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(Department of Biochemistry, Zhongshan School of Medicine，Center for Stem Cell Biology and Tissue Engineering，Sun Yat-sen University, Guangzhou 510080, China)

Optimal timing of autophagy occurrence induced by earle’s balanced salts solution in DLD-1, HCT-116, A2780, CHO, Hep G2 and SMMC7721 cancer cell lines*

ZHANGWenliang,ZHANGMingming,LIUBin,SHIHongshun,WANGHaihe

(Department of Biochemistry, Zhongshan School of Medicine，Center for Stem Cell Biology and Tissue Engineering，Sun Yat-sen University, Guangzhou 510080, China)

Autophagy has emerged as a key effector in tumorgenesis, but its precise function remains elusive. Autophagy flux is too low to be detected in normal culture conditions. Earle’s balanced salts solution (EBSS) is often used to induce autophage with two distinct advantages compared with other inducers： one is only needed to considerate the induction time, rather than the drug concentration; the other is that it exerts only starvation pressure by specifically simulating amino acids deprivation to cells without other undistinguishable cytotoxicity. Given the tolerance threshold of outside stimulus differs in various types of tumor cells, in this study we carried out the autophage induction experiments with several time points in DLD-1 and HCT-116 colon cancer cell lines, A2780 and CHO ovarian cancer cell lines, Hep G2 and SMMC7721 hepatocellular carcinoma cell lines. Our western-blotting and Immunofluorescence staining results demonstrated that EBSS can efficiently induce autophagy in the assayed cells, precisely with 2 hours treatment in DLD-1, HCT-116, Hep G2 and CHO cells, 4 hours in A2780 and 8 hours in SMMC7721 cells, respectively. Our results provide a reference for autophagy induction with EBSS in cancer cells, and would be useful to shorten the time-consuming optimizing process.

autophage induction; EBSS; LC3; SQSTM1/p62; cancer cells

Autophagy is an evolutionarily highly conserved cellular homeostatic process whereby cells maintain their cytoplasmic biomass, organellar abundance and proper distribution, as well as remove potentially harmful protein aggregates and aged organelles[1]. Although the precise function of autophagy remains unclear, more and more evidences have shown that it is an important factor in tumorgenesis and cancer treatment[2]. The current consensus suggests that autophagy may have a dual role in cancer. In normal cells, autophagy functions to prevent the accumulation of damaged or aged organelles and aggregated proteins; by contrast, in cancer cells, autophagy is hijacked to serve as a cell survival mechanism that plays a critical role for established tumors[2]. In normal culture conditions, the detection of autophagy is relatively hard because of its low level[3]. Thus, to elaborate the precise role of autophagy in tumorgenesis and drug resistance, the autophagy activators and inhibitors need to be used to activate and suppress autophagy, respectively. Recently, earle’s balanced salts solution (EBSS) is a saline solution with physiological pH which is often used to induce autophagy[4-10]. At the same time, DLD-1 and HCT-116 as colon cancer[11-13], A2780 and CHO as ovarian cancer[13-15], Hep G2 and SMMC7721 as hepatocellular carcinoma[16-19]have been frequently used to elaborate the mechanism of autophagy in tumorgenesis and drug resistance. However, no detailed information of EBSS starvation duration for autophage induction in those cell lines has been reported yet. In this study, we performed western blotting and Immunofluorescence stainings in DLD-1, HCT-116, A2780, CHO, Hep G2 and SMMC7721 cell lines at several designed time points with EBSS starvation, hoping to find the optimal timing of autophagy occurrence in these tumor cell.

1 Materials and Methods

1.1 Cell culture and treatment

Human colorectal carcinoma cell line DLD-1 and HCT116(ATCC),CHO-K1 cells (ATCC), and human ovarian carcinoma cell line A2780 (ATCC) were maintained in DMEM basic medium(Gbico)supplemented with 10% fetal bovine serum and 1% antibiotics/antimycotics. Hepatocellular carcinoma cell line Hep G2 and SMMC7721 (ATCC) were maintained in DMEM high glucose medium (HyClone) supplemented with 10% fetal bovine serum and 1% antibiotics/antimycotics. All cells were maintained in a 5% CO2incubator at 37 ℃. EBSS was purchased from Life Technologies (Calcium, Magnesium and Phenol Red free, Gibco, 14155-063). Unless otherwise specified, reagents were used at the following final concentrations: chloroquine diphosphate salt (50 μmol/L, C6628, Sigma-Aldrich).

