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Cinobufacini induced MDA-MB-231 cell apoptosis-associated cell cycle arrest and cytoskeleton function
Bioorganic & Medicinal Chemistry Letters 22 (2012) 1459–1463
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Cinobufacini induced MDA-MB-231 cell apoptosis-associated cell cycle arrest and cytoskeleton function Lina Ma a, Bing Song b, Hua Jin a, Jiang Pi a, Li Liu a, Jinhuan Jiang a, Jiye Cai a,⇑ a b
Department of Chemistry and Institute for Nano-Chemistry, Jinan University, Guangzhou 510632, China Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China
a r t i c l e
i n f o
Article history: Received 20 October 2011 Revised 18 November 2011 Accepted 21 November 2011 Available online 30 November 2011 Keywords: Cinobufacini Atomic force microscopy Cell cycle Cytoskeleton Ultrastructure
a b s t r a c t Cinobufacini is a traditional Chinese anti-tumor drug and widely used in clinic experiences. But little is known about its effect on the cells. In this study, the effects of cinobufacini on breast cancer MDAMB-231 cell were evaluated by CCK-8 assay, and the data showed cinobufacini could inhibit the MDAMB-231 cells growth effectively in dose-dependent and time-dependent manners. Cell apoptosis and cell cycle were detected by flow cytometry analysis. After the cells being treated with 50 lg/mL cinobufacini for 48 h, the early apoptosis percentage (20.45 ± 1.46%) is much higher than the normal group (7.73 ± 1.21%). The cell cycle data indicated that cinobufacini caused a cell cycle arrest at S phase. What’s more, cinobufacini can affect the disruption of cytoskeleton, and these alterations changed the cell-surface ultrastructure and the cell morphology which were detected by atomic force microscopy (AFM) at nanoscale level. It indicated that the cell membrane structure and cytoskeleton networks were destroyed and the cell tails were narrowed after the cell being treated with cinobufacini. The present study is to provide valuable new insights to understand the mechanism of the drug in anti-tumor process. Furthermore, the knowledge concerning the signaling of cell cycle is potentially important to clinical utility. Ó 2011 Elsevier Ltd. All rights reserved.
Breast cancer is one of the most common cancers in the world, and its occurrence has been increasing in recent years. Although surgical resection and chemotherapy are common options for carcinoma, surgery is the treatment choice for only the small fraction of patients with localized disease. While chemotherapy drugs can damage normal cells, cytotoxic chemotherapy agents are minimally effective at improving patient survival.1–4 Thus, it is very important to find a new agent for cancer treatment. Cinobufacini is an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor.5,6 It is a traditional Chinese antitumor drug and widely used in clinic experiences. But little is known about its effect on the cell-surface and the interaction between cell-surface and mechanisms of drug action. Previous studies have shown that the cinobufacini can inhibit carcinogenesis in various cell lines, such as gastric, breast, hepatic cancer.7–9 It is reported cinobufacini has a variety of biological effects, such as antineoplastic and immunomodulatory effects.10,11 Recent clinical studies have suggested that cinobufacini combined with docetaxel had a profound effect on a number of cancers, such as gastrointestinal tract carcinomas.12–14 Furthermore, cinobufacini had been reported to have the effects of proliferation inhibi⇑ Corresponding author. Tel./fax: +86 020 85223569. E-mail addresses: [email protected] (L. Ma), [email protected] (B. Song), [email protected] (H. Jin), [email protected] (J. Pi), [email protected] (L. Liu), [email protected] (J. Jiang), [email protected] (J. Cai). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.11.095
tory of cells. For example, it can induce apoptosis of HL-60 cells, and more, it is found that cinobufacini-induced apoptosis was related to the change of the mitochondrial membrane potential.15,16 Lots of articles have been reported that cinobufacini could kill diverse cancers effectively, but the effects on the cell surface are still not clear. Therefore, in this paper, atomic force microscopy and immune fluorescence were used to visualize cell morphology, membrane ultrastructure and cytoskeleton functions. Then the mechanism of cinobufacini was evaluated by CCK-8 assay and flow cytometry. The cinobufacini (Golden Toad Medicine Company head office of Huaibei of Anhui Province) was diluted by culture medium. The MDA-MB-231 cells and 3T3cells were incubated in RPMI1640 medium (Gibco Co.) supplemented with 10% PBS at 37 °C in 95% air and 5% CO2. Then, cells in the logarithmic growth phase were collected for the following experiments. First, the cytotoxicity of cinobufacini effects on MDA-MB-231 cell and 3T3 cell were assessed by CCK-8 assay (Pharmaceutical Company of Shanghai Colleagues). The cells in the logarithmic growth phase were plated at a density of 1 105 cells/mL in 96well plates and incubated for 24 h. The cells were incubated with various concentrations of cinobufacini (0, 12.5, 25, 37.5 and 50 lg/mL) for 48 h and 72 h, separately. Then the cells were collected and 5 mg/ml 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) in phosphate buffered saline (PBS) was added to each well and
