- Email: [email protected]
Antineoplastic effect of calycosin on osteosarcoma through inducing apoptosis showing in vitro and in vivo investigations
Experimental and Molecular Pathology 97 (2014) 17–22
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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Antineoplastic effect of calycosin on osteosarcoma through inducing apoptosis showing in vitro and in vivo investigations Rubiao Qiu a, Gang Ma a, Chenguang Zheng a, Xiaoxia Qiu a, Xinning Li a, Xueyu Li a, Jianlan Mo a, Zhengzhao Li c, Yun Liu d, Linjian Mo b, Guan Bi a, Yu Ye c,⁎ a
Guangxi Maternal and Child Health Hospital, Nanning, Guangxi Zhuang Autonomous Region 530003, PR China Institute of Urology and Nephrology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, PR China Emergency Department, Western Hospital, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530007, PR China d Spine, Osteopathy, Surgery, Division, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, PR China b c
a r t i c l e
i n f o
Article history: Received 22 February 2014 and in revised form 12 April 2014 Available online 4 May 2014 Keywords: Calycosin Osteosarcoma Proliferation Apoptosis
a b s t r a c t Recently, increasing studies have documented that tumorigenesis closely relates to apoptotic processes. Thus, inducing apoptosis is an anti-cancer strategy against osteosarcoma. Here we investigated the anti-proliferative effect of calycosin on human osteosarcoma cell (143B) in vitro. The results showed that calycosin dosedependently inhibited 143B cell proliferation as reflected in tetrazolium salt (MTT) assay (P b 0.01). In addition, calycosin effectively down-regulated cellular mRNA expressions of IκBα, NF-κB p65 and cyclin D1 through RTPCR assay (P b 0.01). Next, calycosin-mediated inhibitory effect on 143B tumor-bearing nude mice and the underlying mechanism were evaluated and discussed. As a result, calycosin administration significantly blocked solid tumor growth in 143B-harbored nude mice (P b 0.01). Furthermore, intracellular Bcl-2 protein expression was effectively reduced in 143B-harbored tumor tissue through western blotting analysis (P b 0.01), while intratumoral Apaf-1 and cleaved Caspase-3 protein levels were up-regulated, respectively (P b 0.01). Taken together, calycosin possesses the anti-osteosarcoma potential, in which the mechanism involved was associated with activation of apoptotic, thus inducing apoptosis. © 2014 Elsevier Inc. All rights reserved.
Introduction Osteosarcoma is accompanied by the characteristic of aggressive progression that results from uncontrolled proliferation in osteoblast and then evolves malposed invasiveness (Ottaviani and Jaffe, 2009). Pathologically, malignant neoplasm that occurred in bone marks the features of subperiosteal lesion and irregular trabeculae calcification, as well as generation of multinucleated osteoclast-like giant cells (Thompson, 2013). Over time, these unfortunate patients suffer sharp pain from the deteriorating bone that may be worse nightly (Sangsin et al., 2013). Commonly, a clinically existing regimen is to apply neoadjuvant chemotherapy combined with surgical resection for maximum effect (Kudawara et al., 2013). Although the achievement of osteosarcoma therapy is remarkable, the use of chemical medication results in lower survival rates due to poor prognosis and marked relapse (Longhi et al., 2012). During cell-division, tumorigenesis results from the uncontrollably changed cell cycle (Li et al., 2012). Therefore, using chemotherapy and radiotherapy in cancer management is primarily through inducing apoptosis on tumor cells. Instead, inhibition of apoptosis can promote growth of various kinds of cancer cell (Dawson and Kouzarides, 2012). Overall, the search ⁎ Corresponding author. Fax: +86 771 3277161. E-mail address: [email protected] (Y. Ye).
http://dx.doi.org/10.1016/j.yexmp.2014.04.014 0014-4800/© 2014 Elsevier Inc. All rights reserved.
for a new alternative that targets osteosarcoma cell with hypotoxicity and effectiveness is an anti-tumor strategy. Trifolium pratense (red clover) naturally contains estrogen-like isoflavones that can mimic the role of endogenous estrogen functioning as certain pharmacological activities (Booth et al., 2006; Chen et al., 2013). The pre-clinical test of calycosin, an extracted bioactive compound, has been shown to relieve menopausal symptoms and manage some cancers (C. Zhang et al., 2012; D. Zhang et al., 2012). More attractively, our previous studies have suggested that calycosin exerts anti-proliferation on estrogen receptor-positive cells through activating estrogen receptors and apoptosis-related signaling pathway in vitro (Chen et al., 2011, 2014). However, the investigation of the calycosin-mediated anti-osteosarcoma role remains limited, especially in the study in vivo. In the present study, we used the cell lines to investigate the anti-proliferative effect of calycosin, and then further evaluated its potential action against 143B-harbored nude mice. Subsequently, the molecular mechanism involved would be discussed. Materials and methods Medication and cell culture Calycosin (purity N98%) was purchased from Tianjin Shilan Technology Co., Ltd (Tianjin, China) and was dissolved in dimethyl sulfoxide
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(DMSO) to adjust as a 250 μg/ml stock solution. The human osteosarcoma cell line (143B) and human osteoblast cell line (hFOB1.19) were provided by Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). These cells were cultivated in DMEM media (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin under a humidified atmosphere with the 37 °C, 5% CO2 condition. 143B cells were subjected with designed concentrations of calycosin (20, 40, 80, 160 μg/ml). Then, cells were incubated with a serum medium and terminated at 24, 48, and 72 h time-points for cell proliferation assessment. MTT assay
growth was observed. After the solid tumor volume reached 0.2 cm3, 143B-harbored nude mice were randomly assigned into five groups: control group (n = 10), ifosfamide positive group (4 mg/kg, n = 10); calycosin administrated groups (2, 4, 8 mg/kg, n = 10). Experiencing 14 day' treatment, the solid tumor of mice was aseptically dissected and harvested. The weight change of tumor was recorded, and neoplastic specimen was stored in −80 °C for further use. The estimation of survival of tumor-bearing nude mice The body weight of inoculated mice was estimated among the groups, and the tumor growth changes were recorded by calycosin administration.
