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AFM-detected apoptosis of hepatocellular carcinoma cells induced by American ginseng root water extract
Micron 104 (2018) 1–7
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Micron journal homepage: www.elsevier.com/locate/micron
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AFM-detected apoptosis of hepatocellular carcinoma cells induced by American ginseng root water extract
MARK
⁎
Yingmin Qua, Zuobin Wanga,b, , Feihu Zhaoc, Jinyun Liua,b, Wenxiao Zhanga, Jingmei Lid, Zhengxun Songa, Hongmei Xua a
International Research Centre for Nano Handling and Manufacturing of China, Changchun University of Science and Technology, Changchun 130022, China Institute for Research in Applicable Computing, University of Bedfordshire, Luton LU1 3JU, UK c Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven 5600 MB, Netherlands d School of Life Sciences, Changchun University of Science and Technology, Changchun 130022, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: American ginseng Atomic force microscope (AFM) Cell apoptosis Cellular stiffness SMMC-7721 cells
American ginseng as a common and traditional herbal medicine has been used in cancer treatment for many years. However, the effect of American ginseng on the cancer cell response (i.e. apoptosis) has not been fully understood yet. Previous studies demonstrated that cellular apoptosis was associated with the changes of mechanical and morphological properties. Therefore, in this study, mechanical and morphological characterizations were carried out by both atomic force microscope (AFM) and inverted optical microscope to investigate the apoptosis of hepatocellular carcinoma (SMMC-7721) cells affected by American ginseng root water extract (AGRWE). The results showed that the cells treated with AGRWE exhibited significantly larger surface roughness, height and elastic modulus values than control group. Moreover, those parameters were upregulated under the higher concentration of AGRWE and longer culture time. Consequently, it indicates that the mechanical and morphological properties can be used as the apoptotic characteristics of SMMC-7721 cells. Also, the increased surface roughness and elastic modulus of cells under the AGRWE treatment have shown that the apoptosis of SMMC-7721 cells can be enhanced by AGRWE. This will provide an important implication for hepatocelluar carcinoma treatment and drug development.
1. Introduction Liver cancer has been found in the second place among the most common cancers, which causes death globally (Ryerson et al., 2016). Moreover, its incidence rate has been increasing yearly. American ginseng (Panax quinquefolius L.) is a popular traditional herbal medicine used for physiological and pharmacological effects on anticancer actions (Yun et al., 1982; Chen et al., 1998; Duda et al., 1999). The anticancer effects of ginsenosides on the cancer cell lines HCT-116 and SW480 were studied at different steaming time periods and temperatures, and the results showed that the cancer cell apoptosis rate increased to 97.8% and 99.6% after the cells cultured with the ginseng berry steamed at 120 °C for 2 h (Wang et al., 2006a). Corbit et al. (2006) found that the American ginseng root was used alone or in combination with contaminant heavy metals could prevent human breast cancer MCF-7 cell proliferation. Microwave processed ginseng can inhibit the growth of three human prostate cancer cell lines with
the IC50 values of 48 μg/ml, 74 μg/ml and 62 μg/ml in PC-3, DU145 and LNCaP cells, respectively. The results showed that the expression of apoptosis-related proteins increased after the treatment with 100 mg/ ml microwave-irradiated ginseng (Park et al., 2016). It was found that the ginseng could present beneficial functions on some cancer cells (Bespalov et al., 1993; Wu et al., 2015). Previous studies found that mechanical properties and cell morphology could be used to describe cell behaviors. The cell stiffening phenomenon during osteogenic differentiation was found through the work on mechanical characterization of human adipose stem cells (Virjula et al., 2017). Hu et al. (2013) characterized the mechanical properties of human adipose stem cells before and after apoptosis, and it was found that the cells were stiffer after apoptosis than the living cells. In a previous report (Liu et al., 2015), we used the AFM to determine the elastic moduli of cancer cells through the analysis of indentation. Xiong et al. (2009) measured the elastic moduli in different areas of aplysia growth cones in the liquid by AFM. Wang et al. (2006b)
Abbreviations: AFM, atomic force microscope; RMSR, root-mean-square roughness; AGRWE, American ginseng root water extract; AR, average roughness ⁎ Corresponding author at: International Research Centre for Nano Handling and Manufacturing of China, Changchun University of Science and Technology, Changchun, 130022, China. E-mail address: [email protected] (Z. Wang). http://dx.doi.org/10.1016/j.micron.2017.10.003 Received 25 August 2017; Received in revised form 10 October 2017; Accepted 10 October 2017 Available online 12 October 2017 0968-4328/ © 2017 Elsevier Ltd. All rights reserved.
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the coverslips were taken out and dried under the sterilization conditions. SMMC-7721 cells were digested with 0.25% trypsin and seeded onto the poly-L-lysine coated coverslips. Afterwards the cells were incubated in 5% CO2 at 37 °C. After 24 h, the following AGRWE concentrations and treatment time periods were employed in cell culture: (i) 0 mg/ml, 3.75 mg/ml and 15 mg/ml for 12 h; (ii) 0 mg/ml and 7.5 mg/ml for 24 h; (iii) 0 mg/ml and 7.5 mg/ml for 36 h. Before the measurements, the coverslips containing the cells were washed with 0.01 M phosphate buffer saline.
investigated the changes in morphology and ultrastructure of HeLa, HepG2 and C6 cells treated with anti-cancer drugs showing the important significance for the diagnosis of apoptosis in cancer cells based on AFM. However, the mechanical and morphological properties of hepatocellular carcinoma cells during apoptosis have not been investigated yet to evaluate the anti-liver cancer effect of AGRWE. In this study, we hypothesized that AGRWE could have the anti-liver cancer effect by enhancing the apoptosis of SMMC-7721 cells. Moreover, SMMC-7721 cell apoptosis would be associated with the changes of the mechanical and morphological properties. Consequently, AFM indentation and inverted optical microscopy were used for characterizing the changes of mechanical and morphological properties of the SMMC-7721 cells treated with AGRWE for different concentrations and time periods. The experiment results have shown that the apoptosis of SMMC-7721 cells can be enhanced by AGRWE. The output of this study will provide an important implication for the treatment of liver cancer at the cellular level and anti-cancer drug development.
