The combination of tephrosin with 2-deoxy-D-glucose enhances the cytotoxicity via accelerating ATP depletion and blunting autophagy in human cancer cells

Abstract

2-Deoxy-D-glucose (2-DG), a synthetic glucose analog that acts as a glycolytic inhibitor, is currently under clinical evaluation for targeting tumor cells. Tephrosin (TSN), a plant rotenoid, is known as an anticancer agent. In this study, we describe that the addition of TSN to 2-DG enhanced the cytotoxic activity of 2-DG against various types of cancer cells by accelerating ATP depletion and blunting autophagy. TSN increased the sensitivity of cancer cells to the cytotoxic effect of 2-DG. The combination of TSN and 2-DG induced acceleration of intracellular ATP depletion and the drastic activation of AMP-activated protein kinase (AMPK), which resulted in the inactivation of the mammalian target of rapamycin (mTOR) pathway. Of particular interest, TSN suppressed 2-DG-induced autophagy, a cell survival process in response to nutrient deprivation. We also showed that TSN inhibited 2-DG-induced activation of elongation factor-2 kinase (eEF-2K), which has been known to regulate 2-DG-induced autophagy. Inhibition of eEF-2K by RNA interference blunted 2-DG-induced autophagy and increased the sensitivity of cancer cells to the cytotoxic effect of 2-DG. The addition of TSN to 2-DG, however, did not enhance the cytotoxic activity of 2-DG by knockdown of eEF-2K, suggesting that inhibition of eEF-2K by tephrsoin could be a critical role in the potentiating effect of TSN on the cytotoxicity of 2-DG. Furthermore, we showed that the blunted autophagy and enhanced cytotoxicity of 2-DG was accompanied by the augmentation of apoptosis. These results show that TSN may be valuable for augmenting the therapeutic efficacy of 2-DG.

Introduction

Cellular metabolism of malignant cells differs significantly from that of normal cells. Whereas normal cells rely on respiration, a process that consumes oxygen and glucose to produce the energy-storing molecule ATP, malignant cells mainly depend on glycolysis, the anaerobic metabolism of glucose into ATP, even in the presence of sufficient oxygen. This increased dependency of malignant cells on glycolysis for ATP production is known as the so-called Warburg effect.^1,2 Due to the high dependence of malignant cells on glycolysis, interference of this metabolic process has recently been proposed as a potentially useful approach for developing new selective cancer therapies.³

2-DG is best known as an inhibitor of glucose metabolism.⁴ Inside a cell, it is converted to phosphorylated 2-DG by hexokinase, the first and rate-limiting enzyme in glycolysis. However, phosphorylated 2-DG cannot be metabolized by the second enzyme in glycolysis, phosphoglucose isomerase.⁵ This leads to trapping and accumulation of phosphorylated 2-DG, which competitively inhibits hexokinase at the rate-limiting step of glycolysis. A direct consequence of 2-DG treatment is intracellular ATP depletion,^6,7 which ultimately suppresses cell proliferation in vitro. Treatment of cancer cells with 2-DG has been shown to inhibit growth and viability of cancer cells.^8-10 Nonetheless, the implementation of 2-DG as an anticancer agent in vivo has been a disappointment. Whereas 2-DG suppresses cell growth in vitro, studies using xenografts indicate that 2-DG treatment, when provided as a single agent, does not inhibit tumor growth.¹¹ Therefore, approaches that can enhance the efficacy of 2-DG may make this agent more useful in the treatment of cancers.

2-DG also induces autophagy.^12,13 Autophagy is a major mechanism by which a starving or stressed cell reallocates nutrients from ancillary processes to more essential ones.^14,15 Mechanistically, autophagy is initiated when the autophagosome, a double-membrane structure, is formed to surround certain targeted cytoplasmic proteins and organelles. This process and the double-membrane structures are associated with the conversion of the microtubule-associated protein light chain 3B-I (LC3-I) to LC3B-II (LC3-II). The protein/organelle containing autophagosome fuses with a lysosome to degrade its inner contents.¹⁴ Autophagy plays a protective role against 2-DG-elicited cell death and blockage of autophagy increases the sensitivity of cancer cells to the cytotoxic effect of 2-DG.^16,17