143B and hFOB1.19 cells were digested by trypsinase and seeded at 3 × 104 cells/well within 96-well plates. After 24 h, calycosin was administrated to the final concentrations in a volume of 100 μl and incubated for 72 h. Then, 10 μl 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, USA) dissolved in phosphate-buffered saline (PBS) was added, incubating for additional 4 h to conduct cell viability testing. Subsequently, 100 μl dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA) was added in the wells after removing the medium, and then the mixtures were shaken for 10 min. At the end of solubilization, the optical density (OD) value of each well was determined using a full-wavelength microplate reader (Molecular Devices Company, USA) at a wavelength of 580 nm. In addition, five independent experiments were conducted repeatedly for calculating the mean value of proliferation inhibitory ratio.
The sections were de-waxed, rehydrated, rinsed with PBS (0.1 mol l− 1 , pH7.2) for three times, digested by freshly diluted proteinase K (20 μg ml −1, pH7.2) in a humidified chamber at 37 °C for 1 h. Subsequently, the sections were quenched in 3% H2O2 in PBS for 15 min at 37 °C prior to treating with the terminal deoxynucleotidyl transferase (TdT) enzyme (TUNEL reaction solution, Roche, Germany). The sections were then rinsed three times with 0.1 M PBS and incubated with anti-digoxigenin conjugate (Roche, Germany) in a humid chamber for 30 min at 37 °C. After being washed three times with fresh PBS, the sections were dyed with diaminobenzidine (DAB) (Zhongshan Goldenbridge Biological Technology, Beijing, China) for 10 min at 37 °C.
Proliferation inhibitory ratio ð%Þ ¼ 1‐ODadministrated =ODcontrol 100%
Western blot analysis for Bcl-2, Apaf-1, and cleaved Caspase-3
RT-PCR assay for target mRNA detection Total RNA was extracted from the cultured 143B cells using Trizol reagent (Invitrogen, USA). The 20 μl reaction solution containing 1.5 μg of total RNA was transformed and synthesized into cDNA through conducting at 37 °C for 30 min and then 92 °C for 5 min. Subsequently, the RT-PCR process was performed using cDNA as a template by adding primer and mixing with RNase-free distilled water. The sequences of forward primer and reverse primer were shown as follows: IκBα 5′-GAA GGA GCG GCT ACT GGA CG-3′ and 5′-AAT TTC TGT GTG GCT GGT TGG TGA-3′, 468 bp; NF-κB p65 5′-ATA GAA GAG ACG CGT GGG GAC T-3′ and 5′-GGA TGA CGT AAA GGG ATA GGG C-3′, 519 bp; cyclin D1 5′-ACG AAG GTC TGC GCG TGT T-3′ and 5′-CCG CTG GCC ATG AAC TAC CT-3′, 324 bp; β-actin 5′-AAG GTC GGA GTC AAC GGA TTT G-3′ and 5′-CTT GAC AAA GTG GTC GTT GAG G-3′, 495 bp, respectively. The reaction process included an initial denaturation at 94 °C for 5 min then followed with 30 cycles of 92 °C for 30 s, 55 °C for 30 s and 70 °C for 10 s, accompanied by a final extension temperature (IκBα at 60 °C; NF-κB p65 at 72 °C; cyclin D1 at 67 °C) for 5 min. The amplification product was visualized onto a 1.5% agarose gel, and the gene bands were analyzed using a sequence detector (Agilent Technologies Co., Ltd., USA). The data was represented by a grey value, and relative grey value ratio was expressed as target gene/β-actin. Animal and medication delivery Sixty BALB/c nude mice, two-months-old, approximately 18–20 g, were originally obtained from Vital River Laboratories (Beijing, China). These mice were housed in a germ-free condition with alternating 12 h periods of light and darkness, a constant temperature of 18–22 °C, and 50–60% humidity. Experimental procedures were conducted in accordance with the guidelines of the Experimental Research Institute of Guangxi Medical University. When cultured cells grow into the logarithmic status, 0.2 ml sterile 143B cells (4 × 106 number/ml) were subcutaneously injected in the dorsum of nude mice, and solid tumor
TUNEL assay
After tumor-bearing nude mice were administered with calycosin for 14 days, solid tumors from each group were dealt with ice-cold lysis buffer. The lysate mixtures were centrifuged at 10,000 ×g for 15 min, and protein contents in obtained supernatant were measured using the BCA protein assay kit. Equivalent numbers of protein (35 μg/lane) were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into 0.45 μm polyvinylidene difluoride (PVDF) membranes (Santa Cruz, USA). These membranes were blocked with TBST (Tris-buffered solution, pH 7.4, 0.02% Tween 80) containing 5 % fat-free milk powder. The membrane was sequentially incubated with primary antibodies including, Bcl-2, Apaf-1, and cleaved Caspase-3 (1:1,000; Santa Cruz, USA) at 4 °C. Then washing with TBST for three times, the blots were incubated with specific secondary antibodies coupled to horseradish peroxidase (HRP) at 37 °C for 1 h and then performed using electrochemiluminescence (ECL) western blot detection reagents (Pierce, UAS). Target protein expressions were represented as compared to the internal control (GAPDH) based on the relative intensities of the band. Statistical analysis The results were presented as the means ± SD, and a statistical analysis was performed with SPSS. Differences were tested using a one-way ANOVA with Student's t-test in vitro and LSD-t test in vivo for multiple comparisons, and differences were considered statistically significant at P-values less than 0.05. Results Calycosin inhibited the proliferation of target cells The data from MTT assay showed that 143B cells in the control group notably grew faster than those in calycosin-administrated groups, exhibiting the characteristic of uncontrolled proliferation. Compared to the control group, calycosin-administered cells effectively blocked cell proliferation in a time–dosage dependent manner (P b 0.01)
