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Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin
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Original article
Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin Du a,1, Yan-Jing Li a,1, Chun-Long Wu b, Jian-Hua Zhou a, Yu Han a, Hong Sui a, Xiao-Li Wei a, Lei Liu , Peng Huang a, Heng-Heng Yuan a, Ting-Ting Zhang a, Wen-Jie Zhang a, Rui Xie a, Xiao-Hui Lang a, De-Xin Jia a, Yu-Xian Bai a,*
Q1 Xiao-Xue a
a
Department of Gastrointestinal Oncology, The Third Affiliated Hospital, Harbin Medical University, Harbin, 150, Haping St, Nangang District, Harbin, 150081, People’s Republic of China b Department of Endoscope, The Third Affiliated Hospital, Harbin Medical University, Harbin, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 January 2013 Accepted 24 January 2013
Dihydroartemisinin (DHA) has recently been shown anti-tumor activity in various cancer cells. However, its effect on esophageal cancer remains unclear. In this study, for the first time, we demonstrated that DHA reduced viability of esophageal cancer cells in a dose-dependent manner. The mechanism was at least partially due to DHA induced apoptosis by upregulating the expression of Bax, downregulating Bcl-2, Bcl-xL and Procaspase-3, and increasing caspase-9 activation, induced cell cycle arrest by downregulating cyclin E, CDK2 and CDK4. Furthermore, we firstly found that DHA induced autophagy in cancer cells. We concluded DHA might be a novel agent against esophageal cancer. ß 2013 Published by Elsevier Masson SAS.
Keywords: Dihydroartemisinin Esophageal cancer Autophagy Apoptosis Cell cycle arrest
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1. Introduction
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Esophageal cancer is the eighth most common cause of cancer worldwide, and it ranks as the fourth leading cause of all cancer deaths in China [1,2]. More than one half of new cases in western countries have adenocarcinoma of the lower esophagus or gastroesophageal junction, while the dominant histologic subtype is squamous cell carcinoma in eastern Asian countries [3]. Despite improvements in surgical and radiotherapy techniques and refinements of chemotherapeutic regimens, the 5-year survival rate is approximately 15% [4]. Thus, it is currently accepted that conventional chemotherapy is not sufficient to manage esophageal cancer, and the development of new chemotherapies is worth investigation to prolong the survival of patients with esophageal cancer. Artemisinin was isolated from a Chinese herb Artemisia annua. Artemisinin and its derivatives are widely used as antimalarial
Abbreviations: AVOs, Autophagic vacuoles; DHA, Dihydroartemisinin; PI, Propidium iodide; ROS, Reactive oxygen species; TEM, Transmission electron microscopy. * Corresponding author. Department of Gastrointestinal Oncology, The Third Affiliated Hospital, Harbin Medical University, Harbin, 150, Haping St, Nangang District, Harbin, 150081, People’s Republic of China. Tel.: +86 451 86298689; fax: +86 451 86298689. E-mail address: [email protected] (Y.-X. Bai). 1 XiaoXue Du and Yanjing Li contributed equally to this work.
agents in clinic and have recently been shown to have anti-tumor activities [5–13]. Dihydroartemisinin (DHA), which is the most potent of artemisinin derivatives, has exhibited anticancer activity in lung cancer [5], breast cancer [6], hepatic cancer [7], pancreatic cancer [8,9], colorectal cancer [10], glioma [11,12], prostate cancer [13,14] and so on. It is proposed that the anti-tumor effects of DHA have been mediated by cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway in lung adenocarcinoma [5], by shifting the immune response towards Th1 in breast cancer [6], by inhibiting cell proliferation and inducing G1-phase arrest in hepatic cancer [7], by cell cycle arrest and inactivation of nuclear factor-kappa B in pancreatic cancer [8,9], by induction of iron-dependent endoplasmic reticulum stress in colorectal carcinoma [10], by inhibiting hypoxia inducible factor1a activation and generating reactive oxygen species (ROS) in glioma [11,12], by suppressing the PI3-K/Akt and ERK cell survival pathways and triggering the induction of death receptor DR5 and activation of extrinsic and intrinsic cell death signalling in prostate cancer [13,14]. Moreover, DHA exerted significantly lower cytotoxicity to normal cells, such as liver cells, prostate epithelia cells [7,13]. These results indicated that DHA might be an appropriate candidate for cancer chemotherapy. However, to our best knowledge, little is known about its effects on esophageal cancers. The purpose of this study, therefore, was to assess the anti-tumor effects of DHA on human esophageal cancer cells, and to investigate the underlying molecular mechanisms.
0753-3322/$ – see front matter ß 2013 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biopha.2013.01.013
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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2. Materials and methods
2.6. Transmission electron microscopy (TEM)
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2.1. Cells
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The human esophageal cancer cell lines Eca109 and Ec9706 (Laboratory of Medical Genetics, Department of Biology, Harbin Medical University) were maintained in RPMI 1640 medium, which was supplemented with 10% heated inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL). Cells were maintained in a humidified atmosphere of 5% CO2 at 37 8C.
Eca109 and Ec9706 cells were treated as above. The cells were harvested, washed and fixed with 2.5% glutaraldehyde (SigmaAldrich, G6257) containing 1% tannic acid for overnight. After washing, the cell pellets were embedded in epon araldite. The ultra-thin sections were observed with a Hitachi-h7650 electron microscope and representative images were analyzed.
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2.7. Immunoblot analysis
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2.2. Materials
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DHA (Sigma–Aldrich, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma) before experiments. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT, Amresco, USA) and propidium iodide (PI) were dissolved to a final concentration of 5 mg/ml and 50 mg/mL in PBS respectively. Annexin V-FITC apoptosis detection kit and ECL Plus system was obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Antibodies against caspase-3, caspase-9, Bcl-xL, Cyclin E, Bax, LC3A were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2, Cdk4, Cdk2, b-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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2.3. Cell viability assay
Eca109 and Ec9706 cells were treated as above. The cells were lysed in RIPA buffer after washing twice with cold PBS. After keeping on ice 8–10 minutes, total proteins were centrifuged at 14,000 rpm for 5 minutes at 4 8C and then quantified by Coomassie brilliant blue G. Protein samples were mixed with the 5 sample buffer. These samples were boiled at 99 8C for 5 minutes. Similar amounts of protein were electrophoresed on a 12% of SDSpolyacriylamide gel and transferred topolyvinylidene difluoride (PVDF) membranes. The membranes were washed with PBSTween and blocked with 5% fat-free milk in PBS-Tween for 1 h at room temperature, and incubated with primary antibody overnight at 4 8C. After washing three times with PBS-Tween, the membranes were incubated for 1 hour at room temperature with secondary antibody and washed again. The blots were visualized using a chemiluminescence detection kit ECL-PLUS. Anti-b-actin was used to ensure equal loading.