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incubated for 2 h at 37 °C in 95% air and 5% CO2. And absorbance was measured in a dual-beam microtiter plate. Cell viability rate = (ODtreatment ODblank/ODcontrol ODblank) 100%. Each experiment group was repeated four times.17 An examination of the effects of cinobufacini on the viability of cancer MDA-MB-231 cells and normal 3T3 cells revealed that cinobufacini was much more cytotoxic to malignant breast cancer cells than normal cells (Fig. 1A). We next investigated whether cinobufacini-induced cell death in malignant breast cancer cells was associated with time. The results in Fig. 1B showed that after the cells being treated with 50 lg/mL cinobufacini, the cell viability have been decreasing to 36.5 ± 2.4% (48 h) and 30.6 ± 1.9% (72 h). That indicated cinobufacini could inhibit the MDA-MB-231 cells growth effectively in dose-dependent and time-dependent manners. For annexin-based FACS analysis, the cells were treated with cinobufacini (0–50 lg/mL) for 48 h, then cells were trypsinized, washed once in ice-cold PBS, and resuspended in 300 lL bing buffer, 5 lL Annexin V and PI (Nanjing KeyGen Biotech. Co. Ltd, China) were added to the cell preparations and incubated for 15 min in the dark. The samples were analyzed by using Cell Quest software on a FACS Aria Flow Cytometer (BD Inc., USA). The results (Fig. 2) showed that both the percentages of early apoptotic cells and late apoptotic cells were increased with the increasing concentrations (0, 25, 37.5 and 50 lg/mL). And at the concentration of 50 lg/mL, the early apoptotic percentage was 20.45 ± 1.46% and the late apoptotic percentage was 12.63% ± 1.89%. The results suggest that cinobufacini can effectively induce cell apoptosis. After the cells were harvested, washed with cold PBS, and processed for cell cycle analysis. The cells were fixed in absolute ethanol and stored for 12 h. Then the fixed cells were centrifuged at 400g force and washed with cold PBS for two times. RNase A (20 lg/mL final concentration) and PI(propidium iodide) staining solution (50 lg/mL final concentration) was added to the cells and incubated for 15 min at 37 °C in the dark. The percentages of the cells in the different phases of the cell cycle were determined by FCM (ELITE, BECKMAN-COULTER, USA).18–21 And the percentages of the cells in the different phases of the cell cycle were determined. The results were shown in Table 1. Before the cells were treated with cinobufacini, the number of S phase was 27.8 ± 1.7%. And after the cells were treated with 50 lg/mL cinobufacini, the number of S phase was 52.3 ± 2.0%. The number (30.7 ± 1.9%) of G1 phase were decreased compared with the normal group (58.00 ± 1.5%) and this decrease was accompanied the increase in
the number of S phase cells. Thus, cinobufacini caused a cell cycle arrest at S phase and the result indicated that cinobufacini inhibited the DNA replication in MDA-MB-231 cell. As a nondestructive surface imaging tool, AFM can obtain images of cell surface at the nanometric scale. It can provide us very important details of the morphology and the cell membrane. MDA-MB-231cells treated with cinobufacini (0–50 lg/mL) for 48 h were fixed with 4% paraformaldehyde for 15 min. Then the cells were washed three times by PBS and air dried. An AFM (Autoprobe CP Research, Veeco, USA) was used in the contact mode to obtain topographic images. The prepared sample was put on the XY scanning station of AFM. The monitor was used to locate scanning area and contact mode was applied for imaging. Then the images were analyzed by onboard software (IP2.1). The results were shown in Fig. 3, the cells in control group had a regular spindle or oval shape and the nuclei were elliptical and plump, and more, the cell surface was relatively intact and smooth (A1–A3). The cell tails were unrolled and the pseudopodium between the cells was connected with each other for information transfer and material exchange. The ultrastructure (A3) showed the cell surface was smooth. After the cells were treated with 25 lg/mL cinobufacini, the cell morphology (B1, B2) and ultrastructure (B3) were changed a little. When cinobufacini concentration increased to 37.5 lg/mL, the cell morphology was deformed. For example, the cells collapsed, cytoplasm leaked and the cell tails also shrank (C1–C3). As cinobufacini concentration increased to 50 lg/mL, the cells vary greatly in size and shape. Such as cell shrinkage, cell size reduction and turn round significantly and tail also disappear (D1–D3). However, some of them were similar to the symbols of necrosis emerging on the cell surface. Eukaryotic cytoskeleton is a cytoplasmic protein complex composed of wire mesh structure.22–26 Firstly, cytoskeleton could maintain the cell morphology. Besides, cytoskeleton also associated with cell movement, cell division and intracellular transportation. So it is important to signal transduction. A large number of the cytoskeletons were composed of threadlike microfilaments, and the threadlike microfilaments were composed mainly of the contractile protein F-actin. Moreover, the apoptosis assay was performed using DAPI staining. DAPI is a powerful fluorescent dye combined with the DNA, and it can penetrate the membrane of living cells. So the nuclear morphology which is a typical apoptosis can be identified by the fluorescence intensity. In this study, the characterizations of apoptosis and cytoskeleton function were
Fig. 1. The growth-inhibiting effects of cinobufacini on MDA-MB-231 cells. (A) MDA-MB-231cells and 3T3 cells were treated with different concentrations (0–50 lg/mL) of cinobufacini for 48 h. (B) MDA-MB-231cells were treated with different concentrations (0–50 lg/mL) of cinobufacini for different times. Each experimental group was repeated for more than three times. Statistical analysis was performed using Student’s t-test. P <0.05 was regarded as statistically significant.
