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XN05, a novel synthesized microtubule inhibitor, exhibits potent activity against human carcinoma cells in vitro
Cancer Letters 285 (2009) 13–22
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
XN05, a novel synthesized microtubule inhibitor, exhibits potent activity against human carcinoma cells in vitro Rui Wu a, Wanjing Ding a, Tao Liu b, Hong Zhu a, Yongzhou Hu b, Bo Yang a,*, Qiaojun He a,* a b
School of Pharmaceutical Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou, Zhejiang 310058, China Zhejiang University-Ecole Normole Superienre Joint Laboratory Medicinal Chemistry, Zhejiang University, Hangzhou 310058, China
a r t i c l e
i n f o
Article history: Received 27 January 2009 Received in revised form 26 March 2009 Accepted 26 April 2009
Keywords: Microtubule inhibitor Antitumor activity Hepatocellular carcinoma cells Cell cycle arrest Apoptosis
a b s t r a c t The present data showed that a novel synthesized compound, N-acetyl-N-(4-(4-methoxyphenyl-3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl)acetamide (XN05), exhibited potent antitumor activity against various cancer cells in vitro. XN05-treatment in human hepatocellular carcinoma cells resulted in the accumulation of G2/M phase cells and finally induced apoptosis assessed by flow cytometry analysis. Western blot and immunofluorescence experiments indicated that XN05 depolymerized microtubules similar to the effect of combretastatin-A4. In addition, XN05-treatment influenced the expression of cell cycle and apoptosis related proteins in BEL-7402 cells, which was associated with the appearance of phosphorylated Bcl-2. Taken together, all the data demonstrated that XN05 exhibited its antitumor activity through disrupting the microtubule assembly, causing cell cycle arrest and consequently inducing apoptosis in BEL-7402 cells. Therefore, the novel compound XN05 is a promising microtubule inhibitor that has great potentials for therapeutic treatment of various malignancies. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Microtubules, a group of cytoskeletal protein, are formed by highly dynamic assemblies of tubulin heterodimers, including a-tubulin and b-tubulin. Microtubules and their dynamics are directly involved in many biological processes, such as mitosis, intracellular transport, maintenance of cell morphology, and signal transduction [1]. Since microtubules play important roles in the regulation of the mitotic progression, disrupting the assembly of microtubules can induce cell cycle arrest in M phase and trigger the signals for programmed cell death [2]. There are two categories of antitubulin compounds used to target highly proliferating malignant cells: promoters and inhibitors of microtubule assembly. The microtubule inhibitors, such as vinca alkaloids, colchicinoids and combretastatin-A4, inhibit tubulin polymerization [3]. In contrast, the * Corresponding authors. Tel./fax: +86 571 88208400 (H. Qiaojun). E-mail addresses: [email protected] (B. Yang), yang924@zju. edu.cn (Q. He). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.04.042
microtubule promoters, such as taxanes and epothilones, promote or stabilize the tubulin polymer form [4]. Combretastatin A-4 (CA-4), a naturally occurring stilbene derived from the South African tree Combretum caffrum [5], shows potent cytotoxicity against a broad spectrum of human cancer cell lines. Despite its strong antimitotic activity by inhibiting microtubule assembly, CA-4 also exhibits notable antiangiogenic activity against the proliferation and survival of cancer cells [6]. Numerous studies on the structure–activity relationships (SAR) of CA4 have confirmed that the cis-orientation between the diaryl groups was essential for its strong cytotoxicity [7]. Based on their SAR, the important 3,4,5-trimethoxyphenyl pharmacophore was retained and its olefinic bond was replaced with non-heterocyclic bridge according to the principal of bioisosterism [8]. In addition, ring B was replaced with substituted phenyl rings or heterodyclic rings [9]. Therefore, such modification produces a series of newly designed and synthesized compounds of CA-4 analogues, some of which have been screened for their antitumor activity. Among these, a novel compound, named XN05
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(Fig. 1), shows its potent antiproliferative activity against human cancer cells. The purpose of this study is to investigate the molecular mechanism underlying XN05’s antitumor action in hepatocellular carcinoma cells. 2. Materials and methods 2.1. Chemicals and antibodies XN05 was synthesized as previously described [10], dissolved in DMSO (22 mM stock solution) and stored at 20 °C. CA-4 was synthesized at the Department of Chemical and Biochemical Engineering, Zhejiang University. 40 ,6Diamidino-2-phenylindole (DAPI) was purchased from Sigma (St. Louis, MO) and dissolved in DMSO (2.5 mg/mL stock solution) and stored at 20 °C. The primary antibodies against cyclin B1, cdc2, CDK7, caspase-3, PARP, Bcl-2, atubulin and b-actin, and HRP-labeled secondary anti-goat, anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ECL, a western blot detection reagent, was purchased from Pierce Biotechnology (Rockford, IL). 2.2. Cell lines All of the cell lines were purchased from the Institute of Cell Biology in Shanghai and grown at 37 °C in a 5% CO2 atmosphere. MCF-7 breast cancer cells were maintained in LG-DMEM (Life Technologies, 2 g/l glucose). PC3 androgen-independent prostate tumor cells, A549 non–small cell lung tumor cells, K562 erythromyeloid tumor cells, SGC-7901 human gastric cells, ECA-109 esophageal cancer cells, BEL-7402 and SMMC-7721 hepatocellular carcinoma cells and HL-7702 human liver cells were maintained in RPMI 1640. All media were supplemented with 10% FCS plus 2 mmol/L of glutamine and 50 units/mL of penicillin. 2.3. Growth inhibition assay The antiproliferative activity of XN05 was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay. Tumor cell lines were seeded in 96-well plates (4 103 per well in 100 ll). After 24 h of incubation in the appropriate medium, cells were treated with different concentrations (4 lM, 2 lM, 1 lM, 0.5 lM, 0.25 lM) of XN05 for another 72 h. Afterwards, MTT solution (5.0 mg/ ml in RPIM-1640, Sigma, St. Louis, MO) was added (20 ll/ well) and then plates were incubated for another 4 h at 37 °C. The purple formazan crystals were dissolved in 100 ll dimethyl sulfoxide (DMSO). After 5 min, the plates were read on a Multiskan Spectrum (Thermo Electron Corporation Marietta, OH) at 570 nm. The IC50 values were obtained using the software of Dose–Effect Analysis with Microcomputers and were defined as concentration of drug causing 50% inhibition in absorbance compared with control cells. Assays were performed in triplicate in three independent experiments. 2.4. Cell-cycle analysis Cell cycle progression was detected using flow cytometry. Hepatocellular cancer cells (5 104 cells/ml, 5 ml) were incubated with various concentrations of XN05 for indicated times. Cells were then collected by centrifugation, washed with phosphate-buffered saline (PBS), and fixed in ice-cold 70% ethanol. The fixed cells were harvested by centrifugation and resuspended in 500 ll of PBS containing 50.0 mg/ml RNase (Amersco, Solon, OH), then incubated at 37 °C for 30 min. After incubation, the cells were stained with 200 mg/ml propidium iodide (PI, Sigma, St. Louis, MO) at 37 °C in dark for 30 min. Flow cytometry was performed on FACScan (BD Biosciences, SanJose, CA), with collection and analysis of data performed using CellQuest software (BD Biosciences). 2.5. Apoptosis analysis The early stage of apoptosis were monitored by annexin V-fluorescein (apoptotic cell marker) and PI (necrotic cell marker) double staining. SMMC-7721 cells were treated with various concentrations of XN05 for 48 h. Then cells were collected and stained with a staining kit (BioVision, Mountain View, CA) according to the manufacture’s instruction. The apoptosis was detected by FACScan flow cytometry (BD Biosciences, SanJose, CA) and analysis of data performed using CellQuest software (BD Biosciences). 2.6. In vitro microtubule polymerization assay
Fig. 1. Chemical structure of CA-4 and its derivative XN05.
