Cordycepin Augments the Chemosensitivity of Human Glioma Cells to

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

pubs.acs.org/molecularpharmaceutics

Cordycepin Augments the Chemosensitivity of Human Glioma Cells to Temozolomide by Activating AMPK and Inhibiting the AKT Signaling Pathway Yiming Bi,† Han Li,‡ Dazhuang Yi,† Yuxue Sun,† Yang Bai,† Sheng Zhong,† Yang Song,† Gang Zhao,*,† and Yong Chen*,† †

Department of Neurosurgery, The First Hospital of Jilin University, 130000 Changchun, China Department of Respiratory Medicine, The Second Hospital of Jilin University, 130000 Changchun, China

Downloaded via KAOHSIUNG MEDICAL UNIV on October 20, 2018 at 05:39:36 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Glioblastoma multiforme (GBM) is the most commonly encountered subtype of deadly brain cancer in human adults. It has a high recurrence rate and shows aggressive proliferation. The novel cytotoxic agent temozolomide (TMZ) is now frequently applied as the first-line chemotherapeutic treatment for GBM; however, a considerable number of patients treated with TMZ turn out to be refractory to this drug. Hence, a more effective therapeutic approach is urgently required to overcome this critical issue. Accumulating evidence has shown that both AMPK and AKT are activated by TMZ, while only AMPK contributes to apoptosis via mammalian target of rapamycin (mTOR) inhibition. Accordingly, AKT increases the tumorigenicity and chemoresistance of various tumor cells. In addition, AKT overexpression increases the resistance of glioma cells to TMZ. Cordycepin, a major bioactive component in Cordyceps militaris, exhibits immunomodulatory, anticancer, antioxidant, and anti-inflammatory activities, among other therapeutic effects. To date, whether GBM sensitivity to TMZ can be enhanced by cordycepin largely remains unknown. In the present study, we evaluated the effect of the combined use of cordycepin and TMZ in the treatment of GBM and explored the molecular mechanisms. Notably, we found that treatment with cordycepin led to inhibition of cellular proliferation, migration, and invasion as well as cellular apoptosis and cell cycle arrest in glioma cell lines in vitro. Likewise, the combined treatment with both cordycepin and TMZ synergistically resulted in inhibition of cellular growth, migration, and tumor metastasis as well as induction of cellular apoptosis and cell cycle arrest. Moreover, we also demonstrated that cordycepin effectively enhanced the activation of AMPK and suppressed the activity of AKT, whose activation was only induced by TMZ. Furthermore, there was an apparent reduction in the expression levels of p-mTOR, p-p70S6K, matrix metalloproteinase (MMP)-2, and MMP-9 in the group treated with both cordycepin and TMZ, in comparison with those in the groups treated with either cordycepin or TMZ alone. In vivo, the combination therapy also obviously reduced the tumor volume as well as prolonged the median survival time of xenograft models. In brief, our results suggested that cordycepin augments TMZ sensitivity in human glioma cells at least partially through activation of AMPK and suppression of the AKT signaling pathway. Overall, the combination therapy of cordycepin and TMZ potentially provides a novel option for a better prognosis of patients with GBM in clinical practice. KEYWORDS: cordycepin, temozolomide, glioma, AMPK, AKT

■

INTRODUCTION Glioblastoma is a common malignant brain tumor that is highly invasive and has a high mortality rate. Approximately 55% of gliomas are glioblastoma multiforme (GBM), which is the most malignant subtype among all three subtypes.1 Currently, newly diagnosed GBM is normally removed by surgical resection, followed by chemotherapy and/or radiotherapy, according to the current standard of care.2 However, malignant gliomas show an enhanced resistance to many options of clinical treatments, such as chemotherapy, radiotherapy, and even some adjunct therapies. Consequently, novel and effective therapeutic options for the better management of patients with glioma are urgently needed. © XXXX American Chemical Society

Cordycepin (Figure 1a), a nucleoside analogue, is the major bioactive component that was discovered, isolated, and purified from Cordyceps militaris; it exhibits numerous biological effects on the modulation of inflammatory responses,3 platelet aggregation,4 and steroidogenesis.5 Cordycepin also has been reported to be involved in regulating protein synthesis as well as cell adhesion.6 Moreover, cordycepin is considered to have an essential influence on suppression of cellular proliferation Received: May 25, 2018 Revised: September 1, 2018 Accepted: October 8, 2018

A

DOI: 10.1021/acs.molpharmaceut.8b00551 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 1. Cordycepin inhibits cell viability and proliferation of glioma cells. (a) The chemical structure of cordycepin. (b) MTT assay showing cordycepin-mediated time- and dose-dependent decreases in the cell viabilities of U87, U251, LN18, and T98G glioma cells as well as its limited toxicity in HUVECs. Cultured cells in 96-well plates were treated with 0−400 μM cordycepin and harvested at 24, 48, and 72 h, respectively. (c) Representative images for colony formation (left) in addition to the related statistical analyses (right) of their colony numbers in LN18 glioma cells treated with cordycepin at each designated concentration. Data are presented as the mean ± SD; n = 3; *p < 0.05 vs the control group.

and invasion as well as tumor metastasis through various signaling pathways.7−10 Temozolomide (TMZ) is one of the most recognized drugs against GBM that is used for chemotherapy. The formation of O6-methylguanine mediates DNA lesions and subsequent cell death, which are initiated by DNA alkylation through TMZ treatment.11 Recent studies suggest that TMZ-mediated activation of AMP-activated protein kinase (AMPK) is involved in cellular apoptosis in glioma cells via mammalian target of rapamycin (mTOR) inhibition.12 Meanwhile, suppression of mTOR expression enhances cytotoxic effects and cellular apoptosis through treatment with TMZ or other compounds, such as rapamycin.13−15 Some reports indicate that TMZ is responsible for enhancing the response from the activation of endogenous protein kinase B (AKT).16,17 Notably, AKT activation is also correlated with tumorigenicity, especially for the overexpression of active AKT, which in turn leads to enhanced chemoresistance to TMZ.18,19 Moreover, cordycepin activates AMPK, thus inhibiting the AKT signaling pathway and, in turn, inducing cell apoptosis.8,20 TMZmediated activation of both AMPK and AKT signaling as well as the suppression of mTOR signaling starts to unravel the undefined role that cordycepin plays in reversing chemoresistance to TMZ in human glioma cells.

