Biotechnol Lett DOI 10.1007/s10529-016-2056-9

ORIGINAL RESEARCH PAPER

Determination of glutathione in apoptotic SMMC-7221 cells induced by xylitol selenite using capillary electrophoresis Xue Wu . Yu Cao . Jian Zhang . Ming Lei . Xiaojie Deng . Kashif Rafiq Zahid . Yanli Liu . Ke Liu . Jihong Yang . Guomei Xiong . Hanchao Yao . Chao Qi

Received: 24 August 2015 / Accepted: 28 January 2016 Ó Springer Science+Business Media Dordrecht 2016

Abstract Objective To determine the glutathione (GSH) content in a human hepatoma cell line (SMMC-7221) treated with xylitol/selenite, providing a part of an investigation of its anti-cancer mechanisms. Results The nuclei of SMMC-7221 cells were stained with Hoechst 33258 in an apoptosis assay, and their morphology subsequently changed from circular to crescent shape. The calibration curve (r2 = 0.992) was established, and GSH content markedly decreased after treated with 0.5 and 1 mg xylitol/selenite l-1 for 12, 36 and 60 h (12 h: from 95.57 ± 19.57 to 29.09 ± 7.74 and 24.27 ± 11.15; 36 h: from 70.73 ± 11.35 to 19.54 ± 6.39 and 9.35 ± 6.69; 60 h: from 72.63 ± 16.94 to 7.432 ± 3.84 and 0). The depletion rate of GSH was more related to the concentration of xylitol/selenite than the

A1 Xue Wu and Yu Cao are co-first authors. A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13

X. Wu
Y. Cao
M. Lei
X. Deng
K. R. Zahid
Y. Liu
K. Liu
J. Yang
G. Xiong
H. Yao
C. Qi (&) Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, China Central Normal University, Wuhan 430079, People’s Republic of China e-mail: [email protected] J. Zhang Shijiazhuang Maternal and Child Health Ultrasonography Department, Shijiazhuang 050011, People’s Republic of China

treatment time (from 69.95 ± 1.87 to 100 % vs. 0.22 ± 0.2 to 100 %). Conclusions Xylitol/selenite is a promising anticancer drug to induce apoptosis in SMMC-7221 cells. It may regulate the apoptosis through the co-action of multiple mechanisms related to GSH depletion. Keywords Apoptosis
Anti-cancer
Capillary electrophoresis
Glutathione
Laser-induce fluorescence
Selenium
Xylitol/selenite

Introduction Selenium (Se), an essential trace element for human and animals, can inhibit carcinogenesis in several cell and animal models. Xylitol/selenite is a new synthetic Se compound, and Lei et al. (2012) first reported that it occurred death of a human hepatoma cell line (SMMC-7221) by causing apoptosis and not necrosis. Glutathione (GSH) acts as a major reducing agent and antioxidant defense by maintaining a tight control of the redox status in cells (Franco and Cidlowski 2012). It behaves as a free radical scavenger and also helps in regenerating other antioxidants, e.g. vitamin E, and ascorbic acid (Camera and Picardo 2002). Moreover, GSH is involved in protein and DNA synthesis and in amino acid transport (Franco and Cidlowski 2012). It also contributes to deactivation

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and elimination of xenobiotics (Musenga et al. 2007). GSH has evolved to serve diverse functions in biological systems, and its content is a determinant of cell death progression (Franco and Cidlowski 2012). Capillary electrophoresis (CE) is an accepted technique to measure GSH levels because of its high resolution and low sample volumes. Different types of electrochemical detection methods (Fang et al. 1995; Jin et al. 2000) have been used for analysis of GSH and related compounds, but laser-induced fluorescence (LIF) detection is better because of its very low limits of detection (Bayle et al. 2004). The GSH content in a variety of biological samples, including red blood cells (Zinellu et al. 2005), plasma (Carru et al. 2004), bacteria (Musenga et al. 2007) and various cell lines (Zinellu et al. 2005), has been measured by CE-LIF. Xylitol selenite is a new anticancer drug, but no description has been reported about its mechanisms of action. Therefore, CE-LIF is used to determine the change of GSH content in SMMC-7221 cells treated with xylitol/selenite, providing more information to help investigate its anticancer mechanisms.

