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A02 through producing reactive oxygen species
European Journal of Pharmacology 718 (2013) 459–468
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
PHII-7 inhibits cell growth and induces apoptosis in leukemia cell line K562 as well as its MDR- counterpart K562/A02 through producing reactive oxygen species Hongwei Peng a,b, Xiangfei Yuan a, Ruizan Shi c, Xiaohua Wei b, Simei Ren d, Cihui Yan a, Yahui Ding a, Yang Lin a, Dongmei Fan a, Ming Yang a,nn, Yanjun Zhang a, Dongsheng Xiong a,n a State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, PR China b Department of Pharmacy, First affiliated Hospital of Nanchang University, Nanchang, Jiangxi, PR China c Department of Pharmacology, Shanxi Medical University, Taiyuan, Shanxi, PR China d Beijing Hospital of the Ministry of Health, National Center for Clinical Laboratories, Beijing, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 January 2013 Received in revised form 13 July 2013 Accepted 16 July 2013 Available online 30 July 2013
Multidrug resistance (MDR) is a major obstacle that hinders the efficacy of chemotherapy in many human malignancies. PHII-7 is a derivative of indirubin, which was designed and synthesized by our laboratory. Our preliminary work indicated its potent antitumor activities in vitro and in vivo. Furthermore, based on the model of MDR cell line, we found its powerful effects in inhibiting the expression of P-glycoprotein (P-gp) and killing multidrug-resistant (MDR) cells with the detailed mechanism remained to be explored. Reactive oxygen species are known for high reactive activity as they possess unmatched electrons. In this study, we showed that PHII-7 generated equal reactive oxygen species in parental K562 and its counterpart MDR K562/A02 cells. Pre-incubation with thiol antioxidants glutathione or N-acetyl-cysteine(NAC) almost abolished the cytotoxicity of PHII-7. Moreover, NAC abrogated DNA damage, cell cycle arrests and apoptosis induced by PHII-7. Our results collectively indicated that reactive oxygen species production induced by PHII-7 contributed to both apoptosis and cell cycle arrets in MDR K562/A02 cells, thus extending our prior related findings. Notably, JNK phosphorylation was also induced by PHII-7 and pre-incubated of K562/A02 cells with NAC or inhibitor of JNK(SP006125) eliminated P-gp downregulation. Taken together, our results may provide a detailed biochemical basis for further clinical application of PHII-7. & 2013 Elsevier B.V. All rights reserved.
Keywords: Reactive oxygen species PHII-7 DNA damage P-glycoprotein (P-gp) Apoptosis Cell cycle arrest
1. Introduction PHII-7 is a derivative of indirubin that designed and synthesized by our laboratory. Our previous study has shown PHII-7 exerted cytotoxic effect in multiple cancer cell lines that had different origins. Interstingly, PHII-7 was also effective in MDR-(multidrug resistance) cancer cell lines (Su et al., 2012; Shi et al., 2011). In this study, we will explore the killing mechanism of PHII-7 in the K562 leukemia cell line, as well as its MDR counterpart K562/A02 cells. Reactive oxygen species, including the superoxide anion, hydrogen peroxide and hydroxyl radical, is known to mediate apoptosis induced by some cancer chemopreventive and therapeutic agents. n
Corresponding author. Tel./fax: +86 22 23909404. Corresponding author. Tel.: +86 22 23909025. E-mail addresses: [email protected] (X. Wei), [email protected] (M. Yang), [email protected], [email protected] (D. Xiong). nn
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.07.038
Intracellular reactive oxygen species may interact with cellular membrane lipids, proteins, DNA and cause oxidative injury (Davis et al., 2001; Valko et al., 2006). Mitochondria is the main source of reactive oxygen species production, and it is also highly enriched with antioxidants and enzymes, such as glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GPx) which are present on both sides of the membranes in order to minimize oxidative stress (Landriscrina et al., 2009; Montero and Jassem, 2011). In normal cells, reactive oxygen species is continuously generated throughout the life span and facilitates cell proliferation, and a redox balance is maintained by the reactive oxygen species system and the anti-oxidative system. However, tumor cells are particularly sensitive to oxidative stress as they typically have persistently higher levels of reactive oxygen species than normal cells. Therefore, as the result of increasing the intracellular reactive oxygen species production or depleting the antioxidant proteins, the dysregulation of redox balance is developed in cancer cells (Ozben, 2007; Reuter et al., 2010). We suppose that the killing effect of PHII-7
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would be related to the reactive oxygen species, and the hypothesis is tested in this research work. Chemotherapy is one of the effective means to treat cancer, especially hematologic maligancies and solid tumor with highly metastasis characteristics. However, the development of multidrug resistance in cancer cells always hinders the efficacy of conventional chemotherapeutic agents in patients. Since more attention had been paid to the anti-MDR activity of PHII-7, we explored its cell killing mechanism deeply as well as the relationship between reactive oxygen species production and its anti-MDR effect in this research work.
2. Materials and methods 2.1. Cell lines and cell culture The human leukemia cell line K562 was obtained from American Type Culture Collection (ATCC, Rockville, MD), and its multidrug resistance counterpart K562/A02 were conserved by our laboratory (Yang et al., 1995). Cells were grown in RPMI 1640 medium supplemented with 10%(v/v) heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin (GIBCO, Grand Island, NY) at 37 1C in a 5% CO2 humidified atmosphere. K562/A02 cells were routingly maintained in medium containing 200 mg/l doxorubicin and incubated in drug-free medium for at least one week prior to experimental use. 2.2. Materials PHII-7 was synthesized in our laboratory and freshly dissolved in dimethyl sulfoxide (DMSO). Before use, PHII-7 was diluted with cultured medium to the desired concentrations. The final concentration of DMSO was less than 0.1%. Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Dojindo Molecular Technologies, Japan). DCFH2-DA, GSH and NAC were purchased from Beyotime (Beyotime Institute of Biotechnology, China). Antibodies against PARP-1, Caspase-3, Caspase-9, Bax, p21, and p27, Phosphorylated H2A.X, H2A.X and GAPDH were obtained from Cell Signaling Techonology (Beverly, MA); antibody against bcl-2 was purchased from Epitomics (Epitomics, Inc, U.S.A.).
selected cells per slide with the Komet 5.5 software (Kinetic Imaging Ltd., Nottinghan, UK) using the parameter olive tail moment (arbitraty units, defined as the product of the percentage of DNA in the tail multiplied by the tail length).
2.6. Apoptosis assesments by Annexin V-FITC and PI staining The Annexin V-FITC-labeled Apoptosis Detection Kit I (BD Biosciences, Pharmingen, San Diego, CA, USA) was used to detect and quantify apoptosis by flow cytometry according to the manufacturer's instructions. In brief, K562 and K562/A02 cells (1 105 cells/ml) were seeded in 6-well plates and then indicated concentrations of PHII-7 were added. After treatment, cells were harvested and washed with ice-cold PBS. The cell pellets were resuspended in 100 ml ice-cold binding buffer. 5 ml annexin V-FITC solution and 5 ml dissolved PI were added to cell suspensions. The samples were mixed gently and incubated at room temperature (25 1C) for 15 min in the dark. Then another 300 ml binding buffer was added and mixed gently before the cell preparations were examined by a flow cytometer (BD, LSRII). Annexin V-positive, PI-negative cells were recorded as apoptotic. Double-stained cells were considered either as necrotic or as late apoptotic (Liu et al., 2010).
2.7. Cell cycle analysis The cell cycle distribution was detected by the Cycle Test TM plus DNA reagent kit (Beckman Dickson, USA) as described by the manufacturer's instruction. Briefly, cells (1 105 cells/ml) were preincubated with or without 5 mM NAC for 30 min before treated with PHII-7. After treatment, cells were harvested, washed the cell pellet with 1 mL Buffer Solution twice. Then 250 μl solution A was added to resuspend the cell pellet. After 10 min-incubation at room temperature, 200 μl solution B was added and mixed gently, incubated at room temperature for 10 min; At last, 200 μl ice-cold solution C was added and incubated at 4 1C in the dark for 10 min. Cell cycle was measured with a flow cytometry (BD, LSRII).
