European Journal of Pharmacology 723 (2014) 148–155
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Induction of apoptosis in cholangiocarcinoma by an andrographolide analogue is mediated through topoisomerase II alpha inhibition Jintapat Nateewattana a,b, Suman Dutta b, Somrudee Reabroi b, Rungnapha Saeeng c, Sakkasem Kasemsook c, Arthit Chairoungdua b, Jittima Weerachayaphorn b, Sopit Wongkham d, Pawinee Piyachaturawat a,b,n a
Toxicology Graduate Program, Faculty of Science, Mahidol University, Bangkok, Thailand Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand c Department of Chemistry, Faculty of Science, Burapha University, Chonburi, Thailand d Department of Biochemistry, Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 June 2013 Received in revised form 4 December 2013 Accepted 4 December 2013 Available online 17 December 2013
Cholangiocarcinoma (CCA), the common primary malignant tumor of bile duct epithelial cells, is unresponsive to most chemotherapeutic drugs. Diagnosis with CCA has a poor prognosis, and therefore urgently requires effective therapeutic agents. In the present study we investigated anti-cancer effects of andrographolide analogue 3A.1 (19-tert-butyldiphenylsilyl-8, 17-epoxy andrographolide) and its mechanism in human CCA cell line KKU-M213 derived from a Thai CCA patient. By 24 h after exposure, the analogue 3A.1 exhibited a potent cytotoxic effect on KKU-M213 cells with an inhibition concentration 50 (IC50) of approximately 8.0 mM. Analogue 3A.1 suppressed DNA topoisomerase II α (Topo II α) protein expression, arrested the cell cycle at sub G0/G1 phase, induced cleavage of DNA repair protein poly (ADP-ribose) polymerases-1 (PARP-1), and enhanced expression of tumor suppressor protein p53 and pro-apoptotic protein Bax. In addition, analogue 3A.1 induced caspase 3 activity and inhibited cyclin D1, CDK6, and COX-2 protein expression. These results suggest that andrographolide analogue 3A.1, a novel topo II inhibitor, has significant potential to be developed as a new anticancer agent for the treatment of CCA. & 2013 Elsevier B.V. All rights reserved.
Keywords: Andrographolide analogue Apoptosis Cholangiocarcinoma KKU-M213 cell line Topoisomerase II α
1. Introduction Cholangiocarcinoma (CCA), the major liver cancer in Southeast Asia (Sripa and Pairojkul, 2008; Songserm et al., 2011), is the most common primary malignant tumor originating from bile duct epithelial cells and is characterized by poor prognosis and unresponsiveness to chemotherapeutic agents (De Groen et al., 1999; Aravindaram and Yang, 2010). Risk for CCA in Thailand is mainly associated with infection by liver fluke (Opisthorchis viverrini) (Srivatanakul et al., 1991). Long term liver fluke infection induces chronic injury and inflammation of the biliary epithelium. Currently, there is no specific treatment for CCA (Diandra, 2008; Judith et al., 2010) and surgical resection only extends lifespan for up to 5 years (Aravindaram and Yang, 2010). Therefore, an alternative treatment with high sensitivity to specific targets is urgently needed. DNA topoisomerases are a group of enzymes involved in the topology of DNA during critical cellular processes including n Corresponding author at: Department of Physiology, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400, Thailand. Tel./Fax : þ 66 02 3547154. E-mail addresses: [email protected], [email protected] (P. Piyachaturawat).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.12.002
replication, recombination, transcription, and repair systems by transiently breaking one or two strands of DNA (Bodley and Liu 1998; Li et al., 2007; Schneider et al., 1990). Inhibition of Topo II α induces DNA damage (Soubeyrand et al., 2010), which then activates expression of the repair system protein PARP-1 (Lori, 2009). DNA damage induces p53 which regulates the cell cycle via cyclin D1 and leads to inhibition of the DNA replication process (Wanitchakool et al., 2012). An increase in p53 expression following unrepaired DNA damage induces Bax activation, which generally triggers apoptotic cell death via activation of caspase 3 (Valkov and Sullivan, 2003). Therefore, DNA topoisomerase inhibitors have been suggested as the most favorable anticancer agents (Bodley and Liu 1998; Li et al., 2007; Schneider et al., 1990). Andrographolide is the major diterpenoid lactone from Andrographis paniculata (Burm.f.) Nees (Acanthaceae) (Thisoda et al., 2006). This naturally occurring compound has anticancer activity in many cancer cell types including Jurkat (lymphocytic), PC-3 (prostate), HepG2 (hepatoma) and Colon 205 cells (Geethangili et al., 2008). A number of semi-synthetic compounds from herbs have been shown striking anticancer activities in both potency and efficacy in various cancer cells (Jada et al., 2006; Nanduri et al., 2004; Sirion et al., 2011). Recently, various andrographolide analogues have been developed
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and their modes of action are being evaluated to find a better regimen for cancer therapy (Jada et al., 2008; Satyanarayana et al., 2004). However, none of these andrographolide analogues have been shown to have Topo II α inhibitory activity. Interestingly, andrographolide analogue 3A.1 has shown Topo II alpha inhibitory activity in an in vitro cell-free system. (Nateewattana et al., 2013). In the present study, we report Topo II α inhibitory activity of andrographolide analogue 3A.1 and the underlying anticancer mechanism in CCA KKU-M213 cells derived from a Thai CCA patient.
2. Materials and methods 2.1. Materials Ham F12 medium and antibiotic–antimycotic agents were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS), RIPA, proteinase inhibitor and SuperSignal West Pico Chemiluminescent Substrate were purchased from Thermoscientific (Cramlington, UK). SDS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), vinblastine, etoposide and propidium iodide (PI) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Annexin-V FITC apoptosis kit was obtained from BD bioscience, USA. CaspaseACE™ colorimetric assay kit and zVADfmk (all caspase inhibitor) were obtained from Promega (Madison, WI, USA). Diamidine-2-phenylindole dihydrochloride (DAPI) was purchased from Roche Ltd (Mannheim, Germany). Doxorubicin was purchased from Guanyu Bio-Tech Co. (Xijan, Republic of China). Cleaved PARP-1, P53, Bax, cyclin D1, CDK-6 and Topo IIα antibodies were procured from Cell Signaling Technology, Inc (Danvers, MA, USA). Cox activity assay kit was purchased from Cayman (Ellsworth, MI, USA). All other chemicals unless otherwise stated were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2.2. Andrographolide analogue 3A.1 Andrographolide was isolated from dried aerial part and root of A. paniculata (Acanthaceae) and andrographolide analogue 3A.1 were prepared as previously described (Nateewattana et al., 2013). The chemical structure is shown in Fig. 1. Purity of the compound was assessed by NMR, IR, and ICPMS spectroscopy and was approximately 99%. 2.3. Cell lines and culture conditions CCA cell line KKU-M213 (adenosquamous carcinoma) derived from a Thai patient, was obtained from Prof. Dr. Banjob Sripa, Faculty of Medicine, Khon Kaen University, Thailand. KKU-M213 cells were cultured in Ham F-12 medium supplemented with 10% (v/v) heat-inactivated FBS, and 1% antibiotic–antimycotic. Cultures were maintained at 37 1C in a CO2 incubator with controlled humidified atmosphere of 95% air and 5% CO2. 2.4. Cytotoxicity assay Cytotoxic activities of andrographolide, andrographolide analogue 3A.1, etoposide, doxorubicin and vinblastine on KKU-M213 cells were assessed by MTT assay. Two hundred microlitre of 3 104 cells/ml was seeded into 96-well micro-plates and routinely cultured. After 24 h, cells were exposed to the test compounds at concentrations ranging from 0 to 50 mM in DMSO for another 24 h. The final concentration of DMSO in the medium was less than 0.1% (v/v). The medium was removed and cells were washed with phosphate buffered saline (PBS). Then fresh medium containing 0.5 mg/ml MTT dye was added to each well and
Fig. 1. Structure and cytotoxicity of 3A.1. (A) Structure of andrographolide analogue 19-tert-butyldiphenylsilyl-8, 17-epoxy andrographolide (3A.1). (B) KKU M-213 cells were treated with parent andrographolide, analogue 3A.1, etoposide, doxorubicin, and vinblastine for 24 h, and cytotoxicity was evaluated by MTT assay. Each value is mean7 S.E.M from three independent experiments in triplicate.