Materials and methods Materials and solutions GSH, tri-n-butylphosphine (TBP), acetonitrile, 5-iodoacetamidofluorescein (5-IAF) were purchased from Sigma. Xylitol/selenite (38 % Se) was obtained from the College of Chemistry (Central China Normal University, Wuhan, China). Cell culture and treatment procedure SMMC-7221 cells were transferred into 24-well plate, and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing antibiotics and 10 % (v/v) fetal bovine serum (FBS) under a humidified atmosphere of 5 % (v/v) CO2 and 95 % (v/v) air at 37 °C for about 14 h to overspread the plate bottom. Confluent cells were treated in serum-free medium in the absence or presence of 0.5 or 1 mg xylitol selenite l-1 for 12, 36 and 60 h. Finally, the harvested cells were trypsinized, washed with phosphate-buffered saline (PBS) and collected.

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Apoptosis assay Apoptosis of SMMC-7221 cells was detected by Hoechst 33258 staining: cells were seeded on sterile cover glasses in the 6-well plates, followed by incubation for about 14 h, and then treated with 0.5 mg xylitol/selenite l-1 for 48 h. Cells were fixed, washed twice with PBS and stained with Hoechst 33258. The nuclei were observed under an inverted fluorescence microscope. Sample preparation SMMC-7221 cells were lysed in 1.2 ml 2 % (v/v) acetonitrile, then 80 ll cell lysates were immediately mixed with 20 ll 50 lM NAC, 20 ll 10 % TBP and 100 ll 400 lM 5-IAF for the quantification of GSH. The mixture was kept in dark for 2 h at room temperature. Finally, 10 ll supernatant was diluted 100 times with deionized water and immediately injected in the capillary electrophoresis (CE) system. Apparatus and CE conditions All analyses were carried out on a Beckman P/ACE MDQ CE instrument (Beckman- Coulter, Fullerton, CA, USA) with laser-induced fluorescence (LIF) detection. Excitation and emission were at 488 and 520 nm, respectively. Experiments were performed in an uncoated fused-silica capillary [50 cm (effective length 43 cm) 9 75 lm ID Yongnian Optical Fiber Co., Hebei, China]. The separation buffer was sodium phosphate (10 mM, pH 11.4). The sample was injected at the pressure of 0.5 psi for 5 s at the anodic end of the capillary. The separation was at 18 kV with normal polarity, and all of the separations were performed at 25 °C. Protein quantification The protein content in cell lysates was determined using bicinchoninic acid protein assay reagent with bovine serum albumin as standard. Statistics Statistical analysis was performed by SPSS software. Data were expressed as mean ± SD. p \ 0.01 and 0.05 were considered significant.

Biotechnol Lett

Results Apoptosis induced by xylitol/selenite To identify the effect of xylitol/selenite, the injured SMMC-7221 cells were stained with Hoechst 33258. The results of fluorescence microscopy revealed that xylitol/selenite induced apoptosis in SMMC-7221 cells. Morphology changed from circular to crescent shape and less nuclei were stained with Hoechst 33258 (Fig. 1a) compared with normal SMMC-7221 cells (Fig. 1b). Optimization of procedure and standard curve The optimal conditions for separating thiol compounds were established by CE. 5-IAF was used to label GSH to form a negatively charged conjugate.

Fig. 1 Apoptosis of SMMC-7221 cells treated with 0.5 mg xylitol/selenite l-1 for 48 h (Hoechst 33258 staining). a control group; b 0.5 mg xylitol/selenite l-1 group. Scale bar 50 lm