1.2 Immunofluorescence staining

Cells were fixed in 4% formaldehyde for 15 min, washed with cold 1 x PBS, permeabilized with 0.2% Triton X-100(BIOSHARP, Ameresco 0694) for 30 min and blocked with 5% BSA (Sigma-Aldrich, 05470) for 1 h at room temperature. Cells were then incubated with primary antibodies overnight at 4 ℃. Anti-LC3 antibody (3868, Cell Signaling Technology) was used at a dilution of 1∶200. After being washed five times with 1 x PBS, cells were incubated with secondary antibody (1∶500 dilution; Life Technologies, A21207) conjugated with appropriate fluorophore for 30 min at room temperature. Slides were mounted with Prolong Gold Antifade Reagent and analyzed immediately by using an LSM710 confocal microscope (Carl Zeiss AG). The averaged LC3 channel pixel intensity value within the cytoplasmic area per cell was determined by Photoshop (Adobe Systems) to represent the overall amount of LC3 puncta accumulation in each cell. Results were presented as mean ± SD.

1.3 Western blotting

The detailed immunoblotting procedures had been described previously[20]. LC3 (3868), SQSTM1/p62 (5114), Anti-rabbit HRP-labeled secondary antibody (7074) and anti-mouse HRP-labeled secondary antibody (7076S) were purchased from Cell Signaling Technology. GAPDH (CW0101A) was purchased from CW Biotech. The Actin (sc-8432) was purchased from Santa Cruz Biotechnology. Immunoreactive bands were visualized by using chemiluminescent substrate (Millipore, MA01821) and photographed on radiography film. Quantification of band intensity was done by Image J software.

1.4 Statistical analysis

All the statistical analyses were performed by GraphPad Prism version 5.00 software for windows (GraphPad Software, San Diego California USA, www.graphpad.com.).One-way ANOVA with Dunnett’s post test was performed for the comparison of means between groups.P< 0.05 was considered as significant in all tests.

2 Results

2.1 EBSS is an easy-to-use autophagy inducer

EBSS is a saline solution with physiological pH that is often used to induce autophagy[4-10]. We searched and summarized the putative reagents utilized for autophagy induction (Table 1). From the above Table, we found that EBSS as a saline solution have two distinct advantages, one is that comparing to other pharmic inducers, when EBSS is used as an autophage inducer, we only need to considerate the induction time, rather than the drug concentration; the other is that by specifically simulating amino acids deprivation, it just exerts starvation pressure to cells without arising other undistinguishable cell toxicities.

2.2 The optimal timing of autophagy induced by EBSS in cancer cells

Microtubule-associated protein 1 light chain 3 (LC3) is the most widely and reliably monitored autophagy-related protein[3]. A hallmark of autophagy initiation is the conversion of the cytosolic LC3 I into the membrane bound LC3 II that associates with autophagosome formation[21]. Upon the autophagy initiation,the ratio of LC3 II/LC3 I and LC3 II level increase because the LC3 II rapidly conversed from LC3 I is more than the LC3 II degraded by autophgolyosome. However, along with the autophagy induction, the ratio of LC3II/LC3 I and LC3 II level are obviously decreased because LC3 II as an autophage substrate is rapidly degraded in autophgolyosome. In addition to LC3, SQSTM1/p62 becomes incorporated into the completed autophagosome and is also significantly degraded in autolysosomes when autophagy is induced[3]. Collectively, the degradation of LC3 and SQSTM1/p62 can be used to monitor the autophagy induction.

Table 1 Reagents used for autophagy induction

2.2.1 In DLD-1 and HCT-116 colon cancer cell lines In colon cancer cells, western blotting results show that upon 1 h EBSS starvation, the ratio of LC3 II/LC3 I has been increased compared with the control group (under normal culture condition) in DLD-1 (Fig.1 A, green box, lane 2 and Fig.1 B) and HCT-116 (Fig.1 E, green box, lane 2 and Fig.1 F), respectively, which means autophagy is initiated by EBSS after one hour starvation. Then, with the starvation time extended to 2 h, the ratios of LC3 II/LC3 I, LC3 II/GAPDH and SQSTM1/p62/GAPDH have both been reduced significantly compared with the control group in DLD-1 ( Fig.1 A, red box, lane 3 and Fig.1 B) and HCT-116 (Fig.1 E, red box, lane 3 and Fig.1 F). In addition, we choose LC3 protein as a marker to trace autophagosome and perform Immunofluorescence (IF) staining. Chloroquine (CQ) is a lysosomal acidification inhibitor that blocks autophagosome related degradation. In colon cancer cell lines, we noticed that DLD-1 and HCT-116 cells treated with EBSS + CQ at 2 h (Fig.1 C, b’ and G, b’) contained more endogenous LC3 puncta per cell than DLD-1 and HCT-116 cells from respective control group (Fig.1 C, a’ and G, a’). Quantification of LC3 puncta intensity confirmed a significantly higher LC3 puncta frequency per cell in EBSS + CQ treated DLD-1 and HCT-116 cells at 2 h compared with normally cultured DLD-1 and HCT-116 cells, respectively (Fig.1 D and H;P<0.001). Collectively, our results suggest that autophage is efficiently induced by EBSS in DLD-1 and HCT-116 colon cancer cells after only 2 hours.