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(Fig. 1A). On the other hand, calycosin infrequently affected the health human osteoblast cell line (hFOB1.19), in which the statistical difference in the inhibitory role was not significant (Fig. 1B). Calycosin down-regulated the expressions of target mRNA in 143B cells As shown in Fig. 2, the findings from PT-PCR indicated that IκBα, NF-κB p65 and cyclin D1 mRNA expressions were at over-expressed levels in 143B-harbored tumor. Strikingly, administration of calycosin effectively down-regulated the mRNA levels of IκBα, NF-κB p65 and cyclin D1 at a dose-dependent relationship compared to control mice (P b 0.01) (Fig. 2). Effect of calycosin on body weight and tumor growth of 143B tumor-bearing nude mice To further assess the therapeutic potency of calycosin, we investigated the intervention in 143B tumor-bearing nude mice. As shown in Fig. 3, the body weight of tumor-bearing mice in ifosfamide and calycosin treatments was effectively attenuated when compared to the classified tumor control (P b 0.01), in which calycosin showed an impact in dose-dependent manner. In addition, the tumor growth was inhibited by calycosin treatment, and the inhibition ratio of tumor exhibited a dose–effect manner (P b 0.01). In particular, the effectiveness in 8 mg/kg calycosin was more significant compared to the model control. Calycosin induced apoptosis in 143B solid tumor cells In the present study, the apoptosis in 143B-harbored tumor was assessed using TUNEL assay. TUNEL-positive cells were observed in 143B solid tumor tissue of each group, and the control group had less apoptotic counts in solid tumor than that in administrated groups. Compared to the control group, the number of TUNEL-positive cells in ifosfamide and calycosin administrated groups was increased in 143B solid tumor (P b 0.01). Notably, the high-dose calycosin exhibited an increase in apoptotic count (Fig. 4). Calycosin elevated the protein expressions of Bcl-2, Apaf-1, and cleaved Caspase-3 in 143B solid tumor cells As shown in Western blot analysis, the endogenous Bcl-2 protein in solid tumor tissue was over-expressed in 143B tumor-bearing nude
Fig. 2. Calycosin reduced target mRNA expressions in 143B cell at the gene level using RT-PCR assay. Results were analyzed with one-way ANOVA followed by Student's t-test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
mice, whereas Apaf-1 and cleaved Caspase-3 expressions were hypoexpressed. However, ifosfamide- and calycosin-administrated tumorbearing mice contributed to reversion of these abnormal changes. Interestingly, calycosin administration resulted in reduction of Bcl-2 level and down-regulation of Apaf-1 and cleaved Caspase-3 expressions compared to those in control mice (P b 0.01), respectively. Similarly, these component levels were reversed by the ifosfamide administration (P b 0.01) (Fig. 5). Discussion Malignant tumor, known for its medically uncontrollable cell differentiation and growth, is an incurable illness that gradually invades important organs of the body (Siegel et al., 2011). Patients living with cancer are clinically employed with a specific regimen, including chemotherapy, radiation therapy and surgery, in order to increase the survival period (Siegel et al., 2013). However, the side effects that occurred in chemotherapy and radiotherapy often lead to cytotoxicity in normal tissue. An alternative substance combating cancer and showing hypotoxicity should be an exciting avenue of tumor management,
Fig. 1. Calycosin inhibited 143B cell proliferation and affected hFOB1.19 cell. Calycosin-administrated 143B cell was determined using MTT assay for assessing cell viability during 24, 48, and 72 h. These independent experiments were repeatedly performed 5 times. Results were analyzed with one-way ANOVA followed by Student's t-test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
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Fig. 3. Effect of calycosin on body weight and tumor growth of 143B tumor-bearing nude mice. Results were analyzed with one-way ANOVA followed by LSD-t test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
which is about to replace certain conventional drugs (Chang et al., 2006). Lately, increasing reports show that cancer patients treated with traditional Chinese medicine (TCM) manifest better prognosis compared to conventional therapy, accompanied by a less adverse
reaction (Smith and Bauer-Wu, 2012). In the present study, the results from MTT assay indicated that calycosin administration exhibited an anti-proliferative effect on 143B cells, whereas the similar dose of calycosin exposed to normal osteoblast cells showed a benign role. We
Fig. 4. Calycosin increased apoptosis-labeled cells in 143B solid tumor using TUNEL staining assay (scale bar: 200 μm). Arrows represent TUNEL-positive cells. Results were analyzed with one-way ANOVA followed by LSD-t test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
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Fig. 5. Calycosin up-regulated the levels of Bcl-2, Apaf-1, and cleaved Caspase-3 in 143B solid tumor using western blotting analysis. Results were analyzed with one-way ANOVA followed by LSD-t test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