2.6. Characterization of morphological and biophysical properties with AFM SMMC-7721 cells were detected by a Nano Wizard III AFM (JPK Instruments, Germany) for cell imaging and elastic modulus measurement in the culture using the quantitative imaging (QI) mode in the experiments. The AFM probe (Bruker, Germany) used in the experiments was made of silicon nitride with a pyramidal shape (the tip radius of 10 nm, and the thickness of 0.55 μm). The probe was mounted on a cantilever (length = 225 μm and width = 20 μm) with a nominal spring constant of 0.03 N/m. A thermal noise method was employed to calibrate the spring constant before experiments (Hutter and Bechhoefer, 1993) [16]. The Hertz model for the pyramid indenters was used to calculate the elastic moduli of cells in this work.
2. Materials and methods 2.1. Preparation of AGRWE The roots of American ginseng were produced by Wisconsin ginseng farm. AGRWE was prepared using 50 g of American ginseng root, and was extracted with 200 ml sterile water at 100 °C for 1 h (Yoo et al., 2006). The heat-processed product was filter-sterilized and kept at 4 °C.
3. Results 3.1. Analysis of cell morphological changes
2.2. Cell culture Fig. 1 shows the experiment results from the SMMC-7721 cells incubated with AGRWE for 12 h. The results were obtained from different concentrations of AGRWE: 0, 20, 40, 60 and 80 mg/ml. The morphological changes of apoptotic cells were observed using an inverted microscope. The control cells without the treatment of AGRWE were full of vitality, as shown in Fig. 1(a). The morphological characteristics of the cells were changed distinctly from the polygonal to round shapes and the cell apoptosis rate was increased when the AGRWE concentration was increased from 20 mg/ml to 80 mg/ml, as shown by arrows in Fig. 1(b)–(e).
In the experiments, SMMC-7721 cells were cultured in the RPMI1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) and incubated in 5% CO2 at 37 °C. The logarithmic phase cells were collected with 0.25% trypsin (Sigma, USA). 2.3. Observation by inverted microscope Cells were collected in the logarithmic phase. Approximately 3 × 105 cells/well were seeded into 6-well plates with 2 ml medium containing 10% FBS (Hyclone, USA), and incubated in 5% CO2 at 37 °C for 24 h. The medium was removed, and then different concentrations of AGRWE were added to each well, except the control group. The final concentrations of AGRWE in each group were 0, 20, 40, 60 and 80 mg/ ml, respectively. After 12 h incubation, the cell morphologies were observed by inverted microscope.
3.2. Analysis of cell viability The inhibition rates of ginseng on the cell growth were determined by MTT assay. The cells were incubated with various ginseng concentrations (0, 20, 40, 60 and 80 mg/ml) for 12 h. As shown in Fig. 2, the viability of the SMMC-7721 cells exposed to AGRWE with 20 mg/ml was reduced to 72.44 ± 1.25%. After the treatment with the ginseng concentration of 80 mg/ml, the cell viability was reduced to 46.47 ± 0.24%. It can be seen that the survival rate of cells is decreased with the increase of concentrations.
2.4. MTT assay 1 × 104 SMMC-7721 cells/well were seeded into 96-well plates in the 200 μl RPMI1640 medium with 10% FBS and incubated for 24 h. AGRWE was added to the culture medium of SMMC-7721 cells to make the different concentrations 0, 20, 40, 60 and 80 mg/ml. After incubating for an additional 12 h, 20 μl of MTT solution with the concentration of 5 mg/ml was individually added to each well. After incubating the cells at 37 °C for 4 h and the solution was removed carefully, and the purple colored formazan product developed in each well was dissolved by DMSO (Solarbio, China). After slow shaking for 10 min, the absorbance was read at the wavelength of 492 nm by microplate reader. Cell viability was expressed as the percentage of control.
3.3. Cellular morphology The variations of the height and membrane roughness of SMMC7721 cells in response to AGRWE were obtained from AFM detection, as shown in Figs. 3–5. It was found that the changes of the height and membrane roughness were dose- and time-dependent. All the experiment results were analyzed with the JPK data processing software. Among the average roughness (AR) and root-mean-square roughness (RMSR) values of cell surfaces, the SMMC-7721 cells cultured without AGRWE had the lowest roughness values, while the cells incubated with the maximum concentration and for the longest time showed the highest roughness values. Minor variations of the membranes of SMMC7721 cells were observed after the cells were treated by 3.75 mg/ml AGRWE for 12 h and 7.5 mg/ml for 24 h. With a higher concentration of 15 mg/ml for 12 h and 7.5 mg/ml for 36 h, the AR and RMSR values
2.5. AGRWE treatments and preparation of AFM imaging samples In order to enhance the cell adherence so as to obtain high quality images in the experiment, the coverslips were coated by poly-L-lysine (Sigma, USA) with the concentration of 0.01 mg/ml. After an overnight, 2
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Fig. 1. Experiment results from the SMMC-7721 cells incubated with AGRWE for 12 h. The AGRWE concentrations in Fig. 1(a)–(e) are 0, 20, 40, 60, and 80 mg/ml, respectively. The arrows show the apoptotic cells.
0.400 ± 0.018 μm and 0.495 ± 0.028 μm, and after the treatment for 36 h they were changed to 0.480 ± 0.045 μm and 0.609 ± 0.065 μm. Figs. 3 (d) and 4 (d) show that the AGRWE can increase the heights of the SMMC-7721 cells. It can be seen from the histograms that the height of SMMC-7721 cells untreated with AGRWE is 3.850 ± 0.520 μm, and that treated with 3.75 mg/ml AGRWE for 12 h is 4.840 ± 0.400 μm, which is lower than those treated with 15 mg/ml AGRWE (5.360 ± 0.420 μm). The height of SMMC-7721 cells with the treatment of 7.5 mg/ml AGRWE for 24 h is 5.285 ± 0.220 μm and it is
were increased with the treatment time periods and concentrations, as shown in Fig. 5(a) and (b). The AR and RMSR of the SMMC-7721 cells untreated with AGRWE were 0.214 ± 0.029 μm and 0.280 ± 0.039 μm. After exposure to the AGRWE with the concentration of 3.75 mg/ml, the AR and RMSR were increased to 0.297 ± 0.079 μm and 0.386 ± 0.097 μm, respectively. They went up to 0.433 ± 0.080 μm and 0.535 ± 0.103 μm after the treatment with 15 mg/ml AGRWE. After the treatment with 7.5 mg/ml AGRWE for 24 h, the AR and RMSR of the SMMC-7721 cells were 3
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(Fig. 6(b)), respectively. The effects of AGRWE on the biophysical properties of SMMC-7721 cells showed that the cell stiffness was increased with the increase of the AGRWE treatment concentration and duration. From the histograms, the average elastic modulus of the control cells is 1.940 ± 0.750 kPa. The stiffness of the SMMC-7721 cells treated with 3.75 mg/ml AGRWE for 12 h is 2.360 ± 0.110 kPa and it is changed to 3.036 ± 0.877 kPa after the treatment with 15 mg/ml AGRWE. The stiffness of SMMC-7721 cells with the treatment of 7.5 mg/ml AGRWE for 24 h is 2.440 ± 0.760 kPa, which are softer than those treated for 36 h. The morphological features and biophysical properties are significantly different due to the different concentrations and durations for the AGRWE treatment of SMMC-7721 cells. 4. Discussion Fig. 2. Evaluation of proliferation of SMMC-7721 cells with different concentrations of AGRWE using the MTT assay. The results were expressed as the mean values and standard errors of three independent experiments.
