Background
Advances in surgery, hormone therapy, and chemotherapy have improved advanced prostate cancer treatments. however, these approaches are limited due to prostate cancer therapy resistance. Thus, there is a critical need to develop treatments against new cellular targets. Recently, regulation of metabolism in cancer therapy is emerging, because rapidly growing cells need plenty of the energy, nutrients, and building blocks that are required to maintain cell survival and proliferation [
1‐
3]. Aggressive cancers consume abundant glucose to produce ATP using glycolysis, to promote the pentose phosphate pathway (PPP) to decrease oxidative stress, and to make many kinds of biomaterials [
4‐
7]. One promising metabolic-control is chemotherapy using 2-deoxyglucose (2DG), which is a well-known glycolysis inhibitor [
8,
9]. 2DG inhibits hexokinase, the rate-limiting enzyme of glycolysis, leading to depleted ATP, antioxidants, and glycolysis intermediates needed for cell survival and maintenance, thereby causing cell growth arrest and death [
10‐
12]. Coincidently, autophagy induction rises in response to intracellular starved conditions and ER stress by 2DG as a cell survival process [
3,
13].
Autophagy has an important role in the catabolic pathways that support intracellular energy sources and building blocks and clears cytotoxins to sustain homeostasis by degrading unfolded or aggregated protein and damaged cytoplasmic components with lysosomal proteases [
14]. In cancer, functioning autophagy is crucial to survival and growth because rapidly proliferating cancer cells need vast energy and biomass to make new proteins, lipids, and intracellular components, and must remove protein aggregates, abnormal cytoplasmic compartments, excess reactive oxygen species, and lipid droplets to maintain the homeostasis that is produced during the development of cancer [
15,
16]. These helpful functions of autophagy produce pro-survival effects in cancer development and increase resistance to chemotherapy [
17,
18]. Therefore, recent reports tried to administer combination chemotherapy of both an anticancer drug and an autophagy inhibitor to block the pro-survival function of autophagy and showed a synergistic anticancer effect [
19‐
22]. However, some groups demonstrated that autophagy contributed to the pro-death function rather than the pro-survival role. Excessive autophagy activation leads to cell death and depends on the cell types and culture environments. It is termed “autophagic cell death,” and arises from unlimited degradation of cytoplasmic components [
23‐
26]. The double-edged sword effects of autophagy on cell survival or death are controversial [
27,
28].
To determine whether autophagy is harmful or helpful for PC3 cells or LNcaP cells survival and growth under nutrient depleted conditions by 2DG, we investigated 2DG’s effect on autophagy regulation in PC3 cells and LNcaP cells, and proved that 2DG significantly suppressed both cells’ growth and promoted intense autophagic flux. Autophagic flux was differentially regulated depending on the exposure time of 2DG. Especially, increased autophagic flux significantly suppressed PC3 cells and LNcaP cells growth, and it would be blocked for cell survival.
Methods
Cell culture
Human prostate cancer cell line PC3 and human embryonic kidney cell line 293 T were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s modified Eagle medium (Gibco, Grand Island, NY, USA) with penicillin-streptomycin (100 U/mL; Gibco) and 10 % fetal bovine serum (Gibco) in a humidified atmosphere of 95 % air and 5 % CO2 at 37 °C.
Cell growth assay
PC3 cells were seeded into 96-well plates (5 × 103 cells per well) and incubated overnight. The next day the culture medium in each well was changed with a different concentration of 2DG containing culture medium and cultured for 3 days. After 2DG treatment, the PC3 cells on the culture plate were counted or we performed an MTT assay. Briefly, PC3 cells on the culture plate were treated with 0.5 mg/mL Thiazolyl blue tetrazolium bromide (MTT, Sigma, Louis, MO, USA) for 4 h and washed with phosphate-buffered saline (PBS). Afterwards, the plates were drained upside down on a paper towel and then solubilized with DMSO. Each well was measured using the optical density at 570 nm.
Confocal microscopy imaging
PC3 cells were seeded on glass-bottomed cell culture dishes (NEST Biotechnology, Shanghai, China) and incubated for 24 h. The culture medium was then changed to 2DG containing culture medium and incubated for 48 h. Cells were fixed with 10 % paraformaldehyde for 30 min and washed three times with PBS. To increase permeabilization, cells were incubated in 0.25 % Triton X-100-containing PBS for 10 min. After washing the cells with a PBS blocking solution (3 % bovine serum albumin, 0.25 % Triton X-100 in PBS), cells were treated for 30 min followed by incubation overnight at 4 °C with diluted primary antibody against microtubule-associated protein 1 light chain-3B (LC3B; 1:200, Cell Signaling Technology, Danvers, MA, USA) or p62 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) in blocking solution. The next day, the cells were washed three times with PBS then incubated with diluted secondary antibody (1:500, Invitrogen, Oregon, USA) in blocking solution for 1 h. After washing the cells in PBS, Hoechst 33258 was used to stain the nucleus. The cells were analyzed using a Carl Zeiss LMS710 confocal microscope (Carl Zeiss, Gottingen, Germany).