The rotenoids such as deguelin and tephrosin (TSN) are known as anticancer agents.^18,19 Deguelin has been well studied for its antitumor activity and has proven to be a promising chemopreventive and therapeutic agent to treat diverse types of cancer.^20,21 TSN has been shown to inhibit mouse skin tumor promotion and invasion of cancer cells.^22,23 We have previously shown that TSN inhibited nuclear factor-κB activity and induced internalization and degradation of epidermal growth factor receptor.^24,25 As part of our continuing search for compounds to enhance the cytotoxic activity of 2-DG from natural products, we found that TSN enhanced the cytotoxic activity of 2-DG against various human cancer cell lines.

In this study, we investigated the combination effect of TSN with 2-DG on the viability of cancer cells. Our data showed that the addition of TSN to 2-DG enhanced depletion of intracellular ATP and suppressed 2-DG-induced autophagy via inhibiting the activation of eEF-2K, thus inducing apoptosis.

Results

The combination of TSN and 2-DG decreases cell viability in various human cancer cell lines

To determine whether TSN modulates the cytotoxic activity of 2-DG, we analyzed the effect of TSN, 2-DG alone or in combination with 2-DG on the cell viability in four cancer cell lines: HeLa, HT-29 and SW-620, and A549 cells (Fig. 1). TSN alone showed a limited cytotoxic effect up to 1 μM in these cell lines. Treatment of 2-DG alone also exhibited only weak cytotoxic activity against these cell lines (Fig. 1). The combination of TSN with 2-DG synergistically decreased cell viability. For example, treatment of HeLa cells with 0.3 μM of TSN or 5 mM of 2-DG alone reduced cell viability to 91 ± 0.4% and 64 ± 1%, respectively of control levels at 48 h. However, combined treatment of 0.3 μM of TSN and 5 mM of 2-DG resulted in the reduction of cell viability to 23 ± 0.5% of control level (Fig. 1A). Similar results were observed in SW-620 (Fig. 1B), HT-29 (Fig. 1C) and A549 cells (Fig. 1D). These results suggested that combined treatment of TSN and 2-DG effectively decreases the cell viability in these human cancer cell lines. Because HeLa cells were the most sensitive to the combination of TSN and 2-DG among these cell lines, we further studied the combined effect of TSN and 2-DG in HeLa cells.

Figure 1.

The combination of TSN with 2-DG exerts a deleterious effect on cancer cell viability in HeLa (A), SW-620 (B), HT-29 (C), and A549 (D). Cells were seeded in triplicate into 96-well plates and cultured with the indicated concentrations of TSN in the presence of 5 mM 2-DG for 48 h. Cell viability was determined by MTT assay. Data are presented as the mean ± SD from two independent experiments, and are expressed as a percentage of the number of control cells.

The combination of TSN and 2-DG aggravates depletion of intracellular ATP

We next determined whether the combination of TSN and 2-DG affects intracellular ATP levels in HeLa cells (Fig. 2). 2-DG alone slightly decreased intracellular ATP concentrations with 24 h of treatment (Fig. 2A). TSN also decreased intracellular ATP concentration in a concentration-dependent manner (Fig. 2B). Importantly, the addition of TSN with 2-DG robustly diminished intracellular ATP concentrations in HeLa cells (Fig. 2C). Similar results were observed in HT-29 cells (Fig. 2D). These results suggested that the combination of TSN and 2-DG could exert a more deleterious effect on cancer cell viability by aggravating intracellular ATP depletion.

Figure 2.

TSN enhances ATP depletion induced by 2-DG. (A and B) HeLa cells were treated with the indicated concentrations of 2-DG (A) or TSN (B) for 24 h and then harvested for the determination of ATP level. (C and D) HeLa (C) or HT-29 (D) cells were treated with 0.3 μM TSN, 5 mM 2-DG, or the combination of both compounds for 24 h and then harvested for the determination of ATP level. Data are presented as the mean ± SD from two independent experiments, and are expressed as a percentage of the number of control cells. Asterisks indicate a significant difference compared the control.