A number of previous studies investigated the effects of ginseng on the apoptosis of cancer cells, and it was found that steamed ginseng could induce apoptotic cell death. Wang et al. (2009) investigated the anticancer effects of steamed ginseng on SW480 human colorectal cancer cells. Additionally, the MDA-MB-231 and MCF-7 cell lines after stimulation with phorbol ester were used to evaluate the anticancer effect of the American ginseng extracted by lyophilized aqueous, and found that ginseng (100 mg/ml) could block the COX-2 protein expression (Peralta et al., 2009). In our study, the American ginseng root water extract was produced with 200 ml sterile water using 50 g of American ginseng root at 100 °C for 1 h. It was found that the
changed to 8.360 ± 1.250 μm after the treatment with AGRWE for 36 h. 3.4. Cellular young’s modulus Fig. 6 shows the average elastic moduli of the control cells and the cells exposed to AGRWE with the concentrations of 3.75 mg/ml and 15 mg/ml for 12 h (Fig. 6(a)) and 7.5 mg/ml for 24 h and 36 h
Fig. 3. Topographies of SMMC-7721 cell surfaces detected by AFM after the treatment of 12 h using different concentrations of AGRWE (mg/ml): (a) 0, (b) 3.75, and (c) 15; (d) Average heights of the control cells and the cells after the treatment using 3.75 and 15 mg/ml AGRWE.
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Fig. 4. Topographies of SMMC-7721 cell surfaces detected by AFM after the treatment with AGRWE for different treatment time periods (h): (a) 0, (b) 24, and (c) 36; (d) Average heights of the control cells and the cells after the treatment of AGRWE for 0 h, 24 h and 36 h.
(Venkatesan et al., 2010). Interestingly, our results showed that the cells in the control group had the polygon shape after 24 h culturing. For the cells treated with the concentrations of 3.75 mg/ml AGRWE for 12 h and 7.5 mg/ml for 24 h, cell shrinking was observed, and the shape became round compared with the control group. Furthermore, the morphologies of the cells, which were exposed to AGRWE with the concentrations of 15 mg/ml for 12 h and 7.5 mg/ml for 36 h, were changed from the polygon to round significantly. According to the literature (Kerr et al., 1972; Bortner and Cidlowski, 1998), the cell shrinkage is a mark of the apoptosis. Therefore, our results indicated
cytotoxicity in SMMC-7721 cells increased with the increase of dose and time of ginseng treatment (Figs. 1 and 2), which was in agreement with the trend of the results reported by Li et al. (2010). Their results found that the steamed American ginseng for 4 h could improve the anticancer effect with more antioxidants or inhibitors. 1 An atomic force microscope can be used as a high resolution tool to study the minor changes in mechanical and morphological properties and the relationship between the cell-surface and the reacting regent (Jin et al., 2011; Cai et al., 2009). It was found that morphologies of the colon cancer cell (HCT-15) displayed cell shrinkage using AFM
Fig. 5. Statistical analyses of the average surface roughness and root mean square values in the selected areas of 10 × 10 μm2 nearby the nuclear of the control cell and the cell after the treatment using AGRWE under the conditions: (a) different concentrations and (b) different treatment time periods with AGRWE. All the results are expressed as the mean values and standard errors. The cell number is 8 for each group.
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Fig. 6. Statistical analyses of average elastic modulus of the control cells and the cells after the treatments using AGRWE under the conditions: (a) 3.75 mg/ml, 12 h and 15 mg/ml, 12 h and (b) 7.5 mg/ml, 24 h and 7.5 mg/ml, 36 h. All the results are expressed as the mean values and standard errors. The cell number is 8 for each group.
Acknowledgments
that the SMMC-7721 cells underwent apoptosis, which was enchanced by the treatment of AGRWE. In addition, the SMMC-7721 cells after treatment with AGRWE had revealed larger roughness (Fig. 5). It was in agreement with the study, in which the cell membrane average roughness increased from 24 to 60 nm during the cell apoptosis induced by H2O2 (Buyukhatipoglu and Clyne, 2011). Cellular stiffness is one of the properties, which is used for evaluating the cellular apoptosis. Previous studies found that cellular apoptosis was associated with the increase of stiffness (Wang et al., 2011). Lam et al. (2007) showed that the elastic modulus was increased for the apoptotic cells induced by chemotherapy. Such cell stiffening phenomenon also agreed with the findings in our study that the elastic modulus and the apoptosis rate of SMMC-7721 cells were increased with the treatment time period and concentration. Moreover, in our study, it was found that the average stiffness of SMMC-7721 cells that were exposed to AGRWE with the concentrations of 15 mg/ml for 12 h and 7.5 mg/ml for 36 h was significantly increased, compared to the elastic moduli of the cells exposed to the AGRWE concentrations of 3.75 mg/ml for 12 h and 7.5 mg/ml for 24 h. The result showed the same tendency with the report (Cross et al., 2011), in which the Young’s modulus of metastatic cells was increased from 0.41 ± 0.18 kPa to 2.54 ± 1.47 kPa after the treatment of green tea for 24 h, and it was concluded that green tea could be associated with the cancer cell apoptosis by upregulating cellular stiffness. Corresponding to the findings in our study, it is indicated that the ginseng can enhance the apoptosis of SMMC-7721 cells. Thus, this work will be useful for prevention and treatment of liver cancer.