2.3. Cytotoxity assays
2.8. Realtime PCR analysis
Cell viability was assessed by CCK-8 kit. Cells were harvested and then seeded at a density of 1 105/ml, ADR, PHII-7, NAC or Vitamine E was added at its required final concentration. After incubation for 24 h, CCK-8 reagent was added, and then read the plate at 450 nm by a microplate hybrid reader (Synergy H4, Biotek, USA). Cell viability was calculated as follows: A450treated/A450control 100%.
Real-time PCR was performed with SYBR Green Master Mix (Applied Biosystems, CA), using a 7500 Real-time PCR System (Applied Biosystems). The quantitative measurement of each gene was normalized to the amount of GAPDH cDNA. Primers used in realtime PCR are as follows: 5′-AGCGAGCAACTGAGAA GC-3′ (sense) and 5′-CGCTGTGAAGCAGAGCTGG-3′ (antisense) for the c-fos gene (to generate an 83 bp fragment); 5′-AGTCAACGGATTTGGTCGTA-3′(sense) and 5′-GGAACATGTAAACCATGTAG-3′ (antisense) for the GAPDH gene (to generate a 122 bp fragment). Primers of MDR1 used for real-time PCR were the same as that for RT-PCR. The GAPDH gene was used as an endogenous control to normalize the mRNA values in each sample. The relative values were determined by the comparative computed tomography analysis method.
2.4. Reactive oxygen species measurement Accumulation of intracellular reactive oxygen species was detected with the probe DCFH2-DA. In brief, cells were preincubated with or without 5 mM NAC preincubated for 30 min. After treated with PHII-7 for indicated time, cells were labeled with 10 mM DCFH2-DA and incubated for additional 30 min at 37 1C in a humidified atmosphere at 5% CO2. The fluorescence intensity was measured by a flow cytometry (BD, LSRII).
2.9. Statistical analysis 2.5. Neutral single cell gel electrophoresis assays DNA DSBs (double strands breaks, DSBs) were evaluated using neutral comet assays as previously described (Olive et al., 1990). Quantitation was achieved by analyzing at least 50 randomly
All results were presented as the means 7S.D. of results from three cultures and the significant difference was analyzed by Student's t-test. Probability values of Pb0.05 were considered to be statistically significant.
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and K562/A02 leukemia cells with IC50 values of 3.45 70.98 and 110.01 715.67 mmol/l respectively, so K562/A02 cells were tested more than 30-fold resistant to ADR in comparison with its parental K562 cells.
3. Results 3.1. Resistant fold of K562/A02 K562 and K562/A02 were treated with different concentrations of ADR for 24 h and the cytotoxicity was determined by CCK-8 assay. As shown in Table 1, ADR exerted cytotoxity against K562 Table 1 Cytotoxicity of adriamycin on K562 and K562/A02. Cell lines
IC50 of ADR
K562 K562/A02
3.45 7 0.98 110.017 15.67nn
nn
po 0.01.
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3.2. Effects of PHII-7 on cell viability Both the two cell lines were treated with PHII-7 at concentrations ranging from 0 to 10 mM for 24 h. Cell viability was determined by CCK-8 assay and appeared to decline gradually in a dose- dependent manner (Fig. 1B). In contrast, both the parental and the MDR cell lines showed no obvious difference to PHII-7 with IC50 values of 2.567 0.23 and 2.08 70.31 mmol/l respectively. These results indicated that PHII-7 had almost the same efficacy against both the chemo-sensitive parental and MDR leukemia cells. Additionally, Fig. 1C clearly demonstrate that the
Fig. 1. The cytotoxicity of PHII-7. (A) The chemical structure of PHII-7. (B) The cytotoxic effects of PHII-7 on K562 as well as its MDR- counterpart K562/A02 (For 24 h PHII-7 treatment). (C) Antioxidants rescued both MDR K562/A02 and parental K562 cells from the cytotoxicity of PHII-7. Cells were incubated with various antioxidants for 30 min, then exposed to 1 mM or 2 mM PHII-7 for another 24 h. Cell viability was assessed by CCK-8 assays. (D) Different concentrations of Vitamine E rescued K562 and K562/A02 cells that treated with PHII-7. Cells were incubated with Vitamine E for 30 min, then exposed to 2 mM PHII-7 for another 24 h. Cell viability was assessed by CCK-8 assays. The data were expressed as expressed as mean7 S.D., n¼3. The concentrations for different antioxidants were: NAC, 5 mM; GSH, 5 mM.
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antioxidants, NAC or GSH, could completely antagonist cell killing effect by PHII-7. Moreover, another antioxidant, Vitamine E, could also block the cytotoxic effect of PHII-7 in the manner of concentration dependently (Fig. 1D). All of these data suggest oxidative stress play roles in cytotoxic effect of PHII-7 in K562 as well as its MDR counterpart K562/A02.
determined by using the DCFH2-DA fluorescence probe. Firstly, the DCF fluorescence gradually increased in a time- dependent manner. The change of fluorescence intensity was visual at 3 h, and reached the peak value at 11 h (Fig. 2A). Pretreatment with NAC significantly reduced reactive oxygen species production by PHII-7 (Fig. 2B). These results indicated PHII-7 increased intracellular reactive oxygen species production in both K562 and K562/A02 cells.
3.3. Measurement of reactive oxygen species production 3.4. DNA damage caused by reactive oxygen species production Based on the fact that NAC and GSH could inhibit the cytotoxic effect of PHII-7, we further detected reactive oxygen species production after PHII-7 treatment. The two cell lines were treated with PHII-7 and the intracellular reactive oxygen species production was
Reactive oxygen species possess high reactive potential for habouring unmatched electron, over production of reactive oxygen species would cause macromolecule damage (Lu et al., 2005;
Fig. 2. PHII-7 equally elicits intracellular Reactive Oxygen Species production and DNA damage in both MDR K562/A02 and parental K562 cells. (A) Cells were exposed to 2 mM PHII-7 for various times(0.5–24 h). Intracellular Reactive Oxygen Species were assessed by flow cytometry with DCFH2-DA staining as described in Materials and Methods. The data from three separate experiments were expressed as mean 7 S.D. Reactive Oxygen Species production increase was visual at 2 h treatment and reached its peak at 11 h treatment. (B) NAC antagonist Reactive Oxygen Species production. Once cells were preincubated with 5 mM NAC for 30 min, Reactive Oxygen Species production increase that induced by 2 mM PHII-7 11 h treatment was significantly reduced. (C) PHII-7 equally induced DNA double strands breaks (DSBs) in both K562 and K562/A02. 2 mM PHII-7 incubated cells for 6 h with or without the pretreatment of 5 mM NAC. DNA DSBs were assessed by neutral single cell gel electrophoresis(comet assay) as described in Material and Methods. Cells were stained with DAPI(1 mg/ml) and photographed by fluorescence micreactive oxygen speciescope. (D) Comet assay analysis. For DNA damage quantification, tail moment in comet assay was assessed by Comet 5.5 software as described in Material and Methods. (E) Western blot analysis confirmed DNA damage. Cells were treated with 2 mM PHII-7 for various times (6–24 h) or various concentrations (0.5–2 mM) of PHII-7 for 24 h. Equal amounts of protein (50 mg) from cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-H2A.X or anti-H2A.X antibody. GAPDH was shown as an internal standard.