TSN inhibits 2-DG-induced autophagy

We next determined whether TSN modulates 2-DG-induced autophagy, which is a cell survival process in response to nutrient deprivation.^14,15 LC3-II was used a marker for autophagy. Treatment of HeLa cells with TSN alone did not significantly modulate expression levels of LC3-II; however, TSN significantly suppressed 2-DG-induced expression of LC3-II in a concentration-dependent manner (Fig. 3A). The ability of TSN to inhibit 2-DG-induced autophagy was confirmed by another widely used method to detect autophagy, namely the LC3 puncta formation assay.²⁶ Punctate GFP-LC3 fluorescence was induced after 2-DG treatment in HeLa cells; however, TSN significantly suppressed the punctate pattern of GFP-LC3 fluorescence induced by 2-DG (Fig. 3B). These results suggested that TSN inhibits 2-DG-induced autophagy.

Figure 3.

TSN inhibits 2-DG-induced autophagy. (A) HeLa cells were treated with the indicated concentrations of TSN, 5 mM 2-DG, or with the combination of both compounds for 24 h and then the expression levels of LC3-I and LC-3-II were determined by protein gel blot analysis and compared with those of α-tubulin. (B) HeLa cells transfected with GFP-LC3 were treated with 0.3 μM TSN, 5 mM 2-DG, or the combination of both compounds for 24 h and then analyzed for puncta formation (autophagosome) by confocal microscopy.

TSN inhibits 2-DG-induced activation of elongation factor-2 kinase

AMP-activated protein kinase (AMPK) is reported to be a key regulator of autophagy via a mechanism that involves inactivation of mammalian target of rapamycin (mTOR) in cancer cells.²⁷ Therefore, we next investigated whether TSN inhibited 2-DG-induced autophagy through the mTOR pathway in HeLa cells (Fig. 4A). Cotreatment of HeLa cells with TSN and 2-DG for 24 h dramatically increased phosphorylation of AMPK. Consistent with this result, the combined treatment of 2-DG with TSN also significantly attenuated the phosphorylation of mTOR and its downstream substrate, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), suggesting that TSN could not inhibit 2-DG-induced autophagy via mTOR pathway.

Figure 4.

The combination of TSN and 2-DG enhances AMPK activation, but inhibits eEF-2K activation. (A and B) HeLa cells were treated with 0.3 μM TSN, 5 mM 2-DG, or the combination of both compounds for 24 h. The whole cell lysates were analyzed by protein gel blotting with the indicated antibodies.

Elongation factor-2 kinase (eEF-2K) has been also implicated in the regulation of 2-DG-induced autophagy.^16,28 We determined whether TSN inhibited the activation of eEF-2K induced by 2-DG. Treatment of HeLa cells with 2-DG induced the activation of eEF-2K, as evidenced by the increased phosphorylation of its substrate, elongation factor-2 (EF-2). TSN completely suppressed 2-DG-induced activation of eEF-2K (Fig. 4B). We further confirmed that TSN suppressed 2-DG-induced autophagy via inhibiting the activation of eEF-2K using an eEF-2K-targeted siRNA (Fig. 5). Knockdown of eEF-2K resulted in a dramatic increase in the cytotoxic activity of 2-DG, as did by the combination of TSN and 2-DG; however, TSN did not further enhance the cytotoxic activity of 2-DG by the knockdown of eEF-2K (Fig. 5A). Furthermore, as previously reported,^28,29 knockdown of eEF-2K suppressed 2-DG-induced autophagy in HeLa cells, as manifested by the increased expression of LC3-II (Fig. 5B). Taken together, these results suggested that eEF-2K could play a critical role in 2-DG-induced autophagic survival pathway and TSN could blunt 2-DG-induced autophagic survival pathway by inhibiting the activation of eEF-2K.

Figure 5.