incubated for an additional 4 h, followed by the addition of 100 μl of DMSO to dissolve the formozan crystals. The plate was analyzed on a microplate reader (Fluostar optima microplate reader, GmbH, Germany) at 540 nm. The inhibition concentration 50 (IC50) values were estimated using GraphPad Prism version 5.01 (GraphPad, Inc, USA). 2.5. Cell cycle analysis Cells were treated with analogue 3A.1 or etoposide and stained with propidium iodide (PI). The proportions of cells at different phases of the cell cycle were studied by flow cytometry. Briefly, 3 104 cells/ml were incubated in 6-well plates for 24 h, after which cells were exposed to the test compounds; analogue 3A.1, etoposide or vinblastine for another 12 h. Cells were collected, washed with PBS, and stained with 50 μg/ml PI for 15 min. Stained cells were analyzed by BD FACS Canto Flow Cytometer (Becton Dickinson, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 620 nm. The data were acquired using the BD CellQuest Pro software on Macs OS 9. Cells were then analyzed with Cell Quest Pro or FlowJo software. For each sample, twentyfive thousand events were collected. Cellular aggregates and debris were excluded from analysis by proper gating. Data were fit to define the G1, S, and G2/M phases by using the Dean–Jett– Fox two population mathematical model of the FlowJo software. 2.6. Analyses of apoptosis and caspase 3 activity To observe nuclear morphology, KKU-M213 cells (3 104 cells/ml) were seeded in 6-well plates and incubated with analogue 3A.1, vinblastine or etoposide for 12 h. The medium was subsequently
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discarded and gently washed with PBS followed by DAPI staining (5 μg/ml) for 20 min at 37 1C and observed under a fluorescence microscope HB-10101AF (Nikon, Japan) and photographed. A total of 500 stained cells/treatment was counted and apoptotic cells were determined by DNA condensation based on fluorescence intensity. Apoptosis was also analyzed by annexin V-FITC and PI staining. KKU-M213 cells were seeded in 6-well plates and incubated with analogue 3A.1, vinblastine or etoposide for 24 h. The medium was discarded and cells were washed with PBS followed by gentle pipetting to detach cells. Cells were then co-incubated with annexin-V and PI for 15 min at room temperature (25 1C) in the dark followed by analysis in BD FACScanto using FACS diva software (BD Bioscience, USA). The activity of caspase 3 was determined using CaspaseACE™ Colorimetric Assay System (Promega, Madison, USA), following the manufacturer's instructions. Briefly, the treated cells were harvested by trypsinization and centrifugation. The pellets were suspended in lysis buffer and lysed by three cycles of freezing and thawing. After centrifugation at 15,000g for 20 min at 4 1C, the supernatant was collected. The caspase 3 activity was measured using a specific substrate and inhibitor in a 96-well assay plate. The reaction was measured at an absorbance wavelength of 405 nm in FLUOstar OPTIMA microplate reader (BMG Labtech GmbH, Germany). To determine whether apoptotic cell death was caspase dependent or independent, an MTT assay with or without an all caspase inhibitor (zVAD-fmk) was performed. Briefly, 3 104 cells/ml were seeded into 96-well microplates and then exposed to analogue 3A.1 (8 μM) with or without zVAD-fmk at a concentration of 10 mM for 24 h, followed by the MTT assay.
2.8. Western blot analysis The proteins involved in cell death induced by analogue 3A.1 were detected by western blot analysis. The expression of Topo II α, p53, PARP-1, Bax, CDK-6, and cyclin D1 proteins were investigated. Briefly, cells (1 106 cells/ml) were seeded in 6-well plates and treated with analogue 3A.1, vinblastine or etposide for a specified period. The treated cells were washed with PBS twice. RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM NaF, 1 mM PMSF, and cocktail protease inhibitor) was then added in a suitable volume. After centrifugation at 14,000 rpm for 30 min at 4 1C, supernatants were collected and concentrations of protein in the supernatants were determined by bicinchoninic acid biuret (BCA) assay. Proteins were separated using 10% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat dried milk, incubated with primary antibodies overnight at 4 1C, and then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein was visualized using the SuperSignals Enhanced Chemiluminescence (ECL) Western blotting detection kit. 2.9. Statistical analyses All data are expressed as means 7the standard error of the mean (S.E.M.). They were analyzed by one-way analysis of variance (ANOVA). Differences among the treatment groups were assessed by Duncan's multiple-range test, using GraphPad Prism version 5.01 (GraphPad Software Inc, CA, and USA). Differences were considered significant at P o0.05.
2.7. COX activity assay
3. Results
The activity of COX was determined using a COX activity assay (Cayman, Ellsworth, USA). The treated cells were harvested and cell lysates were prepared. COX activity was measured using specific a substrate and inhibitor in a 96-well plate assay provided with the kit. The reaction was measured at an absorbance wavelength of 590 nm in a FLUOstar OPTIMA microplate reader (BMG Labtech GmbH, Germany).
3.1. Cytotoxic effects of andrographolide and its analogue on KKU-M213 cells The cytotoxic effects of andrographolide and its analogue 3A.1 on human CCA cells were evaluated by MTT assay. The parent andrographolide compound (50 mM) displays weak cytotoxicity to KKU-M213 cells whereas analogue 3A.1 was highly toxic to the cells
Fig. 2. Inhibitory effect of analogue 3A.1 on Topo II α activity in KKU-M213 cells after exposure to 8 mM analogue 3A.1 for (A) 4 h; (B) 6 h. Total protein (100 μg) was electrophoresed and transferred onto nitrocellulose membranes. The membranes were probed with anti-rabbit Topo II α and anti-mouse β actin antibodies. Topo II α was recognized as the expression of the 190 kDa protein. β actin (42 kDa) was used as an internal loading control. Experiments were repeated three times. *P o0.01, significantly different from control.