concluded that the calycosin-mediated cytostatic role was due to its potent pharmacological activity and low toxicology. As a regulator of cyclin-dependent kinase, cyclin D1 in dysregulation can alter the cell cycle process and then results in tumorigenesis (Suh et al., 2010; C. Zhang et al., 2012; D. Zhang et al., 2012). Functionally, NF-κB plays a vital role in immune modulation, in which its dysregulation is involved in the development of cancer and inflammatory, as well as autoimmune diseases. In a normal physiological condition, NF-κB locates in the cytosol and stably binds with the inhibitory protein of IκBα. Once the IκBα dissociates from NF-κB, the activated NF-κB translocates into the nucleus to further anchor specific sequences of DNA called response elements, leading to transcribing downstream gene for altering cell function (DiDonato et al., 2012; Sun, 2012). Thereby, targeting NF-κB may offer a strategy involved in anti-cancer treatment. These findings suggested that calycosin effectively inhibited endogenous expressions of IκBα and NF-κB p65 mRNA in 143B cells, and down-regulated gene levels were correlated with the benefit of the anti-proliferative role as shown in MTT assay. Together, we extrapolated that NF-κB-dependent signal event dominates the growth of tumor cells. Notably, NF-κB inactivation by calycosin played the secondary effect of regulating associated gene/protein (such as cyclin D1), eventually inhibiting proliferation of tumor cells. In addition, dysregulation of the apoptosis-specific pathway is inclined to induction of tumor formation (Ouyang et al., 2012). It is commonly believed that neoplasm occurrence arises from an outcome that uncontrolled cell proliferation outweighs cell death from tumor tissue (Sun and Peng, 2009). TUNEL is a prevalent method for detecting intracellular DNA fragmentation resulting from apoptotic cascades, in which labeled cell reflects severe DNA damage (Kyrylkova et al., 2012). The results from this study showed that calycosin increased TUNEL-positive cells in 143B-harbored tumor, indicating induction of DNA-destructive cell loss. Moreover, this therapeutic benefit was consistent with the inhibitory effect on tumor cell by calycosin in vitro. Thus, the antiosteosarcoma effect of calycosin was related to inducing apoptosis. Next, the apoptosis-related signaling pathway in calycosin-treated 143B cells should be further investigated. As a result, the malignant neoplasm progressively controls the apoptosis process, in which the antiapoptotic component (such as Bcl-2) is hypo-expressed inactivated in tumors, and target genes (such as Apaf-1 and Caspase-3) transcribe abnormally in diseased cells (Fiandalo, 2012; Riedl and Shi, 2004). Bcl-2 is a regulator protein of the Bcl-2 family that fights off cell death (Youle and Strasser, 2008). Disturbance of Bcl-2 gene expression has been identified as the main nosogenesis of cancer, including osteosarcoma (Kaseta et al., 2008). Apaf-1 encodes a cytoplasmic protein that controls biological processes, such as apoptosis (Cain
et al., 2002). Caspase-3 is activated in apoptotic cell through death ligand and mitochondrial pathways (Fan et al., 2005). As an executioner caspase, the Caspase-3 zymogen is nearly inactive existing in the cell until it is cleaved by an initiator caspase that occurred in apoptotic events (Shi, 2002). Our findings from the present study showed that 143B-bearing tumor nude mice resulted in boosting Bcl-2 activity and reduction of Apaf-1 and cleaved Caspase-3 levels. One theory was that apoptosis could be directed by manipulation of these components, which would have dominant implications in osteosarcoma growth. Following the calycosin administration, these abnormal changes in 143B-harbored tumor were significantly reversed, showing Bcl-2 protein reduction and up-regulated expressions of Apaf-1 and cleaved Caspase-3. Mechanistically, the apoptosis-dependent pathway regulated by calycosin was implicated in the therapeutically molecular mechanisms in vivo, while other identified signaling pathways that targeted apoptosis had indicated calycosin-mediated anti-tumor effect in vitro in our previous studies. When overleaping the Bcl-2 functional pathway, cytochrome c is released and then binded with intracellular apoptotic protease activating factor-1 (Apaf-1) and chelated procaspase-9 to create a protein complex that was known as an apoptosome. The apoptosome selectively cleaved the pro-caspase as Caspase-3, a molecule phenotype, and this effector triggered cascade events that induced apoptosis. We believed that an association between in vitro and in vivo impacts could benefit from calycosin-regulated apoptosis due to its pharmacological activity. Conclusions Overall, these findings demonstrate that calycosin exerts an effective anti-proliferative effect on osteosarcoma, which the underlying mechanism is related to inducing apoptosis in tumor cell. And calycosin may serve as an attractive therapeutic candidate for osteosarcoma treatment. Conflict of interest statement The authors declare that there are no conflicts of interest. References Booth, N.L., Piersen, C.E., Banuvar, S., Geller, S.E., Shulman, L.P., Farnsworth, N.R., 2006. Clinical studies of red clover (Trifolium pratense) dietary supplements in menopause: a literature review. Menopause 13, 251–264. Cain, K., Bratton, S.B., Cohen, G.M., 2002. The Apaf-1 apoptosome: a large caspaseactivating complex. Biochimie 84, 203–214.