The aim of this study was to determine the effect of cordycepin on both AMPK and AKT signaling using in vitro methods to analyze cellular growth and apoptosis in glioma cell lines. In addition, the combination therapy of cordycepin and TMZ was tested in GBM xenograft models. The results of this study will potentially provide a novel option for a better prognosis of patients with GBM in clinical practice.

■

MATERIALS AND METHODS

Reagents. Cordycepin, TMZ, and IGF-1 were purchased from Sigma (St. Louis, MO). The AMPK inhibitor compound C and the AKT-specific inhibitor MK2206 were purchased from Abcam (Cambridge, UK) and dissolved in DMSO. Diluted solutions of both reagents in DMSO stock solutions containing the designated concentration were prepared using DMEM immediately before use. In addition, on the basis of our experimental design, the concentration of DMSO in the media was less than or equal to 0.1%, and it exerted no detectable effect on either cellular growth or death. Primary antibodies against β-actin, Bax, Bcl-2, cleaved caspase-3, poly(ADP-ribose) polymerase-1 (PARP-1), matrix metalloproteinase (MMP)-2, MMP-9, cyclin B1, AMPK, AKT, mTOR, p70S6K, p-AMPK (Thr172), p-AKT (Ser473), p-

B


Article


continuously monitored and recorded at each designated time point under a microscope equipped with a camera. Apoptosis Assays. Cellular apoptosis was assessed using an Annexin V-FITC Apoptosis Detection Kit (BD, USA), according to the manufacturer’s instructions. Briefly, cells were transferred into a 6-well plate at a density of 2 × 105 cells per well during the logarithmic growth phase. After specific treatment in each group, at least 10 000 cells were harvested for fluorescence-activated cell sorting using a FACSCalibur flow cytometer (BD, USA) to determine the proportion of apoptotic and viable cells, respectively. Cell Cycle Analysis. Cells at a density of 2 × 105 cells per well during the logarithmic growth phase were transferred into a 6-well plate, and then the specific treatment was added. After the treatments, the harvested cells were incubated with 70% ethanol overnight at −20 °C. Subsequently, they were washed twice using PBS and incubated with propidium iodide (PI; BD, USA) and RNase (BD, USA) for 25 min. The cells were analyzed by flow cytometry (BD, USA), and the results were statistically compared and automatically visualized using ModFit LT 3.3 software (Verity Software House, USA). Immunoblotting. As reported previously,21 glioma cells were collected and digested to determine the total protein concentration in the cells using a bicinchoninic acid assay kit. Each aliquot of protein solution loaded onto an SDSpolyacrylamide gel was separated by electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membranes were incubated with primary antibodies against β-actin, Bax, Bcl-2, cleaved caspase-3, PARP1, MMP-2, MMP-9, cyclin B1, p-AMPK (Thr172), AMPK, pAKT (Ser473), AKT, p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), or p70S6K, respectively. After being washed (3 × 10 min) with PBS containing Tween 20, the membranes were subsequently incubated with horseradish peroxidase-conjugated IgG secondary antibodies (ZSGB-BIO, China) against mouse or rabbit antigens for 2 h. The membranes were visualized on a Kodak X-omat LS film (Eastman Kodak Company, New Haven, CT). Densitometry was performed with Lane ID image analysis software. Transfection of Small-Interfering RNA (siRNA). LN18 and U87 cells seeded on a 10 cm dish were transfected with siRNA at a concentration of 30 pmol using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. The commercially available vectors for expression of AMPKα1, α2 siRNA (sc-45312), or scrambled siRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA) were transfected into the cells overnight, and then the cells were incubated with various drugs. Immunohistochemistry. Paraffin sections from C6 tumors were deparaffinized. Then, the tissue sections were boiled in citrate buffer for antigen retrieval and treated with 3% goat serum in PBS for 30 min. The sections were incubated overnight at 4 °C with either the p-AMPK (1:100) or p-AKT antibody (1:100), and then they were incubated with their corresponding secondary antibodies conjugated with peroxidase, followed by the addition of the substrate (DAB) as well as counterstaining with Meyer’s hematoxylin. Next, the sections were visualized, and the images were observed and acquired under a microscope (Olympus IX71, Tokyo, Japan). Tumor Xenograft Assay. All of the animal experiments were conducted according to the experimental design following the operational guidelines of the Animal Ethics Committee of the First Affiliated Hospital of Jilin University (Changchun,

mTOR (Ser2448), or p-p70S6K (Thr389) were purchased from Cell Signaling Technology (Beverly, MA). Cell Cultures. Commercially available cell lines obtained from the American Type Culture Collection were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Hyclone, USA), 100 U/mL penicillin−streptomycin (Hyclone, USA), and 2 mM glutamine (Hyclone, USA) in a humidified incubator with 5% CO2 at 37 °C. These cell lines included rat C6 as well as human LN18, T98G, U87, and U251 glioma cells. Using a medium containing 0.25% trypsin and 0.02% EDTA, they were dissociated and serially passaged once every two or 3 days. Human umbilical vein epithelial cells (HUVECs) were purchased from the National Infrastructure of Cell Line Resources in the Chinese Academy of Sciences (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) and were cultured according to the manufacturer’s instructions. Cell Viability Assay. Cells were seeded in a 96-well plate filled with 200 μL of medium per well and cultured overnight. When they reached a density of 5 × 103 cells per well during the logarithmic growth phase, they were transferred to a new plate for treatment. Subsequently, the target compounds serially diluted by DMSO were added at each designated time. The vehicle, herein known as DMSO, served as a control. At 4 h before ending the incubation, 20 μL of 5 mg/mL MTT (Sigma-Aldrich, St. Louis, MO) solution was added, resulting in the formation of formazan, which was isolated from the cells and subsequently dissolved in DMSO. The absorbance of the formazan/DMSO solution was evaluated using a microplate reader (Bio-Tek, USA) at a wavelength of 490 nm. To assess the dose effects of the combination therapy of both cordycepin and TMZ, the Chou−Talalay method was performed after treatment for 48 h using a CalcuSyn 2.0 instrument (Biosoft, Cambridge, UK). To determine whether there was a synergistic effect when applying the combination therapy of the fixed-proportion mixture containing both cordycepin and TMZ to the glioma cell lines, the IC50 value of their combination was obtained as well as the IC50 value of each drug alone. On the basis of the assessment of the combination index (CI), their interactions were categorized into synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1), respectively. Colony Formation Assay. After being cultured for 24 h in a 6-well plate, the medium for glioma cells was then refreshed with new medium supplemented with cordycepin and/or TMZ at the designated concentrations, and the cells were cultured for another 10 days. Following cell fixation with paraformaldehyde solution, crystal violet was utilized to stain the cells. Colonies of >50 cells were counted under a microscope at 4× magnification. Transwell and Wound Healing Assays. A Transwell assay was carried out in a 24-well plate containing a polycarbonate membrane with a pore size of 8 mm. The top side of the membrane was coated with Matrigel (BD, USA) and was placed in the upper chamber. Cells (2 × 104/200 μL) seeded into the top well were kept at 37 °C for 48 h. For those cells growing into the Transwell membrane and beyond, crystal violet was applied for their staining after cell fixation with 4% formaldehyde. For wound healing assays, cells grown to 80% confluency in a 6-well plate were used. To mimic acute wounding, pipet tips were used to gently scratch the cell monolayers, thereby creating a cell-free zone as the wounded area. Cellular migration into the wounded area was C