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Fig. 3. Analysis of cell cycle phases, (A) DNA content histogram showing distribution of KKU-M213 cells at various phases of cell cycle after treatment with vinblastine (20 mM), etoposide (20 mM), and analogue 3A.1. (8 mM) for 12 h. (B) Effect of analogue 3A.1 on CDK6 and cyclin D1 protein expressions 6 h after exposure to analogue 3A.1. Total protein (100 μg) was electrophoresed and transferred onto nitrocellulose membranes. The membranes were probed with anti-rabbit cyclin D1, anti-mouse CDK6, and anti-mouse β actin antibodies. CDK6 was recognized as the expression of the (30 kDa) band and cyclin D1 was recognized as the expression of the (36 kDa) band. β actin (42 kDa) was used as an internal loading control. Experiments were repeated three times.
Fig. 4. Analysis of chromatin condensation and DNA fragmentation in KKU-M213. (A). Changes in nuclear morphology examined by DAPI staining of condensed nuclear chromatin with a fluorescence microscope at a magnification of 400 . (B). Graphical representation of the number of condensed DAPI stained nuclei and fluorescence intensity analyzed using Image J software. nPo 0.01, significantly different from vehicle control.
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having an IC50 of 8.0þ0.1 mM at 24 h (Fig. 1). Etoposide, vinblastine and doxorubicin, which were used as positive controls, had IC50 values of 27.770.1, 6.370.02, and 4.6370.06 mM, respectively. This result also indicates that the potency of the analogue 3A.1 is markedly increased compared to the parent compound. Thereafter, analogue 3A.1 at a dose of 8 mM was chosen to further investigate the mechanism of action of anticancer activity.
3.2. Inhibition of topoisomerase II
α in KKU-M213 cells
At various time points after treatment with analogue 3A.1 at 8 mM, the expression of Topo II α in KKU-M213 cells was evaluated by Western blot. In Fig. 2(A), analogue 3A.1 exhibits dose- and time-dependent decreases in the expression of Topo II α protein (190 kDa) compared to control. Topo II α expression at 4 h after incubation was markedly inhibited, and further decreased at 6 h after incubation (Fig. 2B). Vinblastine and etoposide, two welldefined anticancer drugs, were used as positive controls. Vinblastine (20 mM) did not affect Topo II α in KKU-M213 cells at 4 h, but a slight inhibition was observed at 6 h. Etoposide (20 mM), a well-known topoisomerase poison (Soubeyrand et al., 2010), had less inhibitory effect at 4 h compared to the analogue 3A.1. However, etoposide completely inhibited Topo II α at 6 h after incubation. In contrast,
the parent andrographolide only slightly inhibited the Topo II α level even at 6 h. Therefore, the analogue 3A.1 showed promising Topo II α inhibitory activity. 3.3. Inhibition of cell cycle in KKU-M213 cells The proportion of DNA in KKU-M213 cells at different stages of the cell cycle (sub G0, G1, S and G2) was analyzed by flow cytometer after treatment with analogue 3A.1, vinblastine or etoposide. At 12 h after exposure, vinblastine did not significantly alter the phase distribution of the cell cycle in KKU-M213 cells compared to DMSO treated controls whereas the analogue 3A.1 increased the percentage of cells in the sub G0 phase. The percent distribution of cells at sub G0 phase and G2/M phase were increased from 2.5 7 0.0% to 11.570.2% and from 14.1 70.02 to 23.87 0.01, respectively. Etoposide, a potent topo II inhibitor, also arrested 19.5 7 0.02% of cells at sub G0 phase and 34.3 70.01% of cells at G2/M phase (Fig. 3A). The expressions of cyclin-dependent kinase 6 (CDK6) and cyclin D1, which regulate cell cycle progression and the transition from G1 to S phase, were markedly decreased by analogue 3A.1 in KKU-M213 cells at 6 h after incubation. This result suggests that the analogue 3A.1 indeed induced DNA damage, which in turn led
Fig. 5. Analogue 3A.1 induced apoptosis and activated caspase 3 activity. (A) Cells were treated with analogue 3A.1, and positive controls were then subjected to fluorescence activated cell sorting on a BD FACS Canto machine. Data were analyzed using FACS Diva software. The quadrants represent the percentage of live/apoptotic cells. (B) Caspase 3 activity was measured using specific a substrate and inhibitor in a 96-well assay plate. The reaction was measured absorbance at 405 nm in an ELISA plate reader. (C) KKUM213 cells were treated with 8 mM analogue 3A.1 in the presence or absence of 10 mM all caspase inhibitor (zVAD-fmk). Data are means 7 S.E.M. from two independent experiments which were conducted in triplicates. nP o0.05; significantly different from the vehicle control.
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to cell cycle arrest. Etoposide treatment also decreased cyclin D1 and CDK6 expressions at 6 h after exposure (Fig. 3B). 3.4. Analysis of DNA condensation in KKU-M213 cells The effect of analogue 3A.1 on apoptotic cell death was determined by nuclear morphology using DAPI staining, which is characterized by nuclear condensation and formation of apoptotic bodies. Vinblastine and etoposide were used as positive controls. Vinblastine induced DNA condensation and fragmentation in approximately 75% of the treated cells whereas etoposide induced this effect in only 27% of treated cells as depicted by the fluorescence intensity analysis of apoptotic nuclei using image J software. Analogue 3A.1 markedly induced DAPI staining of condensed chromatin by about 68% (Fig. 4A and B). 3.5. Induction of apoptosis and enhancement of caspase 3 activity in KKU-M213 cells To further examine the apoptotic death process, phosphatidylserine externalization on the outer membrane of cells was assayed by staining with annexin V-FITC and using flow cytometry. As shown in Fig. 5A, treatment with analogue 3A.1 (8 mM) induced apoptosis up to 49.3% of KKU-M213 cells compared to DMSO control (0.9%). Etoposide (20 mM) and vinblastine (20 mM) treatments induced apoptosis in approximately 26.3% and 68.1% of cells, respectively. Analogue 3A.1 also showed a time-dependent increase in caspase 3 activity. At 6 h the activity was not different from control, but activity was significantly increased from 1.070.30 pmol/ml to 470.11 pmol/ml and 6.0970.22 pmol/ml at 12, and 24 h, respectively (Fig. 5B). This effect was abolished by 10 mM zVADfmk (all caspase inhibitor) as the cell viability was increased to the control level (Fig. 5C). This result suggests that the analogue 3A.1-induced cell death is mediated through a caspase 3-dependent pathway.
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is a hallmark of inflammation, and induction of COX-2 is involved in metastasis of cancer (Rattanasinganchan et al., 2006). As shown in Fig. 6, DuP-697 (0.3 mM), a specific COX-2 inhibitor, potently inhibits COX activity whereas SC-560 (0.33 mM), a specific COX-1 inhibitor, has no detectable effect. Analogue 3A.1 (8 mM) significantly inhibited COX2 activity at 24 h after exposure. The analogue 3A.1 decreased COX-2 activity from 17.370.2 pmol/μl to 15.2 7 0.2, and 13.3 70.2 pmol/μl at 12 h and 24 h, respectively. This result suggests that the analogue 3A.1 may also possess antiinflammatory activity. 3.7. Effects of analogue 3A.1 on proteins involved in apoptosis A number of proteins that participate in the apoptosis pathway, particularly those related to inhibition of Topo II, were determined by western blot. At 6 h after exposure, analogue 3A.1 induced cleavage of the PARP-1 protein, which is a hallmark of apoptosis. The analogue 3A.1 also increased expression of p53, a nuclear tumor suppressor protein, in KKU-M213 cells. In addition, expression of BAX protein, which is under the control of p53, was also significantly enhanced after treatment with analogue 3A.1 (Fig. 7), suggesting that apoptosis was induced via a Bax-caspase 3-dependent pathway.