This work was supported by the “111” Project of China (D17017), EU FP7 (BioRA No.612641), China-EU H2020 (FabSurfWAR Nos.2016YFE0112100 and 644971), EU H2020 (MNR4SCell No.734174), and Jilin Provincial Science and Technology Program (Nos.20160520101JH, 20160101318JC and 20160623002TC). References Bespalov, V.G., Aleksandrov, V.A., Davydov, V.V., Limarenko, A.Y., Molokovskii, D.S., Petrov, A.S., Slepyan, L.I., Trilis, Y.G., 1993. Mammary carcinogenesis suppression by ginseng tissue culture biomass tincture. Bull Exp. Biol. Med. 115, 63–65. Bortner, C.D., Cidlowski, J.A., 1998. A necessary role for cell shrinkage in apoptosis. Biochem. Pharmacol. 56, 1549–1559. Buyukhatipoglu, K., Clyne, A.M., 2011. Superparamagnetic iron oxide nanoparticles change endothelial cell morphology and mechanics via reactive oxygen species formation. J. Biomed. Mater. Res. Part A 96 (1), 186–195. Cai, X.F., Gao, S.J., Cai, J.Y., Wu, Y.Z., Deng, H., 2009. Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM. Scanning 31 (2), 83–89. Chen, X.G., Liu, H.Y., Lei, X.H., Fu, Z.D., Li, Y., Tao, L.H., Han, R., 1998. Cancer chemopreventive and therapeutic activities of red ginseng. J. Ethnopharmacol. 60 (1), 71–78. Corbit, R., Ebbs, S., King, M.L., Murphy, L.L., 2006. The influence of lead and arsenite on the inhibition of human breast cancer MCF-7 cell proliferation by American ginseng root (Panax quinquefolius L.). Life Sci. 78, 1336–1340. Cross, S.E., Jin, Y.S., Lu, Q.Y., Rao, J.Y., Gimzewski, J.K., 2011. Green tea extract selectively targets nanomechanics of live metastatic cancer cells. Nanotechnology 22 (21), 215101. Duda, R.B., Zhong, Y., Navas, V., Li, M.Z.C., 1999. American ginseng and breast cancer therapeutic agents synergistically inhibit MCF-7 breast cancer cell growth. J. Surg. Oncol 72 (4), 230–239. Hu, K.X., Zhao, F.H., Wang, Q.K., 2013. Mechanical characterization of living and dead undifferentiated human adipose-derived stem cells by using atomic force microscopy. Proc. IMechE. P. H: J. Eng. Med. 227 (12), 1319–1323. Hutter, J.L., Bechhoefer, J., 1993. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64, 1868–1873. Jin, H., Ma, S.Y., Song, B., Ma, L.N., Pi, J., Chen, X.X., Chen, Y., Cai, J.Y., 2011. Liposome impaired the adhesion and spreading of HEK293 cells: an AFM study. Scanning 33 (6), 413–418. Kerr, J.F.R., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26 (4), 239. Lam, W.A., Rosenbluth, M.J., Fletcher, D.A., 2007. Chemotherapy exposure increases leukemia cell stiffness. Blood 109 (8), 3505–3508. Li, B.H., Wang, C.Z., He, T.C., Yuan, C.S., Du, W., 2010. Antioxidants potentiate American ginseng-induced killing of colorectal cancer cells. Cancer Lett. 289 (1), 62–70. Liu, X.G., Song, Z.X., Qu, Y.M., Wang, G.L., Wang, Z.B., 2015. Mechanical properties study of SW480 cells based on AFM. Cell Biol. Int. 39, 972–977. Park, J.Y., Choi, P., Kim, H.K., Kang, K.S., Ham, J., 2016. Increase in apoptotic effect of Panax ginseng by microwave processing in human prostate cancer cells: in vitro and in vivo studies. J. Gins. Res. 40, 62–67. Peralta, E.A., Murphy, L.L., Minnis, J., Louis, S., Dunnington, G.L., 2009. American Ginseng inhibits induced COX-2 and NFKB activation in breast cancer cells. J. Surg. Res. 157 (2), 261–267. Ryerson, A.B., Eheman, C.R., Altekruse, S.F., Ward, J.W., Jemal, A., Sherman, R.L., Henley, S.J., Holtzman, D., Lake, A., Noone, A.M., Anderson, R.N., Ma, J., Ly, K.N., Cronin, K.A., Penberthy, L., Kohler, B.A., 2016. Annual report to the nation on the status of cancer, 1975–2012, featuring the increasing incidence of liver cancer. Cancer 122 (9), 1312–1337. Venkatesan, P., Das, S., Krishnan, M.M.R., Chakraborty, C., Chaudhury, K., Mandal, M., 2010. Effect of AEE788 and/or Celecoxib on colon cancer cell morphology using advanced microscopic techniques. Micron 41, 247–256.
5. Conclusion In this work, the apoptosis of SMMC-7721 cells induced by American ginseng root water extract was investigated. The optical images and the MTT results showed that the apoptosis rates were increased with the increase culture time and AGRWE concentration. The enhanced apoptosis of SMMC-7721 cells by higher AGRWE concentration and longer culture time was associated with the cellular stiffening phenomenon. This conclusion is in agreement with the results from the morphology and roughness measurements of cells by AFM and the analyses of cell apoptosis with the different AGRWE concentrations and treatment time periods. Also, the elastic modulus can be useful for the further study of the apoptosis-independent growth inhibition mechanism in biomedicine. This work provides a visual method for the detection of early stage apoptosis of SMMC-7721 cells interacted with ginsengs and medicines.
Conflict of interest None.
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Wang, D.C., Chen, K.Y., Tsai, C.H., Chen, G.Y., Chen, C.H., 2011. AFM membrane roughness as a probe to identify oxidative stress-induced cellular apoptosis. J. Biomech. 44, 2790–2794. Wu, K., Li, N., Sun, H.Q., Xu, T., Jin, F., Nie, J.F., 2015. Endoplasmic reticulum stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity. Biochem. Biophys. Res. Commun. 466, 369–375. Xiong, Y., Lee, A.C., Suter, D.M., Lee, G.U., 2009. Topography and nanomechanics of live neuronal growth cones analyzed by atomic force microscopy. Biophys. J. 96, 5060–5072. Yoo, H.H., Yokozawa, T., Satoh, A., Kang, K.S., Kim, H.Y., 2006. Effects of ginseng on the proliferation of human lung fibroblasts. Am. J. Chin. Med. 34, 137–146. Yun, T.K., Yun, Y.S., Han, I.W., 1982. Anticarcinogenic effect of long-term oral administration of red ginseng on newborn mice exposed to various chemical carcinogens. Cancer detect. Prev. 6 (6), 515–525.