Article


Figure 2. Effect of cordycepin on glioma cell apoptosis and cell cycle arrest. (a) Representative flow cytometry images (left) showing the expression levels of annexin V- and PI-labeled LN18 and T98G cells following a 48-h treatment with or without cordycepin at 100, 200, and 400 μM, respectively. Histograms (right) illustrating the number and distribution of apoptotic cells in the total cell population. (b) Western blotting analysis of the expression levels of cleaved PARP-1, caspase-3, Bax, and Bcl-2 in both LN18 and T98G cells following a 48-h treatment with cordycepin at each designated concentration. (c) Representative flow cytometry results evaluating the numbers of LN18 and T98G cells during the G1/S/G2-M phase in both the control and experimental groups treated with 200 μM cordycepin. (d) Representative Western blot results showing the expression level of cyclin B1 in LN18 and T98G cells following a 48-h treatment with cordycepin at each designated concentration. In addition, βactin was used as a reliable internal control. Data are presented as the mean ± SD; n = 3; *p < 0.05 vs the control group.

Twenty-four athymic BALB/c nude mice (aged 6−8 weeks, weight 18−20 g, from Shanghai Laboratory Animal Center, Shanghai, China) were housed in a specific pathogen-free environment and acclimatized to their surroundings for 3 days. A total of 1 × 106 U87 human glioma cells in 100 μL of PBS were subcutaneously injected into the right flank of each mouse. Therapeutic experiments were started when the tumor reached about 150 mm3 after about 7 days. TMZ was orally administered every day for a week. Meanwhile, cordycepin was intraperitoneally injected into the mice every day for a week. On the next day of the last treatment, mice were euthanized by cervical dislocation and their tumor tissues were collected for further analysis. Statistical Analysis. All data obtained from at least three independent experiments were represented as the mean ± standard deviation (SD). One-way analysis of variance was carried out for statistical comparisons. p < 0.05 indicated a

China). Mature male Wistar rats, weighing 160−180 g, were purchased from the animal facilities of Jilin University. Each Wistar rat was housed with an alternating light/dark cycle (12 h/12 h) in a single cage provided with food and water ad libitum. Animals were allowed to acclimatize to their surroundings for the first 3 days. C6 glioma cells cultured in vitro were harvested and placed in suspension. Then, the cells (1 × 106 C6 cells/10 μL) were inserted into the right striatum of Wistar rats with an infusion pump through a Hamilton microsyringe. TMZ was orally administered three times a week. Meanwhile, cordycepin was intraventricularly injected into the rats, whose conditions were subsequently treated with intense care on a regular basis. After the rats died, their brains were resected. Immunohistochemistry was also applied to some tumor tissues normally fixed with 10% paraformaldehyde. Others were simply frozen using liquid nitrogen as soon as possible for the Western blot experiments. D


Article


Figure 3. Cordycepin augments the effects of TMZ in glioma cells. Cytotoxicity was detected by the MTT assay. (a) U87, U251, LN18, and T98G cells were treated with cordycepin and TMZ at each designated concentration (0−400 μM) for 48 h. The fraction affected (Fa)−combination index (CI) plots showing the values of the CI for each treatment combination. Cordycepin exhibited an apparently synergistic effect when combined with TMZ (CI < 1). (b) Clonogenic survival assay showing colony numbers of LN18 cells following treatment with cordycepin (50 μM), TMZ (50 μM), or their combination. Data are presented as the mean ± SD; n = 3; *p < 0.05 vs each group treated with only one drug alone.

significant difference between groups. All data were analyzed using GraphPad Prism 7.0 (San Diego, CA, USA).

The results showed that the number of colony-forming cells in the LN18 cell line was significantly decreased after incubation with 25 μM cordycepin for 10 days. Notably, there was a dosedependent decrease in cells treated with cordycepin, compared to the control group (Figure 1c), suggesting that cordycepin effectively inhibited the viability of glioma cells. Cordycepin Induces Glioma Cell Apoptosis. To determine how cordycepin induces cellular death in cancer cells, LN18, T98G, and C6 glioma cells were treated with cordycepin at each designated concentration for 48 h. Annexin V/PI staining assays were carried out to assess the number of apoptotic cells by using flow cytometry. The results showed that the proportion of cells labeled by PI was obviously increased in a dose-dependent manner (Figures 2a and S1c). Our results showing that 200 μM cordycepin significantly induced glioma cell apoptosis compared to the control group were in agreement with previous studies revealing that cordycepin potentially induces apoptotic cell death.22 To further verify these results, we tested the alterations in the expression levels of apoptosis-related proteins following a 48-h treatment with cordycepin in LN18 and T98G cells. Western blotting analysis (Figure 2b) showed dose-dependent enhanced expression of proapoptotic proteins, such as Bax, as well as reduced expression of antiapoptotic proteins, such as