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Fig. 2. Apoptosis rates of MDA-MB-231 cells with or without cinobufacini treatment detected by flow cytometry. MDA-MB-231 cells were treated with 0, 25, 37.5, and 50 lg/mL cinobufacini for 48 h, respectively. Data are shown by means ± standard deviations (n = 3), P <0.05 was regarded as statistically significant. (A) Annexin V-FITC and PI staining for apoptosis detected by flow cytometry; (B) The graph shows the apoptosis rate of (A). Q2 represents the late apoptosis and dead cells, and Q4 represents the early apoptosis.
Table 1 Analysis of cell cycle in MDA-MB-231 cells treated with cinobufacini Concentration (lg/mL)
G1 (%)
S (%)
G2/M (%)
0 25 37.5 50
58.0 ± 1.5 52.3 ± 2.0 34.1 ± 1.7 30.7 ± 1.9
27.8 ± 1.7 32.5 ± 0.9 46.8 ± 1.6 52.3 ± 1.2
14.2 ± 2.1 15.2 ± 1.1 19.1 ± 2.3 17.0 ± 1.3
Values are expressed as percentage of the cell population in the G1, G2/M and S phase of cell cycle.
evaluated by staining with DAPI (Beyotime Institute of Biotechnology) and rhodamine–phalloidin (shanghai threebio technology Co., Ltd), respectively. The MDA-MB-231 cells with or without cinobufacini treatments were fixed with 4% paraformaldehyde for 20 min and then incubated with 50 lM of DAPI for 10 min and 1 lM rhodamine–phalloidin for 3 h in the dark at room temperature, separately. After that, the cells were washed twice with PBS. At last, the nuclear morphology and organization of cytoskeleton were imaged by a laser scanning confocal microscope (LCM
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Fig. 3. AFM images of MDA-MB-231 cells before and after treatment with cinobufacini (0, 25, 37.5, 50 lg/mL) for 48 h. (A1–D1): Cell morphology; (A2–D2): 2D cell membrane image in black box in (A1–D1), respectively; (A3–D3): 3D images of cell membrane (size: 5 5 lm). These images indicated that there are significant changes appeared on the cell surface treated with cinobufacini. (A1–A3) is control group, (B1–B3) is the 25 lg/mL cinobufacini-treated group; (C1–C3) is the 37.5 lg/mL cinobufacini-treated group; (D1–D3) is the 50 lg/mL cinobufacini-treated group.
510 Meta Duo Scan, Carl Zeiss, Germeny).27–29 Fig. 4A showed the reorganization of F-actin in MDA-MB-231cells. The results (Fig. 4A1) showed the normal cells were rich in F-actin. The F-actins were composed of three-dimensional network structure. We could see the cells exhibited characteristic spindle morphology. After the cells were treated with cinobufacini (50 lg/mL) for 48 h, the cells became irregular in shape. F-actin dispersed in the cells, stress fibers around the nucleus arranged in disorder and the numbers were decreased (Fig. 4B1). Even the cytoskeletal networks disappeared, at last, the cell integrity was damaged. Fig. 4A2 showed that the cells were plump and intact and nuclei morphology was uniform in the normal group. But after the cells were treated with cinobufacini (50 lg/mL, Fig. 4B2), the nuclei had broken, the condensed chromatin gathered at the periphery of the nuclear membrane and the edge of the cells became blurred. This indicated that cinobufacini could significantly inhibit proliferation and induce apoptosis of MDA-MB-231 cells.