Fluorescence-activated cell sorting (FACS) analysis
Chemically pre-treated PC3 cells were trypsinized and harvested. Cells were fixed with 70 % ethanol for 1 h on ice and collected for permeabilization of the cell with 0.25 % Triton X-100. After centrifugation to collect the cells, we removed the supernatant and resuspended the cells with 10 μg/mL RNase and 20 μg/mL propidium iodide-containing PBS for 30 min at room temperature. The DNA content of the cells was measured using an LSRII flow cytometer (BD Bioscience, CA, USA).
Overexpression of LC3B
PC3 cells were seeded into 6-well plates (1 × 104 cells per well) and incubated overnight. LC3B or control gene plasmids were purchased from Addgene (MA, USA). Two mg of each plasmid was transfected into cultured PC3 cells using 0.2 μL/well Lipofectamine2000 (Invitrogen) according to the manufacturer’s protocols. Then LC3B-overexpressing cells were selected in culture medium containing 200 μg/mL Genectin (Invitrogen).
293 T cells were seeded into 60 mm culture dishes (1 × 105 cells) and incubated overnight. Lenti-viral plasmid of Beclin1 or non-targeting shRNA plasmids (1–4 μg; Sigma) were transfected into cultured 293 T cells using 20 μL/well Lipofectamine2000 according to the manufacturer’s protocols to produce virus particles. After 2 days, the culture medium of plasmid transfected 293 T cells was collected and transferred to overnight cultured PC3 cells (1 × 105 cells per 100 mm dish) for virus infection. We removed the viral particle containing medium and replaced it with normal culture medium followed by cell incubation for 2 days. Afterwards, Beclin1 or non-targeting shRNA plasmid infected cells were selected in puromycin containing culture medium (2 μg/mL, Invitrogen).
Western blot analysis
Cells were washed with PBS and lysed with 1 % sodium dodecyl sulfate (SDS) lysis buffer (60 mM Tris–HCl, pH 6.8, 1 % SDS) with protease inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of total protein from each sample were separated by SDS-polyacrylamide gel electrophoresis on 8 % or 12 % gels, and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica MA, USA). Following incubation with primary antibodies against mammalian target of rapamycin (mTOR; 2972), phospho-mTOR (2448), protein kinase B (AKT; 9272), phospho-AKT (9271), AMP-activated protein kinase (AMPK; 2603), phospho-AMPK (2535), cell cycle-regulation antibody (9932, 9870; Cell Signaling Technology, Danvers, MA), p62 (SC28359; Santa Cruz Biotechnology, Dallas, Texas, USA), LC3B (L7543), and actin (A1978; Sigma), membranes were incubated with goat anti-rabbit (sc2004) or anti-mouse (sc2005) IgG horseradish peroxidase (Santa Cruz Biotechnology) as the secondary antibody. Labeled, specific protein bands were visualized using the ECL Kit (Thermo Scientific, Rockford, IL, USA).
Chemicals
CQ (Sigma), a late-phase inhibitor of autophagy, was used at a final concentration of 20 μM for 2 h. Rapamycin (Sigma), blocker of the mTOR signaling pathway, was used as activator of autophagy at various concentrations.
Statistical analysis
Each experiment was carried out in triplicate, and quantitative data were expressed as the mean ± S.D. Statistical analysis was conducted using the GraphPad Prism Software (San Diego, Calif., USA).
Discussion
This study was carried out with the hypothesis that 2DG controls cell growth and survival through autophagy regulation. 2DG triggers intracellular energy deficiency via glycolysis inhibition and increased endoplasmic reticulum (ER) stress, which induces cell death and arrest [
9,
30]. Recent studies reported that genetic or chemical cancer cell metabolism manipulation, including 2DG treatment, showed remarkable antitumor effects [
31,
32]. Cheong et al. demonstrated that 2DG induced cell death and showed synergistic effects using metformin co-treatment conditions [
33]. Our data proved the anticancer effects of 2DG in prostate cancer cells by inhibiting cell proliferation, but did not show strong cell death (Fig.
1). These results were somehow different with the Cheong group study. However, these antitumor effects were dependent on the different types of cancer cells confirmed by other groups. Various types of malignant cancers showed only a slowdown of cell growth rather than cell death, and the administration of only 2DG had no significant effects
in vivo [
8,
34]. However, it is clear that 2DG has a strong inhibition effect on cancer cell growth.
2DG promotes these effects through decreased intracellular ATP levels and ER stress by blocking glycolysis, which leads to autophagy induction. In this study, we found that increased autophagy with rapamycin or LC3B overexpression strongly suppressed PC3 cell growth (Fig.
5c and d). 2DG also increased autophagic flux at the early phase and suppressed the growth of PC3 and LNCaP cells (Fig.
1a and Additional file
1: Figure S1A). Overall, 2DG induced PC3 cell growth inhibition arose by autophagy activation rather than autophagy inhibition.
However, we wondered why increased autophagic flux at the early phase of 2DG treatment was gradually decreased at 48 h of treatment with 2DG although the AMPK signal, an autophagy inducing internal signaling pathway, were constantly activated (Fig.
3).