Knockdown of eEF-2K enhances the cytotoxic effect of 2-DG. (A) HeLa cells were transfected with scramble siRNA or eEF-2K-targeted siRNA were treated with 0.3 μM TSN, 5 mM 2-DG, or the combination of both compounds for 48 h and then cell viability was determined by MTT assay and the whole lysates were analyzed by protein gel blot analysis to determine the expression level of eEF-2K (right panel). Data are presented as the mean ± SD from two independent experiments, and are expressed as a percentage of the number of control cells. Asterisks indicate a significant difference compared the control. (B) HeLa cells transfected with scramble siRNA or eEF-2K-targeted siRNA were treated with 0.3 μM TSN, 5 mM 2-DG for 24 h and then and then the expression levels of LC3-I, LC-3-II and eEF-2K were determined by protein gel blot analysis.

The combination of TSN and 2-DG induces apoptosis

Since it has been known that inhibition of autophagy induces apoptosis,^29,30 we determined whether apoptotic cell death is induced by a combination of TSN and 2-DG in HeLa cells. In order to quantify apoptosis induction, we measured the binding of annexin V-FITC (Fig. 6A and B). A small percentage of untreated HeLa cells bound annexin V-FITC (< 4%). Following treatment with TSN at 0.3 μM, the percentage of annexin V-FITC-binding cells did not significantly increase. Cotreatment of HeLa cells with 0.3 µM TSN and 5 mM 2-DG for 48 h significantly induced apoptosis. To further confirm this result, we analyzed the effect of the combination of TSN and 2-DG for proteolytic processing of procasapse-3 and the caspase-3 substrate PARP (Fig. 6C). Treatment with 0.3 µM of TSN or 5 mM of 2-DG alone only slightly affected the proteolytic processing of procaspase-3 and PARP. However, the combined treatment of TSN and 2-DG resulted in robust procaspase-3 processing and PARP proteolysis.

Figure 6.

The combination of TSN with 2-DG induces apoptosis. (A) HeLa cells were treated with 0.3 μM TSN, 5 mM 2-DG, or the combination of both compounds for 48 h, and subsequently stained with annexin V-FITC and PI, followed by analysis using a flow cytometer. Representative plots of one set of triplicate experiments. Early apoptotic cells (Annexin-V⁺ and PI^-) were displayed in the lower right quadrant and late apoptotic cells (Annexin-V⁺ and PI⁺) were shown in the upper right quadrant. (B) The percentages of apoptotic cells were indicated by Annexin-V⁺ cells shown as means mean ± SD from three independent experiments (*p < 0.01). (C) HeLa cells were treated with 0.3 μM TSN, 5 mM 2-DG, or the combination of both compounds for 48 h. The whole cell lysates were analyzed by protein gel blotting with the indicated antibodies.

Discussion

Recent studies indicate that aberrantly active glycolysis is required for tumorigenesis;^31,32 therefore, aerobic glycolysis is a valid target for cancer therapy. 2-DG is a clinically relevant and the best characterized glycolytic inhibitor. In this study, we demonstrated that TSN, a plant rotenoid, which has been known to possess antitumor activity against various types of human cancer, enhanced the sensitivity of cancer cells to the cytotoxic effect of 2-DG. The addition of TSN to 2-DG accelerated depletion of intracellular ATP and suppressed 2-DG-induced autophagy via the suppression of the activation of eEF-2K, thus inducing apoptosis.

We showed that the combination of TSN and 2-DG synergistically decreased the cell viability in various types of cancer cells: the human colon cancer cell lines (HT-29 and SW-620), a human lung cancer cell line (A549) and a human cervical cancer cell line (HeLa). Therefore, the potentiating effect of TSN on the cytotoxic effect of 2-DG may not be cell-type specific. This effect may be due to the aggravating depletion of intracellular ATP levels as well as the suppression of 2-DG-induced autophagy. It is known that deguelin, a similar compound to TSN, induces a rapid depletion of ATP levels and activates AMPK.^33,34 We showed that TSN also effectively decreased the intracellular ATP levels, and combining TSN and 2-DG led to a drastic reduction of intracellular ATP levels. Consistently, the combination of TSN and 2-DG induced a stronger activation of AMPK and inhibition of phosphorylated 4EBP-1 (a marker of mTOR activity) compared with TSN or 2-DG alone, suggesting that a correlation between ATP depletion and activation of AMPK exists. We also showed that the combination of TSN and 2-DG induced the activation of caspase-3 and apoptosis in HeLa cells. It has been implicated that activation of AMPK positively regulates apoptosis induced by the energetic stress caused by the combination of metformin and 2-DG.^17,35 Thus, a drastic activation of AMPK caused by the combination of TSN and 2-DG may account, in part, for the synergistic decrease of cell viability.