This assay was conducted in a 96-well plate (Corning Costar). Purified bovine tubulin (2 mg/ml) was mixed with indicated concentrations of drugs and incubated at 37 °C in 50 ll of reaction buffer [80 mM PIPES, pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, and 20% glycerol], according to the kit instruction manual (Cytoskeleton, Cat.#BK011). The increase of the relative fluorescence unit (RFU) was measured at excitation of 360 nm, emission of 420 nm for indicated time, using the Fluorence microplate reader (Thermo Electron Corporation Marietta, OH) and analysis with Skanlt software version 2.0 in a kinetic model. The augmentation of the relative fluorescence unit indicated the increase in tubulin polymerization. One-hundred
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percent polymerization was defined as the area under the curve of the untreated control [11]. 2.7. Measurement of in vivo microtubule assembly An established method had been included to measure assembled (polymerized) tubulin. BEL-7402 cells (1 106) were lysed in 50 ll of a hypotonic buffer [1 mM MgCl2, 2 mM EGTA, 0.5% NP40, 2 mM phenlmethysulfonylfluoride, 1 mM orthovanadate, 20 ll of protease inhibitor mixture (Sigma, St. Louis, MO), and 20 mM Tris–HCL, pH 6.8] and centrifuged at 12,000g at 4 °C for 10 min. This yielded soluble tubulin dimers in the supernatant and polymerized microtubules in the pellet. Equal amounts of the two fractions (on a protein basis) were partitioned by SDS–PAGE. Immunoblots were probed with a-tubulin monoclonal antibody and secondary HRP-conjugated antibody [12]. Proteins were visualized using enhanced chemiluminescence (ECL) Western Blotting detection reagents (Amersham Biosciences, Piscataway, NJ). 2.8. Immunofluorescence microscopy BEL-7402 cells grown (4 103 per well in 100 ll) and treated with various concentrations of XN05 in 96-well plates were fixed with 4% formaldehyde in PBS for 15 min, washed two times with PBS, and blocked with 5% Fetal serum for 10 min at room temperature. Microtubules were detected with a monoclonal anti-a-tubulin antibody diluted 1:50 in 0.1% Triton X-100 overnight at 4 °C and FITC conjugated secondary antibody (Sigma, St. Louis, MO) diluted 1:100 in PBS for 1 h at 37 °C. Cells were then washed three times in PBS and stained with DAPI diluted 1:1000 in PBS then imaged with Leica DMI 400B fluorescence microscope. 2.9. Western blot analysis After treatment, cell pellets were collected and lysed in a lysis buffer [150 mM NaCl, 50 mM Tris–HCl pH 8.0, 2 mM ethylene glycol-bis(b-aminoethyl ether), 2 mM EDTA, 25 mM NaF, 25 mM b-glycerophosphate, 0.2% Triton X100, 0.3% Nonidet P-40, and 0.1 mM phenylmethylsulfonyl fluoride]. Total protein concentrations of whole cell lysis were determined using BioRad BCA method (PIERCE, Rockford, IL). Equal amounts of protein sampled from whole cell lysis were subjected to electrophoresis on 10–12% Tris– Glycine pre-cast gels (Novex, San Diego, CA) and electroblotted onto Immobilon-P Transfer Membrane (Millipore Corporation, Billerica, MA), and probed with primary antibodies and then incubated with a horseradish peroxidase (HRP) conjugated secondary antibodies. Proteins were visualized using enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ). Densitometric analyses of expression of cyclin B1 relative to the untreated control was performed using Quantity One software (BD Biosciences).
the SD of the mean was calculated. The significance of differences between the values of the groups was determined with unpaired Student’s t test. Significance levels were set at p < 0.05. 3. Results 3.1. XN05 exhibited antiproliferative activity against human cancer cells MTT assay was used to evaluate the antiproliferative effect of XN05 in various human cancer cells including leukemia (K562), breast cancer (MCF-7), lung cancer (A549), stomach cancer (SGC-7901), esophagus cancer (ECA-109), hepatoma tumor (BEL-7402 and SMMC-7721) cells and human normal liver cells HL-7702. Table 1 showed the IC50 values of XN05 tested in six human cancer cell lines representing several solid tumor cells and leukemia cells. All the tested cancer cell lines showed susceptibility to XN05 with IC50 values ranging from 0.067 to 1.84 lM. Furthermore, IC50 values in Table 2 also indicated that BEL-7402 cells were 10-fold more sensitive to XN05 than to CA-4. However, XN05 exhibited equivalent antiproliferative activity with CA-4 in another hepatocellular carcinoma SMMC-7721 cells. Moreover, XN05 exhibited less effect on normal human liver cells HL-7702 with an IC50 of 14.55 ± 3.56 lM compared with CA-4, an IC50 of 1.82 ± 0.26 lM. Such higher tumor-selective activity of XN05 compared with CA-4 made it a promising compound in treating hepatocellular carcinoma (HCC) cells. 3.2. XN05 caused disruption of tubulin polymerization To investigate whether XN05 inhibits microtubule assembly in vitro, tubulin polymerization assay kit was used to assess microtubule assembly in a cell-free system. Fig. 2A shows that in control samples, the fluorescence intensity increased in a time-dependent manner and finally reached a plateau, reflecting the normal process of polymerization. However, in the presence of 0.75 lM CA-4, or its derivatives XN05, the tubulin polymerization was inhibited compared to that of the control samples. Our data further revealed that XN05 was still effective at dose of 0.2 lM, although its ability to inhibit microtubule assembly at such a low dose was attenuated compared to that of high doses. Therefore, our data demonstrated that XN05 induced tubulin depolymerization in a concentration-dependent manner. The effect of XN05 on the dynamics of microtubule assembly was also examined at the cellular level. As shown in Fig. 2B, the enhanced polymerization of tubulin was observed in BEL-7402 cells treated with 0.5 lM microtubule promoter Taxol for 24 h compared with the control cells, whereas cells treated with 0.5 lM CA-4 or its derivatives XN05 showed a decrease in the polymer form of tubulin. Furthermore, immunofluorenscence techniques was used to evaluate the effect of XN05 on
Table 1 IC50 values in different cell lines measured via MTT assay. Cell line
Cell type
IC50 (lM)
K562 SGC-7901 PC3 ECA-109 MCF-7 A549
Human immortalize myelogenous leukemia Human gastric cell Human prostate cancer cell Esophageal cancer cell Breast cancer cell Non-small cell lung cancer
0.067 0.60 0.25 0.89 1.84 1.04
Table 2 Antiproliferative activity of XN05 against hepatocellular cells. Hepatocellular cells
2.10. Statistical analysis For all parameters measured, the values for all samples in different experimental conditions were averaged, and
SMMC-7721 BEL-7402 HL-7702
Growth inhibition (IC50) (lM) CA-4
XN05
0.60 ± 0.07 10.41 ± 0.58 1.82 ± 0.26
0.47 ± 0.27 1.36 ± 0.16 14.55 ± 3.56
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Fig. 2. XN05 inhibits microtubule polymerization. (A) Effect of XN05 on in vitro microtubule polymerization tested in a cell-free system. Purified bovine tubulin (2 mg/ml) was mixed with special reaction buffer and incubated with XN05 (0.1 lM, 0.2 lM and 0.75 lM) and 0.75 lM CA-4 at 37 °C for almost 60 min. The relative fluorescence unit (RFU) was recorded every indicated time. (B) Effect of XN05 on in vivo microtubule polymerization. Polymerized tubulin (insoluble fraction) and unpolymerized tubulin (soluble fraction) were extracted from BEL-7402 cells treated with indicated concentration of XN05 for 24 h. 0.5 lM Taxol and CA-4 were included as positive controls. Cells were lysed to separate insoluble and soluble fractions as described in ‘‘Materials and methods”. Both polymerized tubulin and unpolymerized ones were loaded on SDS–PAGE. After electrophoresis and transfer to nitrocellulose membrane, a-tubulin was visualized by western blot analysis. (C) Effect of XN05 on the organizations of cellular microtubule network. Immunofluorescent staining was used in BEL-7402 cells treated with XN05 for 24 h. Cells were fixed, permeabilized and stained with anti-a-tubulin monoclonal antibody. Cells were analyzed by fluorescence microscopy (400). DAPI was used for nuclear staining.