4. Discussion The present study demonstrated the underlying anticancer mechanism of the andrographolide analogue 3A.1 in an aggressive CCA cell line isolated from a Thai CCA patient. Analogue 3A.1, but
3.6. Inhibition of COX-2 activity in KKU-M213 cells Cyclooxygenase (COX) is a bifunctional enzyme exhibiting both cyclooxygenase and peroxidase activities. Increased COX 2 activity
Fig. 6. Analogue 3A.1 inhibits COX-2 activity. (A) KKU-M213 cells were treated with analogues 3A.1 and COX activity was measured at various time points. Each value represents mean 7 S.E.M. obtained from three independent experiments which were conducted in triplicate. nPo 0.05; significantly different from the vehicle control.
Fig. 7. Effect of analogue 3A.1 on proteins in the apoptosis pathway; PARP-1, P53, and BAX in KKU-M213 cells. Cells were treated with analogue 3A.1 and expression of different proteins was measured by western blot. PARP-1 protein was recognized as the expression of the cleaved fragment (85 kDa), p53 expressed as the 53 kDa fragment, and BAX protein expressed as the 20 kDa fragment. Total proteins (100 μg) were electrophoresed and transferred onto nitrocellulose membranes. The membranes were probed with antibodies. β actin (42 kDa) was used as an internal loading control. Experiments were repeated three times.
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not its parent compound, potently inhibited KKU-M213 cell proliferation, arrested the cell cycle, suppressed Topo II α protein expression, and induced apoptosis. The inhibitory potency of the analogue 3A.1 on Topo II α at 4 h was comparable to that of etoposide and greater than that of vinblastine, a current therapeutic drug for cancer. To our knowledge, this is the first report showing an inhibition of the Topo II α enzyme in a CCA cell line by an andrographolide analogue that has shown distinct activity from its parent compound. The anti-proliferative effect of analogue 3A.1 on KKU-M213 cells was associated with inhibition of Topo II α enzyme activity and cell cycle arrest which eventually induced apoptosis. Treatment with analogue 3A.1 increased the percentage of cells in sub G0 and G2/M phase (Fig. 3A). This effect was consistent with the expression of p53, which is a nuclear tumor suppressor protein controlling the cell cycle check points at the G1 to S and G2 to M phase (Hainaut, 1995). Topo II α is essential for cell division and is highly produced in S and G2/M phases (Chikamori et al., 2010). Therefore, inhibition of Topo II α arrests the cell cycle at the S and G2/M phases (Li et al., 2007). After DNA damage, p53 protein inhibits TATA box binding protein (TBP) to cause a halt in DNA transcription and suppresses replication protein A (RPA), which inhibits cellular replication (Hainaut, 1995). Indeed, we found that the analogue 3A.1 increased the expression of p53 and down regulated expression of cyclin D1 and CDK6. Apoptosis may be induced by several possible mechanisms (Kerr et al., 1972, and Huang et al., 2004). An inhibition of Topo II α induces DNA damage, which activates expression of repair system proteins including PARP-1 (Valkov and Sullivan, 2003). In the present study, the analogue 3A.1 inhibited expression of Topo II α and increased the cleavage of PARP-1, suggesting that this pathway may be attributed to the apoptosis of KKU-M213 cells. DNA damage also triggers expression of p53, which controls the cell cycle and enhances BAX activation. BAX, which is a member of the intrinsic apoptotic Bcl-2 family of proteins (Yuan and Yankner, 2000) induces apoptosis by producing pores in the mitochondrial membrane (Wanitchakool et al., 2012) from which cell death mediators such as Cytochrome C, Smac/DIABLO and AIF are released into cytosol leading to activation of caspase 9 (Sperandio et al., 2000). Active caspase 9 then activates caspase 3 leading to DNA fragmentation and apoptosis. In the present study, the expression of BAX in KKUM213 cells was substantially increased after treatment with analogue 3A.1. In addition, apoptosis of this cell line was associated with an increase in caspase 3 activity. Overall, our results strongly suggest that the anti-proliferative effect and induction of apoptosis in KKU-M213 cells by analogue 3A.1 are resulted from the inhibition of Topo II α, leading to DNA damage and activation of p53 and PARP1 proteins. Andrographolide analogue 3A.1 also inhibited COX activity, which is in agreement with an earlier report showing that andrographolide inhibits pro-inflammatory cytokines including COX-2 (Jada et al., 2006). Induction of COX-2 has been shown to be involved in cancer metastasis (Asting et al., 2010). Recently, anti-inflammatory activity has been proposed as an important strategy for developing new anticancer regimens from natural products (Rattanasinganchan et al., 2006). Based on this investigation, analogue 3A.1 has significant potential for future development as a new anticancer drug. For the chemical structure of our andrographolide analogue 3A.1 (19-tert-butyldiphenylsilyl-8, 17-epoxy andrographolide), two important sites were modified from the parent compound by adding an epoxide moiety at the core structure C-17, and silicon based molecules, tert-butyldiphenylsilyl (TBDPS), at the C-19 side chain. Previously, it has been reported that compounds containing epoxide is able to inhibit Topo II by covalently binding to the enzyme (Jada et al., 2006; Rattanasinganchan et al., 2006).
Fig. 8. Probable mechanistic pathway of analogue 3A.1 induce KKU-M213 cell apoptosis.
Of special interest, the andrographolide analogue that contained epoxide at the C-17 position had higher potency in inhibiting proliferation of various types of cancer cells (Khan et al., 2003). By analogy, the presence of epoxide in our analogue 3A.1 might contribute to the higher anticancer potency of the compound as well as a unique inhibition of Topo II enzyme. In addition, an appropriate substituent group of a silicon based molecule, which was added at the C-19 side chain, may be another important determinant for the observed increased potency. Therefore, it is likely that both epoxide at the C-17 position and TBDPS at the C-19 position of the andrographolide molecule are required for the inhibitory effect on cell proliferation and induction of cell death. Taken together, andrographolide analogue 3A.1 potently inhibited proliferation and induced apoptosis in the KKU-M213 human CCA cell line whereas the parent andrographolide had no effect. The analogue inhibited topoisomerase II α enzyme activity, arrested the cell cycle, and induced DNA damage, which led to up regulation of PARP-1, p53 and BAX proteins and induced apoptosis via a caspase-dependent pathway. In addition to its anticancer activity, analogue 3A.1 also showed anti-inflammatory activity. We have summarized the probable anticancer mechanistic pathway of analogue 3A.1-induced KKU-M213 cell apoptosis in Fig. 8. Analogue 3A.1 represents a novel class of Topo II α inhibitors and has potential for further development as a new anti-cancer regimen, particularly for CCA.
Acknowledgements This research project is supported by Mahidol University; the Center of Excellence on Environmental Health, Toxicology, Science & Technology Postgraduate Education and Research Development Office (PERDO), Ministry of Education, Thailand; Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (grant PHD/0193/2553 to P.P. and S. R.) and the Office of the Higher Education Commission and Mahidol University under the National Research universities (NRU). We are grateful to Prof. Chumpol Pholpramool and Dr. Chris Viesselmann for their critical reading of the manuscript.