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Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Antineoplastic effect of calycosin on osteosarcoma through inducing apoptosis showing in vitro and in vivo investigations Rubiao Qiu a, Gang Ma a, Chenguang Zheng a, Xiaoxia Qiu a, Xinning Li a, Xueyu Li a, Jianlan Mo a, Zhengzhao Li c, Yun Liu d, Linjian Mo b, Guan Bi a, Yu Ye c,⁎ a
Guangxi Maternal and Child Health Hospital, Nanning, Guangxi Zhuang Autonomous Region 530003, PR China Institute of Urology and Nephrology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, PR China Emergency Department, Western Hospital, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530007, PR China d Spine, Osteopathy, Surgery, Division, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, PR China b c
a r t i c l e
i n f o
Article history: Received 22 February 2014 and in revised form 12 April 2014 Available online 4 May 2014 Keywords: Calycosin Osteosarcoma Proliferation Apoptosis
a b s t r a c t Recently, increasing studies have documented that tumorigenesis closely relates to apoptotic processes. Thus, inducing apoptosis is an anti-cancer strategy against osteosarcoma. Here we investigated the anti-proliferative effect of calycosin on human osteosarcoma cell (143B) in vitro. The results showed that calycosin dosedependently inhibited 143B cell proliferation as reflected in tetrazolium salt (MTT) assay (P b 0.01). In addition, calycosin effectively down-regulated cellular mRNA expressions of IκBα, NF-κB p65 and cyclin D1 through RTPCR assay (P b 0.01). Next, calycosin-mediated inhibitory effect on 143B tumor-bearing nude mice and the underlying mechanism were evaluated and discussed. As a result, calycosin administration significantly blocked solid tumor growth in 143B-harbored nude mice (P b 0.01). Furthermore, intracellular Bcl-2 protein expression was effectively reduced in 143B-harbored tumor tissue through western blotting analysis (P b 0.01), while intratumoral Apaf-1 and cleaved Caspase-3 protein levels were up-regulated, respectively (P b 0.01). Taken together, calycosin possesses the anti-osteosarcoma potential, in which the mechanism involved was associated with activation of apoptotic, thus inducing apoptosis. © 2014 Elsevier Inc. All rights reserved.
Introduction Osteosarcoma is accompanied by the characteristic of aggressive progression that results from uncontrolled proliferation in osteoblast and then evolves malposed invasiveness (Ottaviani and Jaffe, 2009). Pathologically, malignant neoplasm that occurred in bone marks the features of subperiosteal lesion and irregular trabeculae calcification, as well as generation of multinucleated osteoclast-like giant cells (Thompson, 2013). Over time, these unfortunate patients suffer sharp pain from the deteriorating bone that may be worse nightly (Sangsin et al., 2013). Commonly, a clinically existing regimen is to apply neoadjuvant chemotherapy combined with surgical resection for maximum effect (Kudawara et al., 2013). Although the achievement of osteosarcoma therapy is remarkable, the use of chemical medication results in lower survival rates due to poor prognosis and marked relapse (Longhi et al., 2012). During cell-division, tumorigenesis results from the uncontrollably changed cell cycle (Li et al., 2012). Therefore, using chemotherapy and radiotherapy in cancer management is primarily through inducing apoptosis on tumor cells. Instead, inhibition of apoptosis can promote growth of various kinds of cancer cell (Dawson and Kouzarides, 2012). Overall, the search ⁎ Corresponding author. Fax: +86 771 3277161. E-mail address: [email protected] (Y. Ye).
http://dx.doi.org/10.1016/j.yexmp.2014.04.014 0014-4800/© 2014 Elsevier Inc. All rights reserved.
for a new alternative that targets osteosarcoma cell with hypotoxicity and effectiveness is an anti-tumor strategy. Trifolium pratense (red clover) naturally contains estrogen-like isoflavones that can mimic the role of endogenous estrogen functioning as certain pharmacological activities (Booth et al., 2006; Chen et al., 2013). The pre-clinical test of calycosin, an extracted bioactive compound, has been shown to relieve menopausal symptoms and manage some cancers (C. Zhang et al., 2012; D. Zhang et al., 2012). More attractively, our previous studies have suggested that calycosin exerts anti-proliferation on estrogen receptor-positive cells through activating estrogen receptors and apoptosis-related signaling pathway in vitro (Chen et al., 2011, 2014). However, the investigation of the calycosin-mediated anti-osteosarcoma role remains limited, especially in the study in vivo. In the present study, we used the cell lines to investigate the anti-proliferative effect of calycosin, and then further evaluated its potential action against 143B-harbored nude mice. Subsequently, the molecular mechanism involved would be discussed. Materials and methods Medication and cell culture Calycosin (purity N98%) was purchased from Tianjin Shilan Technology Co., Ltd (Tianjin, China) and was dissolved in dimethyl sulfoxide
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(DMSO) to adjust as a 250 μg/ml stock solution. The human osteosarcoma cell line (143B) and human osteoblast cell line (hFOB1.19) were provided by Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). These cells were cultivated in DMEM media (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin under a humidified atmosphere with the 37 °C, 5% CO2 condition. 143B cells were subjected with designed concentrations of calycosin (20, 40, 80, 160 μg/ml). Then, cells were incubated with a serum medium and terminated at 24, 48, and 72 h time-points for cell proliferation assessment. MTT assay
growth was observed. After the solid tumor volume reached 0.2 cm3, 143B-harbored nude mice were randomly assigned into five groups: control group (n = 10), ifosfamide positive group (4 mg/kg, n = 10); calycosin administrated groups (2, 4, 8 mg/kg, n = 10). Experiencing 14 day' treatment, the solid tumor of mice was aseptically dissected and harvested. The weight change of tumor was recorded, and neoplastic specimen was stored in −80 °C for further use. The estimation of survival of tumor-bearing nude mice The body weight of inoculated mice was estimated among the groups, and the tumor growth changes were recorded by calycosin administration.