Virjula, S., Zhao, F.H., Leivo, J., Vanhatupa, S., Kreutzer, J., Vaughan, T.J., Honkala, A.M., Viehrig, M., Mullen, C.A., Kallio, P., McNamara, L.M., Miettinen, S., 2017. The effect of equiaxial stretching on the osteogenic differentiation and mechanical properties of human adipose stem cells. J. Mech. Bech. Behav. Biomed. 72, 38–48. Wang, C.Z., Zhang, B., Song, W.X., Wang, A.B., Ni, M., Luo, X.J., Aung, H.H., Xie, J.T., Tong, R., He, T.C., Yuan, C.S., 2006a. Steamed American ginseng berry: ginsenoside analyses and anticancer activities. J. Agric. Food Chem. 54, 9936–9942. Wang, J.Y., Wan, Z.F., Liu, W.M., Li, L., Ren, L., An, X.Q., Sun, P., Ren, L.L., Zhao, H.Y., Tu, Q., Zhang, Z.Y., Song, N., Zhang, L., 2006b. Atomic force microscope study of tumor cell membranes following treatment with anti-cancer drugs. Biosens. Bioelectron. 25, 721–727. Wang, C.Z., Li, X.L., Wang, Q.F., Mehendale, S.R., Fishbein, A.B., Han, A.H., Sun, S., Yuan, C.S., 2009. The mitochondrial pathway is involved in American ginseng-induced apoptosis of SW-480 colon cancer cells. Oncol. Rep. 21, 577–584.
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Tutorial
AFM-detected apoptosis of hepatocellular carcinoma cells induced by American ginseng root water extract
MARK
⁎
Yingmin Qua, Zuobin Wanga,b, , Feihu Zhaoc, Jinyun Liua,b, Wenxiao Zhanga, Jingmei Lid, Zhengxun Songa, Hongmei Xua a
International Research Centre for Nano Handling and Manufacturing of China, Changchun University of Science and Technology, Changchun 130022, China Institute for Research in Applicable Computing, University of Bedfordshire, Luton LU1 3JU, UK c Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven 5600 MB, Netherlands d School of Life Sciences, Changchun University of Science and Technology, Changchun 130022, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: American ginseng Atomic force microscope (AFM) Cell apoptosis Cellular stiffness SMMC-7721 cells
American ginseng as a common and traditional herbal medicine has been used in cancer treatment for many years. However, the effect of American ginseng on the cancer cell response (i.e. apoptosis) has not been fully understood yet. Previous studies demonstrated that cellular apoptosis was associated with the changes of mechanical and morphological properties. Therefore, in this study, mechanical and morphological characterizations were carried out by both atomic force microscope (AFM) and inverted optical microscope to investigate the apoptosis of hepatocellular carcinoma (SMMC-7721) cells affected by American ginseng root water extract (AGRWE). The results showed that the cells treated with AGRWE exhibited significantly larger surface roughness, height and elastic modulus values than control group. Moreover, those parameters were upregulated under the higher concentration of AGRWE and longer culture time. Consequently, it indicates that the mechanical and morphological properties can be used as the apoptotic characteristics of SMMC-7721 cells. Also, the increased surface roughness and elastic modulus of cells under the AGRWE treatment have shown that the apoptosis of SMMC-7721 cells can be enhanced by AGRWE. This will provide an important implication for hepatocelluar carcinoma treatment and drug development.
1. Introduction Liver cancer has been found in the second place among the most common cancers, which causes death globally (Ryerson et al., 2016). Moreover, its incidence rate has been increasing yearly. American ginseng (Panax quinquefolius L.) is a popular traditional herbal medicine used for physiological and pharmacological effects on anticancer actions (Yun et al., 1982; Chen et al., 1998; Duda et al., 1999). The anticancer effects of ginsenosides on the cancer cell lines HCT-116 and SW480 were studied at different steaming time periods and temperatures, and the results showed that the cancer cell apoptosis rate increased to 97.8% and 99.6% after the cells cultured with the ginseng berry steamed at 120 °C for 2 h (Wang et al., 2006a). Corbit et al. (2006) found that the American ginseng root was used alone or in combination with contaminant heavy metals could prevent human breast cancer MCF-7 cell proliferation. Microwave processed ginseng can inhibit the growth of three human prostate cancer cell lines with
the IC50 values of 48 μg/ml, 74 μg/ml and 62 μg/ml in PC-3, DU145 and LNCaP cells, respectively. The results showed that the expression of apoptosis-related proteins increased after the treatment with 100 mg/ ml microwave-irradiated ginseng (Park et al., 2016). It was found that the ginseng could present beneficial functions on some cancer cells (Bespalov et al., 1993; Wu et al., 2015). Previous studies found that mechanical properties and cell morphology could be used to describe cell behaviors. The cell stiffening phenomenon during osteogenic differentiation was found through the work on mechanical characterization of human adipose stem cells (Virjula et al., 2017). Hu et al. (2013) characterized the mechanical properties of human adipose stem cells before and after apoptosis, and it was found that the cells were stiffer after apoptosis than the living cells. In a previous report (Liu et al., 2015), we used the AFM to determine the elastic moduli of cancer cells through the analysis of indentation. Xiong et al. (2009) measured the elastic moduli in different areas of aplysia growth cones in the liquid by AFM. Wang et al. (2006b)
Abbreviations: AFM, atomic force microscope; RMSR, root-mean-square roughness; AGRWE, American ginseng root water extract; AR, average roughness ⁎ Corresponding author at: International Research Centre for Nano Handling and Manufacturing of China, Changchun University of Science and Technology, Changchun, 130022, China. E-mail address: [email protected] (Z. Wang). http://dx.doi.org/10.1016/j.micron.2017.10.003 Received 25 August 2017; Received in revised form 10 October 2017; Accepted 10 October 2017 Available online 12 October 2017 0968-4328/ © 2017 Elsevier Ltd. All rights reserved.