Our results showed that the decreased mTOR/AKT signaling pathway and increased AMPK signaling pathway were continuously maintained by 2DG until the last time points of sample preparation (Fig.
3b). AMPK is well known as an intracellular energy sensor that has the ability to detect the AMP:ATP ratio. In low levels of ATP due to exercise or metabolic stress, AMPK is activated to inhibit energy consuming anabolic pathways and promote energy producing catabolic pathways like glucose uptake, glycolysis, and beta oxidation to increase ATP levels. In addition, AMPK tightly regulates autophagy through the inhibition of the mTOR signaling pathway [
35,
36].
One possibility for autophagy blocking by 2DG with long-term treatment is lower levels of glucose and ATP in the cytoplasm. ATP is a very important energy source to maintain metabolic homeostasis, and is used as a substrate to transfer energy for transducing signals by kinase, as a material to make nucleic acids, and it participates in biosynthetic processes [
37,
38]. In addition, ATP is a very critical for the process of autophagy maturation [
39]. Autophagy begins with phagophore formation with a small double membrane, and the end of the phagophore is elongated. After then it engulfs the described substrate and encloses the double membrane. This is called the autophagosome. Many autophagy related proteins including ULK2, the autophagy related (ATG) proteins, the focal adhesion kinase family interacting protein FIP200, Beclin1, the human phosphatidylinositol 3 kinase adaptor protein p150, the class III phosphatidylinositol-3 kinase VPS34, and LC3 participate in this process. The autophagosome fuses with the lysosome to construct the autolysosome that degrades the autophagosome contained substrates to recycle biomaterials [
40]. ATP is required for these sequestrations by autophagosome [
41,
42]. ATP has a crucial role in maintenance of V-ATPase activity that produces lysosomal acidification to optimize lysosomal hydrolases within the lysosomal membrane. V-ATPase consists of ATP hydrolysis-acting V
1 and proton translocation-acting V
0. Lower levels of ATP due to limited glucose supply promotes separation of these two components and causes loss of V-ATPase activity, which inhibits autophagic flux and blocks autophagy cargo degradation by lysosomal proteases [
43‐
45]. Similarly, long-term treatment of 2DG prohibits glycolysis and sufficient ATP synthesis, which reduces autolysosome conformation and autophagic flux. This could partially explain the inhibition of autophagic flux by 2DG. Because ATP is synthesized not only by glycolysis, but also beta oxidation in the mitochondria, blocking of glycolysis with 2DG did not perfectly inhibit ATP synthesis. Supplements of glutamine via glutaminolysis and pyruvates to the mitochondria could make ATP [
46]. So, in our experimental conditions, the reason for inhibition of autophagic flux by depletion of ATP with 2DG treatment is not enough.
Another possibility is a defense mechanism for survival in cells. Recent reports use the term "autophagic cell death," which means unlimited autophagy activation. It leads to cell death because autophagy uptakes and degrades not only the properly eliminated proteins or organelles, but also the normal intracellular components that work well in the cell [
28]. Some groups reported the possibility of clinical cancer therapy using autophagy inducers that promote autophagic cell death in cancer cells [
28,
47,
48]. In cancer cells, this autophagic cell death is thoroughly prevented for survival, so does 2DG treated PC3 cells. In our results, blocking of autophagy through chemical or genetic inhibitory manipulation did not have significant anticancer effects compared with normal culture conditions. Knockdown of an autophagy related gene showed only increased autophagic flux blocking and little change in cell cycle related gene expression (Fig.
4). In addition, inhibition of autophagic flux by CQ or shRNA showed no remarkable difference in PC3 cell growth (Fig.
5a and b). In contrast, overactivation of autophagy by both rapamycin and overexpression of LC3B significantly suppressed cancer cell growth, and additional treatment of 2DG into overactivated autophagy conditions strongly decreased PC3 cell viability (Fig.
5c and d). In LNCaP cells, the effect of cell growth inhibition by rapamycin was more intense than in PC3 cells (Additional file
3: Figure S3B).
Although cell death through autophagy was very dependent on cell type and culture environments, recent reports demonstrated that increased autophagic flux has strong anticancer effects [
24,
49,
50]. Zhao et al. report that acetylated Foxo1 interacted with Atg7 and E1-like protein and the oxidative stress increased autophagic flux, leading to cell death. Similar to our results, excessive autophagy activation by increased intracellular ER stress and ROS levels induced cell death [
50]. Taken together, metabolic stress causes autophagy activation and leads to cell death. However, the detailed mechanism for the regulation of autophagic flux in cancer cell survival is not clear and needs to be studied further [
27,
28].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JJ: conception and design of the study, data analysis and interpretation, manuscript writing; SK: data acquisition and data analysis and interpretation; KP: data acquisition; MY: conception and design of the study. All authors read and approved the final manuscript.
1Department of Nuclear Medicine, Severance Hospital, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, 120–752 Rep. of Korea. 2Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, 120–752 Rep. of Korea. 3Severance Biomedical Science Institute, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, 120–752 Rep. of Korea. 4Department of Surgery, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, 120–752 Rep. of Korea.