In the study, we observed the effects of cinobufacini on MDAMB-231 cells by CCK-8 assay, AFM, flow cytometry and immune fluorescence. Since apoptosis or programmed cell death is a normal physiological process, we try to explore the factors associated with apoptosis. The CCK-8 assay and Annexin V-based flow cytometric data showed that cinobufacini could induce apoptosis of MDAMB-231 cells effectively in dose-dependent and time-dependent manners (Fig. 1, Fig. 2). The changes in cell cycle results showed that cinobufacini treatment caused a cell cycle arrest at S phases (Table 1). The results indicated that cinobufacini could block the process of the cell cycle from S phase to G2/M phase, which could block the cell mitosis and make the cell cycle stay at S phases. At last, cinobufacini induced the cell apoptosis. AFM data indicated that cinobufacini could damage the cell membrane and then changed the ultrastructure of cell surface a lot. Simultaneously, the cells shrunk and cell size reduced (Fig. 3). To further confirm these results, we studied clues regarding the relationship between the
L. Ma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1459–1463
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Fig. 4. F-actin in MDA-MB-231 cells was stained with Rhodamine–phalloidin and nuclei was stained with DAPI. (A1) showed the cells were spindle, a large number of F-actin around the nucleus regularly in the cells and the fibers in the cytoplasm arranged as regular rays. (B1) showed the cells after being treated with cinobufacini (50 lg/mL) for 48 h, the cells became irregular in shape. F-actin dispersed in the cells and stress fibers around the nucleus arranged in disorder and the numbers were decreased. A2 showed that the cells were plump and intact, intracellular staining and nuclei morphology were uniform in the normal group. B2 showed the cells were treated with cinobufacini, the nuclei have been deformed and there were some holes in the middle of the nuclei, nuclei changed markedly.
cytoskeletons and functions. The alternations in the immunofluorescence of cytoskeleton and nuclei morphology also showed the apoptosis effects after the cells being treated with cinobufacini (Fig. 4). These results highlight the importance of the cytoskeleton in the spatial signal organization, so it is crucial to determine whether the stress fibers in transformed cells have disappeared after the cells were treated with cinobuficini. Even more, alterations in the cytoskeleton could effect on the signaling pathways. And nuclei morphology also showed the apoptosis induced by cinobufacini. In summary, this research indicated that cinobufacini could inhibit cellular proliferation significantly. And it could induce cell cycle arrest at S phase and induce apoptosis effectively in MDA-MB231 cells and it could provide a further understand of cinobufacini mechanism. Mean while, the structures of the MDA-MB-231 cell changes were detected by AFM at the nanometer scale, it could provide us to understand cinobufacini effect on the cell-surface. So AFM will be a potential sensitive tool for detecting cancer cells. However, more research is required to understand the specific mechanism of cinobufacini. Acknowledgments This work is supported by Ministry of Science and Technology of China (No. 2010CB833603), the National Natural Science Foundation of China (No. 30872404), the Fundamental Research Funds for the Central Universities (No. 30828028, No. 21609305, and No. 21610427), the Key Project of Chinese Ministry of Education (No. 210254) and Specialized Research Fund for the Doctoral Program of Higher Eduction (20104401120004). References and notes 1. Serag, H. B.; Marrero, J. A.; Rudolph, L.; Reddy, K. R. Gastroenterology 2008, 134, 1752.