143B and hFOB1.19 cells were digested by trypsinase and seeded at 3 × 104 cells/well within 96-well plates. After 24 h, calycosin was administrated to the final concentrations in a volume of 100 μl and incubated for 72 h. Then, 10 μl 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, USA) dissolved in phosphate-buffered saline (PBS) was added, incubating for additional 4 h to conduct cell viability testing. Subsequently, 100 μl dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA) was added in the wells after removing the medium, and then the mixtures were shaken for 10 min. At the end of solubilization, the optical density (OD) value of each well was determined using a full-wavelength microplate reader (Molecular Devices Company, USA) at a wavelength of 580 nm. In addition, five independent experiments were conducted repeatedly for calculating the mean value of proliferation inhibitory ratio.
The sections were de-waxed, rehydrated, rinsed with PBS (0.1 mol l− 1 , pH7.2) for three times, digested by freshly diluted proteinase K (20 μg ml −1, pH7.2) in a humidified chamber at 37 °C for 1 h. Subsequently, the sections were quenched in 3% H2O2 in PBS for 15 min at 37 °C prior to treating with the terminal deoxynucleotidyl transferase (TdT) enzyme (TUNEL reaction solution, Roche, Germany). The sections were then rinsed three times with 0.1 M PBS and incubated with anti-digoxigenin conjugate (Roche, Germany) in a humid chamber for 30 min at 37 °C. After being washed three times with fresh PBS, the sections were dyed with diaminobenzidine (DAB) (Zhongshan Goldenbridge Biological Technology, Beijing, China) for 10 min at 37 °C.
Proliferation inhibitory ratio ð%Þ ¼ 1‐ODadministrated =ODcontrol 100%
Western blot analysis for Bcl-2, Apaf-1, and cleaved Caspase-3
RT-PCR assay for target mRNA detection Total RNA was extracted from the cultured 143B cells using Trizol reagent (Invitrogen, USA). The 20 μl reaction solution containing 1.5 μg of total RNA was transformed and synthesized into cDNA through conducting at 37 °C for 30 min and then 92 °C for 5 min. Subsequently, the RT-PCR process was performed using cDNA as a template by adding primer and mixing with RNase-free distilled water. The sequences of forward primer and reverse primer were shown as follows: IκBα 5′-GAA GGA GCG GCT ACT GGA CG-3′ and 5′-AAT TTC TGT GTG GCT GGT TGG TGA-3′, 468 bp; NF-κB p65 5′-ATA GAA GAG ACG CGT GGG GAC T-3′ and 5′-GGA TGA CGT AAA GGG ATA GGG C-3′, 519 bp; cyclin D1 5′-ACG AAG GTC TGC GCG TGT T-3′ and 5′-CCG CTG GCC ATG AAC TAC CT-3′, 324 bp; β-actin 5′-AAG GTC GGA GTC AAC GGA TTT G-3′ and 5′-CTT GAC AAA GTG GTC GTT GAG G-3′, 495 bp, respectively. The reaction process included an initial denaturation at 94 °C for 5 min then followed with 30 cycles of 92 °C for 30 s, 55 °C for 30 s and 70 °C for 10 s, accompanied by a final extension temperature (IκBα at 60 °C; NF-κB p65 at 72 °C; cyclin D1 at 67 °C) for 5 min. The amplification product was visualized onto a 1.5% agarose gel, and the gene bands were analyzed using a sequence detector (Agilent Technologies Co., Ltd., USA). The data was represented by a grey value, and relative grey value ratio was expressed as target gene/β-actin. Animal and medication delivery Sixty BALB/c nude mice, two-months-old, approximately 18–20 g, were originally obtained from Vital River Laboratories (Beijing, China). These mice were housed in a germ-free condition with alternating 12 h periods of light and darkness, a constant temperature of 18–22 °C, and 50–60% humidity. Experimental procedures were conducted in accordance with the guidelines of the Experimental Research Institute of Guangxi Medical University. When cultured cells grow into the logarithmic status, 0.2 ml sterile 143B cells (4 × 106 number/ml) were subcutaneously injected in the dorsum of nude mice, and solid tumor
TUNEL assay
After tumor-bearing nude mice were administered with calycosin for 14 days, solid tumors from each group were dealt with ice-cold lysis buffer. The lysate mixtures were centrifuged at 10,000 ×g for 15 min, and protein contents in obtained supernatant were measured using the BCA protein assay kit. Equivalent numbers of protein (35 μg/lane) were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into 0.45 μm polyvinylidene difluoride (PVDF) membranes (Santa Cruz, USA). These membranes were blocked with TBST (Tris-buffered solution, pH 7.4, 0.02% Tween 80) containing 5 % fat-free milk powder. The membrane was sequentially incubated with primary antibodies including, Bcl-2, Apaf-1, and cleaved Caspase-3 (1:1,000; Santa Cruz, USA) at 4 °C. Then washing with TBST for three times, the blots were incubated with specific secondary antibodies coupled to horseradish peroxidase (HRP) at 37 °C for 1 h and then performed using electrochemiluminescence (ECL) western blot detection reagents (Pierce, UAS). Target protein expressions were represented as compared to the internal control (GAPDH) based on the relative intensities of the band. Statistical analysis The results were presented as the means ± SD, and a statistical analysis was performed with SPSS. Differences were tested using a one-way ANOVA with Student's t-test in vitro and LSD-t test in vivo for multiple comparisons, and differences were considered statistically significant at P-values less than 0.05. Results Calycosin inhibited the proliferation of target cells The data from MTT assay showed that 143B cells in the control group notably grew faster than those in calycosin-administrated groups, exhibiting the characteristic of uncontrolled proliferation. Compared to the control group, calycosin-administered cells effectively blocked cell proliferation in a time–dosage dependent manner (P b 0.01)