As previously reported,^12,13,28 we showed that 2-DG-induced autophagy. When cells are cultured in the absence of nutrients, a survival process is induced to recycle essential metabolites such as lipids and amino acids for fueling their bioenergetic machinery.¹⁴ Here, we showed that combination TSN and 2-DG treatment induces a shift from a survival process to cell death in HeLa and HT-29 cells. In accordance with our observation, inhibition of autophagy by ATG5 siRNA or chemical inhibitors results in tumor cell death by apoptosis both in vitro and in animal models.^29,30 eEF-2K is a calcium/calmodulin-dependent enzyme that regulates protein elongation. This kinase phosphorylates EF-2 that mediates the translocation step in peptide-chain elongation by inducing the transfer of peptidyl-tRNA from the ribosomal A to P site. Phosphorylation of EF-2 at Thr56 by eEF-2K decreases the affinity of this elongation factor for ribosomes and terminates elongation, thereby inhibiting protein synthesis. It is well established that eEF-2K might be a central component of the autophagy pathway that is activated in response to 2-DG.^16,28 Since protein synthesis is a major energy-consuming process, termination of protein synthesis and induction of autophagy via activation of eEF-2K should conserve energy and support cell survival during times of metabolic stress. Knockdown of eEF-2K potentiates the cytotoxic effect of 2-DG by inducing apoptosis against human glioma cells through blunting of autophagy.¹⁶ In this study, we were also able to demonstrate a similar effect. Knockdown of eEF-2K did not induce autophagy in response to 2-DG, but potentiated the cytotoxic effect of 2-DG against HeLa cells. Importantly, we also showed that TSN inhibited 2-DG-induced activation of eEF-2K and the addition of TSN with 2-DG induces a shift from autophagy to apoptosis. Furthermore, TSN did not enhance the cytotoxic effect of 2-DG in eEF-2K knockdown of HeLa cells. Thus, inhibition of 2-DG-induced activation of eEF-2K by TSN could be an important mechanism by which TSN enhances the sensitivity of cancer cells to 2-DG against various cancer cells.

These findings raise intriguing questions as to how TSN inhibits 2-DG-induced activation of eEF-2K. It has been known that eEF-2K is negatively regulated by mTOR signaling.^36-38 Inhibiting of Akt by either novel Akt inhibitor MK-2206 or RNA interference induces eEF-2K-dependent autophagic response via suppressing mTOR/S6 kinase pathway, and inhibition of eEF-2K enhances antitumor activity of MK-2206 by promoting the switch from autophagy to apoptotic cell death.³⁸ Our results showed that TSN suppressed 2-DG-induced activation of mTOR pathway, but inhibited eEF-2K activation. Therefore, TSN may not inhibit the activation eEF-2K via mTOR pathway. Further studies are needed to understand how TSN inhibits 2-DG-induced activation of eEF-2K.

In summary, these studies provided the first evidence that the combination of TSN and 2-DG increased the sensitivity of cancer cells to 2-DG. We also showed that the addition of TSN to 2-DG aggravated intracellular ATP depletion and blocked 2-DG-induced autophagy through inhibition of eEF-2K activation, which resulted in the augmentation of apoptosis. Our study highlights the potential use of the combination of TSN and 2-DG as an anticancer therapy.

Materials and Methods

Cell culture

The human colon cancer cell lines (HT-29 and SW-620), a human lung cancer cell line (A549), and a human cervical cancer cell line (HeLa) were purchased from American Type Culture Collection. Human colon and lung cancer cells were maintained in RPMI 1640 (Hyclone). Human cervical cancer cell were maintained in DMEM (Hyclone). All medium were supplemented with penicillin-streptomycin (Invitrogen) and 10% heat-inactivated fetal bovine serum (Hyclone). All cells were maintained in a humidified 5% CO₂ atmosphere at 37°C.