2. Matthew, J. D.; Wang, Y. M.; Lee, M. Y.; Ashok, R. Science 2004, 306, 876. 3. Bektas, N.; Haaf, A.; Veeck, J.; Wild, P. J.; Luscher, F. J. BMC Cancer 2008, 8, 1. 4. Zhao, Q.; Otavia, L. C.; Samuel, L.; Brian, J. S.; Christian, I.; Sandro, J. S.; Pedro, A.; Dana, B.; Margaret, A. L. Science 2009, 106, 1886. 5. Qi, F. H.; Li, A. Y.; Gao, B.; Tang, W. Drug Discov. Ther. 2010, 45, 318. 6. Guo, J. M. Pharmacopoeia of the People’s Republic of China; China Chemical Industry Press: Beijing, 2010. 7. Zhu, X. Y.; Liu, L. M. Tumor J. World 2006, 4, 272. 8. Emmanuelle, C. J.; Christophe, G.; Flora, I.; Julien, W.; Nathalie, C.; Pascal, F.; Min, H. H.; Mark, E. D. Cancer Res. 2009, 69, 1302. 9. Chen, G. Q.; Zhao, Z. H. W.; Zhou, H. Y.; Liu, Y. J.; Yang, H. J. Med. Oncol. 2010, 27, 406. 10. Lu, C. X.; Nan, K. J.; Lei, Y. Anticancer Drugs 2008, 19, 931. 11. Qi, F. H.; Li, A. Y.; Zhao, L.; Xu, H. L. J. Ethnopharmacol. 2010, 128, 654. 12. Qin, T. J.; Zhao, X. H.; Yun, J.; Zhang, L. X.; Ruan, V.; Pan, B. R. World J. Gastroenterol. 2008, 14, 5210. 13. Gong, H. J. Contemp. Med. 2011, 17, 154. 14. Dominik, F.; Carsten, B.; Gerhard, O.; Volker, D.; Cord, N. J. Cell Biochem. 2008, 103, 270. 15. Ying, H.; Xu, H. l.; Sun, D. J. China Pharmacist. 2008, 10, 1158. 16. Aoki, H.; Takada, Y.; Kondo, S.; Sawaya, R.; Aggarwal, B. B.; Kondo, Y. Mol. Pharmacol. 2007, 72, 29. 17. Angelika, B.; Kathryn, E. S.; Alla, P.; Nandini, K.; Jill, B. Breast Cancer Res. 2011, 13, R3. 18. Thomas, K.; Lin, T.; Paul, G. E.; David, C. S.; Fiona, N.; Ralph, K. L.; Ricky, W. J. Cancer Cell. 2007, 129, 423. 19. Wang, M.; Ruan, Y. X.; Chen, Q.; Li, S. H. P. Eur. J. Pharmacol. 2011, 1, 41. 20. Mor, V.; Kaspa, R. T.; Sheiner, P.; Schwartz, M. Ann. Intern. Med. 1998, 129, 643. 21. Eun, J. C.; Su, M. B.; Woong, S. A. Arch. Pharm. Res. 2008, 31, 1281. 22. Girasole, M.; Pompeo, G.; Cricenti, A.; Longo, G.; Boumis, G.; Bellelli, A.; Amiconi, S. Nanomedicine 2010, 6, 760. 23. Atsushi, I. Adv. Biochem. Engin/Biotechnol. 2010, 119, 47. 24. Hsieh, C. H.; Lin, Y. H.; Lin, S.; WU, J. J.; WU, C. H.; Jiang, C. C. Osteoarthritis Cartilage 2008, 16, 480. 25. Sokolov, I. In Cancer Nanotechnol.; Nalwa, S. H., Webster, H., Eds.; American Scientific Publishers, 2007. Chapter 1. 26. Lilly, Y. W.; Zhu, H.; Shao, L. J.; Chen, Y. W. J. Biol. chem. 2001, 276, 7327. 27. Thomas, M. J. Gastroenterology 2009, 44, 136. 28. Fabio, P.; Rosa, V.; Mariadele, R. O.; Manisha, H. S.; Milena, S. N.; Ivanade, M. I.; Emanuela, P. Cancer Cell 2008, 4, 272. 29. Lilly, Y. W.; Bour, G. Semin. Cancer Biol. 2008, 18, 251.
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Cinobufacini induced MDA-MB-231 cell apoptosis-associated cell cycle arrest and cytoskeleton function Lina Ma a, Bing Song b, Hua Jin a, Jiang Pi a, Li Liu a, Jinhuan Jiang a, Jiye Cai a,⇑ a b
Department of Chemistry and Institute for Nano-Chemistry, Jinan University, Guangzhou 510632, China Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China
a r t i c l e
i n f o
Article history: Received 20 October 2011 Revised 18 November 2011 Accepted 21 November 2011 Available online 30 November 2011 Keywords: Cinobufacini Atomic force microscopy Cell cycle Cytoskeleton Ultrastructure
a b s t r a c t Cinobufacini is a traditional Chinese anti-tumor drug and widely used in clinic experiences. But little is known about its effect on the cells. In this study, the effects of cinobufacini on breast cancer MDAMB-231 cell were evaluated by CCK-8 assay, and the data showed cinobufacini could inhibit the MDAMB-231 cells growth effectively in dose-dependent and time-dependent manners. Cell apoptosis and cell cycle were detected by flow cytometry analysis. After the cells being treated with 50 lg/mL cinobufacini for 48 h, the early apoptosis percentage (20.45 ± 1.46%) is much higher than the normal group (7.73 ± 1.21%). The cell cycle data indicated that cinobufacini caused a cell cycle arrest at S phase. What’s more, cinobufacini can affect the disruption of cytoskeleton, and these alterations changed the cell-surface ultrastructure and the cell morphology which were detected by atomic force microscopy (AFM) at nanoscale level. It indicated that the cell membrane structure and cytoskeleton networks were destroyed and the cell tails were narrowed after the cell being treated with cinobufacini. The present study is to provide valuable new insights to understand the mechanism of the drug in anti-tumor process. Furthermore, the knowledge concerning the signaling of cell cycle is potentially important to clinical utility. Ó 2011 Elsevier Ltd. All rights reserved.