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Induction of apoptosis in cholangiocarcinoma by an andrographolide analogue is mediated through topoisomerase II alpha inhibition Jintapat Nateewattana a,b, Suman Dutta b, Somrudee Reabroi b, Rungnapha Saeeng c, Sakkasem Kasemsook c, Arthit Chairoungdua b, Jittima Weerachayaphorn b, Sopit Wongkham d, Pawinee Piyachaturawat a,b,n a
Toxicology Graduate Program, Faculty of Science, Mahidol University, Bangkok, Thailand Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand c Department of Chemistry, Faculty of Science, Burapha University, Chonburi, Thailand d Department of Biochemistry, Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 June 2013 Received in revised form 4 December 2013 Accepted 4 December 2013 Available online 17 December 2013
Cholangiocarcinoma (CCA), the common primary malignant tumor of bile duct epithelial cells, is unresponsive to most chemotherapeutic drugs. Diagnosis with CCA has a poor prognosis, and therefore urgently requires effective therapeutic agents. In the present study we investigated anti-cancer effects of andrographolide analogue 3A.1 (19-tert-butyldiphenylsilyl-8, 17-epoxy andrographolide) and its mechanism in human CCA cell line KKU-M213 derived from a Thai CCA patient. By 24 h after exposure, the analogue 3A.1 exhibited a potent cytotoxic effect on KKU-M213 cells with an inhibition concentration 50 (IC50) of approximately 8.0 mM. Analogue 3A.1 suppressed DNA topoisomerase II α (Topo II α) protein expression, arrested the cell cycle at sub G0/G1 phase, induced cleavage of DNA repair protein poly (ADP-ribose) polymerases-1 (PARP-1), and enhanced expression of tumor suppressor protein p53 and pro-apoptotic protein Bax. In addition, analogue 3A.1 induced caspase 3 activity and inhibited cyclin D1, CDK6, and COX-2 protein expression. These results suggest that andrographolide analogue 3A.1, a novel topo II inhibitor, has significant potential to be developed as a new anticancer agent for the treatment of CCA. & 2013 Elsevier B.V. All rights reserved.
Keywords: Andrographolide analogue Apoptosis Cholangiocarcinoma KKU-M213 cell line Topoisomerase II α
1. Introduction Cholangiocarcinoma (CCA), the major liver cancer in Southeast Asia (Sripa and Pairojkul, 2008; Songserm et al., 2011), is the most common primary malignant tumor originating from bile duct epithelial cells and is characterized by poor prognosis and unresponsiveness to chemotherapeutic agents (De Groen et al., 1999; Aravindaram and Yang, 2010). Risk for CCA in Thailand is mainly associated with infection by liver fluke (Opisthorchis viverrini) (Srivatanakul et al., 1991). Long term liver fluke infection induces chronic injury and inflammation of the biliary epithelium. Currently, there is no specific treatment for CCA (Diandra, 2008; Judith et al., 2010) and surgical resection only extends lifespan for up to 5 years (Aravindaram and Yang, 2010). Therefore, an alternative treatment with high sensitivity to specific targets is urgently needed. DNA topoisomerases are a group of enzymes involved in the topology of DNA during critical cellular processes including n Corresponding author at: Department of Physiology, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400, Thailand. Tel./Fax : þ 66 02 3547154. E-mail addresses: [email protected], [email protected] (P. Piyachaturawat).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.12.002
replication, recombination, transcription, and repair systems by transiently breaking one or two strands of DNA (Bodley and Liu 1998; Li et al., 2007; Schneider et al., 1990). Inhibition of Topo II α induces DNA damage (Soubeyrand et al., 2010), which then activates expression of the repair system protein PARP-1 (Lori, 2009). DNA damage induces p53 which regulates the cell cycle via cyclin D1 and leads to inhibition of the DNA replication process (Wanitchakool et al., 2012). An increase in p53 expression following unrepaired DNA damage induces Bax activation, which generally triggers apoptotic cell death via activation of caspase 3 (Valkov and Sullivan, 2003). Therefore, DNA topoisomerase inhibitors have been suggested as the most favorable anticancer agents (Bodley and Liu 1998; Li et al., 2007; Schneider et al., 1990). Andrographolide is the major diterpenoid lactone from Andrographis paniculata (Burm.f.) Nees (Acanthaceae) (Thisoda et al., 2006). This naturally occurring compound has anticancer activity in many cancer cell types including Jurkat (lymphocytic), PC-3 (prostate), HepG2 (hepatoma) and Colon 205 cells (Geethangili et al., 2008). A number of semi-synthetic compounds from herbs have been shown striking anticancer activities in both potency and efficacy in various cancer cells (Jada et al., 2006; Nanduri et al., 2004; Sirion et al., 2011). Recently, various andrographolide analogues have been developed
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and their modes of action are being evaluated to find a better regimen for cancer therapy (Jada et al., 2008; Satyanarayana et al., 2004). However, none of these andrographolide analogues have been shown to have Topo II α inhibitory activity. Interestingly, andrographolide analogue 3A.1 has shown Topo II alpha inhibitory activity in an in vitro cell-free system. (Nateewattana et al., 2013). In the present study, we report Topo II α inhibitory activity of andrographolide analogue 3A.1 and the underlying anticancer mechanism in CCA KKU-M213 cells derived from a Thai CCA patient.
2. Materials and methods 2.1. Materials Ham F12 medium and antibiotic–antimycotic agents were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS), RIPA, proteinase inhibitor and SuperSignal West Pico Chemiluminescent Substrate were purchased from Thermoscientific (Cramlington, UK). SDS, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), vinblastine, etoposide and propidium iodide (PI) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Annexin-V FITC apoptosis kit was obtained from BD bioscience, USA. CaspaseACE™ colorimetric assay kit and zVADfmk (all caspase inhibitor) were obtained from Promega (Madison, WI, USA). Diamidine-2-phenylindole dihydrochloride (DAPI) was purchased from Roche Ltd (Mannheim, Germany). Doxorubicin was purchased from Guanyu Bio-Tech Co. (Xijan, Republic of China). Cleaved PARP-1, P53, Bax, cyclin D1, CDK-6 and Topo IIα antibodies were procured from Cell Signaling Technology, Inc (Danvers, MA, USA). Cox activity assay kit was purchased from Cayman (Ellsworth, MI, USA). All other chemicals unless otherwise stated were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2.2. Andrographolide analogue 3A.1 Andrographolide was isolated from dried aerial part and root of A. paniculata (Acanthaceae) and andrographolide analogue 3A.1 were prepared as previously described (Nateewattana et al., 2013). The chemical structure is shown in Fig. 1. Purity of the compound was assessed by NMR, IR, and ICPMS spectroscopy and was approximately 99%. 2.3. Cell lines and culture conditions CCA cell line KKU-M213 (adenosquamous carcinoma) derived from a Thai patient, was obtained from Prof. Dr. Banjob Sripa, Faculty of Medicine, Khon Kaen University, Thailand. KKU-M213 cells were cultured in Ham F-12 medium supplemented with 10% (v/v) heat-inactivated FBS, and 1% antibiotic–antimycotic. Cultures were maintained at 37 1C in a CO2 incubator with controlled humidified atmosphere of 95% air and 5% CO2. 2.4. Cytotoxicity assay Cytotoxic activities of andrographolide, andrographolide analogue 3A.1, etoposide, doxorubicin and vinblastine on KKU-M213 cells were assessed by MTT assay. Two hundred microlitre of 3 104 cells/ml was seeded into 96-well micro-plates and routinely cultured. After 24 h, cells were exposed to the test compounds at concentrations ranging from 0 to 50 mM in DMSO for another 24 h. The final concentration of DMSO in the medium was less than 0.1% (v/v). The medium was removed and cells were washed with phosphate buffered saline (PBS). Then fresh medium containing 0.5 mg/ml MTT dye was added to each well and
Fig. 1. Structure and cytotoxicity of 3A.1. (A) Structure of andrographolide analogue 19-tert-butyldiphenylsilyl-8, 17-epoxy andrographolide (3A.1). (B) KKU M-213 cells were treated with parent andrographolide, analogue 3A.1, etoposide, doxorubicin, and vinblastine for 24 h, and cytotoxicity was evaluated by MTT assay. Each value is mean7 S.E.M from three independent experiments in triplicate.