R. Wu et al. / Cancer Letters 285 (2009) 13–22 microtubule network indirectly. The microtubule network in control cells displayed intact organization and arrangement. Compared with untreated BEL-7402 controls, cells treated with Taxol presented shorter but concentrated microtubules. However, when cells were exposed to various concentrations of XN05 for 24 h, it exhibited filament-like structure and reduced microtubule extent in the cytoplasm (Fig. 2C), which was different from that of Taxol. The microtubule network shrank significantly at 0.25 lM and was disrupted thoroughly at 0.5 lM. These data demonstrated that XN05-treatment could cause the disruption of microtubule assembly.
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3.3. XN05 induced the G2/M arrest and apoptosis BEL-7402 cells were treated with various concentrations of XN05 for 48 h or a single concentration (0.5 lM) from 6 h to 48 h to determine the impact of XN05 on cell cycle progression. We tested the effect of XN05 on cell cycle arrest, using 0.5 lM CA-4 as a positive control for 48 h. As demonstrated in Fig. 3A, CA-4 induced approximately 26.74 ± 1.51% of cells at G2/M phase. In contrast, 0.1–0.5 lM XN05 caused a dramatic increase in the number of cells in G2/M phase, with the percentages rising from 19.43 ± 7.83% to 84.13 ± 1.18% in BEL-7402 cells in a concentration-
A
B
Fig. 3. XN05 induced cell cycle arrest in BEL-7402 cells. (A) XN05 caused G2/M phase arrest in a dose-dependent manner in BEL-7402 cells. Cells were treated with XN05 (0.1, 0.25 and 0.5 lM) for 48 h and analyzed for propidium iodide-stained DNA content by flow cytometry. 0.5 lM CA-4 was involved as a positive control. (B) A time-dependent effect of XN05 on cell cycle progression in BEL-7402 cells. Cells were treated with 0.5 lM XN05 for indicated time (6 h, 12 h, 24 h and 48 h) and analyzed for propidium iodide-stained DNA content by flow cytometry. These results were from one representative experiment of the three independent performance. , p < 0.05, , p < 0.01 versus nontreated control.
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dependent manner. On the other side, treatment of BEL-7402 cells with XN05 (0.5 lM) from 6 h to 48 h induced cell cycle arrest at G2/M phase from 27.31 ± 3.45% to 73.38 ± 4.28% (Fig. 3B). These results suggested that XN05 exerted more potent effect on inhibition of cell growth compared to that of CA-4. Data in Fig. 3 also demonstrates that XN05 induced cell cycle arrest in a concentration- and time-dependent manner in BEL-7402 cells. Similar results were obtained in another hepatocellular cancer cells SMMC-7721 treated with XN05 (Fig. 4A). Treatment with XN05 for 24 h resulted in a concentration-dependent increase of SMMC-7721 cells in the G2/M phase, which was associated with losses of cells in the G1/G0 phase. Previous studies have established that microtubule-inhibiting agents cause G2/M arrest and then trigger apoptosis. Thus, Annexin V-PI double staining method was employed to detect the apoptosis following the cell cycle arrest. Data in Fig. 4B shows that treatment of SMMC-7721 cells with various doses of XN05 increased apoptotic cell population from 16.75 ± 5.67% to 72.47 ± 8.65% in a concentration-dependent manner. The populations of early apoptotic cells and late apoptotic cells both increased and reached a plateau at 0.5 lM. While using PI single staining method data in Fig. 5A also reveals that apoptosis occurred after 72 hexposure to the indicated concentrations of XN05 in BEL-7402 cells, with the percentages of apoptotic cells increasing from 7.74 ± 6.38% to 52.79 ± 0.37%. 3.4. XN05 induced apoptosis through caspase activation Caspase activation plays central role in the execution of apoptosis. Since we showed above that XN05 can cause apoptosis right after cell cycle arrest in two hepatocellular cancer cells, we further examined the expression of the apoptosis related proteins, such as caspase-3, PARP and XIAP in the BEL-7402 cells treated with XN05 (0.1–0.5 lM, 72 h). Results in Fig. 5B shows that the cleavage of the 85-KDa fragment of PARP, a well-known substrate for caspase-3, occured after the concentration of XN05 exceeding 0.25 lM. However, treatment with CA-4 at 0.5 lM did not induce such cleavage form of PARP. In addition, the decrease of procaspase-3 protein and the appearance of its cleaved form (17,19 KDa) were both evident with the concentrations increased from 0.1 lM to 0.5 lM. Moreover, the expression of X-linked inhibitor of apoptosis protein (XIAP), a family of inhibitors of apoptosis proteins (IAPs), was attenuated as compared with that of the control samples. XN05-treated BEL7402 cells showed processing of pro-caspase-3, evident by the detection of the catalytically active fragment of p17. This was associated with increased caspase-3 activity and partial XIAP degradation. However, the XIAP protein content did not change as the concentrations increased. 3.5. XN05 changed the expression of the cell cycle regulatory proteins and Bcl-2 Immunoblotting analysis was used to investigate the underlying mechanisms of cell cycle arrest caused by XN05 in BEL-7402 cells. The cdc2-cyclin B1complex and its activating kinase CDK7 have been reported to play essential roles in G2/M transition. Thus the compounds disturbing G2/M transition probably affect the expression of the regulatory proteins. As shown in Fig. 6A, the protein expression of cyclin B1 gradually elevated upon the treatment of XN05 at the concentrations of 0.1–0.5 lM (48 h), whereas the protein level of cdc2 kinase was decreased, and these two events were associated with the accumulation of cells in the G2/M phase. However, the expression of CDK7, a CDK-activating kinase, was slightly increased under the same condition. Similar results were observed with XN05 at 0.5 lM from 12 h to 48 h (Fig. 6C). After exposure to XN05 with various concentrations or a single concentration (0.5 lM) in different time, the levels of cyclin B1 normalized with the corresponding actin levels were increased according to densitometric analyses (Fig. 6B and 6D). Finally, the phosphorylation of Bcl-2 was also detected after treatment with XN05 at concentrations of 0.25 and 0.5 lM for 48 h, which was accompanied by M phase arrest.