Breast cancer is one of the most common cancers in the world, and its occurrence has been increasing in recent years. Although surgical resection and chemotherapy are common options for carcinoma, surgery is the treatment choice for only the small fraction of patients with localized disease. While chemotherapy drugs can damage normal cells, cytotoxic chemotherapy agents are minimally effective at improving patient survival.1–4 Thus, it is very important to find a new agent for cancer treatment. Cinobufacini is an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor.5,6 It is a traditional Chinese antitumor drug and widely used in clinic experiences. But little is known about its effect on the cell-surface and the interaction between cell-surface and mechanisms of drug action. Previous studies have shown that the cinobufacini can inhibit carcinogenesis in various cell lines, such as gastric, breast, hepatic cancer.7–9 It is reported cinobufacini has a variety of biological effects, such as antineoplastic and immunomodulatory effects.10,11 Recent clinical studies have suggested that cinobufacini combined with docetaxel had a profound effect on a number of cancers, such as gastrointestinal tract carcinomas.12–14 Furthermore, cinobufacini had been reported to have the effects of proliferation inhibi⇑ Corresponding author. Tel./fax: +86 020 85223569. E-mail addresses: [email protected] (L. Ma), [email protected] (B. Song), [email protected] (H. Jin), [email protected] (J. Pi), [email protected] (L. Liu), [email protected] (J. Jiang), [email protected] (J. Cai). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.11.095
tory of cells. For example, it can induce apoptosis of HL-60 cells, and more, it is found that cinobufacini-induced apoptosis was related to the change of the mitochondrial membrane potential.15,16 Lots of articles have been reported that cinobufacini could kill diverse cancers effectively, but the effects on the cell surface are still not clear. Therefore, in this paper, atomic force microscopy and immune fluorescence were used to visualize cell morphology, membrane ultrastructure and cytoskeleton functions. Then the mechanism of cinobufacini was evaluated by CCK-8 assay and flow cytometry. The cinobufacini (Golden Toad Medicine Company head office of Huaibei of Anhui Province) was diluted by culture medium. The MDA-MB-231 cells and 3T3cells were incubated in RPMI1640 medium (Gibco Co.) supplemented with 10% PBS at 37 °C in 95% air and 5% CO2. Then, cells in the logarithmic growth phase were collected for the following experiments. First, the cytotoxicity of cinobufacini effects on MDA-MB-231 cell and 3T3 cell were assessed by CCK-8 assay (Pharmaceutical Company of Shanghai Colleagues). The cells in the logarithmic growth phase were plated at a density of 1 105 cells/mL in 96well plates and incubated for 24 h. The cells were incubated with various concentrations of cinobufacini (0, 12.5, 25, 37.5 and 50 lg/mL) for 48 h and 72 h, separately. Then the cells were collected and 5 mg/ml 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) in phosphate buffered saline (PBS) was added to each well and
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incubated for 2 h at 37 °C in 95% air and 5% CO2. And absorbance was measured in a dual-beam microtiter plate. Cell viability rate = (ODtreatment ODblank/ODcontrol ODblank) 100%. Each experiment group was repeated four times.17 An examination of the effects of cinobufacini on the viability of cancer MDA-MB-231 cells and normal 3T3 cells revealed that cinobufacini was much more cytotoxic to malignant breast cancer cells than normal cells (Fig. 1A). We next investigated whether cinobufacini-induced cell death in malignant breast cancer cells was associated with time. The results in Fig. 1B showed that after the cells being treated with 50 lg/mL cinobufacini, the cell viability have been decreasing to 36.5 ± 2.4% (48 h) and 30.6 ± 1.9% (72 h). That indicated cinobufacini could inhibit the MDA-MB-231 cells growth effectively in dose-dependent and time-dependent manners. For annexin-based FACS analysis, the cells were treated with cinobufacini (0–50 lg/mL) for 48 h, then cells were trypsinized, washed once in ice-cold PBS, and resuspended in 300 lL bing buffer, 5 lL Annexin V and PI (Nanjing KeyGen Biotech. Co. Ltd, China) were added to the cell preparations and incubated for 15 min in the dark. The samples were analyzed by using Cell Quest software on a FACS Aria Flow Cytometer (BD Inc., USA). The results (Fig. 2) showed that both the percentages of early apoptotic cells and late apoptotic cells were increased with the increasing concentrations (0, 25, 37.5 and 50 lg/mL). And at the concentration of 50 lg/mL, the early apoptotic percentage was 20.45 ± 1.46% and the late apoptotic percentage was 12.63% ± 1.89%. The results suggest that cinobufacini can effectively induce cell apoptosis. After the cells were harvested, washed with cold PBS, and processed for cell cycle analysis. The cells were fixed in absolute ethanol and stored for 12 h. Then the fixed cells were centrifuged at 400g force and washed with cold PBS for two times. RNase A (20 lg/mL final concentration) and PI(propidium iodide) staining solution (50 lg/mL final concentration) was added to the cells and incubated for 15 min at 37 °C in the dark. The percentages of the cells in the different phases of the cell cycle were determined by FCM (ELITE, BECKMAN-COULTER, USA).18–21 And the percentages of the cells in the different phases of the cell cycle were determined. The results were shown in Table 1. Before the cells were treated with cinobufacini, the number of S phase was 27.8 ± 1.7%. And after the cells were treated with 50 lg/mL cinobufacini, the number of S phase was 52.3 ± 2.0%. The number (30.7 ± 1.9%) of G1 phase were decreased compared with the normal group (58.00 ± 1.5%) and this decrease was accompanied the increase in