incubated for an additional 4 h, followed by the addition of 100 μl of DMSO to dissolve the formozan crystals. The plate was analyzed on a microplate reader (Fluostar optima microplate reader, GmbH, Germany) at 540 nm. The inhibition concentration 50 (IC50) values were estimated using GraphPad Prism version 5.01 (GraphPad, Inc, USA). 2.5. Cell cycle analysis Cells were treated with analogue 3A.1 or etoposide and stained with propidium iodide (PI). The proportions of cells at different phases of the cell cycle were studied by flow cytometry. Briefly, 3 104 cells/ml were incubated in 6-well plates for 24 h, after which cells were exposed to the test compounds; analogue 3A.1, etoposide or vinblastine for another 12 h. Cells were collected, washed with PBS, and stained with 50 μg/ml PI for 15 min. Stained cells were analyzed by BD FACS Canto Flow Cytometer (Becton Dickinson, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 620 nm. The data were acquired using the BD CellQuest Pro software on Macs OS 9. Cells were then analyzed with Cell Quest Pro or FlowJo software. For each sample, twentyfive thousand events were collected. Cellular aggregates and debris were excluded from analysis by proper gating. Data were fit to define the G1, S, and G2/M phases by using the Dean–Jett– Fox two population mathematical model of the FlowJo software. 2.6. Analyses of apoptosis and caspase 3 activity To observe nuclear morphology, KKU-M213 cells (3 104 cells/ml) were seeded in 6-well plates and incubated with analogue 3A.1, vinblastine or etoposide for 12 h. The medium was subsequently
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discarded and gently washed with PBS followed by DAPI staining (5 μg/ml) for 20 min at 37 1C and observed under a fluorescence microscope HB-10101AF (Nikon, Japan) and photographed. A total of 500 stained cells/treatment was counted and apoptotic cells were determined by DNA condensation based on fluorescence intensity. Apoptosis was also analyzed by annexin V-FITC and PI staining. KKU-M213 cells were seeded in 6-well plates and incubated with analogue 3A.1, vinblastine or etoposide for 24 h. The medium was discarded and cells were washed with PBS followed by gentle pipetting to detach cells. Cells were then co-incubated with annexin-V and PI for 15 min at room temperature (25 1C) in the dark followed by analysis in BD FACScanto using FACS diva software (BD Bioscience, USA). The activity of caspase 3 was determined using CaspaseACE™ Colorimetric Assay System (Promega, Madison, USA), following the manufacturer's instructions. Briefly, the treated cells were harvested by trypsinization and centrifugation. The pellets were suspended in lysis buffer and lysed by three cycles of freezing and thawing. After centrifugation at 15,000g for 20 min at 4 1C, the supernatant was collected. The caspase 3 activity was measured using a specific substrate and inhibitor in a 96-well assay plate. The reaction was measured at an absorbance wavelength of 405 nm in FLUOstar OPTIMA microplate reader (BMG Labtech GmbH, Germany). To determine whether apoptotic cell death was caspase dependent or independent, an MTT assay with or without an all caspase inhibitor (zVAD-fmk) was performed. Briefly, 3 104 cells/ml were seeded into 96-well microplates and then exposed to analogue 3A.1 (8 μM) with or without zVAD-fmk at a concentration of 10 mM for 24 h, followed by the MTT assay.
2.8. Western blot analysis The proteins involved in cell death induced by analogue 3A.1 were detected by western blot analysis. The expression of Topo II α, p53, PARP-1, Bax, CDK-6, and cyclin D1 proteins were investigated. Briefly, cells (1 106 cells/ml) were seeded in 6-well plates and treated with analogue 3A.1, vinblastine or etposide for a specified period. The treated cells were washed with PBS twice. RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM NaF, 1 mM PMSF, and cocktail protease inhibitor) was then added in a suitable volume. After centrifugation at 14,000 rpm for 30 min at 4 1C, supernatants were collected and concentrations of protein in the supernatants were determined by bicinchoninic acid biuret (BCA) assay. Proteins were separated using 10% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat dried milk, incubated with primary antibodies overnight at 4 1C, and then incubated with horseradish peroxidase-conjugated secondary antibodies. Protein was visualized using the SuperSignals Enhanced Chemiluminescence (ECL) Western blotting detection kit. 2.9. Statistical analyses All data are expressed as means 7the standard error of the mean (S.E.M.). They were analyzed by one-way analysis of variance (ANOVA). Differences among the treatment groups were assessed by Duncan's multiple-range test, using GraphPad Prism version 5.01 (GraphPad Software Inc, CA, and USA). Differences were considered significant at P o0.05.
2.7. COX activity assay
3. Results
The activity of COX was determined using a COX activity assay (Cayman, Ellsworth, USA). The treated cells were harvested and cell lysates were prepared. COX activity was measured using specific a substrate and inhibitor in a 96-well plate assay provided with the kit. The reaction was measured at an absorbance wavelength of 590 nm in a FLUOstar OPTIMA microplate reader (BMG Labtech GmbH, Germany).
3.1. Cytotoxic effects of andrographolide and its analogue on KKU-M213 cells The cytotoxic effects of andrographolide and its analogue 3A.1 on human CCA cells were evaluated by MTT assay. The parent andrographolide compound (50 mM) displays weak cytotoxicity to KKU-M213 cells whereas analogue 3A.1 was highly toxic to the cells
Fig. 2. Inhibitory effect of analogue 3A.1 on Topo II α activity in KKU-M213 cells after exposure to 8 mM analogue 3A.1 for (A) 4 h; (B) 6 h. Total protein (100 μg) was electrophoresed and transferred onto nitrocellulose membranes. The membranes were probed with anti-rabbit Topo II α and anti-mouse β actin antibodies. Topo II α was recognized as the expression of the 190 kDa protein. β actin (42 kDa) was used as an internal loading control. Experiments were repeated three times. *P o0.01, significantly different from control.