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Lee et al., 2009). Neutral single cell gel electrophoresis assays (also known as comet assay) have been used to assess DNA DSBs (double strands breaks) at individual cell levels. In our experiment, both the two cell lines were treated with 2 mM PHII-7 for 6 h and analyzed with the ‘comet assay’. Using this assay, we found that
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PHII-7 produced similar levels of DNA DSBs in both MDR K562/A02 cells and parental K562 cells (Fig. 2C). The parameter used to evaluate DNA damage was tail moment. Fig. 2D showed that PHII-7 treatment caused an increase in tail moments (Po0.05), indicating DNA damage in PHII-7-treated cells. As a hallmark of DNA damage
Fig. 3. PHII-7 induced apoptosis in K562 and K562/A02 and NAC antagonist this effect. (A) Apoptosis analysis. Cells were treated with 2 mM PHII-7 for various times and stained by PI/Annexin V and then detected by flow cytometry. (B) Western blot analysis confirmed apoptotic signaling initiation. Cells were treated with 2 mM PHII-7 for different concentrations. Equal amounts of protein (50 mg) from cell lysates were separated by SDS-PAGE and immunoblotted with anti-PARP, caspase-9, bax, bcl-2 antibody. GAPDH was shown as an internal control. (C) NAC antagonist apoptosis induced by PHII-7. 2 mM PHII-7 incubated for 24 h induced significantly apoptosis while NAC pretreatment antagonist apoptosis in K562 as well as K562/A02. (D) Confocal micreactive oxygen speciescope photograph clearly showed apoptotic body after 2 mM PHII-7 24 h treatment and NAC pretreatment for 30 min could block this effect. (E) Western blot perfectly explained that NAC pretreatment before 2 mM PHII-7 incubation antagonist apoptosis signaling. (F) CCK-8 assay demonstrated that antioxidants GSH and NAC(precursor of GSH) totally rescued both MDR K562/A02 and parental K562 cells from the cytotoxicity of PHII-7 while caspase inhibitor could partly rescued cells, indicating besides apoptosis, there is another pathway involved in cytotoxic effect of PHII-7.
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(Burma et al., 2001), the phosphorylation of Ser139-H2A.X was presented a dose- and time- dependent accumulation in the treated K562/A02 and its parental K562 cells by PHII-7. Taken together, these findings suggest that PHII-7 causes DNA damage leading to serine 139 phosphorylation of H2A.X, which is also known as an early marker of apoptosis induction. 3.5. Apoptosis initiation through reactive oxygen species production In order to confirm whether PHII-7 could induce apoptosis in cells, we employed flow cytometry and western blot analysis. According to Fig. 3A and B, PHII-7 induced apoptosis in a time- and dose- dependent manner both in K562 and K562/A02 cell lines and initiated apoptotic signaling. Next, we investigated the relationship between reactive oxygen species and apoptosis both induced by PHII-7. As shown in Fig. 3C, when treated with PHII-7 alone, obvious and almost equally apoptosis was observed in K562/A02 and K562 cells. Once pretreated with NAC, apoptotic rate was greatly reduced. In addition, NAC alone has no toxicity to both the two cell lines. Apoptotic bodies were observed with confocal microscope when treated with 2 mM PHII-7 for 24 h. When pretreated with NAC, apoptotic bodies were greatly diminished (Fig. 3D). And the caspase activation by PHII-7 was reversed by NAC too (Fig. 3E). These results indicated NAC abrogated apoptotic cell death induced by PHII-7. Moreover, we compared the influence of caspase inhibitor and reactive oxygen species scanverger on the cytotoxicity of PHII-7. According to Fig. 3F, NAC could completely abrogate the cytotoxicity of PHII-7 while the caspase inhibitor could only partly block the effect. These results suggest that besides apoptosis, there were other signaling pathways involved in the cytotoxic effect of PHII-7, and increased reactive oxygen species production may be the main contributor to cell growth inhibition by PHII-7. 3.6. Reactive oxygen species production was correlated with cell cycle arrest Cell cycle distribution was evaluated by flow cytometry. As shown in Fig. 4A, PHII-7 treatment caused S-phase arrest in K562/ A02 and K562 cell lines in a dose- and time- dependent manner. Moreover, the expression of cell cycle related protein, c-Myc, p21, p27 were altered. As shown in Fig. 4B, PHII-7 increased the expression of p21 and p27, decreased the expression of c-Myc concentration dependently. In addition, NAC was also employed to analyze the relationship between cell cycle arrest and reactive oxygen species production. As shown in Fig. 4C and D, pretreatment with NAC could greatly block S-phase arrest caused by PHII-7 and abrogate the regulation of c-Myc, p21 and p27 by PHII-7, which was also in accordance with our previous results that NAC protected cells from cell growth inhibition and DNA damage (Fig. 1C and Fig. 2C). Above all, we presumed S-phase arrest caused by PHII-7 closely related to reactive oxygen species production and DNA damage. 3.7. PHII-7 induces P-gp downregulation through its reactive oxygen species generation in MDR K562/A02 cells In our preliminary results, P-gp was reported to be overexpressed in the MDR- leukemia cell line K562/A02. Although PHII-7 could down-regulated the expression of P-gp in MDRcancer cell line (Su et al., 2012; Shi et al., 2011), the mechanism remained to be elucidated. According to our results (Fig. 5), the relative mRNA level of mdr-1 decreased on dependence of PHII-7, while NAC could antagonist the effect. JNK was known to mediate the expression of P-gp. Our results unveiled the fact that PHII-7 activated JNK, while NAC inhibited this effect, suggesting reactive
oxygen species may serve as a leading role in JNK activation. And the changes of mean-fluorescence-integrity(MFI) of P-gp(MDR1) detected by flow cytometry confirmed that PHII-7 effectively reduced P-gp expression in a dose- dependent manner while pretreatment with NAC or JNK inhibitor SP600125 could inhibit the effect. More importantly, PHII-7 treatment for 24 h largely inhibited the function of P-gp and thus enhanced rodamine123 uptake while NAC and SP600125 antagonist the above effect. Those results suggest reactive oxygen species may play a vital role in P-gp modulation by PHII-7.
4. Discussion Here, we report for the first time that PHII-7, an indirubin derivative, has shown great potential in reactive oxygen species production. The reactive oxygen species production was evident after 3 h treatment and reached its peak at the point of 1 h treatment in both K562 and K562/A02 (MDR) cell lines. When supplied with GSH (a pan reactive oxygen species scavenger) or NAC (a precursor of GSH), the reactive oxygen species production was inhibited markably. And following this, the cell killing activity of PHII-7 was antagonist in the two cell lines. These results indicated that reactive oxygen species played a critical role in the cytotoxicity of PHII-7. As mentioned above, overproduction of intracellular reactive oxygen species is toxic to cells as they interact with macromolecules, such as proteins, lipids and DNA, and thus initiate cell signaling in a broad variety of cellular processes (Ray et al., 2012; Rassool et al., 2007). In cell models, DNA damage activates ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) proteins, which then activate downstream checkpoint kinases, such as cyclin-dependent kinase 1 (CHK1) and CHK2. In mammals, H2A.X would be phosphorylated by CHK1/CHK2, thus facilitating the recruitment of repair or damage-signaling factors to the sites of DNA damage so that phosphorylation of H2A.X deemed as a hallmark of DNA damage (Podhorecka et al., 2010; Reuter et al., 2010). Above all, these effectors are aimed to stop the progression of cell cycle and to activate proteins responsible for DNA repair. DNA DSBs (double strands breaks, DSBs) is one of the most severe DNA damage and is the prototype of DNA damage caused by reactive oxygen species production (Cai et al., 2008; Olive et al., 1991). Neutral single cell gel electrophoresis assays (also known as comet assay) have been revealed to be a reliable, reproducible and sensitive visual fluorescent technique for assessing DNA DSBs at individual cell levels (Collins, 2004; Gartel and Tyner, 2002). In the two leukemia cell lines, PHII-7 induced DNA DSB after 6 h treatment, right after upregulating the intracellular reactive oxygen species level. And the phosphor-H2A.X also increased in a time- and dose- dependent manner. Similarly as mentioned above, NAC could attenuate DNA DSB occurrence from PHII-7. These results indicated reactive oxygen species production by PHII-7 led to DNA damage. Following DNA damage, the cyclin-dependent kinase (CDK) inhibitor, p21Cip/Waf1 (referred to p21) would be activated, which would block cyclinA-D1, D2, and E-CDK2 activities so that cell cycle would be halted at certain stage(Abbas and Dutta, 2009; O’Reilly, 2005; Masqras et al., 2012; Passos et al., 2010). We then examined the expression of p21 after PHII-7 treatment, and the level of p21 was up-regulated in a time- and dose- dependent manner and the cell cycle arrest was obvious after 6 h 2uM PHII-7 treatment. This confirmed our hypothesis that PHII-7 could alter cell cycle distribution through p53-independent induction of p21 pathway, because p53 was lost in K562 cells (Tian et al., 2009). Once DNA damage occurred, except for p21, p27 would be upregulated to block the interaction and halt the cell cycle.