The optimum derivatization time was 2 h (Fig. 2), electrophoretic buffer was sodium phosphate (10 mM, pH 11.4) (Fig. 3) and separation voltage was 18 kV (Fig. 4). Some crucial parameters, such as the concentration of 5-IAF, cartridge temperatures and injection time, were also considered (data not shown). An eight-point calibration curve (Fig. 5) was obtained, and it provided a coefficient of determination (r2) of 0.992. Effects of xylitol/selenite on the GSH levels As shown in Fig. 6a, the levels of GSH markedly decreased after treated with 0.5 and 1 mg xylitol/ selenite l-1 for 12, 36 and 60 h (12 h: from 95.57 ± 19.57 to 29.09 ± 7.74 and 24.27 ± 11.15; 36 h: from 70.73 ± 11.35 to 19.54 ± 6.39 and 9.35 ± 6.69; 60 h: from 72.63 ± 16.94 to 7.432 ± 3.84 and 0. The data were from control to 0.5 mg/l group and 1 mg/l group). (**p \ 0.01, Fig. 6a). 1 mg xylitol/selenite l-1 caused a slight decrease in the GSH content at every time point compared with 0.5 mg xylitol/selenite l-1, but this was not significant (Fig. 6a, b). The depletion rate of GSH was more related to the concentration of xylitol/selenite than the treatment time (depletion rate was from 69.95 ± 1.87 to 100 % vs. 0.22 ± 0.2 to 100 %) (Fig. 6b, c).

Fig. 2 Derivatization time profile. 80 ll 32 lM GSH, 20 ll 50 lM NAC, 20 ll 10 % TBP derivatized with 100 ll 400 lM 5-IAF at room temperature for different time. Peak areas obtained are plotted against the derivatization time. CE conditions uncoated fused silica capillary (effective length 43 cm; id 75 lm); 10 mM pH 11.4 phosphate buffer; voltage 18 kV; cartridge temperature 25 °C; LIF detection (laser wavelength 488 nm); injection by pressure (0.5 psi for 5 s)

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Fig. 3 Electropherogram obtained from analysis of Buffer pH. Upper panel pH 9.4; middle panel pH 10.4; bottom panel pH 11.4. Other conditions as for Fig. 2. Sodium phosphate buffer (10 mM, pH 11.4) showed a better separated degree and repeatability

Fig. 4 Electropherogram obtained from analysis of separation voltage. Upper panel 14 kV; middle panel 18 kV; bottom panel 22 kV. Other conditions as for Fig. 2. 18 kV showed a relatively short migration time and a good steady current

Discussion In the previous study, Lei et al. (2012) found that xylitol/selenite occurred death of SMMC-7221 cells by causing apoptosis and not necrosis, and 0.5 mg xylitol/selenite l-1 gained half-maximal inhibition, therefore, 0.5 mg xylitol/selenite l-1 was used in the apoptosis assay to identify its effect. Markedly changed in morphology demonstrated that 0.5 mg xylitol/selenite l-1 induced apoptosis in SMMC-7221.

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Fig. 5 Relationship between GSH/NAC peak-area ratios and GSH concentrations. GSH standard solutions with the concentrations ranging between 5 and 70 lM were analyzed in triplicate. Other conditions as for Fig. 2. The regression equation obtained was y = 0.05729x ? 0.01761 (r2 = 0.992)

GSH is a determinant of cell death progression. Its depletion is central signaling event that regulates the activation of cell death pathways (Franco and Cidlowski 2012). In our work, we tested whether xylitol/ selenite affect the GSH content in the process of apoptosis in SMMC-7221 cells. Compared with the control group, GSH levels markedly decreased at every time point by inducing of 0.5 and 1 mg xylitol/ selenite l-1, which indicated that the apoptosis induced by xylitol/selenite was related to GSH depletion. Several studies have revealed that GSH depletion is necessary for the formation of apoptosome (Sato et al. 2004). It could also trigger the permeability transition pore of the mitochondria, and some apoptotic receptors translocate to the mitochondria and promote the release of cytochrome C (Cyt C), which could activate some executioner caspases (Aon et al. 2007; Armstrong and Jones 2002). GSH depletion could induce redox imbalance and precede reactive oxygen species (ROS) accumulation (Franco and Cidlowski 2012). Furthermore, GSH depletion up-regulates antiapoptotic proteins such as B cell lymphoma 2(Bcl-2), heat shock proteins, and nuclear factor-kappa B (NF-jB), as well as other antioxidant systems, including the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 that might inhibit cell death progression (Franco and Cidlowski 2012). Therefore, xylitol/selenite may induce apoptosis through the co-

Biotechnol Lett b Fig. 6 The effect of xylitol/selenite on GSH level in SMMC-

7221 cells. a The GSH level in SMMC-7221 cells treated with different concentrations and treatment time of xylitol/selenite; b the depletion rate of GSH content under different concentrations of xylitol/selenite; c the depletion rate of GSH content under different culture time. **p \ 0.01, NS not significant (n = 3)