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(Fig. 1A). On the other hand, calycosin infrequently affected the health human osteoblast cell line (hFOB1.19), in which the statistical difference in the inhibitory role was not significant (Fig. 1B). Calycosin down-regulated the expressions of target mRNA in 143B cells As shown in Fig. 2, the findings from PT-PCR indicated that IκBα, NF-κB p65 and cyclin D1 mRNA expressions were at over-expressed levels in 143B-harbored tumor. Strikingly, administration of calycosin effectively down-regulated the mRNA levels of IκBα, NF-κB p65 and cyclin D1 at a dose-dependent relationship compared to control mice (P b 0.01) (Fig. 2). Effect of calycosin on body weight and tumor growth of 143B tumor-bearing nude mice To further assess the therapeutic potency of calycosin, we investigated the intervention in 143B tumor-bearing nude mice. As shown in Fig. 3, the body weight of tumor-bearing mice in ifosfamide and calycosin treatments was effectively attenuated when compared to the classified tumor control (P b 0.01), in which calycosin showed an impact in dose-dependent manner. In addition, the tumor growth was inhibited by calycosin treatment, and the inhibition ratio of tumor exhibited a dose–effect manner (P b 0.01). In particular, the effectiveness in 8 mg/kg calycosin was more significant compared to the model control. Calycosin induced apoptosis in 143B solid tumor cells In the present study, the apoptosis in 143B-harbored tumor was assessed using TUNEL assay. TUNEL-positive cells were observed in 143B solid tumor tissue of each group, and the control group had less apoptotic counts in solid tumor than that in administrated groups. Compared to the control group, the number of TUNEL-positive cells in ifosfamide and calycosin administrated groups was increased in 143B solid tumor (P b 0.01). Notably, the high-dose calycosin exhibited an increase in apoptotic count (Fig. 4). Calycosin elevated the protein expressions of Bcl-2, Apaf-1, and cleaved Caspase-3 in 143B solid tumor cells As shown in Western blot analysis, the endogenous Bcl-2 protein in solid tumor tissue was over-expressed in 143B tumor-bearing nude
Fig. 2. Calycosin reduced target mRNA expressions in 143B cell at the gene level using RT-PCR assay. Results were analyzed with one-way ANOVA followed by Student's t-test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
mice, whereas Apaf-1 and cleaved Caspase-3 expressions were hypoexpressed. However, ifosfamide- and calycosin-administrated tumorbearing mice contributed to reversion of these abnormal changes. Interestingly, calycosin administration resulted in reduction of Bcl-2 level and down-regulation of Apaf-1 and cleaved Caspase-3 expressions compared to those in control mice (P b 0.01), respectively. Similarly, these component levels were reversed by the ifosfamide administration (P b 0.01) (Fig. 5). Discussion Malignant tumor, known for its medically uncontrollable cell differentiation and growth, is an incurable illness that gradually invades important organs of the body (Siegel et al., 2011). Patients living with cancer are clinically employed with a specific regimen, including chemotherapy, radiation therapy and surgery, in order to increase the survival period (Siegel et al., 2013). However, the side effects that occurred in chemotherapy and radiotherapy often lead to cytotoxicity in normal tissue. An alternative substance combating cancer and showing hypotoxicity should be an exciting avenue of tumor management,
Fig. 1. Calycosin inhibited 143B cell proliferation and affected hFOB1.19 cell. Calycosin-administrated 143B cell was determined using MTT assay for assessing cell viability during 24, 48, and 72 h. These independent experiments were repeatedly performed 5 times. Results were analyzed with one-way ANOVA followed by Student's t-test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
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Fig. 3. Effect of calycosin on body weight and tumor growth of 143B tumor-bearing nude mice. Results were analyzed with one-way ANOVA followed by LSD-t test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
which is about to replace certain conventional drugs (Chang et al., 2006). Lately, increasing reports show that cancer patients treated with traditional Chinese medicine (TCM) manifest better prognosis compared to conventional therapy, accompanied by a less adverse
reaction (Smith and Bauer-Wu, 2012). In the present study, the results from MTT assay indicated that calycosin administration exhibited an anti-proliferative effect on 143B cells, whereas the similar dose of calycosin exposed to normal osteoblast cells showed a benign role. We
Fig. 4. Calycosin increased apoptosis-labeled cells in 143B solid tumor using TUNEL staining assay (scale bar: 200 μm). Arrows represent TUNEL-positive cells. Results were analyzed with one-way ANOVA followed by LSD-t test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
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Fig. 5. Calycosin up-regulated the levels of Bcl-2, Apaf-1, and cleaved Caspase-3 in 143B solid tumor using western blotting analysis. Results were analyzed with one-way ANOVA followed by LSD-t test, and data were expressed as the mean ± SD. Notes: *P b 0.01 vs. control group.