4. Discussion Hepatocellular carcinoma (HCC) is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death, and its incidence is rising
in many countries [13,14]. It is a phenotypically and genetically heterogeneous polyclonal disease and resistant to most conventional chemotherapy [15]. Microtubules are an attractive target for chemotherapeutic agents [16]. This study investigated the effect of XN05, a newly synthesized compound derived from CA-4, on microtubule assembly and hepatocellular carcinoma cell growth. According to the studies of structure–activity relationship, XN05 retained the 3,4,5-trimethyloxyphenyl pharmacophore as its fundamental structure. The restricted rotation of rings A and B of CA-4 can be maintained by introducing suitable conformationally restricted heterocycles, such as imidazole, isoxazole, and indole, many of which showed potent cytotoxicity against various cancer cell lines. A series of cis restricted analogues with imidazol-2-one instead of the cis double bond in CA-4 have been synthesized, among which XN05 is considered to be the most effective compound. Its efficacy was predicted based on the structural analogue of CA-4 that is well-known as a microtubule inhibitor. The potent antiproliferative activity of XN05 was verified in eight human cancer cell lines including leukemia and solid tumors. In hepatocellular cancer cells, XN05 exhibits equivalent or even better antitumor activity compared with CA-4. Notably, XN05 shows less effect on human liver cells in contrast to that of CA-4, which indicating XN05 a novel outstanding CA-4 derivative in treating hepatocellular carcinoma cell in vitro. Drug resistance is a serious problem that limits the clinical application of microtubule interfering drugs. Although the taxanes and the Vinca alkaloids are effective in the treatment of various human cancers, their potentials are limited by the emergence of drug resistance owing to the overexpression of drug efflux pump including P-gp, the decrease of drug accumulation inside the cancer cells and the genetic mutations or post-translational modifications caused structural and functional alterations in the a- and b-tubulins [17]. Therefore, the antitumor activities against those resistant cancer cells of XN05 needs be further investigated. XN05, as a CA-4 derivative, theoretically targets microtubules. To test this hypothesis, the in vivo and in vitro measurements of its effects on microtubule assembly were conducted. Our data clearly demonstrate that XN05 strongly inhibits tubulin polymerization. In addition, western blot and immunofluorescence microscopy, using the anti a-tubulin antibody, confirms that XN05 induces the depolymerization of microtubules and degradation of the microtubule network in liver cancer cells. Thus, XN05 can be classified as a microtubule-depolymerizing agent. Our data also indicate that like the other microtubule inhibitors, XN05 firstly inhibits tubulin polymerization, subsequently induces cell cycle arrest and finally triggers apoptosis in the two tested hepatocellular cancer cells. A concentration- and time-dependent G2/M blockade induced by XN05 is associated with the changed expression of the cell cycle regulatory proteins and the appearance of phosphorylated form of Bcl-2, a marker of M phase events [18]. Regulation of Cdc2 activity is controlled by cyclin B1 binding, phosphorylation of Thr161 by Cdk7,
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A
B
Fig. 4. Cell cycle arrest and apoptosis were detected in another hepatocellular carcinoma cells SMMC-7721. (A) XN05 caused G2/M phase arrest in a dosedependent manner in SMMC-7721 cells. Cells were treated with XN05 (0.1, 0.5 and 1 lM) for 24 h and analyzed for propidium iodide-stained DNA content by flow cytometry. (B) Cells were incubated with XN05 (0.1, 0.5 and 1 lM) for 48 h and apoptosis was analyzed by staining phosphatidylserine translocation with FITC-Annexin V. Annexin V+ cells were represented on the X-axis and the number of cells counted is represented on the Y-axis. Simple vertical bars represent the mean apoptotic rate of SMMC-7721; error bars represent SD Data are mean ± SD (n = 3). , p < 0.05, , p < 0.01, , p < 0.001 versus nontreated control.
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A
B
Fig. 5. Apoptosis was detected in BEL-7402 cells right after the cell cycle arrest caused by XN05. (A) Cells were treated with XN05 (0.1, 0.25 and 0.5 lM) for 72 h and analyzed for propidium iodide-stained DNA content by flow cytometry. Simple vertical bars represent the mean rate of G2/M arrest; error bars represent SD Data are mean ± SD (n = 3). (B) Apoptotic proteins were activated by XN05-treatment. Cells treated with (0.1, 0.25 and 0.5 lM) for 72 h were prepared and loaded on SDS–PAGE. After electrophoresis and transfer to nitrocellulose membrane, proteins were probed with caspase-3, cleaved caspase-3, PARP, XIAP and internal control b-actin antibodies. , p < 0.05, , p < 0.01 versus nontreated control.
and dephosphorylation of Tyr14 and Tyr15 by Cdc25C. Activated cdc2/cyclin B1 complex assembly is necessary for cells in G2/M phase transition and entry into mitosis during the cell cycle progression [19]. There are two steps for cdc2 activation—cyclin-binding and T-loop phosphorylation. Blocking any of these steps may lead to cell cycle arrest and subsequent apoptosis. In this complicated cell cycle signaling pathways, proteins interact actively with each other. In this study, we find that treatment with XN05 from 0.1 to 0.5 lM for 48 h decreases the expression of cdc2, and increases the accumulation of cyclin B1 and cdc2-activating kinase Cdk. Cdk7, a well-known CDK-activating kinase, is also a part of the transcription machinery in the cell cycle. Theoretically, inhibiting Cdk7 in G2 blocks
entry to mitosis, and disrupts Cdk1/cyclin B complex assembly. However, in this study, XN05-treatment slightly increases the expression of Cdk7. This is probably due to the complex interaction between the cell cycle related proteins. Further studies will be required to address the increased expression of Cdk7 by XN05. In addition, we also notice that when BEL-7402 cells were exposed to XN05 at 0.25 lM for 48 h, the phosphorylation of Bcl-2 occurred, which was associated with the increased number of G2/M cells detected by flow cytometry. At a higher concentration of 0.5 lM, XN05-induced phosphorylation of Bcl-2 was still sustained. Although there are always two slower migrating bands existing after treatment with many microtubule-target agents, suggesting the multiple phosphorylation states,
R. Wu et al. / Cancer Letters 285 (2009) 13–22
A
B
C
D
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Fig. 6. (A and C) Change of the expression of cell cycle related proteins in BEL-7402 cells after XN05-treatment. Cells treated with XN05 (0.1, 0.25 and 0.5 lM) for 48 h or 0.5 lM XN05 for indicated time (12 h, 24 h and 48 h) were prepared and loaded on SDS–PAGE. After electrophoresis and transfer to nitrocellulose membrane, proteins were probed with Bcl-2, cyclin B1, cdc2, Cdk7 and the internal control b-actin. (B and D) Densitometric analyses of expression of cyclin B1 relative to the untreated control. p < 0.05 versus nontreated control.
this was not observed in XN05-treated cells, indicating the possibility of subtle differences in Bcl-2 phosphorylation among the microtubule-target agents [20]. It is well accepted that microtubule inhibitors can induce apoptosis caused by cell cycle arrest. Here, our data also reveal that besides its induction of cell cycle arrest, XN05 promotes apoptosis, as indicated by an increase in the sub-G1 population and annexin-V positive. IAPs such as X-linked IAP (XIAP) inhibit the activity of caspases, thereby blocking apoptosis [21]. Caspase-3, a effector caspase, is activated by caspase-8 and -9 to form p19 and p17 subunits, and inhibited by XIAP via their binding on p19–p12 of caspase-3 complex through the BIR2 domain of XIAP [22,23]. However, autocatalytic cleavage of the p19 caspase-3 subunit to a p17-form results in removal of the XIAP-binding site and attenuates XIAP-mediated inhibition of caspase-3 [24]. Here, our results show that XN05-treatment causes the cleavage of the downstream executioner caspase-3 into its active form (p19 and p17), resulting in the cleavage of PARP. In addition, the downregulation of XIAP provides additional evidence of the activation of caspase-3. Therefore, we demonstrate that XN05 evoked apoptosis is mainly executed by the caspase activation. Taxol, a microtubule target agent, has been used in clinical therapy for years. However, its potential is limited by the drug resistance and its adverse effects. No matter what triggers drug resistance, tumors become refractory to a
variety of structurally diverse anticancer drugs [25]. Therefore, drug combinations have been employed to significantly improve treatment outcomes in cancer. In recent years, combinations of Taxol and some other conventional chemotherapeutic drugs (such as cisplatin, doxorubicin, TRAIL) [26,27] have been studied in same cases, they have already used in clinical trials. Combination therapy aims to solve the problem of drug resistance and use every agent at its maximum tolerated dose with lowest toxicity [28]. So based on the above theory, our next goal is to investigate whether XN05, also a microtubule target agent, could be used in drug combination therapy. In conclusion, we have provided convincing evidence to demonstrate that XN05, a novel synthesized compound, possesses potent antitumor activity against human tumor cells via disruption of microtubule. Compared with CA-4, a positive control, XN05 exhibits equivalent or even better antiproliferative activity against human hepatocellular carcinoma cells, indicating that XN05 can be a promising compound against human cancer cells. To confirm the potent of XN05, further experiments will be required to address the anti-MDR property and its drug combination in chemotherapy.