Reagents, antibodies, siRNA and plasmid

TSN was isolated from the aerial parts of Amorpha fruticosa.^24,25 The purity of TSN is more than 98% in HPLC analysis. 2-DG, and anti-α-tubulin antibody were purchased from Sigma-Aldrich. All these compounds were solubilized in 100% dimethyl sulfoxide and used at a final concentration of less than 0.05% dimethyl sulfoxide. Antibodies for caspase-3 (9662), PARP (9542), LC3 (2775), AMPKα (2603), phospho-AMPKα (2535), mTOR (2972), phospho-mTOR (2971), 4EBP1 (9452), phospho-4EBP1 (9451), eEF-2K (3692), EF-2 (2332), and phospho-EF-2 (2331) were purchased from Cell Signaling Technology. Green fluorescent protein (GFP)-LC3 expression vector was kindly provided by Prof. Kim YM (Kangwon National University). Scramble small interfering RNA (siRNA) and eEF-2K siRNA (sc-39011) were purchased from Santa Cruz Technology.

Cell viability assay

For the determination of cell viability, 1 × 10⁴ cells per well were seeded in 96-well plates and allowed to grow to the plate for 24 h. TSN was added to the wells at the indicated concentrations either alone or in combination with 5 mM of 2-DG for all cell lines, and incubated for an additional 48 h. Cell viability was determined by MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide]-based colorimetric assay. Data are expressed as the mean ± SD of triplicate determinations from at least three independent experiments.

Apoptosis assay

The extent of apoptosis was evaluated with annexin V-staining. Annexin V-staining was performed using annexin V-FITC (fluorescein isothiocyanate) apoptosis detection kit (BD Biosciences) following the instructions of the manufacturer. Briefly, after incubation, cells were harvested, washed with PBS (pH 7.4), centrifuged, and stained with annexin V-FITC and 2 μg/ml propidium iodide (PI) in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) for 15 min at 37°C in the dark. The samples were analyzed by flow cytometry using a FACScan flow cytometer. The CellQuest software was used to analyze the data (Becton-Dickinson).

Transfection with GFP-LC3 and confocal analysis

HeLa cells were transfected with GFP-LC3 expression vector using Lipofectamine Plus reagent according to the manufacturer’s instructions (Invitrogen), treated with agents for 24 h, fixed, and analyzed by confocal microscope. Confocal images were acquired using an OLYMPUS FV1000 inverted laser scanning confocal microscope equipped with an external argon, HeNe laser Green, and HeNe laser Red. Using a UPLSAPO 60X NA1.35 oil immersion objective (Olympus), images were captured at the colony midsection.

Protein gel blot analysis

Proteins were extracted from cells in ice-cold lysis buffer [50 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 5 mM sodium orthovanadate, 1X protease inhibitor cocktail (BD Biosciences)]. Fifty micrograms of protein per lane was separated by sodium dodecyl sulfate (SDS)-PAGE (PAGE) and followed by transferring to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% BSA or 5% skim milk, and then incubated with the corresponding antibody. After binding of an appropriate secondary antibody coupled to horseradish peroxidase, proteins were visualized by enhanced chemiluminescence according to the instructions of the manufacturer (Animal Genetics).

Measurement of intracellular ATP

Cells were plated in 60 mm plates and treated with the indicated compounds for 24 h. ATP concentrations were measured by luciferase activity using the ATP bioluminescent somatic cell assay kit according to the manufacturer’s protocol (Sigma-Aldrich).

Statistical Analysis

Statistical differences were determined by paired Student’s t-test. A probability of p < 0.05 was considered significance.

Glossary

Abbreviations:

AMPK

AMP-activated protein kinase

2-DG

2-Deoxy-D-glucose

EF-2

elongation factor-2

eEF-2K

elongation factor-2 kinase

4EBP1

eukaryotic translation initiation factor 4E binding protein 1

FITC

fluorescein isothiocynate

GFP

green fluorescent protein

LC3

microtubule-associated protein light chain 3B

PARP

poly ADP-ribose polymerase

mTOR

mammalian target of rapamycin

PI

propidium iodide

siRNA

small interfering RNA

TSN

tephrosin

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.