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Eca109 (4 103 cells/well) and Ec9706 (5 103 cells/well) cells were seeded into 96-well plates, and cultured overnight. The cells were treated with different concentrations of DHA. Cells treated with DMSO were used as untreated control. After incubation for 48 h and 72 hours respectively, MTT (20 mL) was added to each well with 3–4 hours incubation at 37 8C. The medium was discarded, and DMSO (150 mL) was added into each well at room temperature for 10 minutes with shaking. The OD490 nm was measured on a plate reader (BioTek, USA), and the difference of absorbance between the treated and untreated control groups was calculated to determine cell viability. The experiments were repeated thrice.
2.8. Statistical analysis
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All results were expressed as mean values standard deviation, except the results from western blot assay. One-way ANOVA was used for statistical analysis. A value of less than 0.05 (P < 0.05) was used for statistical significance.
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3. Results
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3.1. DHA reduces viability of esophageal cancer cells
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2.4. Cell apoptosis assay
For esophageal cancer cell lines, Eca109 and Ec9706 were exposed to various concentrations of DHA (2.5–120 mmol/L) for 48 and 72 hours respectively. The effect of DHA on the viability of two types of cells was detected with MTT assay. As shown in Fig. 1, DHA reduced the viability of two types of cells in vitro in a dosedependent manner, compared with untreated cells. These results suggested that DHA was cytotoxic to human esophageal cancer cells. The concentration of DHA IC50 values for Eca109 and Ec9706 were 76.86 mM, 93.81 mM respectively.
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Eca109 and Ec9706 cells were treated as above. The cells were harvested and washed thrice with ice-cold PBS. Cells were suspended in 195 mL binding buffer, 5 mL of Annexin V and 10 mL of PI were added, and incubated for 20 minutes at room temperature in dark. After diluted with 190 mL binding buffer, the cells were measured within 1 hour. After filtration, the single-cell suspensions were analyzed on the flow cytometer (Epics Altra II, Beckman Coulter, USA). Apoptotic and necrotic cells were quantified by counting the relative amount of fragmented blue or red nuclei (early or late apoptosis) and non-fragmented red nuclei (necrosis). The apoptotic or necrotic cells were reported as percentages of total cells. The experiment was repeated thrice.
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2.5. Cell Cycle Analysis
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Eca109 and Ec9706 cells treated with DHA were cultured respectively for 48 h and 72 h. The cells were harvested, washed with ice-cold PBS twice, and fixed in ice-cold 70% (v/v) ethanol for overnight at 4 8C Prior to analysis, the ethanol was removed by centrifugation, samples were washed with PBS again and cells were re-suspended in PBS containing PI (50 mg/mL) and RNase A (10 mg/mL) and then incubated at 37 8C in the dark for 30 minutes. The percentage of cells at G0/G1, S, or G2/M phase was thereby documented.
Fig. 1. Dihydroartemisinin (DHA) inhibits the proliferation of human esophageal cancer cells in vitro. Two human esophageal cancer cells Eca109 and Ec9706 were exposed to various concentrations of DHA (2.5–120 mmol/L) for 48 and 72 hours respectively. The viability of cells was assessed by the MTT method to calculate the viability index (%). Values represented mean SD.
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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Fig. 2. Eca109 and Ec9706 cells were exposed to various concentrations of Dihydroartemisinin (DHA) (0, 10, 40, and 80 mM) for 48 and 72 hours respectively followed by apoptosis assay: representative histograms were from cytometrically analyzed Eca109 (upper panel) or Ec9706 (lower panel) cells incubated with control medium (control) or media containing 10 or 80 mmol/L of DHA (a); DHA induced a significant apoptosis in the two types of esophageal cancer cells in a dose-dependent manner. Values represented mean SD (n = 3) (b). * P < 0.05 versus control.
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3.2. DHA induces apoptosis of esophageal cancer cells
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After Eca109 and Ec9706 were exposed to various concentrations of DHA (2.5-120 mmol/L) for 48 and 72 h respectively, DHAinduced apoptotic changes were detected by flow cytometric analysis using AnnexinV-FITC and PI staining (Fig. 2). As shown in Fig. 2b, DHA treatment induced a significant apoptosis in the two types of esophageal cancer cells in a dose-dependent manner. The representative histograms of flow cytometry showed that the mean apoptosis rates of Eca109 cells were 3.55, 5.26, 14.61% when they were treated with DHA at concentrations at 0, 10, 80 mmol/L respectively, and that of Ec9706 cells were 1.1, 1.8 and 16.15% (Fig. 2a).
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3.3. DHA induces G0/G1 phase cell cycle arrest in human esophageal cancer cells
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In our study, we performed DNA cell cycle analysis to assess the effect of DHA treatment on the distribution of cells in the cell cycle. As shown in Fig. 3a, significant changes in cell cycle were noted in the two types of esophageal cancer cells. As compared with the control group, the ratio of cells in S phase gradually decreased
while the ratio of cells in G0/G1 phase gradually increased. And the effects occurred in a dose-dependent manner (Fig. 3b). These data suggested that DHA induced cell cycle arrest in G0/G1 phase in human esophageal cancer cells.