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Fig. 4. NAC rescued cells from cell cycle arrest caused by PHII-7. (A) PHII-7 induced cell cycle arrest. Cells were treated with various concentrations (0.5–2 mM ) of PHII-7 for 24 h or 2 mM PHII-7 for various time (6–24 h), and then detected cell cycle distribution as described in Materials and Methods. The percentage of cell cycle distribution in G1, S, G2/M were showed as bars. (B) PHII-7 altered the expression of cell-cycle related protein. Cells were treated with various concentrations of PHII-7 and total protein (50 mg) from cell lysates were separated by SDS-PAGE and immunoblotted with c-Myc, p21, p27 antibody. GAPDH was shown as an internal control. (C) PHII-7 alone (2 mM, 24 h) caused severe S-phase arrest in both K562 and K562/A02 while pretreatment with NAC rescued cells from S-phase arrest. (D) Western bolt analysis explained that NAC blocked c-Myc downregulation and p21, p27 upregulation induced by PHII-7.
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Fig. 5. Reactive oxygen species and phosphorylation of JNK contribute to P-gp downregulation. (A) Realtime PCR analysis of relative mdr-1 expression on K562/A02 under different condition. (B) JNK was activated in a dose-dependent manner and western blot analysis showed NAC could block JNK activation in both K562 and K562/A02 cells. Cells were treated with PHII-7(2 mM, 24 h) or preincubated with 5 mM NAC.Total protein (50 mg) from cell lysates were treated as above. (C) Flow cytometry analysis of P-gp expression on K562/A02 in different conditions(0.5–2 mM PHII-7 for 24 h, or 2 mM PHII-7 pretreatment with 5 mM NAC, 5 mM NAC alone and 2 mM PHII-7 pretreatment with JNK inhibitor SP600125). (D) Effect of PHII-7 on the intracellular accumulation of Rhodamine 123. Cells were treated with verapamil(2 mM), PHII-7(0.5, 2 mM) for 24 h, or 2 mM PHII-7 before 5 mM NAC or 10 mM SP600125 treatment for 30 min.
Nevertheless, c-Myc could antagonist the function of p21 and p27, thus promote cell cycle progression in spite of DNA damage (Mitchell and El-Deiry, 1999; Yang et al., 2001). Our research work demonstrated that PHII-7 down-regulated c-Myc, which may facilitate block DNA mutagenesis and halt cell cycle progression in leukemia cells (Lutz et al., 2002). Furthermore, NAC antagonist this effect. Combined with the above results, we presumed c-Myc down-regulation, p21 and p27 up-regulation was all correlated with reactive oxygen species generation by PHII-7. Above all, this suggest reactive oxygen species production is the leading source that caused cell cycle arrest as well as apoptosis induction and thus inhibit cell growth. Taken together, the killing mechanism of PHII-7 which we had identified was summarized in a scheme (Fig. 6). Apoptosis inevitablely occured after continuous reactive oxygen species burst. As it is well known, Bax is a Bcl-2 family member, and can form oligomer on mitochondrial outer membrane. Generally, Bcl-2, an anti-apoptotic protein, neutralizes the function of Bax by binding it in the cytoplasm. Following the increase of reactive oxygen species production, Bax released from Bcl2-Bax heterodimer, then inserted into the outer mitochondrial membrane and oligomerized to presumably form dynamic lipid pores through which lethal proteins were released from the mitochondrial intermembranous space (Jurečeková et al., 2011; Marinou and Youle, 2011; Heitz et al., 2010). As a result, the caspase cascade was activated. Our studies demonstrated Bax was up-regulated after PHII-7 treatment in a time- and dose- dependent manner while Bcl-2 was down-regulated. Bcl-2 is an important anti-apoptosis protein, and previous research work unveiled that reactive oxygen species
overproduction could decrease the level of this protein and thus facilitated apoptosis initiation (Hildeman et al., 2003). Our results were correlated with these above findings. The cellular protein of Bcl-2 gradually decreased after PHII-7 treatment and NAC antagonist this effect. Caspases cleavage triggered by PHII-7 is evident right after 12 h treatment. These results are consistent with our previous results that apoptosis was induced right after reactive oxygen species burst. Additionally, the fact that NAC could rescue K562 and K562/A02 cells from apoptosis, perfectly reflect that reactive oxygen species production was highly correlated with the apoptosis triggered by PHII-7. However, according to our results, there may be other pathways involved, as caspase inhibitor could only partly antagonist cytotoxicity of PHII-7. Especially, our data showed that the reactive oxygen species production by PHII-7 equally contributed to inhibit the cell growth both in the K562 and its MDR counterpart cell line, K562/A02, which indicated intrinsic relationship between reactive oxygen species production and MDR inhibition. JNK is an important member of MAPK superfamily, recently researchers have unveiled the relationship between JNK activation and P-gp regulation (Miao and Ding, 2003; Cai et al., 2007). Our results demostrated that PHII-7 down-regulated the expression of P-gp through JNK activation by increasing reactive oxygen species production. To summarize, in this paper, we have identified the anti-tumor mechanism of PHII-7 in two leukemia cell lines (K562 and its MDR- counterpart, K562/A02), and the reactive oxygen species production played a vital role in it. Particularly, through demonstrating the contribution of reactive oxygen species induced by PHII-7 to its anti-MDR activity, the current study has extended our
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Fig. 6. A detailed model for PHII-7as an antitumor agent.
prior related work and given new insights into the multifaceted features of this compound for cancer therapy. Although it is well accepted that many chemo-therapeutic agents could increase reactive oxygen species production, which may help in killing cancer cells (Flora, 2011), constitutive up-regulation of reactive oxygen species may cause DNA damage and ultimately lead to genome instability that not only results in carcinogenesis and also induces resistance to conventional chemo-therapeutic agents (Ozben, 2007). PHII-7 seems not having this kind of problem, with the mechanism remained to be explored. As a natural compound which has dual roles, intracellular reactive oxygen species production and P-gp inhibition, fully displaying cytotoxic effect of PHII-7 would not only benefit in further clinical application but also in developmenting new strategies in cancer treatment.
Acknowledgments This research was supported by the National Natural Science Foundation of China (Nos. 30873091, 30971291). Conceived and designed the experiments: HP, XY, DF, YZ, DX. Performed the experiments: HP, XY, SR, RS, CY, YD, YL. Drafting the article and revising it critically for important intellectual content: HP, FY, DX. Final approval for the version to be submitted: YZ, DX. All the authors declare no conflict of interests. References Abbas, T., Dutta, A., 2009. p21 in cancer: intricate networks and multiple activites. Nature Reviews Cancer 9, 400–414.