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Fig. 3. Analysis of cell cycle phases, (A) DNA content histogram showing distribution of KKU-M213 cells at various phases of cell cycle after treatment with vinblastine (20 mM), etoposide (20 mM), and analogue 3A.1. (8 mM) for 12 h. (B) Effect of analogue 3A.1 on CDK6 and cyclin D1 protein expressions 6 h after exposure to analogue 3A.1. Total protein (100 μg) was electrophoresed and transferred onto nitrocellulose membranes. The membranes were probed with anti-rabbit cyclin D1, anti-mouse CDK6, and anti-mouse β actin antibodies. CDK6 was recognized as the expression of the (30 kDa) band and cyclin D1 was recognized as the expression of the (36 kDa) band. β actin (42 kDa) was used as an internal loading control. Experiments were repeated three times.
Fig. 4. Analysis of chromatin condensation and DNA fragmentation in KKU-M213. (A). Changes in nuclear morphology examined by DAPI staining of condensed nuclear chromatin with a fluorescence microscope at a magnification of 400 . (B). Graphical representation of the number of condensed DAPI stained nuclei and fluorescence intensity analyzed using Image J software. nPo 0.01, significantly different from vehicle control.
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having an IC50 of 8.0þ0.1 mM at 24 h (Fig. 1). Etoposide, vinblastine and doxorubicin, which were used as positive controls, had IC50 values of 27.770.1, 6.370.02, and 4.6370.06 mM, respectively. This result also indicates that the potency of the analogue 3A.1 is markedly increased compared to the parent compound. Thereafter, analogue 3A.1 at a dose of 8 mM was chosen to further investigate the mechanism of action of anticancer activity.
3.2. Inhibition of topoisomerase II
α in KKU-M213 cells
At various time points after treatment with analogue 3A.1 at 8 mM, the expression of Topo II α in KKU-M213 cells was evaluated by Western blot. In Fig. 2(A), analogue 3A.1 exhibits dose- and time-dependent decreases in the expression of Topo II α protein (190 kDa) compared to control. Topo II α expression at 4 h after incubation was markedly inhibited, and further decreased at 6 h after incubation (Fig. 2B). Vinblastine and etoposide, two welldefined anticancer drugs, were used as positive controls. Vinblastine (20 mM) did not affect Topo II α in KKU-M213 cells at 4 h, but a slight inhibition was observed at 6 h. Etoposide (20 mM), a well-known topoisomerase poison (Soubeyrand et al., 2010), had less inhibitory effect at 4 h compared to the analogue 3A.1. However, etoposide completely inhibited Topo II α at 6 h after incubation. In contrast,
the parent andrographolide only slightly inhibited the Topo II α level even at 6 h. Therefore, the analogue 3A.1 showed promising Topo II α inhibitory activity. 3.3. Inhibition of cell cycle in KKU-M213 cells The proportion of DNA in KKU-M213 cells at different stages of the cell cycle (sub G0, G1, S and G2) was analyzed by flow cytometer after treatment with analogue 3A.1, vinblastine or etoposide. At 12 h after exposure, vinblastine did not significantly alter the phase distribution of the cell cycle in KKU-M213 cells compared to DMSO treated controls whereas the analogue 3A.1 increased the percentage of cells in the sub G0 phase. The percent distribution of cells at sub G0 phase and G2/M phase were increased from 2.5 7 0.0% to 11.570.2% and from 14.1 70.02 to 23.87 0.01, respectively. Etoposide, a potent topo II inhibitor, also arrested 19.5 7 0.02% of cells at sub G0 phase and 34.3 70.01% of cells at G2/M phase (Fig. 3A). The expressions of cyclin-dependent kinase 6 (CDK6) and cyclin D1, which regulate cell cycle progression and the transition from G1 to S phase, were markedly decreased by analogue 3A.1 in KKU-M213 cells at 6 h after incubation. This result suggests that the analogue 3A.1 indeed induced DNA damage, which in turn led
Fig. 5. Analogue 3A.1 induced apoptosis and activated caspase 3 activity. (A) Cells were treated with analogue 3A.1, and positive controls were then subjected to fluorescence activated cell sorting on a BD FACS Canto machine. Data were analyzed using FACS Diva software. The quadrants represent the percentage of live/apoptotic cells. (B) Caspase 3 activity was measured using specific a substrate and inhibitor in a 96-well assay plate. The reaction was measured absorbance at 405 nm in an ELISA plate reader. (C) KKUM213 cells were treated with 8 mM analogue 3A.1 in the presence or absence of 10 mM all caspase inhibitor (zVAD-fmk). Data are means 7 S.E.M. from two independent experiments which were conducted in triplicates. nP o0.05; significantly different from the vehicle control.
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to cell cycle arrest. Etoposide treatment also decreased cyclin D1 and CDK6 expressions at 6 h after exposure (Fig. 3B). 3.4. Analysis of DNA condensation in KKU-M213 cells The effect of analogue 3A.1 on apoptotic cell death was determined by nuclear morphology using DAPI staining, which is characterized by nuclear condensation and formation of apoptotic bodies. Vinblastine and etoposide were used as positive controls. Vinblastine induced DNA condensation and fragmentation in approximately 75% of the treated cells whereas etoposide induced this effect in only 27% of treated cells as depicted by the fluorescence intensity analysis of apoptotic nuclei using image J software. Analogue 3A.1 markedly induced DAPI staining of condensed chromatin by about 68% (Fig. 4A and B). 3.5. Induction of apoptosis and enhancement of caspase 3 activity in KKU-M213 cells To further examine the apoptotic death process, phosphatidylserine externalization on the outer membrane of cells was assayed by staining with annexin V-FITC and using flow cytometry. As shown in Fig. 5A, treatment with analogue 3A.1 (8 mM) induced apoptosis up to 49.3% of KKU-M213 cells compared to DMSO control (0.9%). Etoposide (20 mM) and vinblastine (20 mM) treatments induced apoptosis in approximately 26.3% and 68.1% of cells, respectively. Analogue 3A.1 also showed a time-dependent increase in caspase 3 activity. At 6 h the activity was not different from control, but activity was significantly increased from 1.070.30 pmol/ml to 470.11 pmol/ml and 6.0970.22 pmol/ml at 12, and 24 h, respectively (Fig. 5B). This effect was abolished by 10 mM zVADfmk (all caspase inhibitor) as the cell viability was increased to the control level (Fig. 5C). This result suggests that the analogue 3A.1-induced cell death is mediated through a caspase 3-dependent pathway.
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is a hallmark of inflammation, and induction of COX-2 is involved in metastasis of cancer (Rattanasinganchan et al., 2006). As shown in Fig. 6, DuP-697 (0.3 mM), a specific COX-2 inhibitor, potently inhibits COX activity whereas SC-560 (0.33 mM), a specific COX-1 inhibitor, has no detectable effect. Analogue 3A.1 (8 mM) significantly inhibited COX2 activity at 24 h after exposure. The analogue 3A.1 decreased COX-2 activity from 17.370.2 pmol/μl to 15.2 7 0.2, and 13.3 70.2 pmol/μl at 12 h and 24 h, respectively. This result suggests that the analogue 3A.1 may also possess antiinflammatory activity. 3.7. Effects of analogue 3A.1 on proteins involved in apoptosis A number of proteins that participate in the apoptosis pathway, particularly those related to inhibition of Topo II, were determined by western blot. At 6 h after exposure, analogue 3A.1 induced cleavage of the PARP-1 protein, which is a hallmark of apoptosis. The analogue 3A.1 also increased expression of p53, a nuclear tumor suppressor protein, in KKU-M213 cells. In addition, expression of BAX protein, which is under the control of p53, was also significantly enhanced after treatment with analogue 3A.1 (Fig. 7), suggesting that apoptosis was induced via a Bax-caspase 3-dependent pathway.