Conflicts of interest None declared.
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Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
XN05, a novel synthesized microtubule inhibitor, exhibits potent activity against human carcinoma cells in vitro Rui Wu a, Wanjing Ding a, Tao Liu b, Hong Zhu a, Yongzhou Hu b, Bo Yang a,*, Qiaojun He a,* a b
School of Pharmaceutical Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou, Zhejiang 310058, China Zhejiang University-Ecole Normole Superienre Joint Laboratory Medicinal Chemistry, Zhejiang University, Hangzhou 310058, China
a r t i c l e
i n f o
Article history: Received 27 January 2009 Received in revised form 26 March 2009 Accepted 26 April 2009
Keywords: Microtubule inhibitor Antitumor activity Hepatocellular carcinoma cells Cell cycle arrest Apoptosis
a b s t r a c t The present data showed that a novel synthesized compound, N-acetyl-N-(4-(4-methoxyphenyl-3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl)acetamide (XN05), exhibited potent antitumor activity against various cancer cells in vitro. XN05-treatment in human hepatocellular carcinoma cells resulted in the accumulation of G2/M phase cells and finally induced apoptosis assessed by flow cytometry analysis. Western blot and immunofluorescence experiments indicated that XN05 depolymerized microtubules similar to the effect of combretastatin-A4. In addition, XN05-treatment influenced the expression of cell cycle and apoptosis related proteins in BEL-7402 cells, which was associated with the appearance of phosphorylated Bcl-2. Taken together, all the data demonstrated that XN05 exhibited its antitumor activity through disrupting the microtubule assembly, causing cell cycle arrest and consequently inducing apoptosis in BEL-7402 cells. Therefore, the novel compound XN05 is a promising microtubule inhibitor that has great potentials for therapeutic treatment of various malignancies. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Microtubules, a group of cytoskeletal protein, are formed by highly dynamic assemblies of tubulin heterodimers, including a-tubulin and b-tubulin. Microtubules and their dynamics are directly involved in many biological processes, such as mitosis, intracellular transport, maintenance of cell morphology, and signal transduction [1]. Since microtubules play important roles in the regulation of the mitotic progression, disrupting the assembly of microtubules can induce cell cycle arrest in M phase and trigger the signals for programmed cell death [2]. There are two categories of antitubulin compounds used to target highly proliferating malignant cells: promoters and inhibitors of microtubule assembly. The microtubule inhibitors, such as vinca alkaloids, colchicinoids and combretastatin-A4, inhibit tubulin polymerization [3]. In contrast, the * Corresponding authors. Tel./fax: +86 571 88208400 (H. Qiaojun). E-mail addresses: [email protected] (B. Yang), yang924@zju. edu.cn (Q. He). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.04.042
microtubule promoters, such as taxanes and epothilones, promote or stabilize the tubulin polymer form [4]. Combretastatin A-4 (CA-4), a naturally occurring stilbene derived from the South African tree Combretum caffrum [5], shows potent cytotoxicity against a broad spectrum of human cancer cell lines. Despite its strong antimitotic activity by inhibiting microtubule assembly, CA-4 also exhibits notable antiangiogenic activity against the proliferation and survival of cancer cells [6]. Numerous studies on the structure–activity relationships (SAR) of CA4 have confirmed that the cis-orientation between the diaryl groups was essential for its strong cytotoxicity [7]. Based on their SAR, the important 3,4,5-trimethoxyphenyl pharmacophore was retained and its olefinic bond was replaced with non-heterocyclic bridge according to the principal of bioisosterism [8]. In addition, ring B was replaced with substituted phenyl rings or heterodyclic rings [9]. Therefore, such modification produces a series of newly designed and synthesized compounds of CA-4 analogues, some of which have been screened for their antitumor activity. Among these, a novel compound, named XN05
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(Fig. 1), shows its potent antiproliferative activity against human cancer cells. The purpose of this study is to investigate the molecular mechanism underlying XN05’s antitumor action in hepatocellular carcinoma cells. 2. Materials and methods 2.1. Chemicals and antibodies XN05 was synthesized as previously described [10], dissolved in DMSO (22 mM stock solution) and stored at 20 °C. CA-4 was synthesized at the Department of Chemical and Biochemical Engineering, Zhejiang University. 40 ,6Diamidino-2-phenylindole (DAPI) was purchased from Sigma (St. Louis, MO) and dissolved in DMSO (2.5 mg/mL stock solution) and stored at 20 °C. The primary antibodies against cyclin B1, cdc2, CDK7, caspase-3, PARP, Bcl-2, atubulin and b-actin, and HRP-labeled secondary anti-goat, anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ECL, a western blot detection reagent, was purchased from Pierce Biotechnology (Rockford, IL). 2.2. Cell lines All of the cell lines were purchased from the Institute of Cell Biology in Shanghai and grown at 37 °C in a 5% CO2 atmosphere. MCF-7 breast cancer cells were maintained in LG-DMEM (Life Technologies, 2 g/l glucose). PC3 androgen-independent prostate tumor cells, A549 non–small cell lung tumor cells, K562 erythromyeloid tumor cells, SGC-7901 human gastric cells, ECA-109 esophageal cancer cells, BEL-7402 and SMMC-7721 hepatocellular carcinoma cells and HL-7702 human liver cells were maintained in RPMI 1640. All media were supplemented with 10% FCS plus 2 mmol/L of glutamine and 50 units/mL of penicillin. 2.3. Growth inhibition assay The antiproliferative activity of XN05 was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay. Tumor cell lines were seeded in 96-well plates (4 103 per well in 100 ll). After 24 h of incubation in the appropriate medium, cells were treated with different concentrations (4 lM, 2 lM, 1 lM, 0.5 lM, 0.25 lM) of XN05 for another 72 h. Afterwards, MTT solution (5.0 mg/ ml in RPIM-1640, Sigma, St. Louis, MO) was added (20 ll/ well) and then plates were incubated for another 4 h at 37 °C. The purple formazan crystals were dissolved in 100 ll dimethyl sulfoxide (DMSO). After 5 min, the plates were read on a Multiskan Spectrum (Thermo Electron Corporation Marietta, OH) at 570 nm. The IC50 values were obtained using the software of Dose–Effect Analysis with Microcomputers and were defined as concentration of drug causing 50% inhibition in absorbance compared with control cells. Assays were performed in triplicate in three independent experiments. 2.4. Cell-cycle analysis Cell cycle progression was detected using flow cytometry. Hepatocellular cancer cells (5 104 cells/ml, 5 ml) were incubated with various concentrations of XN05 for indicated times. Cells were then collected by centrifugation, washed with phosphate-buffered saline (PBS), and fixed in ice-cold 70% ethanol. The fixed cells were harvested by centrifugation and resuspended in 500 ll of PBS containing 50.0 mg/ml RNase (Amersco, Solon, OH), then incubated at 37 °C for 30 min. After incubation, the cells were stained with 200 mg/ml propidium iodide (PI, Sigma, St. Louis, MO) at 37 °C in dark for 30 min. Flow cytometry was performed on FACScan (BD Biosciences, SanJose, CA), with collection and analysis of data performed using CellQuest software (BD Biosciences). 2.5. Apoptosis analysis The early stage of apoptosis were monitored by annexin V-fluorescein (apoptotic cell marker) and PI (necrotic cell marker) double staining. SMMC-7721 cells were treated with various concentrations of XN05 for 48 h. Then cells were collected and stained with a staining kit (BioVision, Mountain View, CA) according to the manufacture’s instruction. The apoptosis was detected by FACScan flow cytometry (BD Biosciences, SanJose, CA) and analysis of data performed using CellQuest software (BD Biosciences). 2.6. In vitro microtubule polymerization assay
Fig. 1. Chemical structure of CA-4 and its derivative XN05.