concluded that the calycosin-mediated cytostatic role was due to its potent pharmacological activity and low toxicology. As a regulator of cyclin-dependent kinase, cyclin D1 in dysregulation can alter the cell cycle process and then results in tumorigenesis (Suh et al., 2010; C. Zhang et al., 2012; D. Zhang et al., 2012). Functionally, NF-κB plays a vital role in immune modulation, in which its dysregulation is involved in the development of cancer and inflammatory, as well as autoimmune diseases. In a normal physiological condition, NF-κB locates in the cytosol and stably binds with the inhibitory protein of IκBα. Once the IκBα dissociates from NF-κB, the activated NF-κB translocates into the nucleus to further anchor specific sequences of DNA called response elements, leading to transcribing downstream gene for altering cell function (DiDonato et al., 2012; Sun, 2012). Thereby, targeting NF-κB may offer a strategy involved in anti-cancer treatment. These findings suggested that calycosin effectively inhibited endogenous expressions of IκBα and NF-κB p65 mRNA in 143B cells, and down-regulated gene levels were correlated with the benefit of the anti-proliferative role as shown in MTT assay. Together, we extrapolated that NF-κB-dependent signal event dominates the growth of tumor cells. Notably, NF-κB inactivation by calycosin played the secondary effect of regulating associated gene/protein (such as cyclin D1), eventually inhibiting proliferation of tumor cells. In addition, dysregulation of the apoptosis-specific pathway is inclined to induction of tumor formation (Ouyang et al., 2012). It is commonly believed that neoplasm occurrence arises from an outcome that uncontrolled cell proliferation outweighs cell death from tumor tissue (Sun and Peng, 2009). TUNEL is a prevalent method for detecting intracellular DNA fragmentation resulting from apoptotic cascades, in which labeled cell reflects severe DNA damage (Kyrylkova et al., 2012). The results from this study showed that calycosin increased TUNEL-positive cells in 143B-harbored tumor, indicating induction of DNA-destructive cell loss. Moreover, this therapeutic benefit was consistent with the inhibitory effect on tumor cell by calycosin in vitro. Thus, the antiosteosarcoma effect of calycosin was related to inducing apoptosis. Next, the apoptosis-related signaling pathway in calycosin-treated 143B cells should be further investigated. As a result, the malignant neoplasm progressively controls the apoptosis process, in which the antiapoptotic component (such as Bcl-2) is hypo-expressed inactivated in tumors, and target genes (such as Apaf-1 and Caspase-3) transcribe abnormally in diseased cells (Fiandalo, 2012; Riedl and Shi, 2004). Bcl-2 is a regulator protein of the Bcl-2 family that fights off cell death (Youle and Strasser, 2008). Disturbance of Bcl-2 gene expression has been identified as the main nosogenesis of cancer, including osteosarcoma (Kaseta et al., 2008). Apaf-1 encodes a cytoplasmic protein that controls biological processes, such as apoptosis (Cain
et al., 2002). Caspase-3 is activated in apoptotic cell through death ligand and mitochondrial pathways (Fan et al., 2005). As an executioner caspase, the Caspase-3 zymogen is nearly inactive existing in the cell until it is cleaved by an initiator caspase that occurred in apoptotic events (Shi, 2002). Our findings from the present study showed that 143B-bearing tumor nude mice resulted in boosting Bcl-2 activity and reduction of Apaf-1 and cleaved Caspase-3 levels. One theory was that apoptosis could be directed by manipulation of these components, which would have dominant implications in osteosarcoma growth. Following the calycosin administration, these abnormal changes in 143B-harbored tumor were significantly reversed, showing Bcl-2 protein reduction and up-regulated expressions of Apaf-1 and cleaved Caspase-3. Mechanistically, the apoptosis-dependent pathway regulated by calycosin was implicated in the therapeutically molecular mechanisms in vivo, while other identified signaling pathways that targeted apoptosis had indicated calycosin-mediated anti-tumor effect in vitro in our previous studies. When overleaping the Bcl-2 functional pathway, cytochrome c is released and then binded with intracellular apoptotic protease activating factor-1 (Apaf-1) and chelated procaspase-9 to create a protein complex that was known as an apoptosome. The apoptosome selectively cleaved the pro-caspase as Caspase-3, a molecule phenotype, and this effector triggered cascade events that induced apoptosis. We believed that an association between in vitro and in vivo impacts could benefit from calycosin-regulated apoptosis due to its pharmacological activity. Conclusions Overall, these findings demonstrate that calycosin exerts an effective anti-proliferative effect on osteosarcoma, which the underlying mechanism is related to inducing apoptosis in tumor cell. And calycosin may serve as an attractive therapeutic candidate for osteosarcoma treatment. Conflict of interest statement The authors declare that there are no conflicts of interest. References Booth, N.L., Piersen, C.E., Banuvar, S., Geller, S.E., Shulman, L.P., Farnsworth, N.R., 2006. Clinical studies of red clover (Trifolium pratense) dietary supplements in menopause: a literature review. Menopause 13, 251–264. Cain, K., Bratton, S.B., Cohen, G.M., 2002. The Apaf-1 apoptosome: a large caspaseactivating complex. Biochimie 84, 203–214.
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