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the coverslips were taken out and dried under the sterilization conditions. SMMC-7721 cells were digested with 0.25% trypsin and seeded onto the poly-L-lysine coated coverslips. Afterwards the cells were incubated in 5% CO2 at 37 °C. After 24 h, the following AGRWE concentrations and treatment time periods were employed in cell culture: (i) 0 mg/ml, 3.75 mg/ml and 15 mg/ml for 12 h; (ii) 0 mg/ml and 7.5 mg/ml for 24 h; (iii) 0 mg/ml and 7.5 mg/ml for 36 h. Before the measurements, the coverslips containing the cells were washed with 0.01 M phosphate buffer saline.
investigated the changes in morphology and ultrastructure of HeLa, HepG2 and C6 cells treated with anti-cancer drugs showing the important significance for the diagnosis of apoptosis in cancer cells based on AFM. However, the mechanical and morphological properties of hepatocellular carcinoma cells during apoptosis have not been investigated yet to evaluate the anti-liver cancer effect of AGRWE. In this study, we hypothesized that AGRWE could have the anti-liver cancer effect by enhancing the apoptosis of SMMC-7721 cells. Moreover, SMMC-7721 cell apoptosis would be associated with the changes of the mechanical and morphological properties. Consequently, AFM indentation and inverted optical microscopy were used for characterizing the changes of mechanical and morphological properties of the SMMC-7721 cells treated with AGRWE for different concentrations and time periods. The experiment results have shown that the apoptosis of SMMC-7721 cells can be enhanced by AGRWE. The output of this study will provide an important implication for the treatment of liver cancer at the cellular level and anti-cancer drug development.
2.6. Characterization of morphological and biophysical properties with AFM SMMC-7721 cells were detected by a Nano Wizard III AFM (JPK Instruments, Germany) for cell imaging and elastic modulus measurement in the culture using the quantitative imaging (QI) mode in the experiments. The AFM probe (Bruker, Germany) used in the experiments was made of silicon nitride with a pyramidal shape (the tip radius of 10 nm, and the thickness of 0.55 μm). The probe was mounted on a cantilever (length = 225 μm and width = 20 μm) with a nominal spring constant of 0.03 N/m. A thermal noise method was employed to calibrate the spring constant before experiments (Hutter and Bechhoefer, 1993) [16]. The Hertz model for the pyramid indenters was used to calculate the elastic moduli of cells in this work.
2. Materials and methods 2.1. Preparation of AGRWE The roots of American ginseng were produced by Wisconsin ginseng farm. AGRWE was prepared using 50 g of American ginseng root, and was extracted with 200 ml sterile water at 100 °C for 1 h (Yoo et al., 2006). The heat-processed product was filter-sterilized and kept at 4 °C.
3. Results 3.1. Analysis of cell morphological changes
2.2. Cell culture Fig. 1 shows the experiment results from the SMMC-7721 cells incubated with AGRWE for 12 h. The results were obtained from different concentrations of AGRWE: 0, 20, 40, 60 and 80 mg/ml. The morphological changes of apoptotic cells were observed using an inverted microscope. The control cells without the treatment of AGRWE were full of vitality, as shown in Fig. 1(a). The morphological characteristics of the cells were changed distinctly from the polygonal to round shapes and the cell apoptosis rate was increased when the AGRWE concentration was increased from 20 mg/ml to 80 mg/ml, as shown by arrows in Fig. 1(b)–(e).
In the experiments, SMMC-7721 cells were cultured in the RPMI1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) and incubated in 5% CO2 at 37 °C. The logarithmic phase cells were collected with 0.25% trypsin (Sigma, USA). 2.3. Observation by inverted microscope Cells were collected in the logarithmic phase. Approximately 3 × 105 cells/well were seeded into 6-well plates with 2 ml medium containing 10% FBS (Hyclone, USA), and incubated in 5% CO2 at 37 °C for 24 h. The medium was removed, and then different concentrations of AGRWE were added to each well, except the control group. The final concentrations of AGRWE in each group were 0, 20, 40, 60 and 80 mg/ ml, respectively. After 12 h incubation, the cell morphologies were observed by inverted microscope.
3.2. Analysis of cell viability The inhibition rates of ginseng on the cell growth were determined by MTT assay. The cells were incubated with various ginseng concentrations (0, 20, 40, 60 and 80 mg/ml) for 12 h. As shown in Fig. 2, the viability of the SMMC-7721 cells exposed to AGRWE with 20 mg/ml was reduced to 72.44 ± 1.25%. After the treatment with the ginseng concentration of 80 mg/ml, the cell viability was reduced to 46.47 ± 0.24%. It can be seen that the survival rate of cells is decreased with the increase of concentrations.
2.4. MTT assay 1 × 104 SMMC-7721 cells/well were seeded into 96-well plates in the 200 μl RPMI1640 medium with 10% FBS and incubated for 24 h. AGRWE was added to the culture medium of SMMC-7721 cells to make the different concentrations 0, 20, 40, 60 and 80 mg/ml. After incubating for an additional 12 h, 20 μl of MTT solution with the concentration of 5 mg/ml was individually added to each well. After incubating the cells at 37 °C for 4 h and the solution was removed carefully, and the purple colored formazan product developed in each well was dissolved by DMSO (Solarbio, China). After slow shaking for 10 min, the absorbance was read at the wavelength of 492 nm by microplate reader. Cell viability was expressed as the percentage of control.
3.3. Cellular morphology The variations of the height and membrane roughness of SMMC7721 cells in response to AGRWE were obtained from AFM detection, as shown in Figs. 3–5. It was found that the changes of the height and membrane roughness were dose- and time-dependent. All the experiment results were analyzed with the JPK data processing software. Among the average roughness (AR) and root-mean-square roughness (RMSR) values of cell surfaces, the SMMC-7721 cells cultured without AGRWE had the lowest roughness values, while the cells incubated with the maximum concentration and for the longest time showed the highest roughness values. Minor variations of the membranes of SMMC7721 cells were observed after the cells were treated by 3.75 mg/ml AGRWE for 12 h and 7.5 mg/ml for 24 h. With a higher concentration of 15 mg/ml for 12 h and 7.5 mg/ml for 36 h, the AR and RMSR values
2.5. AGRWE treatments and preparation of AFM imaging samples In order to enhance the cell adherence so as to obtain high quality images in the experiment, the coverslips were coated by poly-L-lysine (Sigma, USA) with the concentration of 0.01 mg/ml. After an overnight, 2
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Fig. 1. Experiment results from the SMMC-7721 cells incubated with AGRWE for 12 h. The AGRWE concentrations in Fig. 1(a)–(e) are 0, 20, 40, 60, and 80 mg/ml, respectively. The arrows show the apoptotic cells.