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
PHII-7 inhibits cell growth and induces apoptosis in leukemia cell line K562 as well as its MDR- counterpart K562/A02 through producing reactive oxygen species Hongwei Peng a,b, Xiangfei Yuan a, Ruizan Shi c, Xiaohua Wei b, Simei Ren d, Cihui Yan a, Yahui Ding a, Yang Lin a, Dongmei Fan a, Ming Yang a,nn, Yanjun Zhang a, Dongsheng Xiong a,n a State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, PR China b Department of Pharmacy, First affiliated Hospital of Nanchang University, Nanchang, Jiangxi, PR China c Department of Pharmacology, Shanxi Medical University, Taiyuan, Shanxi, PR China d Beijing Hospital of the Ministry of Health, National Center for Clinical Laboratories, Beijing, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 21 January 2013 Received in revised form 13 July 2013 Accepted 16 July 2013 Available online 30 July 2013
Multidrug resistance (MDR) is a major obstacle that hinders the efficacy of chemotherapy in many human malignancies. PHII-7 is a derivative of indirubin, which was designed and synthesized by our laboratory. Our preliminary work indicated its potent antitumor activities in vitro and in vivo. Furthermore, based on the model of MDR cell line, we found its powerful effects in inhibiting the expression of P-glycoprotein (P-gp) and killing multidrug-resistant (MDR) cells with the detailed mechanism remained to be explored. Reactive oxygen species are known for high reactive activity as they possess unmatched electrons. In this study, we showed that PHII-7 generated equal reactive oxygen species in parental K562 and its counterpart MDR K562/A02 cells. Pre-incubation with thiol antioxidants glutathione or N-acetyl-cysteine(NAC) almost abolished the cytotoxicity of PHII-7. Moreover, NAC abrogated DNA damage, cell cycle arrests and apoptosis induced by PHII-7. Our results collectively indicated that reactive oxygen species production induced by PHII-7 contributed to both apoptosis and cell cycle arrets in MDR K562/A02 cells, thus extending our prior related findings. Notably, JNK phosphorylation was also induced by PHII-7 and pre-incubated of K562/A02 cells with NAC or inhibitor of JNK(SP006125) eliminated P-gp downregulation. Taken together, our results may provide a detailed biochemical basis for further clinical application of PHII-7. & 2013 Elsevier B.V. All rights reserved.
Keywords: Reactive oxygen species PHII-7 DNA damage P-glycoprotein (P-gp) Apoptosis Cell cycle arrest
1. Introduction PHII-7 is a derivative of indirubin that designed and synthesized by our laboratory. Our previous study has shown PHII-7 exerted cytotoxic effect in multiple cancer cell lines that had different origins. Interstingly, PHII-7 was also effective in MDR-(multidrug resistance) cancer cell lines (Su et al., 2012; Shi et al., 2011). In this study, we will explore the killing mechanism of PHII-7 in the K562 leukemia cell line, as well as its MDR counterpart K562/A02 cells. Reactive oxygen species, including the superoxide anion, hydrogen peroxide and hydroxyl radical, is known to mediate apoptosis induced by some cancer chemopreventive and therapeutic agents. n
Corresponding author. Tel./fax: +86 22 23909404. Corresponding author. Tel.: +86 22 23909025. E-mail addresses: [email protected] (X. Wei), [email protected] (M. Yang), [email protected], [email protected] (D. Xiong). nn
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.07.038
Intracellular reactive oxygen species may interact with cellular membrane lipids, proteins, DNA and cause oxidative injury (Davis et al., 2001; Valko et al., 2006). Mitochondria is the main source of reactive oxygen species production, and it is also highly enriched with antioxidants and enzymes, such as glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GPx) which are present on both sides of the membranes in order to minimize oxidative stress (Landriscrina et al., 2009; Montero and Jassem, 2011). In normal cells, reactive oxygen species is continuously generated throughout the life span and facilitates cell proliferation, and a redox balance is maintained by the reactive oxygen species system and the anti-oxidative system. However, tumor cells are particularly sensitive to oxidative stress as they typically have persistently higher levels of reactive oxygen species than normal cells. Therefore, as the result of increasing the intracellular reactive oxygen species production or depleting the antioxidant proteins, the dysregulation of redox balance is developed in cancer cells (Ozben, 2007; Reuter et al., 2010). We suppose that the killing effect of PHII-7
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would be related to the reactive oxygen species, and the hypothesis is tested in this research work. Chemotherapy is one of the effective means to treat cancer, especially hematologic maligancies and solid tumor with highly metastasis characteristics. However, the development of multidrug resistance in cancer cells always hinders the efficacy of conventional chemotherapeutic agents in patients. Since more attention had been paid to the anti-MDR activity of PHII-7, we explored its cell killing mechanism deeply as well as the relationship between reactive oxygen species production and its anti-MDR effect in this research work.
2. Materials and methods 2.1. Cell lines and cell culture The human leukemia cell line K562 was obtained from American Type Culture Collection (ATCC, Rockville, MD), and its multidrug resistance counterpart K562/A02 were conserved by our laboratory (Yang et al., 1995). Cells were grown in RPMI 1640 medium supplemented with 10%(v/v) heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin (GIBCO, Grand Island, NY) at 37 1C in a 5% CO2 humidified atmosphere. K562/A02 cells were routingly maintained in medium containing 200 mg/l doxorubicin and incubated in drug-free medium for at least one week prior to experimental use. 2.2. Materials PHII-7 was synthesized in our laboratory and freshly dissolved in dimethyl sulfoxide (DMSO). Before use, PHII-7 was diluted with cultured medium to the desired concentrations. The final concentration of DMSO was less than 0.1%. Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Dojindo Molecular Technologies, Japan). DCFH2-DA, GSH and NAC were purchased from Beyotime (Beyotime Institute of Biotechnology, China). Antibodies against PARP-1, Caspase-3, Caspase-9, Bax, p21, and p27, Phosphorylated H2A.X, H2A.X and GAPDH were obtained from Cell Signaling Techonology (Beverly, MA); antibody against bcl-2 was purchased from Epitomics (Epitomics, Inc, U.S.A.).
selected cells per slide with the Komet 5.5 software (Kinetic Imaging Ltd., Nottinghan, UK) using the parameter olive tail moment (arbitraty units, defined as the product of the percentage of DNA in the tail multiplied by the tail length).
2.6. Apoptosis assesments by Annexin V-FITC and PI staining The Annexin V-FITC-labeled Apoptosis Detection Kit I (BD Biosciences, Pharmingen, San Diego, CA, USA) was used to detect and quantify apoptosis by flow cytometry according to the manufacturer's instructions. In brief, K562 and K562/A02 cells (1 105 cells/ml) were seeded in 6-well plates and then indicated concentrations of PHII-7 were added. After treatment, cells were harvested and washed with ice-cold PBS. The cell pellets were resuspended in 100 ml ice-cold binding buffer. 5 ml annexin V-FITC solution and 5 ml dissolved PI were added to cell suspensions. The samples were mixed gently and incubated at room temperature (25 1C) for 15 min in the dark. Then another 300 ml binding buffer was added and mixed gently before the cell preparations were examined by a flow cytometer (BD, LSRII). Annexin V-positive, PI-negative cells were recorded as apoptotic. Double-stained cells were considered either as necrotic or as late apoptotic (Liu et al., 2010).
2.7. Cell cycle analysis The cell cycle distribution was detected by the Cycle Test TM plus DNA reagent kit (Beckman Dickson, USA) as described by the manufacturer's instruction. Briefly, cells (1 105 cells/ml) were preincubated with or without 5 mM NAC for 30 min before treated with PHII-7. After treatment, cells were harvested, washed the cell pellet with 1 mL Buffer Solution twice. Then 250 μl solution A was added to resuspend the cell pellet. After 10 min-incubation at room temperature, 200 μl solution B was added and mixed gently, incubated at room temperature for 10 min; At last, 200 μl ice-cold solution C was added and incubated at 4 1C in the dark for 10 min. Cell cycle was measured with a flow cytometry (BD, LSRII).