■

RESULTS Cordycepin Inhibits Glioma Cell Viability. To investigate the toxic effect of cordycepin on malignant glioma cells, we treated U87, U251, LN18, and T98G glioma cell lines with cordycepin at various concentrations for 24, 48, or 72 h. The MTT assay results (Figure 1b) showed that cordycepin treatment resulted in dose- and time-dependent growth inhibition in glioma cells, compared to that in the control group. Cordycepin had the same effects in rat C6 glioma cells (Figure S1a). Thus, the IC50 values of cordycepin at 48 h were 153.4 μM in C6 cells, 184.2 μM in U87 cells, 141.3 μM in U251 cells, 267.4 μM in LN18 cells, and 308.6 μM in T98G cells. To evaluate the toxicity of cordycepin in normal cells, HUVECs were treated with cordycepin at various concentrations for 24, 48, or 72 h. According to the MTT assay results, the inhibitory effect of cordycepin on HUVECs was significantly decreased, compared to that in glioma cells (Figure 1b). In contrast, cordycepin exhibited a limited inhibitory effect on normal cells. Additionally, colony formation assays were performed to assess the long-term toxicity of cordycepin against the proliferation of glioma cells. E


Article


Figure 4. Effect of cordycepin and TMZ on glioma cell apoptosis and cell cycle arrest. LN18 and T98G cells were cultured in medium supplemented with cordycepin (200 μM), TMZ (200 μM), or their combination for 48 h. (a, left) Representative results showing the levels of annexin V- and propidium iodide-labeled LN18 and T98G cells. (a, right) Statistical analysis for the time course of the apoptosis rate in both cell lines. (b) Western blots revealing the effects of the combination therapy on the expression levels of cleaved PARP-1, caspase-3, Bax, and Bcl-2 in LN18 and T98G cells. (c) Representative results showing the numbers of LN18 and T98G cells in the G1/S/G2-M phase. (d) Western blots showing the expression level of cyclin B1 in LN18 and T98G cells. Compared to treatment with each drug alone, the combination therapy of both cordycepin and TMZ enhanced glioma cell apoptosis and cell cycle arrest. β-Actin was considered as a reliable internal control. Data are presented as the mean ± SD; n = 3; *p < 0.05 vs each group treated with only one drug.

Bcl-2 in LN18 and T98G cells. We also detected an apparent elevation of PARP-1 and caspase-3 cleavage (Figure 2b), which was consistent with an increased incidence of apoptosis. Therefore, our results suggested that cordycepin induced caspase-dependent apoptosis in glioma cells. Cordycepin Induces Cell Cycle Arrest in the G2-M Phase. To further explore the underlying mechanism of the cordycepin-mediated antiproliferative effect in glioma cells, we examined whether cordycepin treatment affected cell cycle arrest in glioma cells. LN18 and T98G cells were treated with cordycepin at each designated concentration for 48 h, followed by the examination of the cell number distribution in each phase of the cell cycle. Cordycepin treatment led to an increased number of cells in the G2-M phase, compared to that

in the control group (Figure 2c). Cordycepin had the same effects in rat C6 glioma cells (Figure S1d). To explore the underlying mechanism of cordycepininduced G2-M phase arrest, Western blotting analysis was performed to evaluate the expression level of cyclin B1. Following treatment with cordycepin for 48 h, the results showed that cordycepin treatment exhibited a dose-dependent decrease in the expression level of cyclin B1, suggesting failure of the transition from the G2 phase to the M phase (Figure 2d). Cordycepin Enhances the Synergistic Effects of TMZ on Inhibition of Glioma Cell Proliferation as well as on Induction of Cell Apoptosis and Cell Cycle Arrest. In order to determine whether cordycepin augments TMZF


Article


Figure 5. Cordycepin sensitizes human glioma cells to TMZ by activating the AMPK signaling pathway. (a) Western blots revealing the expression levels of AMPK and p-AMPK. LN18 and U87 cells were treated with cordycepin at each designated concentration for 48 h. (b, upper) Western blotting analysis showing that compound C or AMPK siRNA reduced the expression level of p-AMPK in LN18 and U87 cells. (b, bottom) Western blotting analysis showing that compound C or AMPK siRNA blocked cordycepin-induced AMPK activation in LN18 and U87 cells. (c) LN18 and U87 cells were treated with cordycepin (200 μM), TMZ (200 μM), or their combination for 48 h. Cordycepin enhanced TMZ-induced activation of p-AMPK. (d) Western blotting analysis showing that the expression level of p-AMPK was significantly decreased in AMPK-inhibited cells compared to that in cells treated with both cordycepin and TMZ. (e) Cell viability of LN18 and U87 cells by using the MTT assay. β-Actin was used as a reliable internal control. Data are presented as the mean ± SD; n = 3; *p < 0.05, cordycepin (COR) vs COR + compound C; Δ*p < 0.05, TMZ + COR + compound C vs COR + TMZ; #p < 0.05, COR + AMPK siRNA vs COR; Δ#p < 0.05, TMZ + COR + AMPK siRNA vs TMZ + COR.

mediated inhibitory effects on cell proliferation in glioma cells, the MTT assay was performed in U87, U251, LN18, and T98G cells treated with cordycepin and/or TMZ at each designated concentration according to their own IC50 values for 48 h. The combination therapy of both cordycepin and TMZ led to enhanced inhibition of cellular proliferation compared to that in the groups treated with either of the drugs alone (Figure 3a). The combination therapy of both cordycepin and TMZ had the same effects in rat C6 glioma cells (Figure S1b). Colony formation was also obviously suppressed in the LN18 cells following a 10-day treatment with both cordycepin (50 μM) and TMZ (50 μM) (Figure 3b). To determine whether the combination therapy had a synergistic, additive, or antagonistic effect, the CI was calculated. The results demonstrated that cordycepin had a synergistic effect

with TMZ (CI < 1) on the inhibition of glioma cell proliferation following a 48-h treatment (Figure 3a). To explore the underlying mechanism of the combined antiproliferative effect, we detected cell apoptosis by using annexin V/PI staining with flow cytometry. In addition, we examined the expression levels of cleaved caspase-3, cleaved PARP-1, Bcl-2, and Bax by Western blotting analysis in LN18 and T98G cells treated with both cordycepin and TMZ for 48 h, respectively. The combination therapy for 48 h obviously enhanced the cellular apoptosis of LN18, T98G, and C6 cells (Figures 4a and S1c). Notably, the combination therapy obviously induced cellular apoptosis in glioma cells, compared to that in the groups treated with either drug alone. The results also revealed that in LN18 and T98G cells, cleaved PARP-1, cleaved caspase-3, and Bax were highly activated by the G