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3.4. DHA induces ultrastructural morphologic changes in human esophageal cancer cells
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In the control group, no obvious cell damage was observed in Eca109 (Fig. 4a) and Ec9706 cells (Fig. 4c). After exposed to various concentrations of DHA, swollen mitochondria were found in the treated cells. Even some cells showed typical characteristics of apoptotic and necrotic cells such as apoptotic body, nuclear condensation and broken cell membranes with nuclear lysis (Fig. 4b, d). At present, TEM observation is the only absolute test of autophagy. A defining feature of the autophagosome is doublemembraned autophagic vacuoles (AVOs). The cells in control group were intact with rich cytoplasm, mitochondria were integrated, and very few vacuoles were observed in the cytoplasm (Fig. 4e). The cell morphology was greatly changed, and many AVOs were observed in the cytoplasm of cells after treated with DHA. As
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Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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Fig. 3. Eca109 cells were exposed to various concentrations of Dihydroartemisinin (DHA) (0, 10, 40, and 80 mM) for 48 h respectively followed by cell cycle distribution assay: representative histograms were from cytometrically analyzed Eca109 cells incubated with control medium (control) or media containing 10, 40, 80 mmol/L of DHA (a); the percent of cells in G0/G1, S, and G2/M phases of the cell cycle are shown as mean SD (n = 3) (b). *P < 0.05 versus control.
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shown in Fig. 4f, it clearly showed some enclosed vacuoles, which contained what appeared to be mitochondria or other cellular contents (black arrow indicated).
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3.5. DHA modulates the expression of apoptosis-related proteins in human esophageal cancer cells
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To characterize the potential mechanism by which DHA induced apoptosis of human esophageal cancer cells, the effect of DHA on the expression of apoptosis-related proteins was examined. DHA treatment upregulated the expression of Bax and downregulated the expression of Bcl-2, Bcl-xL and Procaspase-3, and also increased the activation of caspase-9 in the two types of cells. Taken together, these data suggested that DHA upregulated
the expression of pro-apoptotic proteins to activate caspases, thus promoted apoptosis of esophageal cancer cells.
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3.6. DHA modulates the expression of cell cycle-related proteins in human esophageal cancer cells
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To investigate the potential mechanism by which DHA regulated the cell cycle of esophageal cancer cells, the effect of DHA on the expression of G0/G1 phase -related proteins, such as cyclin E, CDK2 and CDK4 was examined. As shown in Fig. 5, DHA treatment resulted in downregulation of cyclin E, CDK2 and CDK4. Our results documented the modulation effects of DHA on key regulators in G0/G1 phase cell cycle progression in esophageal cancer cells.
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Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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Fig. 4. Ec9706 cells were exposed to various concentrations of Dihydroartemisinin (DHA) (0, 10, 40, and 80 mM) for 72 hours respectively followed by cell cycle distribution assay: representative histograms were from cytometrically analyzed Ec9706 cells incubated with control medium (control) or media containing 10, 40, 80 mmol/L of DHA (a); the percent of cells in G0/G1, S, and G2/M phases of the cell cycle are shown as mean SD (n = 3) (b). *P < 0.05 versus control.
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3.7. DHA induces the expression of LC3-II in human esophageal cancer cells
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The amount of LC3-II is correlated with the extent of AVOs formation, and conversion of LC3-I to LC3-II has been used as a marker for autophagy [15,16]. As shown in Fig. 5, DHA treatment resulted in a conversion of LC3-I to LC3-II in two types of esophageal cancer cell lines.
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4. Discussion and conclusion
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Recent studies suggested a potential use of artemisinin and its active metabolite DHA as anticancer agents [5–13]. However, the effect of DHA on esophageal cancer cells remains unclear. Furthermore, to our best knowledge, the phenomenon of autophagy has not been investigated so far in DHA treatment on cancer cells. In this study, it was first found that in human esophageal cancer cells, DHA could induce apoptosis by upregulating Bax, downregulating Bcl-2, Bcl-xL and
Procaspase-3, and increasing the activation of caspase-9, induce G0/G1 phase arrest by downregulaing cyclin E, CDK2 and CDK4. We also found the formation of AVOs by TEM and conversion of LC3-I to LC3-II by immunoblot analysis. For the first time, our study demonstrated that DHA could induce autophagy in cancer cells. Apoptosis, which does not trigger immune reactions, is considered as a better way to kill cancer cells than instant cell death and necrosis [17]. Members of the Bcl-2 family, including pro-apoptosis proteins (Bax, Bad) and anti-apoptotic proteins (Bcl-2 and Bcl-xL), are the most prominent regulators of mitochondrial-dependent apoptosis [18]. It has been demonstrated that Bcl-xL could inhibit membrane permeabilization and cytochrome c release by blocking the activation of Bax and Bak, and improve the ability of tumor cells to escape from therapy [19,20]. Bcl-2 is located on the membrane of mitochondria, whereas Bax directly binds to Bcl-2 and inhibits its function [21]. An increase in the ratio of Bax/Bcl-2 stimulates the release of cytochrome c from the mitochondria into the cytosol and the cytosolic cytochrome c
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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Fig. 5. Eca109 and Ec9706 cells were exposed to various concentrations of Dihydroartemisinin (DHA) (0, 10, 40, and 80 mM) for 48 and 72 hours respectively followed by Transmission electron microscopy (TEM) observation. No obvious cell damage was observed in the control group. Apoptosis, necrosis and autophagy occurred in the DHA treatment group: the control untreated Eca109 cells (a, e); the control untreated Ec9706 cells (c); Eca109 cells treated with 40 mmol/L DHA (arrow indicated autophagosome) (b, f); Ec9706 cells treated with 40 mmol/L DHA (d).
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then binds to Apaf-1, leading to the activation of procaspase-9, 3 which results in apoptosis [19–21]. Several studies have indicated that induction of apoptosis of cancer cells by DHA might be an important mechanism responsible for the impact on tumor cell viability. Jiao et al. showed that DHA induced apoptosis in human ovarian cancer cells accompanied by a decrease of Bcl-xL and Bcl-2 and an increase of Bax and Bad [22].