This assay was conducted in a 96-well plate (Corning Costar). Purified bovine tubulin (2 mg/ml) was mixed with indicated concentrations of drugs and incubated at 37 °C in 50 ll of reaction buffer [80 mM PIPES, pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, and 20% glycerol], according to the kit instruction manual (Cytoskeleton, Cat.#BK011). The increase of the relative fluorescence unit (RFU) was measured at excitation of 360 nm, emission of 420 nm for indicated time, using the Fluorence microplate reader (Thermo Electron Corporation Marietta, OH) and analysis with Skanlt software version 2.0 in a kinetic model. The augmentation of the relative fluorescence unit indicated the increase in tubulin polymerization. One-hundred
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percent polymerization was defined as the area under the curve of the untreated control [11]. 2.7. Measurement of in vivo microtubule assembly An established method had been included to measure assembled (polymerized) tubulin. BEL-7402 cells (1 106) were lysed in 50 ll of a hypotonic buffer [1 mM MgCl2, 2 mM EGTA, 0.5% NP40, 2 mM phenlmethysulfonylfluoride, 1 mM orthovanadate, 20 ll of protease inhibitor mixture (Sigma, St. Louis, MO), and 20 mM Tris–HCL, pH 6.8] and centrifuged at 12,000g at 4 °C for 10 min. This yielded soluble tubulin dimers in the supernatant and polymerized microtubules in the pellet. Equal amounts of the two fractions (on a protein basis) were partitioned by SDS–PAGE. Immunoblots were probed with a-tubulin monoclonal antibody and secondary HRP-conjugated antibody [12]. Proteins were visualized using enhanced chemiluminescence (ECL) Western Blotting detection reagents (Amersham Biosciences, Piscataway, NJ). 2.8. Immunofluorescence microscopy BEL-7402 cells grown (4 103 per well in 100 ll) and treated with various concentrations of XN05 in 96-well plates were fixed with 4% formaldehyde in PBS for 15 min, washed two times with PBS, and blocked with 5% Fetal serum for 10 min at room temperature. Microtubules were detected with a monoclonal anti-a-tubulin antibody diluted 1:50 in 0.1% Triton X-100 overnight at 4 °C and FITC conjugated secondary antibody (Sigma, St. Louis, MO) diluted 1:100 in PBS for 1 h at 37 °C. Cells were then washed three times in PBS and stained with DAPI diluted 1:1000 in PBS then imaged with Leica DMI 400B fluorescence microscope. 2.9. Western blot analysis After treatment, cell pellets were collected and lysed in a lysis buffer [150 mM NaCl, 50 mM Tris–HCl pH 8.0, 2 mM ethylene glycol-bis(b-aminoethyl ether), 2 mM EDTA, 25 mM NaF, 25 mM b-glycerophosphate, 0.2% Triton X100, 0.3% Nonidet P-40, and 0.1 mM phenylmethylsulfonyl fluoride]. Total protein concentrations of whole cell lysis were determined using BioRad BCA method (PIERCE, Rockford, IL). Equal amounts of protein sampled from whole cell lysis were subjected to electrophoresis on 10–12% Tris– Glycine pre-cast gels (Novex, San Diego, CA) and electroblotted onto Immobilon-P Transfer Membrane (Millipore Corporation, Billerica, MA), and probed with primary antibodies and then incubated with a horseradish peroxidase (HRP) conjugated secondary antibodies. Proteins were visualized using enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ). Densitometric analyses of expression of cyclin B1 relative to the untreated control was performed using Quantity One software (BD Biosciences).
the SD of the mean was calculated. The significance of differences between the values of the groups was determined with unpaired Student’s t test. Significance levels were set at p < 0.05. 3. Results 3.1. XN05 exhibited antiproliferative activity against human cancer cells MTT assay was used to evaluate the antiproliferative effect of XN05 in various human cancer cells including leukemia (K562), breast cancer (MCF-7), lung cancer (A549), stomach cancer (SGC-7901), esophagus cancer (ECA-109), hepatoma tumor (BEL-7402 and SMMC-7721) cells and human normal liver cells HL-7702. Table 1 showed the IC50 values of XN05 tested in six human cancer cell lines representing several solid tumor cells and leukemia cells. All the tested cancer cell lines showed susceptibility to XN05 with IC50 values ranging from 0.067 to 1.84 lM. Furthermore, IC50 values in Table 2 also indicated that BEL-7402 cells were 10-fold more sensitive to XN05 than to CA-4. However, XN05 exhibited equivalent antiproliferative activity with CA-4 in another hepatocellular carcinoma SMMC-7721 cells. Moreover, XN05 exhibited less effect on normal human liver cells HL-7702 with an IC50 of 14.55 ± 3.56 lM compared with CA-4, an IC50 of 1.82 ± 0.26 lM. Such higher tumor-selective activity of XN05 compared with CA-4 made it a promising compound in treating hepatocellular carcinoma (HCC) cells. 3.2. XN05 caused disruption of tubulin polymerization To investigate whether XN05 inhibits microtubule assembly in vitro, tubulin polymerization assay kit was used to assess microtubule assembly in a cell-free system. Fig. 2A shows that in control samples, the fluorescence intensity increased in a time-dependent manner and finally reached a plateau, reflecting the normal process of polymerization. However, in the presence of 0.75 lM CA-4, or its derivatives XN05, the tubulin polymerization was inhibited compared to that of the control samples. Our data further revealed that XN05 was still effective at dose of 0.2 lM, although its ability to inhibit microtubule assembly at such a low dose was attenuated compared to that of high doses. Therefore, our data demonstrated that XN05 induced tubulin depolymerization in a concentration-dependent manner. The effect of XN05 on the dynamics of microtubule assembly was also examined at the cellular level. As shown in Fig. 2B, the enhanced polymerization of tubulin was observed in BEL-7402 cells treated with 0.5 lM microtubule promoter Taxol for 24 h compared with the control cells, whereas cells treated with 0.5 lM CA-4 or its derivatives XN05 showed a decrease in the polymer form of tubulin. Furthermore, immunofluorenscence techniques was used to evaluate the effect of XN05 on
Table 1 IC50 values in different cell lines measured via MTT assay. Cell line
Cell type
IC50 (lM)
K562 SGC-7901 PC3 ECA-109 MCF-7 A549
Human immortalize myelogenous leukemia Human gastric cell Human prostate cancer cell Esophageal cancer cell Breast cancer cell Non-small cell lung cancer
0.067 0.60 0.25 0.89 1.84 1.04
Table 2 Antiproliferative activity of XN05 against hepatocellular cells. Hepatocellular cells
2.10. Statistical analysis For all parameters measured, the values for all samples in different experimental conditions were averaged, and
SMMC-7721 BEL-7402 HL-7702
Growth inhibition (IC50) (lM) CA-4
XN05
0.60 ± 0.07 10.41 ± 0.58 1.82 ± 0.26
0.47 ± 0.27 1.36 ± 0.16 14.55 ± 3.56
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Fig. 2. XN05 inhibits microtubule polymerization. (A) Effect of XN05 on in vitro microtubule polymerization tested in a cell-free system. Purified bovine tubulin (2 mg/ml) was mixed with special reaction buffer and incubated with XN05 (0.1 lM, 0.2 lM and 0.75 lM) and 0.75 lM CA-4 at 37 °C for almost 60 min. The relative fluorescence unit (RFU) was recorded every indicated time. (B) Effect of XN05 on in vivo microtubule polymerization. Polymerized tubulin (insoluble fraction) and unpolymerized tubulin (soluble fraction) were extracted from BEL-7402 cells treated with indicated concentration of XN05 for 24 h. 0.5 lM Taxol and CA-4 were included as positive controls. Cells were lysed to separate insoluble and soluble fractions as described in ‘‘Materials and methods”. Both polymerized tubulin and unpolymerized ones were loaded on SDS–PAGE. After electrophoresis and transfer to nitrocellulose membrane, a-tubulin was visualized by western blot analysis. (C) Effect of XN05 on the organizations of cellular microtubule network. Immunofluorescent staining was used in BEL-7402 cells treated with XN05 for 24 h. Cells were fixed, permeabilized and stained with anti-a-tubulin monoclonal antibody. Cells were analyzed by fluorescence microscopy (400). DAPI was used for nuclear staining.