Article


Figure 6. Cordycepin increases glioma cell sensitivity to TMZ by downregulating the AKT signaling pathway. (a) Western blotting analysis showing that cordycepin inhibited p-AKT, p-mTOR, and p-p70S6K in LN18 and U87 cells. (b, upper) Western blotting analysis showing that IGF1 increased the expression level of p-AKT, and that MK2206 inhibited the expression level of p-AKT. (b, bottom) Western blotting analysis showing that IGF-1 enhanced the expression level of AKT and that MK2206 blocked IGF-1-induced AKT activation in LN18 and U87 cells. (c) Western blotting analysis showing that cordycepin reversed the enhanced expression of p-AKT induced by TMZ and enhanced TMZ-induced suppression of p-mTOR and p-p70S6K. (d) Western blotting analysis showing that IGF-1 reversed the reduction of AKT induced by the combined use of both cordycepin and TMZ. Furthermore, MK2206 blocked AKT activation induced by the combined use of cordycepin, TMZ, and IGF-1. (e) Cell viability of LN18 and U87 cells by using the MTT assay. β-Actin was used as a reliable internal control. Data are presented as the mean ± SD; n = 3; *p < 0.05, cordycepin (COR) vs COR + IGF-1; Δ*p < 0.05, TMZ + COR + IGF-1 vs TMZ + COR; #p < 0.05, COR + IGF-1 vs COR + IGF-1 + MK2206; Δ#p < 0.05, TMZ + COR + IGF-1 vs TMZ + COR + IGF-1 + MK2206.

combined use of both cordycepin and TMZ, compared to the cells treated with each drug alone. Meanwhile, the antiapoptotic protein Bcl-2 was decreased in cells treated with cordycepin and TMZ, compared to treatment with each drug alone (Figure 4b). In summary, the combination therapy of both cordycepin and TMZ remarkably promoted the cellular apoptosis of glioma cells. Following the 48-h treatment with cordycepin and/or TMZ, we then evaluated the cell number distribution in different phases of the cell cycle. We found that the percentage of cells

in the G2-M phase was remarkably elevated in cells treated with both cordycepin and TMZ, compared to that in the other groups treated with each drug alone (Figures 4c and S1c). Meanwhile, the expression of cyclin B1 was significantly decreased in cells treated with both cordycepin and TMZ, compared to that in the other groups treated with each drug alone (Figure 4d), indicating that the combined use of both cordycepin and TMZ markedly promoted cell cycle arrest. Cordycepin Sensitizes Human Glioma Cells to TMZ by Activating the AMPK Signaling Pathway. Cordycepin H


Article


Figure 7. Cordycepin regulates cellular migration and invasion of glioma cells. A wound healing assay was conducted to evaluate the cellular migration of LN18 cells. (a, left) Representative images showing the growth into wounded areas of LN18 cells treated with cordycepin and/or TMZ at each designated concentration, i.e., 0, 50, 100, and 200 μM, at either 0 or 24 h following the scratching. (a, right) Histogram revealing the distribution of cells observed in three random fields under each condition. (b) The invasive capabilities of LN18 cells were inhibited by cordycepin at each designated concentration. LN18 cells were also treated with cordycepin (50 μM), TMZ (50 μM), or their combination for 48 h, followed by the Transwell assay. (c) Western blotting analysis showing the protein expression levels of MMP-2 and MMP-9 in LN18 and U87 cells treated with cordycepin at each designated concentration. (d) Western blotting analysis showing the protein expression levels of MMP-2 and MMP-9 in LN18 and U87 cells treated with vehicle (CON), cordycepin (200 μM), TMZ (200 μM), or their combination. β-Actin was used as a reliable internal control. Data are presented as the mean ± SD; n = 3; *p < 0.05 vs the control group; Δ*p < 0.05 vs each group treated with only one drug.

inhibits intracellular lipid accumulation by activating AMPK via its interaction with the γ1 subunit.23 To determine whether the growth inhibitory effect of glioma cells treated with

cordycepin and TMZ was mediated by activation of the AMPK signaling pathway, we performed Western blotting analysis and found that cordycepin upregulated p-AMPK in a doseI


Article


plays an important role in cordycepin-mediated repression of glioma cell proliferation. TMZ treatment has been shown to mediate the increase in the activation of AKT signaling, followed by dephosphorylation of mTOR as well as its downstream signaling pathways.16 However, overexpression of constitutively activated AKT increased cell resistance to TMZ. Furthermore, suppression of AKT decreased cell resistance to TMZ as well. To investigate whether cordycepin augments the cytotoxicity of TMZ by downregulating AKT signaling, the alterations in cell signaling were examined following treatment with both cordycepin and TMZ in LN18 and U87 cells for 48 h. The Western blot results showed that the combined use of cordycepin and TMZ led to an apparent decrease in AKT, mTOR, and p70S6K phosphorylation, compared to controls and other groups treated with each drug alone (Figure 6c). The combination therapy of both cordycepin and TMZ had the same effects in rat C6 glioma cells (Figure S1f). To further assess whether AKT signaling mediates the synergistic effect of the combination therapy, IGF-1 and/or MK2206 were applied in LN18 and U87 cells. Following pretreatment with IGF-1 (400 ng/mL), IGF-1 reversed the reduction of AKT induced by the combination therapy of cordycepin and TMZ in LN18 and U87 cells (Figure 6d). In addition, IGF-1 largely attenuated the cell viability reduction induced by cordycepin and TMZ (Figure 6e). Furthermore, MK2206 (2 μM) blocked AKT activation induced by the combination therapy of cordycepin, TMZ, and IGF-1 (Figure 6d) as well as reversed the increase in cell viability in LN18 and U87 cells (Figure 6e). Altogether, these data demonstrated that cordycepin at least partially suppressed TMZ-induced pAKT expression, resulting in enhanced cell sensitivity to TMZ. Besides, cordycepin and TMZ may also act synergistically to downregulate the expression levels of p-mTOR and p-p70S6K, leading to the induction of cell death and growth inhibition. Combination Therapy of Both Cordycepin and TMZ Inhibits Cellular Migration and Invasion of Glioma Cells by Downregulating MMP-2 and MMP-9. Previous studies have shown that activated AKT is responsible for the cellular invasion of glioma cells as well as their migration. Nevertheless, cordycepin significantly reduced the cellular migration and invasion of LN18 cells in a dose-dependent manner (Figure 7a,b). To determine whether cordycepin enhanced TMZmediated inhibition in glioma cells, wound healing assays were performed. We found that cordycepin significantly promoted the TMZ-mediated inhibition of cellular migration and invasion of LN18 cells (Figure 7a,b). Western blotting analyses showed that the expression levels of MMP-2 and MMP-9 were apparently decreased in a dose-dependent manner in glioma cells treated with cordycepin for 48 h (Figure 7c). Furthermore, the combination therapy of both cordycepin and TMZ led to an enhanced reduction of MMP-2 and MMP9 expression, compared to that in the control group and in the other groups treated with each drug alone (Figure 7d). Cordycepin Exhibits Synergistic Activity with TMZ in a Tumor Xenograft Assay in Vivo. To determine whether the combination therapy of both cordycepin and TMZ exhibits synergistic antitumor effects in vivo, we established a gliomabearing Wistar rat model that was implanted intracranially with C6 cells. Seven days following the tumor implantation, the rats were randomly subdivided into four groups, with six rats in each group, as follows: control group, cordycepin treatment group (20 mg/kg), TMZ treatment group (20 mg/kg), or the