Hou et al. suggested that DHA induced apoptosis, activated caspase-3, increased the Bax/Bcl-2 in human hepatoma cells [7]. Similarly, Chen et al. reported that DHA induced apoptosis by reducing the ratio of Bcl-2/Bax and increasing the activation of caspase-9 in a dose-dependent manner [9]. Similar to the reports above, in our study, DHA treatment induced a significant apoptosis in the two types of esophageal cancer cells in a dose-dependent
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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manner. And we also found that DHA upregulated the expression of Bax and downregulated the expression of Bcl-2, Bcl-xL and Procaspase-3, and also increased the activation of caspase-9. Our data indicated that DHA induced apoptosis by regulating Bcl-2 family members and activating caspases that lead to apoptosis. Obstruction of cell cycle progression in cancer cells is considered as one of the most effective strategies for the control of tumor growth [23,24]. In our study, we demonstrated that the ratio of cells in S phase gradually decreased while the ratio of cells in G0/G1 phase gradually increased compared with the control group. Furthermore, our study demonstrated that DHA treatment resulted in the downregulation of cyclin E, CDK2 and CDK4. Cyclin– Cdk heterodimers play an important role in the control of cell cycle progression and cell cycle checkpoints in mammalian cells. And cyclin E, CDK2 and CDK4 are important complexes responsible for the progression of cells through the G1 phase of cell cycle and initiation of DNA replication [25]. These data indicated that block of G0/G1 transition might be one mechanism responsible for the significant viability reduction of human esophageal cancer cells induced by DHA, which induced cell arrest partly through downregulating cyclin E, CDK2 and CDK4. Consistent with our result, some investigators found that DHA could induce G0/G1 phase arrest in pancreatic cancer and hepatoma [7,26]. However, in contrast, cell arrest in G2/M phase was induced in osteosarcoma and ovarian cancer [22,27]. These data suggested that DHA induced cell arrest depending on the cell lines. Autophagy is a process whereby cytosol and organelles become encased in vacuoles termed autophagosomes. It is a regulated process of degradation and recycling of cellular constituents,
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participating in organelle turnover and the bioenergetic manage- 293 ment of starvation [28,29]. In our study, in human esophageal 294 cancer cells treated by DHA, we found the formation of AVOs and 295 conversion of LC3-I to LC3-II. Conversion of LC3-I to LC3-II has been 296 used as a marker for autophagy [15]. To the best of our knowledge, 297 for the first time, we found that DHA could induce autophagy in 298 299 cancer cells. Recent evidence indicated that autophagy played an important 300 role in tumor development, cell death and survival. Some authors 301 thought that autophagy might lead to cell death through excessive 302 self-digestion and degradation of essential cellular constituents 303 and could proceed in the absence of caspase signaling or even be 304 activated under conditions of caspase inhibition [15]. Autophagic 305 cell death, which is different from both apoptosis and necrosis, has 306 independent morphological and biochemical features [16]. So it is 307 considered to be a second type of programmed cell death 308 (sometimes referred to ‘type II cell death’). Other authors thought 309 that autophagy might support cancer cell survival by providing an 310 ‘escape route’ and protection from proteotoxicity. Similarly, it was 311 reported recently that autophagy occured in chemotherapeutic 312 treatment and contributed to drug resistance [30]. Furthermore, it 313 was reported that there was an equilibrium established between 314 autophagy and apoptosis, with inhibition of either process leading 315 to enhanced activity by the other [31]. In our study, DHA induced 316 apoptosis and autophagy in human esophageal cancer cells. 317 Therefore, how about the effect of autophagy in cancer cells 318 treated by DHA? How about the detailed mechanism between 319 apoptosis and autophagy? These mechanisms are unknown and 320 further investigation is required in the future (Fig. 6). Q2321
Fig. 6. DHA modulated the expression of apoptosis-related, cell cycle-related and autophagy-related proteins in esophageal cancer cells. Eca109 and Ec9706 cells were exposed to various concentrations of DHA (0, 10, 40, and 80 mM) for 48 and 72 hours respectively followed by western blot analysis.
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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In conclusion, we first investigated the anticancer effects of DHA on human esophageal cancer cells in vitro. We demonstrated that DHA induced apoptosis, cell cycle arrest and autophagy in esophageal cancer cells. Our study indicated that DHA might be a potent and promising agent to combat esophageal cancer. However, these results are only based on studies in vitro, and further studies in vivo are necessary to investigate.
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Disclosure of interest
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The authors declare that they have no conflicts of interest concerning this article.
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Acknowledgements
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We are grateful to the teachers of central cancer laboratory of Harbin Medical University for their kind help. And the authors thank Yuyan Ma and Yangmei Yang for their technical assistance.
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[11] Huang XJ, Li CT, Zhang WP, et al. Dihydroartemisinin potentiates the cytotoxic effect of temozolomide in rat C6 glioma cells. Pharmacology 2008;82: 1–9. [12] Huang XJ, Ma ZQ, Zhang WP, et al. Dihydroartemisinin exerts cytotoxic effects and inhibits hypoxia inducible factor-1alpha activation in C6 glioma cells. J Pharm Pharmacol 2007;59:849–56. [13] He Q, Shi J, Shen XL, et al. Dihydroartemisinin upregulates death receptor 5 expression and cooperates with TRAIL to induce apoptosis inhuman prostate cancer cells. Cancer Biol Ther 2010;9:819–24. [14] Morrissey C, Gallis B, Solazzi JW, et al. Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells. Anticancer Drugs 2010;21: 423–32. [15] Reiners Jr JJ, Agostinis P, Berg K, et al. Assessing autophagy in the context of photodynamic therapy. Autophagy 2010;6:7–18. [16] Kroemer G, Galluzzi L, Vandenabeele P, et al. Classification of cell death: Recommendations of the nomenclature committee on cell death. Cell Death Differ 2009;16:3–11. [17] Feril Jr LB, Kondo T, Cui ZG, et al. Apoptosis induced by the sonomechanical effects of low intensity pulsed ultrasound in a human leukemia cell line. Cancer Lett 2005;221:145–52. [18] Gustafsson AB, Gottlieb RA. Bcl-2 family members and apoptosis, taken to heart. Am J Physiol Cell Physiol 2007;292:C45–51. [19] Wakabayashi K, Saito H, Ebinuma H, et al. Bcl-2 related proteins are dramatically induced at the early stage of differentiation in human liver cancer cells by a histone deacetylase inhibitor projecting an anti-apoptotic role during this period. Oncol Rep 2000;7:285–8. [20] Adams JM, Cory S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 2001;26:61–6. [21] Schwarz CS, Evert BO, Seyfried J, et al. Overexpression of bcl-2 results in reduction of cytochrome c content and inhibition of complex I activity. Biochem Biophys Res Commun 2001;280:1021–7. [22] Jiao Y, Ge CM, Meng QH, et al. Dihydroartemisinin is an inhibitor of ovarian cancer cell growth. Acta Pharmacol Sin 2007;28:1045–56. [23] Nam W, Tak J, Ryu JK, et al. Effects of artemisinin and its derivatives on growth inhibition and apoptosis of oral cancer cells. Head Neck 2007;29:335–40. [24] Lin X, Tang M, Tao Y, et al. Epstein-Barr virus-encoded LMP1 triggers regulation of the ERK-mediated Op18/stathmin signaling pathway in association with cell cycle. Cancer Sci 2012;103:993–9. [25] Sherr CJ, Roberts JM. Living with or without cyclins and cyclin-dependent kinases. Genes 2004;18:2699–711. [26] Wang SJ, Gao Y, Chen H, et al. Dihydroartemisinin inactivates NF-kappaB and potentiates the anti-tumor effect of gemcitabine on pancreatic cancer both in vitro and in vivo. Cancer Lett 2010;293:99–108. [27] Ji Y, Zhang YC, Pei LB, et al. Anti-tumor effects of dihydroartemisinin on human osteosarcoma. Mol Cell Biochem 2011;351:99–108. [28] Lu CH, Liu YW, Hua SC, et al. Autophagy induction of reversine on human follicular thyroid cancer cells. Biomed Pharmacother 2012;66:642–7. [29] Zhuang W, Long L, Zheng B, et al. Curcumin promotes differentiation of glioma-initiating cells by inducing autophagy. Cancer Sci 2012;103:684–90. [30] Amaravadi RK, Lippincott-Schwartz J, Yin XM, et al. Principles and current strategies for targeting autophagy for cancer treatment. Clin Cancer Res 2011;17:654–66. [31] Kessel D, Vicente MG, Reiners Jr JJ. Initiation of apoptosis and autophagy by photodynamic therapy. Lasers Surg Med 2006;38:482–8.
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Original article
Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin Du a,1, Yan-Jing Li a,1, Chun-Long Wu b, Jian-Hua Zhou a, Yu Han a, Hong Sui a, Xiao-Li Wei a, Lei Liu , Peng Huang a, Heng-Heng Yuan a, Ting-Ting Zhang a, Wen-Jie Zhang a, Rui Xie a, Xiao-Hui Lang a, De-Xin Jia a, Yu-Xian Bai a,*
Q1 Xiao-Xue a
a
Department of Gastrointestinal Oncology, The Third Affiliated Hospital, Harbin Medical University, Harbin, 150, Haping St, Nangang District, Harbin, 150081, People’s Republic of China b Department of Endoscope, The Third Affiliated Hospital, Harbin Medical University, Harbin, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 January 2013 Accepted 24 January 2013
Dihydroartemisinin (DHA) has recently been shown anti-tumor activity in various cancer cells. However, its effect on esophageal cancer remains unclear. In this study, for the first time, we demonstrated that DHA reduced viability of esophageal cancer cells in a dose-dependent manner. The mechanism was at least partially due to DHA induced apoptosis by upregulating the expression of Bax, downregulating Bcl-2, Bcl-xL and Procaspase-3, and increasing caspase-9 activation, induced cell cycle arrest by downregulating cyclin E, CDK2 and CDK4. Furthermore, we firstly found that DHA induced autophagy in cancer cells. We concluded DHA might be a novel agent against esophageal cancer. ß 2013 Published by Elsevier Masson SAS.
Keywords: Dihydroartemisinin Esophageal cancer Autophagy Apoptosis Cell cycle arrest
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1. Introduction
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Esophageal cancer is the eighth most common cause of cancer worldwide, and it ranks as the fourth leading cause of all cancer deaths in China [1,2]. More than one half of new cases in western countries have adenocarcinoma of the lower esophagus or gastroesophageal junction, while the dominant histologic subtype is squamous cell carcinoma in eastern Asian countries [3]. Despite improvements in surgical and radiotherapy techniques and refinements of chemotherapeutic regimens, the 5-year survival rate is approximately 15% [4]. Thus, it is currently accepted that conventional chemotherapy is not sufficient to manage esophageal cancer, and the development of new chemotherapies is worth investigation to prolong the survival of patients with esophageal cancer. Artemisinin was isolated from a Chinese herb Artemisia annua. Artemisinin and its derivatives are widely used as antimalarial
Abbreviations: AVOs, Autophagic vacuoles; DHA, Dihydroartemisinin; PI, Propidium iodide; ROS, Reactive oxygen species; TEM, Transmission electron microscopy. * Corresponding author. Department of Gastrointestinal Oncology, The Third Affiliated Hospital, Harbin Medical University, Harbin, 150, Haping St, Nangang District, Harbin, 150081, People’s Republic of China. Tel.: +86 451 86298689; fax: +86 451 86298689. E-mail address: [email protected] (Y.-X. Bai). 1 XiaoXue Du and Yanjing Li contributed equally to this work.
agents in clinic and have recently been shown to have anti-tumor activities [5–13]. Dihydroartemisinin (DHA), which is the most potent of artemisinin derivatives, has exhibited anticancer activity in lung cancer [5], breast cancer [6], hepatic cancer [7], pancreatic cancer [8,9], colorectal cancer [10], glioma [11,12], prostate cancer [13,14] and so on. It is proposed that the anti-tumor effects of DHA have been mediated by cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway in lung adenocarcinoma [5], by shifting the immune response towards Th1 in breast cancer [6], by inhibiting cell proliferation and inducing G1-phase arrest in hepatic cancer [7], by cell cycle arrest and inactivation of nuclear factor-kappa B in pancreatic cancer [8,9], by induction of iron-dependent endoplasmic reticulum stress in colorectal carcinoma [10], by inhibiting hypoxia inducible factor1a activation and generating reactive oxygen species (ROS) in glioma [11,12], by suppressing the PI3-K/Akt and ERK cell survival pathways and triggering the induction of death receptor DR5 and activation of extrinsic and intrinsic cell death signalling in prostate cancer [13,14]. Moreover, DHA exerted significantly lower cytotoxicity to normal cells, such as liver cells, prostate epithelia cells [7,13]. These results indicated that DHA might be an appropriate candidate for cancer chemotherapy. However, to our best knowledge, little is known about its effects on esophageal cancers. The purpose of this study, therefore, was to assess the anti-tumor effects of DHA on human esophageal cancer cells, and to investigate the underlying molecular mechanisms.