the number of S phase cells. Thus, cinobufacini caused a cell cycle arrest at S phase and the result indicated that cinobufacini inhibited the DNA replication in MDA-MB-231 cell. As a nondestructive surface imaging tool, AFM can obtain images of cell surface at the nanometric scale. It can provide us very important details of the morphology and the cell membrane. MDA-MB-231cells treated with cinobufacini (0–50 lg/mL) for 48 h were fixed with 4% paraformaldehyde for 15 min. Then the cells were washed three times by PBS and air dried. An AFM (Autoprobe CP Research, Veeco, USA) was used in the contact mode to obtain topographic images. The prepared sample was put on the XY scanning station of AFM. The monitor was used to locate scanning area and contact mode was applied for imaging. Then the images were analyzed by onboard software (IP2.1). The results were shown in Fig. 3, the cells in control group had a regular spindle or oval shape and the nuclei were elliptical and plump, and more, the cell surface was relatively intact and smooth (A1–A3). The cell tails were unrolled and the pseudopodium between the cells was connected with each other for information transfer and material exchange. The ultrastructure (A3) showed the cell surface was smooth. After the cells were treated with 25 lg/mL cinobufacini, the cell morphology (B1, B2) and ultrastructure (B3) were changed a little. When cinobufacini concentration increased to 37.5 lg/mL, the cell morphology was deformed. For example, the cells collapsed, cytoplasm leaked and the cell tails also shrank (C1–C3). As cinobufacini concentration increased to 50 lg/mL, the cells vary greatly in size and shape. Such as cell shrinkage, cell size reduction and turn round significantly and tail also disappear (D1–D3). However, some of them were similar to the symbols of necrosis emerging on the cell surface. Eukaryotic cytoskeleton is a cytoplasmic protein complex composed of wire mesh structure.22–26 Firstly, cytoskeleton could maintain the cell morphology. Besides, cytoskeleton also associated with cell movement, cell division and intracellular transportation. So it is important to signal transduction. A large number of the cytoskeletons were composed of threadlike microfilaments, and the threadlike microfilaments were composed mainly of the contractile protein F-actin. Moreover, the apoptosis assay was performed using DAPI staining. DAPI is a powerful fluorescent dye combined with the DNA, and it can penetrate the membrane of living cells. So the nuclear morphology which is a typical apoptosis can be identified by the fluorescence intensity. In this study, the characterizations of apoptosis and cytoskeleton function were
Fig. 1. The growth-inhibiting effects of cinobufacini on MDA-MB-231 cells. (A) MDA-MB-231cells and 3T3 cells were treated with different concentrations (0–50 lg/mL) of cinobufacini for 48 h. (B) MDA-MB-231cells were treated with different concentrations (0–50 lg/mL) of cinobufacini for different times. Each experimental group was repeated for more than three times. Statistical analysis was performed using Student’s t-test. P <0.05 was regarded as statistically significant.
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Fig. 2. Apoptosis rates of MDA-MB-231 cells with or without cinobufacini treatment detected by flow cytometry. MDA-MB-231 cells were treated with 0, 25, 37.5, and 50 lg/mL cinobufacini for 48 h, respectively. Data are shown by means ± standard deviations (n = 3), P <0.05 was regarded as statistically significant. (A) Annexin V-FITC and PI staining for apoptosis detected by flow cytometry; (B) The graph shows the apoptosis rate of (A). Q2 represents the late apoptosis and dead cells, and Q4 represents the early apoptosis.
Table 1 Analysis of cell cycle in MDA-MB-231 cells treated with cinobufacini Concentration (lg/mL)
G1 (%)
S (%)
G2/M (%)
0 25 37.5 50
58.0 ± 1.5 52.3 ± 2.0 34.1 ± 1.7 30.7 ± 1.9
27.8 ± 1.7 32.5 ± 0.9 46.8 ± 1.6 52.3 ± 1.2
14.2 ± 2.1 15.2 ± 1.1 19.1 ± 2.3 17.0 ± 1.3
Values are expressed as percentage of the cell population in the G1, G2/M and S phase of cell cycle.
evaluated by staining with DAPI (Beyotime Institute of Biotechnology) and rhodamine–phalloidin (shanghai threebio technology Co., Ltd), respectively. The MDA-MB-231 cells with or without cinobufacini treatments were fixed with 4% paraformaldehyde for 20 min and then incubated with 50 lM of DAPI for 10 min and 1 lM rhodamine–phalloidin for 3 h in the dark at room temperature, separately. After that, the cells were washed twice with PBS. At last, the nuclear morphology and organization of cytoskeleton were imaged by a laser scanning confocal microscope (LCM
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Fig. 3. AFM images of MDA-MB-231 cells before and after treatment with cinobufacini (0, 25, 37.5, 50 lg/mL) for 48 h. (A1–D1): Cell morphology; (A2–D2): 2D cell membrane image in black box in (A1–D1), respectively; (A3–D3): 3D images of cell membrane (size: 5 5 lm). These images indicated that there are significant changes appeared on the cell surface treated with cinobufacini. (A1–A3) is control group, (B1–B3) is the 25 lg/mL cinobufacini-treated group; (C1–C3) is the 37.5 lg/mL cinobufacini-treated group; (D1–D3) is the 50 lg/mL cinobufacini-treated group.