dependent manner in LN18 and U87 cells (Figure 5a). In addition, cordycepin had the same effects in rat C6 glioma cells (Figure S1e). To further explore the underlying mechanism responsible for the cordycepin-mediated inhibitory effect on cellular growth through activation of the AMPK signaling pathway in glioma cells, the AMPK-specific inhibitor compound C and AMPK-specific siRNA were applied to LN18 and U87 cells for the suppression of AMPK expression. Following pretreatment with 2 μM compound C (Figure 5b), cordycepin-induced AMPK activation was obviously blocked in LN18 and U87 cells. Moreover, it also contributed to the attenuation of cordycepin-induced cell viability reduction (Figure 5e). Similarly, the molecular knockdown of AMPK abrogated cordycepin-induced AMPK activation in LN18 and U87 cells (Figure 5b). Cordycepin-induced cytotoxicity was also largely ameliorated by the AMPK-specific siRNA (Figure 5e), further suggesting that AMPK activation was indeed required for cordycepin-mediated glioma cell death. Our results also showed enhanced activation of AMPK when the combination therapy was conducted for 48 h, compared to the other groups treated with each drug alone (Figures 5c and S1f). To further determine the involvement of AMPK in the synergistic effect of the combination therapy, compound C (2 μM) and the AMPK-specific siRNA were applied to suppress AMPK activity. We found that the combination therapy of cordycepin, TMZ, and compound C led to enhanced cell viability in LN18 and U87 cells, compared to the other groups treated with either cordycepin or TMZ alone (Figure 5e). Meanwhile, cell death induced by the combined use of cordycepin, TMZ, and AMPK siRNA was largely reversed in LN18 and U87 cells, compared to that in the group treated with cordycepin and TMZ. The expression level of p-AMPK was significantly decreased in AMPK-inhibited cells, compared to those cells without compound C or AMPK siRNA treatment (Figure 5d), indicating the AMPK signaling pathway was indeed involved in cordycepin-mediated sensitization to TMZ in human glioma cells. Cordycepin Increases Glioma Cell Sensitivity to TMZ through Suppression of the AKT Signaling Pathway. To determine whether the cordycepin-induced inhibitory effect on cellular growth was mediated by suppressing the AKT signaling pathway in a dose-dependent manner, LN18 and U87 cells were treated with cordycepin at each designated concentration (0, 100, 200, and 400 μM) for 48 h. We found that cordycepin downregulated the expression levels of p-AKT, p-mTOR, and p-p70S6K in LN18 and U87 cells (Figure 6a). Meanwhile, cordycepin downregulated p-AKT in rat C6 glioma cells (Figure S1e). To further determine the potential candidate responsible for the cordycepin-mediated inhibitory effect on the cellular growth of glioma cells, IGF-1 and the AKT-specific inhibitor MK2206 were used in LN18 and U87 cells. Following pretreatment with IGF-1 (400 ng/mL), IGF-1 reversed the cordycepin-induced reduction of AKT in LN18 and U87 cells (Figure 6b). In addition, it largely attenuated the cordycepininduced cell viability reduction (Figure 6e). Furthermore, MK2206 (2 μM) blocked IGF-1-induced AKT activation in LN18 and U87 cells (Figure 6b) as well as reversed the increased viability seen in IGF-1-induced cells (Figure 6e). These results confirmed that cordycepin inhibits glioma cell proliferation by suppressing p-AKT expression, and that AKT J


Article


Figure 8. Cordycepin and TMZ synergize to inhibit glioma growth in vivo. (a) Kaplan−Meier survival curves for rats bearing a brain tumor. (b) Statistical analysis of tumor volumes in different groups. The tumor volume was calculated as 0.5 × A × B2, where A represents the length of the tumor, and B represents the width of the tumor. (c) Representative images showing the tumor volumes in different groups. (d) Immunohistochemistry showing that cordycepin treatment enhanced the expression level of p-AMPK induced by TMZ in vivo; magnification, 20×. (e) Immunohistochemistry revealing that cordycepin reduced the expression level of p-AKT induced by TMZ in vivo. (f) Western blotting analysis showing that cordycepin enhanced the activation of AMPK and inhibited the activation of AKT induced by TMZ in the C6 xenograft-bearing rats. β-Actin was used as a reliable internal control. Data are presented as the mean ± SD; n = 3; *p < 0.05 vs the control group; Δ*p < 0.05 vs each group treated with only one drug.

also exhibited synergistic activity in the U87 xenograft-bearing mice (Figure S1g,h). In brief, the combination therapy of both cordycepin and TMZ synergizes to reduce tumor growth and extend survival in these glioma models in vivo.

combined treatment group with both cordycepin (20 mg/kg) and TMZ (20 mg/kg). Kaplan−Meier survival analysis (Figure 8a) showed that the combination therapy apparently prolonged the median survival time of rats bearing tumors. In addition, the rats that received the combination therapy displayed a decreased tumor volume, compared to that in the control group and in the other groups treated with each drug alone (Figure 8b,c). Immunohistochemical analysis revealed that cordycepin enhanced the activation of AMPK (Figure 8d) and suppressed the TMZ-induced activation of AKT in vivo (Figure 8e). Likewise, Western blotting analysis demonstrated that cordycepin enhanced the activation of AMPK and suppressed the TMZ-induced activation of AKT in the C6 xenograft-bearing rats (Figure 8f). Furthermore, we also used nude mice bearing subcutaneous U87 glioma xenografts to assess the synergistic antiglioma effect of the combination therapy of both cordycepin and TMZ. Additionally, cordycepin not only suppressed the growth of glioma cells in vivo, but it