0753-3322/$ – see front matter ß 2013 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biopha.2013.01.013
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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2. Materials and methods
2.6. Transmission electron microscopy (TEM)
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2.1. Cells
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The human esophageal cancer cell lines Eca109 and Ec9706 (Laboratory of Medical Genetics, Department of Biology, Harbin Medical University) were maintained in RPMI 1640 medium, which was supplemented with 10% heated inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL). Cells were maintained in a humidified atmosphere of 5% CO2 at 37 8C.
Eca109 and Ec9706 cells were treated as above. The cells were harvested, washed and fixed with 2.5% glutaraldehyde (SigmaAldrich, G6257) containing 1% tannic acid for overnight. After washing, the cell pellets were embedded in epon araldite. The ultra-thin sections were observed with a Hitachi-h7650 electron microscope and representative images were analyzed.
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2.7. Immunoblot analysis
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2.2. Materials
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DHA (Sigma–Aldrich, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma) before experiments. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT, Amresco, USA) and propidium iodide (PI) were dissolved to a final concentration of 5 mg/ml and 50 mg/mL in PBS respectively. Annexin V-FITC apoptosis detection kit and ECL Plus system was obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Antibodies against caspase-3, caspase-9, Bcl-xL, Cyclin E, Bax, LC3A were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2, Cdk4, Cdk2, b-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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2.3. Cell viability assay
Eca109 and Ec9706 cells were treated as above. The cells were lysed in RIPA buffer after washing twice with cold PBS. After keeping on ice 8–10 minutes, total proteins were centrifuged at 14,000 rpm for 5 minutes at 4 8C and then quantified by Coomassie brilliant blue G. Protein samples were mixed with the 5 sample buffer. These samples were boiled at 99 8C for 5 minutes. Similar amounts of protein were electrophoresed on a 12% of SDSpolyacriylamide gel and transferred topolyvinylidene difluoride (PVDF) membranes. The membranes were washed with PBSTween and blocked with 5% fat-free milk in PBS-Tween for 1 h at room temperature, and incubated with primary antibody overnight at 4 8C. After washing three times with PBS-Tween, the membranes were incubated for 1 hour at room temperature with secondary antibody and washed again. The blots were visualized using a chemiluminescence detection kit ECL-PLUS. Anti-b-actin was used to ensure equal loading.
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Eca109 (4 103 cells/well) and Ec9706 (5 103 cells/well) cells were seeded into 96-well plates, and cultured overnight. The cells were treated with different concentrations of DHA. Cells treated with DMSO were used as untreated control. After incubation for 48 h and 72 hours respectively, MTT (20 mL) was added to each well with 3–4 hours incubation at 37 8C. The medium was discarded, and DMSO (150 mL) was added into each well at room temperature for 10 minutes with shaking. The OD490 nm was measured on a plate reader (BioTek, USA), and the difference of absorbance between the treated and untreated control groups was calculated to determine cell viability. The experiments were repeated thrice.
2.8. Statistical analysis
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All results were expressed as mean values standard deviation, except the results from western blot assay. One-way ANOVA was used for statistical analysis. A value of less than 0.05 (P < 0.05) was used for statistical significance.
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3. Results
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3.1. DHA reduces viability of esophageal cancer cells
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2.4. Cell apoptosis assay
For esophageal cancer cell lines, Eca109 and Ec9706 were exposed to various concentrations of DHA (2.5–120 mmol/L) for 48 and 72 hours respectively. The effect of DHA on the viability of two types of cells was detected with MTT assay. As shown in Fig. 1, DHA reduced the viability of two types of cells in vitro in a dosedependent manner, compared with untreated cells. These results suggested that DHA was cytotoxic to human esophageal cancer cells. The concentration of DHA IC50 values for Eca109 and Ec9706 were 76.86 mM, 93.81 mM respectively.
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Eca109 and Ec9706 cells were treated as above. The cells were harvested and washed thrice with ice-cold PBS. Cells were suspended in 195 mL binding buffer, 5 mL of Annexin V and 10 mL of PI were added, and incubated for 20 minutes at room temperature in dark. After diluted with 190 mL binding buffer, the cells were measured within 1 hour. After filtration, the single-cell suspensions were analyzed on the flow cytometer (Epics Altra II, Beckman Coulter, USA). Apoptotic and necrotic cells were quantified by counting the relative amount of fragmented blue or red nuclei (early or late apoptosis) and non-fragmented red nuclei (necrosis). The apoptotic or necrotic cells were reported as percentages of total cells. The experiment was repeated thrice.
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2.5. Cell Cycle Analysis
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Eca109 and Ec9706 cells treated with DHA were cultured respectively for 48 h and 72 h. The cells were harvested, washed with ice-cold PBS twice, and fixed in ice-cold 70% (v/v) ethanol for overnight at 4 8C Prior to analysis, the ethanol was removed by centrifugation, samples were washed with PBS again and cells were re-suspended in PBS containing PI (50 mg/mL) and RNase A (10 mg/mL) and then incubated at 37 8C in the dark for 30 minutes. The percentage of cells at G0/G1, S, or G2/M phase was thereby documented.
Fig. 1. Dihydroartemisinin (DHA) inhibits the proliferation of human esophageal cancer cells in vitro. Two human esophageal cancer cells Eca109 and Ec9706 were exposed to various concentrations of DHA (2.5–120 mmol/L) for 48 and 72 hours respectively. The viability of cells was assessed by the MTT method to calculate the viability index (%). Values represented mean SD.
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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Fig. 2. Eca109 and Ec9706 cells were exposed to various concentrations of Dihydroartemisinin (DHA) (0, 10, 40, and 80 mM) for 48 and 72 hours respectively followed by apoptosis assay: representative histograms were from cytometrically analyzed Eca109 (upper panel) or Ec9706 (lower panel) cells incubated with control medium (control) or media containing 10 or 80 mmol/L of DHA (a); DHA induced a significant apoptosis in the two types of esophageal cancer cells in a dose-dependent manner. Values represented mean SD (n = 3) (b). * P < 0.05 versus control.