510 Meta Duo Scan, Carl Zeiss, Germeny).27–29 Fig. 4A showed the reorganization of F-actin in MDA-MB-231cells. The results (Fig. 4A1) showed the normal cells were rich in F-actin. The F-actins were composed of three-dimensional network structure. We could see the cells exhibited characteristic spindle morphology. After the cells were treated with cinobufacini (50 lg/mL) for 48 h, the cells became irregular in shape. F-actin dispersed in the cells, stress fibers around the nucleus arranged in disorder and the numbers were decreased (Fig. 4B1). Even the cytoskeletal networks disappeared, at last, the cell integrity was damaged. Fig. 4A2 showed that the cells were plump and intact and nuclei morphology was uniform in the normal group. But after the cells were treated with cinobufacini (50 lg/mL, Fig. 4B2), the nuclei had broken, the condensed chromatin gathered at the periphery of the nuclear membrane and the edge of the cells became blurred. This indicated that cinobufacini could significantly inhibit proliferation and induce apoptosis of MDA-MB-231 cells.
In the study, we observed the effects of cinobufacini on MDAMB-231 cells by CCK-8 assay, AFM, flow cytometry and immune fluorescence. Since apoptosis or programmed cell death is a normal physiological process, we try to explore the factors associated with apoptosis. The CCK-8 assay and Annexin V-based flow cytometric data showed that cinobufacini could induce apoptosis of MDAMB-231 cells effectively in dose-dependent and time-dependent manners (Fig. 1, Fig. 2). The changes in cell cycle results showed that cinobufacini treatment caused a cell cycle arrest at S phases (Table 1). The results indicated that cinobufacini could block the process of the cell cycle from S phase to G2/M phase, which could block the cell mitosis and make the cell cycle stay at S phases. At last, cinobufacini induced the cell apoptosis. AFM data indicated that cinobufacini could damage the cell membrane and then changed the ultrastructure of cell surface a lot. Simultaneously, the cells shrunk and cell size reduced (Fig. 3). To further confirm these results, we studied clues regarding the relationship between the
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Fig. 4. F-actin in MDA-MB-231 cells was stained with Rhodamine–phalloidin and nuclei was stained with DAPI. (A1) showed the cells were spindle, a large number of F-actin around the nucleus regularly in the cells and the fibers in the cytoplasm arranged as regular rays. (B1) showed the cells after being treated with cinobufacini (50 lg/mL) for 48 h, the cells became irregular in shape. F-actin dispersed in the cells and stress fibers around the nucleus arranged in disorder and the numbers were decreased. A2 showed that the cells were plump and intact, intracellular staining and nuclei morphology were uniform in the normal group. B2 showed the cells were treated with cinobufacini, the nuclei have been deformed and there were some holes in the middle of the nuclei, nuclei changed markedly.
cytoskeletons and functions. The alternations in the immunofluorescence of cytoskeleton and nuclei morphology also showed the apoptosis effects after the cells being treated with cinobufacini (Fig. 4). These results highlight the importance of the cytoskeleton in the spatial signal organization, so it is crucial to determine whether the stress fibers in transformed cells have disappeared after the cells were treated with cinobuficini. Even more, alterations in the cytoskeleton could effect on the signaling pathways. And nuclei morphology also showed the apoptosis induced by cinobufacini. In summary, this research indicated that cinobufacini could inhibit cellular proliferation significantly. And it could induce cell cycle arrest at S phase and induce apoptosis effectively in MDA-MB231 cells and it could provide a further understand of cinobufacini mechanism. Mean while, the structures of the MDA-MB-231 cell changes were detected by AFM at the nanometer scale, it could provide us to understand cinobufacini effect on the cell-surface. So AFM will be a potential sensitive tool for detecting cancer cells. However, more research is required to understand the specific mechanism of cinobufacini. Acknowledgments This work is supported by Ministry of Science and Technology of China (No. 2010CB833603), the National Natural Science Foundation of China (No. 30872404), the Fundamental Research Funds for the Central Universities (No. 30828028, No. 21609305, and No. 21610427), the Key Project of Chinese Ministry of Education (No. 210254) and Specialized Research Fund for the Doctoral Program of Higher Eduction (20104401120004). References and notes 1. Serag, H. B.; Marrero, J. A.; Rudolph, L.; Reddy, K. R. Gastroenterology 2008, 134, 1752.
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