2.3. Cytotoxity assays
2.8. Realtime PCR analysis
Cell viability was assessed by CCK-8 kit. Cells were harvested and then seeded at a density of 1 105/ml, ADR, PHII-7, NAC or Vitamine E was added at its required final concentration. After incubation for 24 h, CCK-8 reagent was added, and then read the plate at 450 nm by a microplate hybrid reader (Synergy H4, Biotek, USA). Cell viability was calculated as follows: A450treated/A450control 100%.
Real-time PCR was performed with SYBR Green Master Mix (Applied Biosystems, CA), using a 7500 Real-time PCR System (Applied Biosystems). The quantitative measurement of each gene was normalized to the amount of GAPDH cDNA. Primers used in realtime PCR are as follows: 5′-AGCGAGCAACTGAGAA GC-3′ (sense) and 5′-CGCTGTGAAGCAGAGCTGG-3′ (antisense) for the c-fos gene (to generate an 83 bp fragment); 5′-AGTCAACGGATTTGGTCGTA-3′(sense) and 5′-GGAACATGTAAACCATGTAG-3′ (antisense) for the GAPDH gene (to generate a 122 bp fragment). Primers of MDR1 used for real-time PCR were the same as that for RT-PCR. The GAPDH gene was used as an endogenous control to normalize the mRNA values in each sample. The relative values were determined by the comparative computed tomography analysis method.
2.4. Reactive oxygen species measurement Accumulation of intracellular reactive oxygen species was detected with the probe DCFH2-DA. In brief, cells were preincubated with or without 5 mM NAC preincubated for 30 min. After treated with PHII-7 for indicated time, cells were labeled with 10 mM DCFH2-DA and incubated for additional 30 min at 37 1C in a humidified atmosphere at 5% CO2. The fluorescence intensity was measured by a flow cytometry (BD, LSRII).
2.9. Statistical analysis 2.5. Neutral single cell gel electrophoresis assays DNA DSBs (double strands breaks, DSBs) were evaluated using neutral comet assays as previously described (Olive et al., 1990). Quantitation was achieved by analyzing at least 50 randomly
All results were presented as the means 7S.D. of results from three cultures and the significant difference was analyzed by Student's t-test. Probability values of Pb0.05 were considered to be statistically significant.
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and K562/A02 leukemia cells with IC50 values of 3.45 70.98 and 110.01 715.67 mmol/l respectively, so K562/A02 cells were tested more than 30-fold resistant to ADR in comparison with its parental K562 cells.
3. Results 3.1. Resistant fold of K562/A02 K562 and K562/A02 were treated with different concentrations of ADR for 24 h and the cytotoxicity was determined by CCK-8 assay. As shown in Table 1, ADR exerted cytotoxity against K562 Table 1 Cytotoxicity of adriamycin on K562 and K562/A02. Cell lines
IC50 of ADR
K562 K562/A02
3.45 7 0.98 110.017 15.67nn
nn
po 0.01.
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3.2. Effects of PHII-7 on cell viability Both the two cell lines were treated with PHII-7 at concentrations ranging from 0 to 10 mM for 24 h. Cell viability was determined by CCK-8 assay and appeared to decline gradually in a dose- dependent manner (Fig. 1B). In contrast, both the parental and the MDR cell lines showed no obvious difference to PHII-7 with IC50 values of 2.567 0.23 and 2.08 70.31 mmol/l respectively. These results indicated that PHII-7 had almost the same efficacy against both the chemo-sensitive parental and MDR leukemia cells. Additionally, Fig. 1C clearly demonstrate that the
Fig. 1. The cytotoxicity of PHII-7. (A) The chemical structure of PHII-7. (B) The cytotoxic effects of PHII-7 on K562 as well as its MDR- counterpart K562/A02 (For 24 h PHII-7 treatment). (C) Antioxidants rescued both MDR K562/A02 and parental K562 cells from the cytotoxicity of PHII-7. Cells were incubated with various antioxidants for 30 min, then exposed to 1 mM or 2 mM PHII-7 for another 24 h. Cell viability was assessed by CCK-8 assays. (D) Different concentrations of Vitamine E rescued K562 and K562/A02 cells that treated with PHII-7. Cells were incubated with Vitamine E for 30 min, then exposed to 2 mM PHII-7 for another 24 h. Cell viability was assessed by CCK-8 assays. The data were expressed as expressed as mean7 S.D., n¼3. The concentrations for different antioxidants were: NAC, 5 mM; GSH, 5 mM.
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antioxidants, NAC or GSH, could completely antagonist cell killing effect by PHII-7. Moreover, another antioxidant, Vitamine E, could also block the cytotoxic effect of PHII-7 in the manner of concentration dependently (Fig. 1D). All of these data suggest oxidative stress play roles in cytotoxic effect of PHII-7 in K562 as well as its MDR counterpart K562/A02.
determined by using the DCFH2-DA fluorescence probe. Firstly, the DCF fluorescence gradually increased in a time- dependent manner. The change of fluorescence intensity was visual at 3 h, and reached the peak value at 11 h (Fig. 2A). Pretreatment with NAC significantly reduced reactive oxygen species production by PHII-7 (Fig. 2B). These results indicated PHII-7 increased intracellular reactive oxygen species production in both K562 and K562/A02 cells.
3.3. Measurement of reactive oxygen species production 3.4. DNA damage caused by reactive oxygen species production Based on the fact that NAC and GSH could inhibit the cytotoxic effect of PHII-7, we further detected reactive oxygen species production after PHII-7 treatment. The two cell lines were treated with PHII-7 and the intracellular reactive oxygen species production was
Reactive oxygen species possess high reactive potential for habouring unmatched electron, over production of reactive oxygen species would cause macromolecule damage (Lu et al., 2005;
Fig. 2. PHII-7 equally elicits intracellular Reactive Oxygen Species production and DNA damage in both MDR K562/A02 and parental K562 cells. (A) Cells were exposed to 2 mM PHII-7 for various times(0.5–24 h). Intracellular Reactive Oxygen Species were assessed by flow cytometry with DCFH2-DA staining as described in Materials and Methods. The data from three separate experiments were expressed as mean 7 S.D. Reactive Oxygen Species production increase was visual at 2 h treatment and reached its peak at 11 h treatment. (B) NAC antagonist Reactive Oxygen Species production. Once cells were preincubated with 5 mM NAC for 30 min, Reactive Oxygen Species production increase that induced by 2 mM PHII-7 11 h treatment was significantly reduced. (C) PHII-7 equally induced DNA double strands breaks (DSBs) in both K562 and K562/A02. 2 mM PHII-7 incubated cells for 6 h with or without the pretreatment of 5 mM NAC. DNA DSBs were assessed by neutral single cell gel electrophoresis(comet assay) as described in Material and Methods. Cells were stained with DAPI(1 mg/ml) and photographed by fluorescence micreactive oxygen speciescope. (D) Comet assay analysis. For DNA damage quantification, tail moment in comet assay was assessed by Comet 5.5 software as described in Material and Methods. (E) Western blot analysis confirmed DNA damage. Cells were treated with 2 mM PHII-7 for various times (6–24 h) or various concentrations (0.5–2 mM) of PHII-7 for 24 h. Equal amounts of protein (50 mg) from cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-H2A.X or anti-H2A.X antibody. GAPDH was shown as an internal standard.