■

DISCUSSION The inherent properties of GBM with chemo- and radioresistance largely predict the fundamental basis of its aggressive progression and outcomes with poor efficacy for many clinical treatments. TMZ, well-known as the first-line treatment for patients with GBM, still shows chemoresistance in most subtypes of GBM. Hence, this urgent medical problem demands a comprehensive solution.24 Cordycepin has been reported to have outstanding antitumor effects, including induction of cellular apoptosis as well as inhibition of cellular proliferation, migration, and tumor metastasis.7−10 To determine whether cordycepin can reverse chemoresistance to TMZ in human glioma cells, we evaluated the influence of K


Article


IGF-1 reversed the reduction of AKT induced by the combined use of both cordycepin and TMZ in LN18 and U87 cells, in which the cordycepin/TMZ-mediated reduction of cell viability was also largely reversed. MK2206 blocked AKT activation induced by the combined use of cordycepin, TMZ, and IGF-1 as well as reversed the increase in cell viability of LN18 and U87 cells. Consequently, we demonstrated that cordycepin augmented TMZ sensitivity in human glioma cells, at least partially, by activating AMPK, while blocking the AKT signaling pathway. In order to verify this effect in vivo, we established a xenograft assay in an animal model. We found that the combination therapy in vivo significantly reduced the tumor growth and prolonged the median survival times of the rats bearing tumors. According to previous studies, cordycepin can protect the integrity of the blood−brain barrier following traumatic brain injury.30 Whether cordycepin can penetrate the blood−brain barrier requires further investigations in the future. Multiple studies have focused on sensitizing TMZ to maximize the overall survival rate of GBM patients. For example, metformin, in combination with TMZ, synergistically inhibits glioma stem cell proliferation through suppression of the AKT/mTOR signaling pathway.31 Likewise, NVP-BEZ235, in combination with TMZ, also acts synergistically to inhibit cellular growth in glioma cells through suppression of the PI3K/AKT/mTOR signaling pathway.14 It is commonly known that p70S6K and 4EBP1 are the major downstream targets modulated by mTOR. The combined use of both NVPBEZ235 and TMZ significantly suppresses the formation and related activities of p-p70S6K and/or p-4EBP1. Nevertheless, the combined use of both cordycepin and TMZ reduces pp70S6k, while showing no effect on p-4EBP1. Cordycepin induces cell-cycle arrest in the S phase and cellular apoptosis in leukemia cells as well as human gallbladder cancer cells.7,32 In our study, however, cordycepin inhibited glioma proliferation by inducing cell-cycle arrest in the G2-M phase. Therefore, we speculate that there are different mechanisms underlying the pharmacological effect of cordycepin in various types of tumor cells. Other combination therapies using cordycepin with other anticancer drugs also have been extensively explored in most commercially available cancer cell lines and/or animal models. For instance, cordycepin facilitated the antitumor efficacy of some chemotherapeutic treatments, including gemcitabine and 5-fluorouracil in GBC-SD cells.20 Notably, it is reasonable to suggest that cordycepin-induced AMPK activation potentially underlies these combination therapies, e.g., the combined use of cordycepin and TMZ in glioma cells. Additionally, the combination therapy of both cisplatin and cordycepin synergistically exhibits an apoptotic effect in the human OC3 oral cancer cell line by activating the JNK/caspase-7/PARP signaling pathway.33 Notably, cordycepin apparently enhances the efficiency of Epstein−Barr virus (EBV) reactivation, thereby leading to the improved elimination of low-dose doxorubicin in EBV-positive tumor cells.34 Likewise, cordycepin combined with cisplatin shows an enhanced apoptotic effect, namely, improved activation of caspase-related cascades, possibly through the MAPK signaling pathway in head and neck squamous cell carcinoma cells.35 In addition, the combined use of cordycepin with cisplatin and/or paclitaxel potentially has an additive effect as well on apoptosis in MA-10 cells through the activation of caspase-related cascades, including the MAPK and/or p53 signaling pathways.36 To

the cordycepin treatment on the activity of glioma cells using both in vitro and in vivo methods. Accordingly, we tested the synergistic effect of the combination therapy. Empirical evidence suggests that the conventional tumor treatments supplemented with cordycepin had obviously improved primary efficacy outcomes in patients with quite a few tumor subtypes. 25 Hence, we demonstrated that cordycepin apparently induced cellular apoptosis and cell cycle arrest in these glioma cells as well as significantly inhibited not only cellular proliferation and migration but also tumor invasion. Besides, cordycepin exerted its inhibitory effect in a dose-dependent manner. Our Western blotting analysis results also revealed that cordycepin remarkably decreased the expression levels of Bcl-2, cyclin B1, MMP-2, and MMP-9 and increased the levels of Bax, cleaved caspase-3, and cleaved PARP-1 in glioma cell lines. In addition, cordycepin mediated the activation of AMPK and suppressed the AKT signaling pathway, which were shown by the reduced growth of glioma cells. Thus, cordycepin could be an effective anticancer agent in a preclinical model of GBM. AMPK acts as an energy sensor and hence plays an essential role in the regulation of cellular growth and metabolism.26 TMZ induces cellular apoptosis among other types of cell death through activation of AMPK signaling in glioma cells, and AMPK, in turn, downregulates the mTOR signaling pathway.12 In addition, the activity of the PI3K/AKT signaling pathway is widely considered to be consistently associated with the resistance of tumor cells in response to chemotherapies.27,28 AKT is normally activated when tumor cells are treated with TMZ, which thus exerts protection against drug-induced cytotoxicity.16 Moreover, the AKT signaling pathway is almost indispensable for tumor metastasis through regulation of MMP-2 and/or MMP-9 expression in cellular invasion.29 Accordingly, our results strongly indicated that the combined use of both TMZ and cordycepin was potentially more beneficial to improve the efficacy outcome through activation of AMPK in addition to suppression of the AKT signaling pathway. Although some studies also have suggested the molecular basis of cordycepin-induced effects, the mechanism underlying the combination therapy of both cordycepin and TMZ remains unclear. In this study, it was well demonstrated that cordycepin combined with TMZ inhibited cellular proliferation, migration, and invasion as well as induced cellular apoptosis and cell cycle arrest with better effectiveness compared to the use of either cordycepin or TMZ alone. The Western blotting analysis results also showed that the expression levels of cleaved PARP-1, cleaved caspase-3, and Bax were upregulated, while the expression levels of Bcl-2, cyclin B1, MMP-2, and MMP-9 were downregulated in the combination therapy group compared to the groups treated with each drug alone. Furthermore, our Western blotting analysis results also revealed that cordycepin effectively enhanced AMPK activation and suppressed AKT activation, which were induced by TMZ treatment. The combination therapy led to an apparent increase in the expression level of p-AMPK but a decrease in the expression levels of p-AKT, p-mTOR, and p-p70S6K. However, when compound C or AMPK siRNA was utilized to activate AMPK in the cancer cells, the cell viability was obviously improved in comparison to the cells without compound C or AMPK siRNA treatment. Accordingly, the expression level of p-AMPK was significantly decreased in AMPK-inhibited cells than in those without compound C or AMPK siRNA treatment. In addition, L