4. Discussion The present study demonstrated the underlying anticancer mechanism of the andrographolide analogue 3A.1 in an aggressive CCA cell line isolated from a Thai CCA patient. Analogue 3A.1, but
3.6. Inhibition of COX-2 activity in KKU-M213 cells Cyclooxygenase (COX) is a bifunctional enzyme exhibiting both cyclooxygenase and peroxidase activities. Increased COX 2 activity
Fig. 6. Analogue 3A.1 inhibits COX-2 activity. (A) KKU-M213 cells were treated with analogues 3A.1 and COX activity was measured at various time points. Each value represents mean 7 S.E.M. obtained from three independent experiments which were conducted in triplicate. nPo 0.05; significantly different from the vehicle control.
Fig. 7. Effect of analogue 3A.1 on proteins in the apoptosis pathway; PARP-1, P53, and BAX in KKU-M213 cells. Cells were treated with analogue 3A.1 and expression of different proteins was measured by western blot. PARP-1 protein was recognized as the expression of the cleaved fragment (85 kDa), p53 expressed as the 53 kDa fragment, and BAX protein expressed as the 20 kDa fragment. Total proteins (100 μg) were electrophoresed and transferred onto nitrocellulose membranes. The membranes were probed with antibodies. β actin (42 kDa) was used as an internal loading control. Experiments were repeated three times.
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not its parent compound, potently inhibited KKU-M213 cell proliferation, arrested the cell cycle, suppressed Topo II α protein expression, and induced apoptosis. The inhibitory potency of the analogue 3A.1 on Topo II α at 4 h was comparable to that of etoposide and greater than that of vinblastine, a current therapeutic drug for cancer. To our knowledge, this is the first report showing an inhibition of the Topo II α enzyme in a CCA cell line by an andrographolide analogue that has shown distinct activity from its parent compound. The anti-proliferative effect of analogue 3A.1 on KKU-M213 cells was associated with inhibition of Topo II α enzyme activity and cell cycle arrest which eventually induced apoptosis. Treatment with analogue 3A.1 increased the percentage of cells in sub G0 and G2/M phase (Fig. 3A). This effect was consistent with the expression of p53, which is a nuclear tumor suppressor protein controlling the cell cycle check points at the G1 to S and G2 to M phase (Hainaut, 1995). Topo II α is essential for cell division and is highly produced in S and G2/M phases (Chikamori et al., 2010). Therefore, inhibition of Topo II α arrests the cell cycle at the S and G2/M phases (Li et al., 2007). After DNA damage, p53 protein inhibits TATA box binding protein (TBP) to cause a halt in DNA transcription and suppresses replication protein A (RPA), which inhibits cellular replication (Hainaut, 1995). Indeed, we found that the analogue 3A.1 increased the expression of p53 and down regulated expression of cyclin D1 and CDK6. Apoptosis may be induced by several possible mechanisms (Kerr et al., 1972, and Huang et al., 2004). An inhibition of Topo II α induces DNA damage, which activates expression of repair system proteins including PARP-1 (Valkov and Sullivan, 2003). In the present study, the analogue 3A.1 inhibited expression of Topo II α and increased the cleavage of PARP-1, suggesting that this pathway may be attributed to the apoptosis of KKU-M213 cells. DNA damage also triggers expression of p53, which controls the cell cycle and enhances BAX activation. BAX, which is a member of the intrinsic apoptotic Bcl-2 family of proteins (Yuan and Yankner, 2000) induces apoptosis by producing pores in the mitochondrial membrane (Wanitchakool et al., 2012) from which cell death mediators such as Cytochrome C, Smac/DIABLO and AIF are released into cytosol leading to activation of caspase 9 (Sperandio et al., 2000). Active caspase 9 then activates caspase 3 leading to DNA fragmentation and apoptosis. In the present study, the expression of BAX in KKUM213 cells was substantially increased after treatment with analogue 3A.1. In addition, apoptosis of this cell line was associated with an increase in caspase 3 activity. Overall, our results strongly suggest that the anti-proliferative effect and induction of apoptosis in KKU-M213 cells by analogue 3A.1 are resulted from the inhibition of Topo II α, leading to DNA damage and activation of p53 and PARP1 proteins. Andrographolide analogue 3A.1 also inhibited COX activity, which is in agreement with an earlier report showing that andrographolide inhibits pro-inflammatory cytokines including COX-2 (Jada et al., 2006). Induction of COX-2 has been shown to be involved in cancer metastasis (Asting et al., 2010). Recently, anti-inflammatory activity has been proposed as an important strategy for developing new anticancer regimens from natural products (Rattanasinganchan et al., 2006). Based on this investigation, analogue 3A.1 has significant potential for future development as a new anticancer drug. For the chemical structure of our andrographolide analogue 3A.1 (19-tert-butyldiphenylsilyl-8, 17-epoxy andrographolide), two important sites were modified from the parent compound by adding an epoxide moiety at the core structure C-17, and silicon based molecules, tert-butyldiphenylsilyl (TBDPS), at the C-19 side chain. Previously, it has been reported that compounds containing epoxide is able to inhibit Topo II by covalently binding to the enzyme (Jada et al., 2006; Rattanasinganchan et al., 2006).
Fig. 8. Probable mechanistic pathway of analogue 3A.1 induce KKU-M213 cell apoptosis.
Of special interest, the andrographolide analogue that contained epoxide at the C-17 position had higher potency in inhibiting proliferation of various types of cancer cells (Khan et al., 2003). By analogy, the presence of epoxide in our analogue 3A.1 might contribute to the higher anticancer potency of the compound as well as a unique inhibition of Topo II enzyme. In addition, an appropriate substituent group of a silicon based molecule, which was added at the C-19 side chain, may be another important determinant for the observed increased potency. Therefore, it is likely that both epoxide at the C-17 position and TBDPS at the C-19 position of the andrographolide molecule are required for the inhibitory effect on cell proliferation and induction of cell death. Taken together, andrographolide analogue 3A.1 potently inhibited proliferation and induced apoptosis in the KKU-M213 human CCA cell line whereas the parent andrographolide had no effect. The analogue inhibited topoisomerase II α enzyme activity, arrested the cell cycle, and induced DNA damage, which led to up regulation of PARP-1, p53 and BAX proteins and induced apoptosis via a caspase-dependent pathway. In addition to its anticancer activity, analogue 3A.1 also showed anti-inflammatory activity. We have summarized the probable anticancer mechanistic pathway of analogue 3A.1-induced KKU-M213 cell apoptosis in Fig. 8. Analogue 3A.1 represents a novel class of Topo II α inhibitors and has potential for further development as a new anti-cancer regimen, particularly for CCA.
Acknowledgements This research project is supported by Mahidol University; the Center of Excellence on Environmental Health, Toxicology, Science & Technology Postgraduate Education and Research Development Office (PERDO), Ministry of Education, Thailand; Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. Program (grant PHD/0193/2553 to P.P. and S. R.) and the Office of the Higher Education Commission and Mahidol University under the National Research universities (NRU). We are grateful to Prof. Chumpol Pholpramool and Dr. Chris Viesselmann for their critical reading of the manuscript.
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