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Lee et al., 2009). Neutral single cell gel electrophoresis assays (also known as comet assay) have been used to assess DNA DSBs (double strands breaks) at individual cell levels. In our experiment, both the two cell lines were treated with 2 mM PHII-7 for 6 h and analyzed with the ‘comet assay’. Using this assay, we found that
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PHII-7 produced similar levels of DNA DSBs in both MDR K562/A02 cells and parental K562 cells (Fig. 2C). The parameter used to evaluate DNA damage was tail moment. Fig. 2D showed that PHII-7 treatment caused an increase in tail moments (Po0.05), indicating DNA damage in PHII-7-treated cells. As a hallmark of DNA damage
Fig. 3. PHII-7 induced apoptosis in K562 and K562/A02 and NAC antagonist this effect. (A) Apoptosis analysis. Cells were treated with 2 mM PHII-7 for various times and stained by PI/Annexin V and then detected by flow cytometry. (B) Western blot analysis confirmed apoptotic signaling initiation. Cells were treated with 2 mM PHII-7 for different concentrations. Equal amounts of protein (50 mg) from cell lysates were separated by SDS-PAGE and immunoblotted with anti-PARP, caspase-9, bax, bcl-2 antibody. GAPDH was shown as an internal control. (C) NAC antagonist apoptosis induced by PHII-7. 2 mM PHII-7 incubated for 24 h induced significantly apoptosis while NAC pretreatment antagonist apoptosis in K562 as well as K562/A02. (D) Confocal micreactive oxygen speciescope photograph clearly showed apoptotic body after 2 mM PHII-7 24 h treatment and NAC pretreatment for 30 min could block this effect. (E) Western blot perfectly explained that NAC pretreatment before 2 mM PHII-7 incubation antagonist apoptosis signaling. (F) CCK-8 assay demonstrated that antioxidants GSH and NAC(precursor of GSH) totally rescued both MDR K562/A02 and parental K562 cells from the cytotoxicity of PHII-7 while caspase inhibitor could partly rescued cells, indicating besides apoptosis, there is another pathway involved in cytotoxic effect of PHII-7.
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(Burma et al., 2001), the phosphorylation of Ser139-H2A.X was presented a dose- and time- dependent accumulation in the treated K562/A02 and its parental K562 cells by PHII-7. Taken together, these findings suggest that PHII-7 causes DNA damage leading to serine 139 phosphorylation of H2A.X, which is also known as an early marker of apoptosis induction. 3.5. Apoptosis initiation through reactive oxygen species production In order to confirm whether PHII-7 could induce apoptosis in cells, we employed flow cytometry and western blot analysis. According to Fig. 3A and B, PHII-7 induced apoptosis in a time- and dose- dependent manner both in K562 and K562/A02 cell lines and initiated apoptotic signaling. Next, we investigated the relationship between reactive oxygen species and apoptosis both induced by PHII-7. As shown in Fig. 3C, when treated with PHII-7 alone, obvious and almost equally apoptosis was observed in K562/A02 and K562 cells. Once pretreated with NAC, apoptotic rate was greatly reduced. In addition, NAC alone has no toxicity to both the two cell lines. Apoptotic bodies were observed with confocal microscope when treated with 2 mM PHII-7 for 24 h. When pretreated with NAC, apoptotic bodies were greatly diminished (Fig. 3D). And the caspase activation by PHII-7 was reversed by NAC too (Fig. 3E). These results indicated NAC abrogated apoptotic cell death induced by PHII-7. Moreover, we compared the influence of caspase inhibitor and reactive oxygen species scanverger on the cytotoxicity of PHII-7. According to Fig. 3F, NAC could completely abrogate the cytotoxicity of PHII-7 while the caspase inhibitor could only partly block the effect. These results suggest that besides apoptosis, there were other signaling pathways involved in the cytotoxic effect of PHII-7, and increased reactive oxygen species production may be the main contributor to cell growth inhibition by PHII-7. 3.6. Reactive oxygen species production was correlated with cell cycle arrest Cell cycle distribution was evaluated by flow cytometry. As shown in Fig. 4A, PHII-7 treatment caused S-phase arrest in K562/ A02 and K562 cell lines in a dose- and time- dependent manner. Moreover, the expression of cell cycle related protein, c-Myc, p21, p27 were altered. As shown in Fig. 4B, PHII-7 increased the expression of p21 and p27, decreased the expression of c-Myc concentration dependently. In addition, NAC was also employed to analyze the relationship between cell cycle arrest and reactive oxygen species production. As shown in Fig. 4C and D, pretreatment with NAC could greatly block S-phase arrest caused by PHII-7 and abrogate the regulation of c-Myc, p21 and p27 by PHII-7, which was also in accordance with our previous results that NAC protected cells from cell growth inhibition and DNA damage (Fig. 1C and Fig. 2C). Above all, we presumed S-phase arrest caused by PHII-7 closely related to reactive oxygen species production and DNA damage. 3.7. PHII-7 induces P-gp downregulation through its reactive oxygen species generation in MDR K562/A02 cells In our preliminary results, P-gp was reported to be overexpressed in the MDR- leukemia cell line K562/A02. Although PHII-7 could down-regulated the expression of P-gp in MDRcancer cell line (Su et al., 2012; Shi et al., 2011), the mechanism remained to be elucidated. According to our results (Fig. 5), the relative mRNA level of mdr-1 decreased on dependence of PHII-7, while NAC could antagonist the effect. JNK was known to mediate the expression of P-gp. Our results unveiled the fact that PHII-7 activated JNK, while NAC inhibited this effect, suggesting reactive
oxygen species may serve as a leading role in JNK activation. And the changes of mean-fluorescence-integrity(MFI) of P-gp(MDR1) detected by flow cytometry confirmed that PHII-7 effectively reduced P-gp expression in a dose- dependent manner while pretreatment with NAC or JNK inhibitor SP600125 could inhibit the effect. More importantly, PHII-7 treatment for 24 h largely inhibited the function of P-gp and thus enhanced rodamine123 uptake while NAC and SP600125 antagonist the above effect. Those results suggest reactive oxygen species may play a vital role in P-gp modulation by PHII-7.
4. Discussion Here, we report for the first time that PHII-7, an indirubin derivative, has shown great potential in reactive oxygen species production. The reactive oxygen species production was evident after 3 h treatment and reached its peak at the point of 1 h treatment in both K562 and K562/A02 (MDR) cell lines. When supplied with GSH (a pan reactive oxygen species scavenger) or NAC (a precursor of GSH), the reactive oxygen species production was inhibited markably. And following this, the cell killing activity of PHII-7 was antagonist in the two cell lines. These results indicated that reactive oxygen species played a critical role in the cytotoxicity of PHII-7. As mentioned above, overproduction of intracellular reactive oxygen species is toxic to cells as they interact with macromolecules, such as proteins, lipids and DNA, and thus initiate cell signaling in a broad variety of cellular processes (Ray et al., 2012; Rassool et al., 2007). In cell models, DNA damage activates ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) proteins, which then activate downstream checkpoint kinases, such as cyclin-dependent kinase 1 (CHK1) and CHK2. In mammals, H2A.X would be phosphorylated by CHK1/CHK2, thus facilitating the recruitment of repair or damage-signaling factors to the sites of DNA damage so that phosphorylation of H2A.X deemed as a hallmark of DNA damage (Podhorecka et al., 2010; Reuter et al., 2010). Above all, these effectors are aimed to stop the progression of cell cycle and to activate proteins responsible for DNA repair. DNA DSBs (double strands breaks, DSBs) is one of the most severe DNA damage and is the prototype of DNA damage caused by reactive oxygen species production (Cai et al., 2008; Olive et al., 1991). Neutral single cell gel electrophoresis assays (also known as comet assay) have been revealed to be a reliable, reproducible and sensitive visual fluorescent technique for assessing DNA DSBs at individual cell levels (Collins, 2004; Gartel and Tyner, 2002). In the two leukemia cell lines, PHII-7 induced DNA DSB after 6 h treatment, right after upregulating the intracellular reactive oxygen species level. And the phosphor-H2A.X also increased in a time- and dose- dependent manner. Similarly as mentioned above, NAC could attenuate DNA DSB occurrence from PHII-7. These results indicated reactive oxygen species production by PHII-7 led to DNA damage. Following DNA damage, the cyclin-dependent kinase (CDK) inhibitor, p21Cip/Waf1 (referred to p21) would be activated, which would block cyclinA-D1, D2, and E-CDK2 activities so that cell cycle would be halted at certain stage(Abbas and Dutta, 2009; O’Reilly, 2005; Masqras et al., 2012; Passos et al., 2010). We then examined the expression of p21 after PHII-7 treatment, and the level of p21 was up-regulated in a time- and dose- dependent manner and the cell cycle arrest was obvious after 6 h 2uM PHII-7 treatment. This confirmed our hypothesis that PHII-7 could alter cell cycle distribution through p53-independent induction of p21 pathway, because p53 was lost in K562 cells (Tian et al., 2009). Once DNA damage occurred, except for p21, p27 would be upregulated to block the interaction and halt the cell cycle.