2.2.2 In A2780 and CHO ovarian cancer cell lines Noteworthily, upon 2 h EBSS starvation in A2780 cell line, the ratios of LC3 II/LC3 I and LC3 II/GAPDH have been obviously increased compared with the control group (Fig.2 A, green box, lane 2 and Fig.2 B). The increased ratios of LC3 II/LC3 I and LC3 II/GAPDH indicate that autophagy is initiated by EBSS starvation at 2 h in A2780. When the starvation reaches 4 h, the ratios of LC3 II/GAPDH and SQSTM1/p62/GAPDH are both obviously decreased compared with the control group in A2780 (Fig.2 A, red box, lane 3 and Fig.2 B). In addition, the IF results show that A2780 cells treated with EBSS + CQ at 4 h (Fig.2 C, b’) contained more endogenous LC3 puncta per cell than A2780 cells from control group (Fig.2 C, a’). Quantification of LC3 puncta intensity confirmed a significantly higher LC3 puncta frequency per cell in EBSS + CQ treated A2780 cells at 4 h compared with normally cultured A2780 cells, respectively (Fig.2 D;P<0.001 ). Our results suggest that EBSS can efficiently induce autophage in A2780 when the starvation time is extended to 4 h. However, in CHO cell line, upon 2 h EBSS starvation, the ratios of LC3II/LC3 I, LC3 II/GAPDH and SQSTM1/p62/GAPDH have been significantly reduced compared with the control group (Fig.2 E, red box, lane 2 and Fig.2 F). Those results demonstrate that the autophgy is induced in CHO by EBSS starvation after only 2 hours. In addition, the IF results showed that CHO cells treated with EBSS + CQ at 2 h (Fig.2 G, b’) contained more endogenous LC3 puncta per cell than CHO cells from control group (Fig.2 G, a’). Quantification of LC3 puncta intensity confirmed a significantly higher LC3 puncta frequency per cell in EBSS + CQ treated CHO cells at 2 h compared with normally cultured CHO cells, respectively (Fig.2 H;P<0.001 ). Thus, our results confirm that 2 h EBSS starvation can efficiently induce autophage in CHO cells.

2.2.3 In HepG2 and SMMC7721 hepatocarcinoma cell lines In HepG2 cell line, the western blotting result indicates that upon 1 h EBSS starvation, the ratios of LC3 II/LC3 I and LC3 II/GAPDH have been both increased compared with the control group (Fig.3 A, boxed lane 2 and Fig.3 B). The increased ratios of LC3 II/LC3 I and LC3 II/GAPDH also confirm that autophagy is initiated by 1 h EBSS starvation in Hep G2. With the starvation extended to 2 h, the ratios of LC3 II/GAPDH and SQSTM1/p62/GAPDH are both significantly reduced compared with the control group (Fig.3 A, boxed lane 3 and Fig.3 B). It should also be noted that 2 h EBSS + CQ treatment clearly increase the LC3 II level compared with the control group in Hep G2 (Fig.3 C, boxed lane 3). Thus, the above results suggest that the autophage is induced by only 2 h EBSS starvation in Hep G2. However, in SMMC7721 cell line, upon 4 h EBSS starvation, the LC3II/GAPDH and LC3 II / LC3 I are not reduced compared with the control group (Fig.3 D, boxed lane 2 and Fig.3 E). When the starvation is extended to 8 h in SMMC7721, the ratios of LC3 II/GAPDH and SQSTM1/p62/GAPDH rapidly decrease compared with the respective control group (Fig.3 D, boxed lane 3 and Fig.3 E). Similarly, we noticed that 8 h EBSS + CQ treatment significantly increased the LC3 II level compared with the control group in SMMC7721 (Fig.3 F, boxed lane 3). Our results show that the efficient induction time of SMMC7721 by EBSS starvation should be 8 h. Collectively, EBSS can also efficiently induce autophagy in hepatocarcinoma cells and it’s possible that the EBSS optimal induction time of autophagy is dependent on specific tumor cell types.