R. Wu et al. / Cancer Letters 285 (2009) 13–22 microtubule network indirectly. The microtubule network in control cells displayed intact organization and arrangement. Compared with untreated BEL-7402 controls, cells treated with Taxol presented shorter but concentrated microtubules. However, when cells were exposed to various concentrations of XN05 for 24 h, it exhibited filament-like structure and reduced microtubule extent in the cytoplasm (Fig. 2C), which was different from that of Taxol. The microtubule network shrank significantly at 0.25 lM and was disrupted thoroughly at 0.5 lM. These data demonstrated that XN05-treatment could cause the disruption of microtubule assembly.
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3.3. XN05 induced the G2/M arrest and apoptosis BEL-7402 cells were treated with various concentrations of XN05 for 48 h or a single concentration (0.5 lM) from 6 h to 48 h to determine the impact of XN05 on cell cycle progression. We tested the effect of XN05 on cell cycle arrest, using 0.5 lM CA-4 as a positive control for 48 h. As demonstrated in Fig. 3A, CA-4 induced approximately 26.74 ± 1.51% of cells at G2/M phase. In contrast, 0.1–0.5 lM XN05 caused a dramatic increase in the number of cells in G2/M phase, with the percentages rising from 19.43 ± 7.83% to 84.13 ± 1.18% in BEL-7402 cells in a concentration-
A
B
Fig. 3. XN05 induced cell cycle arrest in BEL-7402 cells. (A) XN05 caused G2/M phase arrest in a dose-dependent manner in BEL-7402 cells. Cells were treated with XN05 (0.1, 0.25 and 0.5 lM) for 48 h and analyzed for propidium iodide-stained DNA content by flow cytometry. 0.5 lM CA-4 was involved as a positive control. (B) A time-dependent effect of XN05 on cell cycle progression in BEL-7402 cells. Cells were treated with 0.5 lM XN05 for indicated time (6 h, 12 h, 24 h and 48 h) and analyzed for propidium iodide-stained DNA content by flow cytometry. These results were from one representative experiment of the three independent performance. , p < 0.05, , p < 0.01 versus nontreated control.
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dependent manner. On the other side, treatment of BEL-7402 cells with XN05 (0.5 lM) from 6 h to 48 h induced cell cycle arrest at G2/M phase from 27.31 ± 3.45% to 73.38 ± 4.28% (Fig. 3B). These results suggested that XN05 exerted more potent effect on inhibition of cell growth compared to that of CA-4. Data in Fig. 3 also demonstrates that XN05 induced cell cycle arrest in a concentration- and time-dependent manner in BEL-7402 cells. Similar results were obtained in another hepatocellular cancer cells SMMC-7721 treated with XN05 (Fig. 4A). Treatment with XN05 for 24 h resulted in a concentration-dependent increase of SMMC-7721 cells in the G2/M phase, which was associated with losses of cells in the G1/G0 phase. Previous studies have established that microtubule-inhibiting agents cause G2/M arrest and then trigger apoptosis. Thus, Annexin V-PI double staining method was employed to detect the apoptosis following the cell cycle arrest. Data in Fig. 4B shows that treatment of SMMC-7721 cells with various doses of XN05 increased apoptotic cell population from 16.75 ± 5.67% to 72.47 ± 8.65% in a concentration-dependent manner. The populations of early apoptotic cells and late apoptotic cells both increased and reached a plateau at 0.5 lM. While using PI single staining method data in Fig. 5A also reveals that apoptosis occurred after 72 hexposure to the indicated concentrations of XN05 in BEL-7402 cells, with the percentages of apoptotic cells increasing from 7.74 ± 6.38% to 52.79 ± 0.37%. 3.4. XN05 induced apoptosis through caspase activation Caspase activation plays central role in the execution of apoptosis. Since we showed above that XN05 can cause apoptosis right after cell cycle arrest in two hepatocellular cancer cells, we further examined the expression of the apoptosis related proteins, such as caspase-3, PARP and XIAP in the BEL-7402 cells treated with XN05 (0.1–0.5 lM, 72 h). Results in Fig. 5B shows that the cleavage of the 85-KDa fragment of PARP, a well-known substrate for caspase-3, occured after the concentration of XN05 exceeding 0.25 lM. However, treatment with CA-4 at 0.5 lM did not induce such cleavage form of PARP. In addition, the decrease of procaspase-3 protein and the appearance of its cleaved form (17,19 KDa) were both evident with the concentrations increased from 0.1 lM to 0.5 lM. Moreover, the expression of X-linked inhibitor of apoptosis protein (XIAP), a family of inhibitors of apoptosis proteins (IAPs), was attenuated as compared with that of the control samples. XN05-treated BEL7402 cells showed processing of pro-caspase-3, evident by the detection of the catalytically active fragment of p17. This was associated with increased caspase-3 activity and partial XIAP degradation. However, the XIAP protein content did not change as the concentrations increased. 3.5. XN05 changed the expression of the cell cycle regulatory proteins and Bcl-2 Immunoblotting analysis was used to investigate the underlying mechanisms of cell cycle arrest caused by XN05 in BEL-7402 cells. The cdc2-cyclin B1complex and its activating kinase CDK7 have been reported to play essential roles in G2/M transition. Thus the compounds disturbing G2/M transition probably affect the expression of the regulatory proteins. As shown in Fig. 6A, the protein expression of cyclin B1 gradually elevated upon the treatment of XN05 at the concentrations of 0.1–0.5 lM (48 h), whereas the protein level of cdc2 kinase was decreased, and these two events were associated with the accumulation of cells in the G2/M phase. However, the expression of CDK7, a CDK-activating kinase, was slightly increased under the same condition. Similar results were observed with XN05 at 0.5 lM from 12 h to 48 h (Fig. 6C). After exposure to XN05 with various concentrations or a single concentration (0.5 lM) in different time, the levels of cyclin B1 normalized with the corresponding actin levels were increased according to densitometric analyses (Fig. 6B and 6D). Finally, the phosphorylation of Bcl-2 was also detected after treatment with XN05 at concentrations of 0.25 and 0.5 lM for 48 h, which was accompanied by M phase arrest.