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Fig. 4. NAC rescued cells from cell cycle arrest caused by PHII-7. (A) PHII-7 induced cell cycle arrest. Cells were treated with various concentrations (0.5–2 mM ) of PHII-7 for 24 h or 2 mM PHII-7 for various time (6–24 h), and then detected cell cycle distribution as described in Materials and Methods. The percentage of cell cycle distribution in G1, S, G2/M were showed as bars. (B) PHII-7 altered the expression of cell-cycle related protein. Cells were treated with various concentrations of PHII-7 and total protein (50 mg) from cell lysates were separated by SDS-PAGE and immunoblotted with c-Myc, p21, p27 antibody. GAPDH was shown as an internal control. (C) PHII-7 alone (2 mM, 24 h) caused severe S-phase arrest in both K562 and K562/A02 while pretreatment with NAC rescued cells from S-phase arrest. (D) Western bolt analysis explained that NAC blocked c-Myc downregulation and p21, p27 upregulation induced by PHII-7.
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Fig. 5. Reactive oxygen species and phosphorylation of JNK contribute to P-gp downregulation. (A) Realtime PCR analysis of relative mdr-1 expression on K562/A02 under different condition. (B) JNK was activated in a dose-dependent manner and western blot analysis showed NAC could block JNK activation in both K562 and K562/A02 cells. Cells were treated with PHII-7(2 mM, 24 h) or preincubated with 5 mM NAC.Total protein (50 mg) from cell lysates were treated as above. (C) Flow cytometry analysis of P-gp expression on K562/A02 in different conditions(0.5–2 mM PHII-7 for 24 h, or 2 mM PHII-7 pretreatment with 5 mM NAC, 5 mM NAC alone and 2 mM PHII-7 pretreatment with JNK inhibitor SP600125). (D) Effect of PHII-7 on the intracellular accumulation of Rhodamine 123. Cells were treated with verapamil(2 mM), PHII-7(0.5, 2 mM) for 24 h, or 2 mM PHII-7 before 5 mM NAC or 10 mM SP600125 treatment for 30 min.
Nevertheless, c-Myc could antagonist the function of p21 and p27, thus promote cell cycle progression in spite of DNA damage (Mitchell and El-Deiry, 1999; Yang et al., 2001). Our research work demonstrated that PHII-7 down-regulated c-Myc, which may facilitate block DNA mutagenesis and halt cell cycle progression in leukemia cells (Lutz et al., 2002). Furthermore, NAC antagonist this effect. Combined with the above results, we presumed c-Myc down-regulation, p21 and p27 up-regulation was all correlated with reactive oxygen species generation by PHII-7. Above all, this suggest reactive oxygen species production is the leading source that caused cell cycle arrest as well as apoptosis induction and thus inhibit cell growth. Taken together, the killing mechanism of PHII-7 which we had identified was summarized in a scheme (Fig. 6). Apoptosis inevitablely occured after continuous reactive oxygen species burst. As it is well known, Bax is a Bcl-2 family member, and can form oligomer on mitochondrial outer membrane. Generally, Bcl-2, an anti-apoptotic protein, neutralizes the function of Bax by binding it in the cytoplasm. Following the increase of reactive oxygen species production, Bax released from Bcl2-Bax heterodimer, then inserted into the outer mitochondrial membrane and oligomerized to presumably form dynamic lipid pores through which lethal proteins were released from the mitochondrial intermembranous space (Jurečeková et al., 2011; Marinou and Youle, 2011; Heitz et al., 2010). As a result, the caspase cascade was activated. Our studies demonstrated Bax was up-regulated after PHII-7 treatment in a time- and dose- dependent manner while Bcl-2 was down-regulated. Bcl-2 is an important anti-apoptosis protein, and previous research work unveiled that reactive oxygen species
overproduction could decrease the level of this protein and thus facilitated apoptosis initiation (Hildeman et al., 2003). Our results were correlated with these above findings. The cellular protein of Bcl-2 gradually decreased after PHII-7 treatment and NAC antagonist this effect. Caspases cleavage triggered by PHII-7 is evident right after 12 h treatment. These results are consistent with our previous results that apoptosis was induced right after reactive oxygen species burst. Additionally, the fact that NAC could rescue K562 and K562/A02 cells from apoptosis, perfectly reflect that reactive oxygen species production was highly correlated with the apoptosis triggered by PHII-7. However, according to our results, there may be other pathways involved, as caspase inhibitor could only partly antagonist cytotoxicity of PHII-7. Especially, our data showed that the reactive oxygen species production by PHII-7 equally contributed to inhibit the cell growth both in the K562 and its MDR counterpart cell line, K562/A02, which indicated intrinsic relationship between reactive oxygen species production and MDR inhibition. JNK is an important member of MAPK superfamily, recently researchers have unveiled the relationship between JNK activation and P-gp regulation (Miao and Ding, 2003; Cai et al., 2007). Our results demostrated that PHII-7 down-regulated the expression of P-gp through JNK activation by increasing reactive oxygen species production. To summarize, in this paper, we have identified the anti-tumor mechanism of PHII-7 in two leukemia cell lines (K562 and its MDR- counterpart, K562/A02), and the reactive oxygen species production played a vital role in it. Particularly, through demonstrating the contribution of reactive oxygen species induced by PHII-7 to its anti-MDR activity, the current study has extended our
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Fig. 6. A detailed model for PHII-7as an antitumor agent.
prior related work and given new insights into the multifaceted features of this compound for cancer therapy. Although it is well accepted that many chemo-therapeutic agents could increase reactive oxygen species production, which may help in killing cancer cells (Flora, 2011), constitutive up-regulation of reactive oxygen species may cause DNA damage and ultimately lead to genome instability that not only results in carcinogenesis and also induces resistance to conventional chemo-therapeutic agents (Ozben, 2007). PHII-7 seems not having this kind of problem, with the mechanism remained to be explored. As a natural compound which has dual roles, intracellular reactive oxygen species production and P-gp inhibition, fully displaying cytotoxic effect of PHII-7 would not only benefit in further clinical application but also in developmenting new strategies in cancer treatment.
Acknowledgments This research was supported by the National Natural Science Foundation of China (Nos. 30873091, 30971291). Conceived and designed the experiments: HP, XY, DF, YZ, DX. Performed the experiments: HP, XY, SR, RS, CY, YD, YL. Drafting the article and revising it critically for important intellectual content: HP, FY, DX. Final approval for the version to be submitted: YZ, DX. All the authors declare no conflict of interests. References Abbas, T., Dutta, A., 2009. p21 in cancer: intricate networks and multiple activites. Nature Reviews Cancer 9, 400–414.
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