Fig.1 The optimal timing of autophagy induction by EBSS in DLD-1 and HCT-116 colon cancer cell lines

Fig.2 The optimal timing of autophagy induction by EBSS in A2780 and CHO ovarian cancer cell lines

Fig.3 The optimal timing of autophagy induction by EBSS in Hep G2 and SMMC7721 hepatocarcinoma cell lines

3 Discussions

Autophagy has emerged as a key effecter in tumorgenesis and cancer treatment, although its precise function remains elusive[2].The DLD-1, HCT-116, A2780, CHO, Hep G2 and SMMC7721 cell lines are often used as models to elaborate the precise role of autophagy in tumorgenesis and drug resistance[11-19]. EBSS is often used as an autophage inducer in related studies[4-10], but its usage in DLD-1, HCT-116, A2780, CHO, Hep G2 and SMMC7721 for autophagy researches has not been explored yet. In this study, firstly we analyzed several common inducers of autophagy and pointed out that EBSS is easier to use in tumor cells than others, secondly we demonstrated that tumor cells can be efficiently induced by EBSS starvation in just a few hours. We also found that the optimal timing of autophagy occurrence varies in different cell types. Most importantly, we have obtained the optimal induction time of DLD-1, HCT-116, A2780, CHO, Hep G2, and SMMC7721 cells through western blotting and IF results acquired from several specific induction time points. To sum up, our results provide practical usage data of EBSS as an autophagy inducer in several cancer cell lines, which would possibly be useful for other researchers to optimize their autophagy experiments and spare the time-consuming process of exploring the proper inducer and finding the appropriate induction time.

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2016-01-11

國家自然科學(xué)基金資助項(xiàng)目 (81171947，31271481)

張文亮(1991年生)，男；研究方向：細(xì)胞自噬與腫瘤發(fā)生；通信作者：王海河;E-mail:wanghaih@mail.sysu.edu.cn

Q-331

A

0529-6579(2016)04-0108-10

Earle’s鹽平衡液饑餓誘導(dǎo)DLD-1, HCT-116, A2780, CHO,Hep G2和 SMMC7721腫瘤細(xì)胞系自噬發(fā)生的時(shí)間優(yōu)化

張文亮， 張明明，劉 斌，石宏順，王海河

(中山大學(xué)中山醫(yī)學(xué)院生物化學(xué)系∥中山大學(xué)干細(xì)胞與組織工程中心，廣東 廣州 510080)

腫瘤細(xì)胞在正常培養(yǎng)條件下自噬水平非常低，很難被監(jiān)測到。與藥物性自噬誘導(dǎo)劑相比，Earle’s鹽平衡液(EBSS)應(yīng)用簡便，除產(chǎn)生饑餓誘導(dǎo)自噬外，無其他細(xì)胞毒性，有利于研究細(xì)胞自噬的分子機(jī)制和功能。本文應(yīng)用蛋白免疫印記和細(xì)胞免疫熒光技術(shù)探討了EBSS對DLD-1和HCT-116結(jié)直腸癌、A2780和CHO卵巢癌、HepG2和SMMC7721肝癌細(xì)胞系的自噬誘導(dǎo)效果和有效誘導(dǎo)時(shí)間。結(jié)果顯示：EBSS短短幾小時(shí)處理，能夠非常有效地誘導(dǎo)上述6株腫瘤細(xì)胞發(fā)生自噬，但不同細(xì)胞系之間的有效誘導(dǎo)時(shí)間卻明顯不同。其中，DLD-1、HCT-116、CHO和HepG2細(xì)胞系的有效誘導(dǎo)時(shí)間僅需2 h；A2780細(xì)胞需要稍微延長到4 h；然而SMMC7721卻要延長到8 h左右。本研究為其他研究者在應(yīng)用EBSS誘導(dǎo)腫瘤細(xì)胞自噬時(shí)提供了實(shí)踐依據(jù)和參考。

細(xì)胞自噬誘導(dǎo)；Earle’s鹽平衡液；LC3； SQSTM1/p62；癌細(xì)胞

10.13471/j.cnki.acta.snus.2016.04.018