4. Discussion Hepatocellular carcinoma (HCC) is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death, and its incidence is rising
in many countries [13,14]. It is a phenotypically and genetically heterogeneous polyclonal disease and resistant to most conventional chemotherapy [15]. Microtubules are an attractive target for chemotherapeutic agents [16]. This study investigated the effect of XN05, a newly synthesized compound derived from CA-4, on microtubule assembly and hepatocellular carcinoma cell growth. According to the studies of structure–activity relationship, XN05 retained the 3,4,5-trimethyloxyphenyl pharmacophore as its fundamental structure. The restricted rotation of rings A and B of CA-4 can be maintained by introducing suitable conformationally restricted heterocycles, such as imidazole, isoxazole, and indole, many of which showed potent cytotoxicity against various cancer cell lines. A series of cis restricted analogues with imidazol-2-one instead of the cis double bond in CA-4 have been synthesized, among which XN05 is considered to be the most effective compound. Its efficacy was predicted based on the structural analogue of CA-4 that is well-known as a microtubule inhibitor. The potent antiproliferative activity of XN05 was verified in eight human cancer cell lines including leukemia and solid tumors. In hepatocellular cancer cells, XN05 exhibits equivalent or even better antitumor activity compared with CA-4. Notably, XN05 shows less effect on human liver cells in contrast to that of CA-4, which indicating XN05 a novel outstanding CA-4 derivative in treating hepatocellular carcinoma cell in vitro. Drug resistance is a serious problem that limits the clinical application of microtubule interfering drugs. Although the taxanes and the Vinca alkaloids are effective in the treatment of various human cancers, their potentials are limited by the emergence of drug resistance owing to the overexpression of drug efflux pump including P-gp, the decrease of drug accumulation inside the cancer cells and the genetic mutations or post-translational modifications caused structural and functional alterations in the a- and b-tubulins [17]. Therefore, the antitumor activities against those resistant cancer cells of XN05 needs be further investigated. XN05, as a CA-4 derivative, theoretically targets microtubules. To test this hypothesis, the in vivo and in vitro measurements of its effects on microtubule assembly were conducted. Our data clearly demonstrate that XN05 strongly inhibits tubulin polymerization. In addition, western blot and immunofluorescence microscopy, using the anti a-tubulin antibody, confirms that XN05 induces the depolymerization of microtubules and degradation of the microtubule network in liver cancer cells. Thus, XN05 can be classified as a microtubule-depolymerizing agent. Our data also indicate that like the other microtubule inhibitors, XN05 firstly inhibits tubulin polymerization, subsequently induces cell cycle arrest and finally triggers apoptosis in the two tested hepatocellular cancer cells. A concentration- and time-dependent G2/M blockade induced by XN05 is associated with the changed expression of the cell cycle regulatory proteins and the appearance of phosphorylated form of Bcl-2, a marker of M phase events [18]. Regulation of Cdc2 activity is controlled by cyclin B1 binding, phosphorylation of Thr161 by Cdk7,
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A
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Fig. 4. Cell cycle arrest and apoptosis were detected in another hepatocellular carcinoma cells SMMC-7721. (A) XN05 caused G2/M phase arrest in a dosedependent manner in SMMC-7721 cells. Cells were treated with XN05 (0.1, 0.5 and 1 lM) for 24 h and analyzed for propidium iodide-stained DNA content by flow cytometry. (B) Cells were incubated with XN05 (0.1, 0.5 and 1 lM) for 48 h and apoptosis was analyzed by staining phosphatidylserine translocation with FITC-Annexin V. Annexin V+ cells were represented on the X-axis and the number of cells counted is represented on the Y-axis. Simple vertical bars represent the mean apoptotic rate of SMMC-7721; error bars represent SD Data are mean ± SD (n = 3). , p < 0.05, , p < 0.01, , p < 0.001 versus nontreated control.
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A
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Fig. 5. Apoptosis was detected in BEL-7402 cells right after the cell cycle arrest caused by XN05. (A) Cells were treated with XN05 (0.1, 0.25 and 0.5 lM) for 72 h and analyzed for propidium iodide-stained DNA content by flow cytometry. Simple vertical bars represent the mean rate of G2/M arrest; error bars represent SD Data are mean ± SD (n = 3). (B) Apoptotic proteins were activated by XN05-treatment. Cells treated with (0.1, 0.25 and 0.5 lM) for 72 h were prepared and loaded on SDS–PAGE. After electrophoresis and transfer to nitrocellulose membrane, proteins were probed with caspase-3, cleaved caspase-3, PARP, XIAP and internal control b-actin antibodies. , p < 0.05, , p < 0.01 versus nontreated control.
and dephosphorylation of Tyr14 and Tyr15 by Cdc25C. Activated cdc2/cyclin B1 complex assembly is necessary for cells in G2/M phase transition and entry into mitosis during the cell cycle progression [19]. There are two steps for cdc2 activation—cyclin-binding and T-loop phosphorylation. Blocking any of these steps may lead to cell cycle arrest and subsequent apoptosis. In this complicated cell cycle signaling pathways, proteins interact actively with each other. In this study, we find that treatment with XN05 from 0.1 to 0.5 lM for 48 h decreases the expression of cdc2, and increases the accumulation of cyclin B1 and cdc2-activating kinase Cdk. Cdk7, a well-known CDK-activating kinase, is also a part of the transcription machinery in the cell cycle. Theoretically, inhibiting Cdk7 in G2 blocks
entry to mitosis, and disrupts Cdk1/cyclin B complex assembly. However, in this study, XN05-treatment slightly increases the expression of Cdk7. This is probably due to the complex interaction between the cell cycle related proteins. Further studies will be required to address the increased expression of Cdk7 by XN05. In addition, we also notice that when BEL-7402 cells were exposed to XN05 at 0.25 lM for 48 h, the phosphorylation of Bcl-2 occurred, which was associated with the increased number of G2/M cells detected by flow cytometry. At a higher concentration of 0.5 lM, XN05-induced phosphorylation of Bcl-2 was still sustained. Although there are always two slower migrating bands existing after treatment with many microtubule-target agents, suggesting the multiple phosphorylation states,
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Fig. 6. (A and C) Change of the expression of cell cycle related proteins in BEL-7402 cells after XN05-treatment. Cells treated with XN05 (0.1, 0.25 and 0.5 lM) for 48 h or 0.5 lM XN05 for indicated time (12 h, 24 h and 48 h) were prepared and loaded on SDS–PAGE. After electrophoresis and transfer to nitrocellulose membrane, proteins were probed with Bcl-2, cyclin B1, cdc2, Cdk7 and the internal control b-actin. (B and D) Densitometric analyses of expression of cyclin B1 relative to the untreated control. p < 0.05 versus nontreated control.
this was not observed in XN05-treated cells, indicating the possibility of subtle differences in Bcl-2 phosphorylation among the microtubule-target agents [20]. It is well accepted that microtubule inhibitors can induce apoptosis caused by cell cycle arrest. Here, our data also reveal that besides its induction of cell cycle arrest, XN05 promotes apoptosis, as indicated by an increase in the sub-G1 population and annexin-V positive. IAPs such as X-linked IAP (XIAP) inhibit the activity of caspases, thereby blocking apoptosis [21]. Caspase-3, a effector caspase, is activated by caspase-8 and -9 to form p19 and p17 subunits, and inhibited by XIAP via their binding on p19–p12 of caspase-3 complex through the BIR2 domain of XIAP [22,23]. However, autocatalytic cleavage of the p19 caspase-3 subunit to a p17-form results in removal of the XIAP-binding site and attenuates XIAP-mediated inhibition of caspase-3 [24]. Here, our results show that XN05-treatment causes the cleavage of the downstream executioner caspase-3 into its active form (p19 and p17), resulting in the cleavage of PARP. In addition, the downregulation of XIAP provides additional evidence of the activation of caspase-3. Therefore, we demonstrate that XN05 evoked apoptosis is mainly executed by the caspase activation. Taxol, a microtubule target agent, has been used in clinical therapy for years. However, its potential is limited by the drug resistance and its adverse effects. No matter what triggers drug resistance, tumors become refractory to a
variety of structurally diverse anticancer drugs [25]. Therefore, drug combinations have been employed to significantly improve treatment outcomes in cancer. In recent years, combinations of Taxol and some other conventional chemotherapeutic drugs (such as cisplatin, doxorubicin, TRAIL) [26,27] have been studied in same cases, they have already used in clinical trials. Combination therapy aims to solve the problem of drug resistance and use every agent at its maximum tolerated dose with lowest toxicity [28]. So based on the above theory, our next goal is to investigate whether XN05, also a microtubule target agent, could be used in drug combination therapy. In conclusion, we have provided convincing evidence to demonstrate that XN05, a novel synthesized compound, possesses potent antitumor activity against human tumor cells via disruption of microtubule. Compared with CA-4, a positive control, XN05 exhibits equivalent or even better antiproliferative activity against human hepatocellular carcinoma cells, indicating that XN05 can be a promising compound against human cancer cells. To confirm the potent of XN05, further experiments will be required to address the anti-MDR property and its drug combination in chemotherapy.
Conflicts of interest None declared.
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