0.400 ± 0.018 μm and 0.495 ± 0.028 μm, and after the treatment for 36 h they were changed to 0.480 ± 0.045 μm and 0.609 ± 0.065 μm. Figs. 3 (d) and 4 (d) show that the AGRWE can increase the heights of the SMMC-7721 cells. It can be seen from the histograms that the height of SMMC-7721 cells untreated with AGRWE is 3.850 ± 0.520 μm, and that treated with 3.75 mg/ml AGRWE for 12 h is 4.840 ± 0.400 μm, which is lower than those treated with 15 mg/ml AGRWE (5.360 ± 0.420 μm). The height of SMMC-7721 cells with the treatment of 7.5 mg/ml AGRWE for 24 h is 5.285 ± 0.220 μm and it is
were increased with the treatment time periods and concentrations, as shown in Fig. 5(a) and (b). The AR and RMSR of the SMMC-7721 cells untreated with AGRWE were 0.214 ± 0.029 μm and 0.280 ± 0.039 μm. After exposure to the AGRWE with the concentration of 3.75 mg/ml, the AR and RMSR were increased to 0.297 ± 0.079 μm and 0.386 ± 0.097 μm, respectively. They went up to 0.433 ± 0.080 μm and 0.535 ± 0.103 μm after the treatment with 15 mg/ml AGRWE. After the treatment with 7.5 mg/ml AGRWE for 24 h, the AR and RMSR of the SMMC-7721 cells were 3
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(Fig. 6(b)), respectively. The effects of AGRWE on the biophysical properties of SMMC-7721 cells showed that the cell stiffness was increased with the increase of the AGRWE treatment concentration and duration. From the histograms, the average elastic modulus of the control cells is 1.940 ± 0.750 kPa. The stiffness of the SMMC-7721 cells treated with 3.75 mg/ml AGRWE for 12 h is 2.360 ± 0.110 kPa and it is changed to 3.036 ± 0.877 kPa after the treatment with 15 mg/ml AGRWE. The stiffness of SMMC-7721 cells with the treatment of 7.5 mg/ml AGRWE for 24 h is 2.440 ± 0.760 kPa, which are softer than those treated for 36 h. The morphological features and biophysical properties are significantly different due to the different concentrations and durations for the AGRWE treatment of SMMC-7721 cells. 4. Discussion Fig. 2. Evaluation of proliferation of SMMC-7721 cells with different concentrations of AGRWE using the MTT assay. The results were expressed as the mean values and standard errors of three independent experiments.
A number of previous studies investigated the effects of ginseng on the apoptosis of cancer cells, and it was found that steamed ginseng could induce apoptotic cell death. Wang et al. (2009) investigated the anticancer effects of steamed ginseng on SW480 human colorectal cancer cells. Additionally, the MDA-MB-231 and MCF-7 cell lines after stimulation with phorbol ester were used to evaluate the anticancer effect of the American ginseng extracted by lyophilized aqueous, and found that ginseng (100 mg/ml) could block the COX-2 protein expression (Peralta et al., 2009). In our study, the American ginseng root water extract was produced with 200 ml sterile water using 50 g of American ginseng root at 100 °C for 1 h. It was found that the
changed to 8.360 ± 1.250 μm after the treatment with AGRWE for 36 h. 3.4. Cellular young’s modulus Fig. 6 shows the average elastic moduli of the control cells and the cells exposed to AGRWE with the concentrations of 3.75 mg/ml and 15 mg/ml for 12 h (Fig. 6(a)) and 7.5 mg/ml for 24 h and 36 h
Fig. 3. Topographies of SMMC-7721 cell surfaces detected by AFM after the treatment of 12 h using different concentrations of AGRWE (mg/ml): (a) 0, (b) 3.75, and (c) 15; (d) Average heights of the control cells and the cells after the treatment using 3.75 and 15 mg/ml AGRWE.
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Fig. 4. Topographies of SMMC-7721 cell surfaces detected by AFM after the treatment with AGRWE for different treatment time periods (h): (a) 0, (b) 24, and (c) 36; (d) Average heights of the control cells and the cells after the treatment of AGRWE for 0 h, 24 h and 36 h.
(Venkatesan et al., 2010). Interestingly, our results showed that the cells in the control group had the polygon shape after 24 h culturing. For the cells treated with the concentrations of 3.75 mg/ml AGRWE for 12 h and 7.5 mg/ml for 24 h, cell shrinking was observed, and the shape became round compared with the control group. Furthermore, the morphologies of the cells, which were exposed to AGRWE with the concentrations of 15 mg/ml for 12 h and 7.5 mg/ml for 36 h, were changed from the polygon to round significantly. According to the literature (Kerr et al., 1972; Bortner and Cidlowski, 1998), the cell shrinkage is a mark of the apoptosis. Therefore, our results indicated
cytotoxicity in SMMC-7721 cells increased with the increase of dose and time of ginseng treatment (Figs. 1 and 2), which was in agreement with the trend of the results reported by Li et al. (2010). Their results found that the steamed American ginseng for 4 h could improve the anticancer effect with more antioxidants or inhibitors. 1 An atomic force microscope can be used as a high resolution tool to study the minor changes in mechanical and morphological properties and the relationship between the cell-surface and the reacting regent (Jin et al., 2011; Cai et al., 2009). It was found that morphologies of the colon cancer cell (HCT-15) displayed cell shrinkage using AFM
Fig. 5. Statistical analyses of the average surface roughness and root mean square values in the selected areas of 10 × 10 μm2 nearby the nuclear of the control cell and the cell after the treatment using AGRWE under the conditions: (a) different concentrations and (b) different treatment time periods with AGRWE. All the results are expressed as the mean values and standard errors. The cell number is 8 for each group.
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Fig. 6. Statistical analyses of average elastic modulus of the control cells and the cells after the treatments using AGRWE under the conditions: (a) 3.75 mg/ml, 12 h and 15 mg/ml, 12 h and (b) 7.5 mg/ml, 24 h and 7.5 mg/ml, 36 h. All the results are expressed as the mean values and standard errors. The cell number is 8 for each group.