Table 1 GSH content in different cells Cell

GSH (nmol/mg protein)

V79cl cells

14.7 ± 1.3

Erythrocytes

5.89 ± 0.36

Jukat T cells

33.2 ± 3.6

Peripheral blood mononuclear cells

74.4 ± 21.2

in a variety of cells (Camera and Picardo 2002), including V79 cl cells, erythrocytes, Jurkat T cells and peripheral blood mononuclear cells (Table 1), is in the range of the level in control and experimental groups cells (from 0 to 95.57 ± 11.30 nmol/mg protein). Furthermore, it was reported that GSH level in cancer cells was much higher than that in normal tissues and cells (Xie et al. 2015), supporting the credibility of the CE-LIF data presented here. Xylitol selenite is a new kind of anti-cancer drug, and no apoptotic mechanisms have been reported in the past researches. In this paper, the mechanisms were studied from the aspect of GSH depletion, and whether some other apoptotic pathways are involved or not, a great deal of researches should be conducted. Acknowledgments This research was supported by the Natural Science Foundation of China (No. 31300629), selfdetermined research funds of CCNU from the college’s basic research and operation of MOE (No. CCNU14F01006), and the project of Hubei Key Laboratory of Genetic Regulation and Integrative Biology (GRIB201505).

References

action of multiple mechanisms related to GSH depletion. This is the first report that examines GSH content in SMMC-7221 cells. The mean of GSH level measured

Aon MA, Cortassa S, Maack C, O’Rourke B (2007) Sequential opening of mitochondrial ion channels as a function of glutathione redox thiol status. J Biol Chem 282:21889– 21900 Armstrong JS, Jones DP (2002) Glutathione depletion enforces the mitochondrial permeability transition and causes cell death in Bcl-2 overexpressing HL60 cells. FASEB J 16:1263–1265

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Biotechnol Lett Bayle C, Causse´ E, Couderc F (2004) Determination of aminothiols in body fluids, cells, and tissues by capillary electrophoresis. Electrophoresis 25:1457–1472 Camera E, Picardo M (2002) Analytical methods to investigate glutathione and related compounds in biological and pathological processes. J Chromatogr B 781:181–206 Carru C, Deiana L, Sotgia S, Pes GM, Zinellu A (2004) Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis. Electrophoresis 25:882–889 Fang YZ, Fang XM, Ye JN (1995) Determination of polyhydroxy antibiotics by capillary electrophoresis with amperometric detection at a copper electrode. Chem Res Chin Univ 16:1514–1518 Franco R, Cidlowski JA (2012) Glutathione efflux and cell death. Antioxid Redox Sign 17:1694–1713 Lei M, Chen DD, Deng XJ, Liu J, Chen LY, Liu YL, Li B, Yao HC, Xiong GM, Cao Y, Yang JH, Qi C (2012) Dynamic sieving capillary electrophoresis analysis of xylitol selenite-induced apoptosis in SMMC-7221 cells. Biotechnol Lett 39:1617–1621 Musenga A, Mandrioli R, Bonifazi P, Kenndler E, Pompei A, Raggi MA (2007) Sensitive and selective determination of

123

glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence. Anal Bioanal Chem 387:917–924 Sato T, Machida T, Takahashi S, Iyama S, Sato Y, Kuribayashi K, Takada K, Oku T, Kawano Y, Okamoto T, Takimoto R, Matsunaga T, Takayama T, Takahashi M, Kato J, Niitsu Y (2004) Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells. J Immunol 173:285–296 Xie X, Zhao Y, Ma CY, Xu XM, Zhang YQ, Wang CG, Jin J, Shen X, Gao JL, Li N, Sun ZJ, Dong DL (2015) Dimethyl fumarate induces necroptosis in colon cancer cells through glutathione depletion/ROS increase/MAPKs activation pathway. Br J Pharmacol 172:3929–3943 Zinellu A, Sotgia S, Posadino AM, Pasciu V, Perino MG, Tadolini B, Deiana L, Carru C (2005) Highly sensitive simultaneous detection of cultured cellular thiols by laser induced fluorescence-capillary electrophoresis. Electrophoresis 26:1063–1070