declared.
Acknowledgements
This study was financially supported by the grants from Show Chwan Memorial Hospital, Taiwan, ROC (Grant No. MI97003 and MI99002). The authors would like to thank Dr. Yang SF for his tech-nical assistance and Mr. Ted Knoy for his editorial assistance. References
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# #Fig. 8. Attenuation of norcantharidin-induced BCa cytotoxicity by blockage of kinase activity. HS-578T cells cultivated in a 96-well plate were treated with either 0.1% DMSO as solvent control or the selective kinase inhibitors, including 20lM U0126 for MAPK/ERK kinase (ERK), 20lM SP600125 for c-Jun N-terminal kinase (JNK), or 20lM SB203580 for p38MAPK, at 2 h before adding 20 and 30lg/ml norcantharidin in HS-578T and MCF-7 cells, respectively. After being treated with norcantharidin 24 h, the drug-driven cytotoxicity was evaluated by MTT assay. Data are presented as mean ± SD.#
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