Acknowledgments
that the SMMC-7721 cells underwent apoptosis, which was enchanced by the treatment of AGRWE. In addition, the SMMC-7721 cells after treatment with AGRWE had revealed larger roughness (Fig. 5). It was in agreement with the study, in which the cell membrane average roughness increased from 24 to 60 nm during the cell apoptosis induced by H2O2 (Buyukhatipoglu and Clyne, 2011). Cellular stiffness is one of the properties, which is used for evaluating the cellular apoptosis. Previous studies found that cellular apoptosis was associated with the increase of stiffness (Wang et al., 2011). Lam et al. (2007) showed that the elastic modulus was increased for the apoptotic cells induced by chemotherapy. Such cell stiffening phenomenon also agreed with the findings in our study that the elastic modulus and the apoptosis rate of SMMC-7721 cells were increased with the treatment time period and concentration. Moreover, in our study, it was found that the average stiffness of SMMC-7721 cells that were exposed to AGRWE with the concentrations of 15 mg/ml for 12 h and 7.5 mg/ml for 36 h was significantly increased, compared to the elastic moduli of the cells exposed to the AGRWE concentrations of 3.75 mg/ml for 12 h and 7.5 mg/ml for 24 h. The result showed the same tendency with the report (Cross et al., 2011), in which the Young’s modulus of metastatic cells was increased from 0.41 ± 0.18 kPa to 2.54 ± 1.47 kPa after the treatment of green tea for 24 h, and it was concluded that green tea could be associated with the cancer cell apoptosis by upregulating cellular stiffness. Corresponding to the findings in our study, it is indicated that the ginseng can enhance the apoptosis of SMMC-7721 cells. Thus, this work will be useful for prevention and treatment of liver cancer.
This work was supported by the “111” Project of China (D17017), EU FP7 (BioRA No.612641), China-EU H2020 (FabSurfWAR Nos.2016YFE0112100 and 644971), EU H2020 (MNR4SCell No.734174), and Jilin Provincial Science and Technology Program (Nos.20160520101JH, 20160101318JC and 20160623002TC). References Bespalov, V.G., Aleksandrov, V.A., Davydov, V.V., Limarenko, A.Y., Molokovskii, D.S., Petrov, A.S., Slepyan, L.I., Trilis, Y.G., 1993. Mammary carcinogenesis suppression by ginseng tissue culture biomass tincture. Bull Exp. Biol. Med. 115, 63–65. Bortner, C.D., Cidlowski, J.A., 1998. A necessary role for cell shrinkage in apoptosis. Biochem. Pharmacol. 56, 1549–1559. Buyukhatipoglu, K., Clyne, A.M., 2011. Superparamagnetic iron oxide nanoparticles change endothelial cell morphology and mechanics via reactive oxygen species formation. J. Biomed. Mater. Res. Part A 96 (1), 186–195. Cai, X.F., Gao, S.J., Cai, J.Y., Wu, Y.Z., Deng, H., 2009. Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM. Scanning 31 (2), 83–89. Chen, X.G., Liu, H.Y., Lei, X.H., Fu, Z.D., Li, Y., Tao, L.H., Han, R., 1998. Cancer chemopreventive and therapeutic activities of red ginseng. J. Ethnopharmacol. 60 (1), 71–78. Corbit, R., Ebbs, S., King, M.L., Murphy, L.L., 2006. The influence of lead and arsenite on the inhibition of human breast cancer MCF-7 cell proliferation by American ginseng root (Panax quinquefolius L.). Life Sci. 78, 1336–1340. Cross, S.E., Jin, Y.S., Lu, Q.Y., Rao, J.Y., Gimzewski, J.K., 2011. Green tea extract selectively targets nanomechanics of live metastatic cancer cells. Nanotechnology 22 (21), 215101. Duda, R.B., Zhong, Y., Navas, V., Li, M.Z.C., 1999. American ginseng and breast cancer therapeutic agents synergistically inhibit MCF-7 breast cancer cell growth. J. Surg. Oncol 72 (4), 230–239. Hu, K.X., Zhao, F.H., Wang, Q.K., 2013. Mechanical characterization of living and dead undifferentiated human adipose-derived stem cells by using atomic force microscopy. Proc. IMechE. P. H: J. Eng. Med. 227 (12), 1319–1323. Hutter, J.L., Bechhoefer, J., 1993. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64, 1868–1873. Jin, H., Ma, S.Y., Song, B., Ma, L.N., Pi, J., Chen, X.X., Chen, Y., Cai, J.Y., 2011. Liposome impaired the adhesion and spreading of HEK293 cells: an AFM study. Scanning 33 (6), 413–418. Kerr, J.F.R., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26 (4), 239. Lam, W.A., Rosenbluth, M.J., Fletcher, D.A., 2007. Chemotherapy exposure increases leukemia cell stiffness. Blood 109 (8), 3505–3508. Li, B.H., Wang, C.Z., He, T.C., Yuan, C.S., Du, W., 2010. Antioxidants potentiate American ginseng-induced killing of colorectal cancer cells. Cancer Lett. 289 (1), 62–70. Liu, X.G., Song, Z.X., Qu, Y.M., Wang, G.L., Wang, Z.B., 2015. Mechanical properties study of SW480 cells based on AFM. Cell Biol. Int. 39, 972–977. Park, J.Y., Choi, P., Kim, H.K., Kang, K.S., Ham, J., 2016. Increase in apoptotic effect of Panax ginseng by microwave processing in human prostate cancer cells: in vitro and in vivo studies. J. Gins. Res. 40, 62–67. Peralta, E.A., Murphy, L.L., Minnis, J., Louis, S., Dunnington, G.L., 2009. American Ginseng inhibits induced COX-2 and NFKB activation in breast cancer cells. J. Surg. Res. 157 (2), 261–267. Ryerson, A.B., Eheman, C.R., Altekruse, S.F., Ward, J.W., Jemal, A., Sherman, R.L., Henley, S.J., Holtzman, D., Lake, A., Noone, A.M., Anderson, R.N., Ma, J., Ly, K.N., Cronin, K.A., Penberthy, L., Kohler, B.A., 2016. Annual report to the nation on the status of cancer, 1975–2012, featuring the increasing incidence of liver cancer. Cancer 122 (9), 1312–1337. Venkatesan, P., Das, S., Krishnan, M.M.R., Chakraborty, C., Chaudhury, K., Mandal, M., 2010. Effect of AEE788 and/or Celecoxib on colon cancer cell morphology using advanced microscopic techniques. Micron 41, 247–256.
5. Conclusion In this work, the apoptosis of SMMC-7721 cells induced by American ginseng root water extract was investigated. The optical images and the MTT results showed that the apoptosis rates were increased with the increase culture time and AGRWE concentration. The enhanced apoptosis of SMMC-7721 cells by higher AGRWE concentration and longer culture time was associated with the cellular stiffening phenomenon. This conclusion is in agreement with the results from the morphology and roughness measurements of cells by AFM and the analyses of cell apoptosis with the different AGRWE concentrations and treatment time periods. Also, the elastic modulus can be useful for the further study of the apoptosis-independent growth inhibition mechanism in biomedicine. This work provides a visual method for the detection of early stage apoptosis of SMMC-7721 cells interacted with ginsengs and medicines.
Conflict of interest None.
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