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3.2. DHA induces apoptosis of esophageal cancer cells
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After Eca109 and Ec9706 were exposed to various concentrations of DHA (2.5-120 mmol/L) for 48 and 72 h respectively, DHAinduced apoptotic changes were detected by flow cytometric analysis using AnnexinV-FITC and PI staining (Fig. 2). As shown in Fig. 2b, DHA treatment induced a significant apoptosis in the two types of esophageal cancer cells in a dose-dependent manner. The representative histograms of flow cytometry showed that the mean apoptosis rates of Eca109 cells were 3.55, 5.26, 14.61% when they were treated with DHA at concentrations at 0, 10, 80 mmol/L respectively, and that of Ec9706 cells were 1.1, 1.8 and 16.15% (Fig. 2a).
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3.3. DHA induces G0/G1 phase cell cycle arrest in human esophageal cancer cells
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In our study, we performed DNA cell cycle analysis to assess the effect of DHA treatment on the distribution of cells in the cell cycle. As shown in Fig. 3a, significant changes in cell cycle were noted in the two types of esophageal cancer cells. As compared with the control group, the ratio of cells in S phase gradually decreased
while the ratio of cells in G0/G1 phase gradually increased. And the effects occurred in a dose-dependent manner (Fig. 3b). These data suggested that DHA induced cell cycle arrest in G0/G1 phase in human esophageal cancer cells.
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3.4. DHA induces ultrastructural morphologic changes in human esophageal cancer cells
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In the control group, no obvious cell damage was observed in Eca109 (Fig. 4a) and Ec9706 cells (Fig. 4c). After exposed to various concentrations of DHA, swollen mitochondria were found in the treated cells. Even some cells showed typical characteristics of apoptotic and necrotic cells such as apoptotic body, nuclear condensation and broken cell membranes with nuclear lysis (Fig. 4b, d). At present, TEM observation is the only absolute test of autophagy. A defining feature of the autophagosome is doublemembraned autophagic vacuoles (AVOs). The cells in control group were intact with rich cytoplasm, mitochondria were integrated, and very few vacuoles were observed in the cytoplasm (Fig. 4e). The cell morphology was greatly changed, and many AVOs were observed in the cytoplasm of cells after treated with DHA. As
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Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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Fig. 3. Eca109 cells were exposed to various concentrations of Dihydroartemisinin (DHA) (0, 10, 40, and 80 mM) for 48 h respectively followed by cell cycle distribution assay: representative histograms were from cytometrically analyzed Eca109 cells incubated with control medium (control) or media containing 10, 40, 80 mmol/L of DHA (a); the percent of cells in G0/G1, S, and G2/M phases of the cell cycle are shown as mean SD (n = 3) (b). *P < 0.05 versus control.
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shown in Fig. 4f, it clearly showed some enclosed vacuoles, which contained what appeared to be mitochondria or other cellular contents (black arrow indicated).
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3.5. DHA modulates the expression of apoptosis-related proteins in human esophageal cancer cells
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To characterize the potential mechanism by which DHA induced apoptosis of human esophageal cancer cells, the effect of DHA on the expression of apoptosis-related proteins was examined. DHA treatment upregulated the expression of Bax and downregulated the expression of Bcl-2, Bcl-xL and Procaspase-3, and also increased the activation of caspase-9 in the two types of cells. Taken together, these data suggested that DHA upregulated
the expression of pro-apoptotic proteins to activate caspases, thus promoted apoptosis of esophageal cancer cells.
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3.6. DHA modulates the expression of cell cycle-related proteins in human esophageal cancer cells
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To investigate the potential mechanism by which DHA regulated the cell cycle of esophageal cancer cells, the effect of DHA on the expression of G0/G1 phase -related proteins, such as cyclin E, CDK2 and CDK4 was examined. As shown in Fig. 5, DHA treatment resulted in downregulation of cyclin E, CDK2 and CDK4. Our results documented the modulation effects of DHA on key regulators in G0/G1 phase cell cycle progression in esophageal cancer cells.
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Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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Fig. 4. Ec9706 cells were exposed to various concentrations of Dihydroartemisinin (DHA) (0, 10, 40, and 80 mM) for 72 hours respectively followed by cell cycle distribution assay: representative histograms were from cytometrically analyzed Ec9706 cells incubated with control medium (control) or media containing 10, 40, 80 mmol/L of DHA (a); the percent of cells in G0/G1, S, and G2/M phases of the cell cycle are shown as mean SD (n = 3) (b). *P < 0.05 versus control.
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3.7. DHA induces the expression of LC3-II in human esophageal cancer cells
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The amount of LC3-II is correlated with the extent of AVOs formation, and conversion of LC3-I to LC3-II has been used as a marker for autophagy [15,16]. As shown in Fig. 5, DHA treatment resulted in a conversion of LC3-I to LC3-II in two types of esophageal cancer cell lines.
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4. Discussion and conclusion
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Recent studies suggested a potential use of artemisinin and its active metabolite DHA as anticancer agents [5–13]. However, the effect of DHA on esophageal cancer cells remains unclear. Furthermore, to our best knowledge, the phenomenon of autophagy has not been investigated so far in DHA treatment on cancer cells. In this study, it was first found that in human esophageal cancer cells, DHA could induce apoptosis by upregulating Bax, downregulating Bcl-2, Bcl-xL and
Procaspase-3, and increasing the activation of caspase-9, induce G0/G1 phase arrest by downregulaing cyclin E, CDK2 and CDK4. We also found the formation of AVOs by TEM and conversion of LC3-I to LC3-II by immunoblot analysis. For the first time, our study demonstrated that DHA could induce autophagy in cancer cells. Apoptosis, which does not trigger immune reactions, is considered as a better way to kill cancer cells than instant cell death and necrosis [17]. Members of the Bcl-2 family, including pro-apoptosis proteins (Bax, Bad) and anti-apoptotic proteins (Bcl-2 and Bcl-xL), are the most prominent regulators of mitochondrial-dependent apoptosis [18]. It has been demonstrated that Bcl-xL could inhibit membrane permeabilization and cytochrome c release by blocking the activation of Bax and Bak, and improve the ability of tumor cells to escape from therapy [19,20]. Bcl-2 is located on the membrane of mitochondria, whereas Bax directly binds to Bcl-2 and inhibits its function [21]. An increase in the ratio of Bax/Bcl-2 stimulates the release of cytochrome c from the mitochondria into the cytosol and the cytosolic cytochrome c
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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Fig. 5. Eca109 and Ec9706 cells were exposed to various concentrations of Dihydroartemisinin (DHA) (0, 10, 40, and 80 mM) for 48 and 72 hours respectively followed by Transmission electron microscopy (TEM) observation. No obvious cell damage was observed in the control group. Apoptosis, necrosis and autophagy occurred in the DHA treatment group: the control untreated Eca109 cells (a, e); the control untreated Ec9706 cells (c); Eca109 cells treated with 40 mmol/L DHA (arrow indicated autophagosome) (b, f); Ec9706 cells treated with 40 mmol/L DHA (d).
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then binds to Apaf-1, leading to the activation of procaspase-9, 3 which results in apoptosis [19–21]. Several studies have indicated that induction of apoptosis of cancer cells by DHA might be an important mechanism responsible for the impact on tumor cell viability. Jiao et al. showed that DHA induced apoptosis in human ovarian cancer cells accompanied by a decrease of Bcl-xL and Bcl-2 and an increase of Bax and Bad [22].
Hou et al. suggested that DHA induced apoptosis, activated caspase-3, increased the Bax/Bcl-2 in human hepatoma cells [7]. Similarly, Chen et al. reported that DHA induced apoptosis by reducing the ratio of Bcl-2/Bax and increasing the activation of caspase-9 in a dose-dependent manner [9]. Similar to the reports above, in our study, DHA treatment induced a significant apoptosis in the two types of esophageal cancer cells in a dose-dependent
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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manner. And we also found that DHA upregulated the expression of Bax and downregulated the expression of Bcl-2, Bcl-xL and Procaspase-3, and also increased the activation of caspase-9. Our data indicated that DHA induced apoptosis by regulating Bcl-2 family members and activating caspases that lead to apoptosis. Obstruction of cell cycle progression in cancer cells is considered as one of the most effective strategies for the control of tumor growth [23,24]. In our study, we demonstrated that the ratio of cells in S phase gradually decreased while the ratio of cells in G0/G1 phase gradually increased compared with the control group. Furthermore, our study demonstrated that DHA treatment resulted in the downregulation of cyclin E, CDK2 and CDK4. Cyclin– Cdk heterodimers play an important role in the control of cell cycle progression and cell cycle checkpoints in mammalian cells. And cyclin E, CDK2 and CDK4 are important complexes responsible for the progression of cells through the G1 phase of cell cycle and initiation of DNA replication [25]. These data indicated that block of G0/G1 transition might be one mechanism responsible for the significant viability reduction of human esophageal cancer cells induced by DHA, which induced cell arrest partly through downregulating cyclin E, CDK2 and CDK4. Consistent with our result, some investigators found that DHA could induce G0/G1 phase arrest in pancreatic cancer and hepatoma [7,26]. However, in contrast, cell arrest in G2/M phase was induced in osteosarcoma and ovarian cancer [22,27]. These data suggested that DHA induced cell arrest depending on the cell lines. Autophagy is a process whereby cytosol and organelles become encased in vacuoles termed autophagosomes. It is a regulated process of degradation and recycling of cellular constituents,
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participating in organelle turnover and the bioenergetic manage- 293 ment of starvation [28,29]. In our study, in human esophageal 294 cancer cells treated by DHA, we found the formation of AVOs and 295 conversion of LC3-I to LC3-II. Conversion of LC3-I to LC3-II has been 296 used as a marker for autophagy [15]. To the best of our knowledge, 297 for the first time, we found that DHA could induce autophagy in 298 299 cancer cells. Recent evidence indicated that autophagy played an important 300 role in tumor development, cell death and survival. Some authors 301 thought that autophagy might lead to cell death through excessive 302 self-digestion and degradation of essential cellular constituents 303 and could proceed in the absence of caspase signaling or even be 304 activated under conditions of caspase inhibition [15]. Autophagic 305 cell death, which is different from both apoptosis and necrosis, has 306 independent morphological and biochemical features [16]. So it is 307 considered to be a second type of programmed cell death 308 (sometimes referred to ‘type II cell death’). Other authors thought 309 that autophagy might support cancer cell survival by providing an 310 ‘escape route’ and protection from proteotoxicity. Similarly, it was 311 reported recently that autophagy occured in chemotherapeutic 312 treatment and contributed to drug resistance [30]. Furthermore, it 313 was reported that there was an equilibrium established between 314 autophagy and apoptosis, with inhibition of either process leading 315 to enhanced activity by the other [31]. In our study, DHA induced 316 apoptosis and autophagy in human esophageal cancer cells. 317 Therefore, how about the effect of autophagy in cancer cells 318 treated by DHA? How about the detailed mechanism between 319 apoptosis and autophagy? These mechanisms are unknown and 320 further investigation is required in the future (Fig. 6). Q2321
Fig. 6. DHA modulated the expression of apoptosis-related, cell cycle-related and autophagy-related proteins in esophageal cancer cells. Eca109 and Ec9706 cells were exposed to various concentrations of DHA (0, 10, 40, and 80 mM) for 48 and 72 hours respectively followed by western blot analysis.
Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013
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In conclusion, we first investigated the anticancer effects of DHA on human esophageal cancer cells in vitro. We demonstrated that DHA induced apoptosis, cell cycle arrest and autophagy in esophageal cancer cells. Our study indicated that DHA might be a potent and promising agent to combat esophageal cancer. However, these results are only based on studies in vitro, and further studies in vivo are necessary to investigate.
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Disclosure of interest
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The authors declare that they have no conflicts of interest concerning this article.
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Acknowledgements
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We are grateful to the teachers of central cancer laboratory of Harbin Medical University for their kind help. And the authors thank Yuyan Ma and Yangmei Yang for their technical assistance.
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References
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Please cite this article in press as: Du X-X, et al. Initiation of apoptosis, cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.01.013