Article


Temozolomide for glioblastoma. N. Engl. J. Med. 2005, 352 (10), 987−96. (3) Jeong, J. W.; Jin, C. Y.; Kim, G. Y.; Lee, J. D.; Park, C.; Kim, G. D.; Kim, W. J.; Jung, W. K.; Seo, S. K.; Choi, I. W.; Choi, Y. H. Antiinflammatory effects of cordycepin via suppression of inflammatory mediators in BV2 microglial cells. Int. Immunopharmacol. 2010, 10 (12), 1580−6. (4) Cho, H. J.; Cho, J. Y.; Rhee, M. H.; Park, H. J. Cordycepin (3′deoxyadenosine) inhibits human platelet aggregation in a cyclic AMPand cyclic GMP-dependent manner. Eur. J. Pharmacol. 2007, 558 (1− 3), 43−51. (5) Pao, H. Y.; Pan, B. S.; Leu, S. F.; Huang, B. M. Cordycepin stimulated steroidogenesis in MA-10 mouse Leydig tumor cells through the protein kinase C Pathway. J. Agric. Food Chem. 2012, 60 (19), 4905−13. (6) Wong, Y. Y.; Moon, A.; Duffin, R.; Barthet-Barateig, A.; Meijer, H. A.; Clemens, M. J.; de Moor, C. H. Cordycepin inhibits protein synthesis and cell adhesion through effects on signal transduction. J. Biol. Chem. 2010, 285 (4), 2610−21. (7) Liao, Y.; Ling, J.; Zhang, G.; Liu, F.; Tao, S.; Han, Z.; Chen, S.; Chen, Z.; Le, H. Cordycepin induces cell cycle arrest and apoptosis by inducing DNA damage and up-regulation of p53 in Leukemia cells. Cell Cycle 2015, 14 (5), 761−71. (8) Pan, B. S.; Wang, Y. K.; Lai, M. S.; Mu, Y. F.; Huang, B. M. Cordycepin induced MA-10 mouse Leydig tumor cell apoptosis by regulating p38 MAPKs and PI3K/AKT signaling pathways. Sci. Rep. 2015, 5, 13372. (9) Chen, Y.; Yang, S. H.; Hueng, D. Y.; Syu, J. P.; Liao, C. C.; Wu, Y. C. Cordycepin induces apoptosis of C6 glioma cells through the adenosine 2A receptor-p53-caspase-7-PARP pathway. Chem.-Biol. Interact. 2014, 216, 17−25. (10) Baik, J. S.; Mun, S. W.; Kim, K. S.; Park, S. J.; Yoon, H. K.; Kim, D. H.; Park, M. K.; Kim, C. H.; Lee, Y. C. Apoptotic Effects of Cordycepin Through the Extrinsic Pathway and p38 MAPK Activation in Human Glioblastoma U87MG Cells. J. Microbiol. Biotechnol. 2016, 26 (2), 309−14. (11) Roos, W. P.; Batista, L. F.; Naumann, S. C.; Wick, W.; Weller, M.; Menck, C. F.; Kaina, B. Apoptosis in malignant glioma cells triggered by the Temozolomide-induced DNA lesion O6-methylguanine. Oncogene 2007, 26 (2), 186−97. (12) Zhang, W. B.; Wang, Z.; Shu, F.; Jin, Y. H.; Liu, H. Y.; Wang, Q. J.; Yang, Y. Activation of AMP-activated protein kinase by Temozolomide contributes to apoptosis in glioblastoma cells via p53 activation and mTORC1 inhibition. J. Biol. Chem. 2010, 285 (52), 40461−71. (13) Burckel, H.; Josset, E.; Denis, J. M.; Gueulette, J.; Slabbert, J.; Noel, G.; Bischoff, P. Combination of the mTOR inhibitor RAD001 with Temozolomide and radiation effectively inhibits the growth of glioblastoma cells in culture. Oncol. Rep. 2015, 33 (1), 471−7. (14) Yu, Z.; Xie, G.; Zhou, G.; Cheng, Y.; Zhang, G.; Yao, G.; Chen, Y.; Li, Y.; Zhao, G. NVP-BEZ235, a novel dual PI3K-mTOR inhibitor displays anti-glioma activity and reduces chemoresistance to Temozolomide in human glioma cells. Cancer Lett. 2015, 367 (1), 58−68. (15) Dai, C.; Zhang, B.; Liu, X.; Ma, S.; Yang, Y.; Yao, Y.; Feng, M.; Bao, X.; Li, G.; Wang, J.; Guo, K.; Ma, W.; Xing, B.; Lian, W.; Xiao, J.; Cai, F.; Zhang, H.; Wang, R. Inhibition of PI3K/AKT/mTOR pathway enhances Temozolomide-induced cytotoxicity in pituitary adenoma cell lines in vitro and xenografted pituitary adenoma in female nude mice. Endocrinology 2013, 154 (3), 1247−59. (16) Caporali, S.; Levati, L.; Starace, G.; Ragone, G.; Bonmassar, E.; Alvino, E.; D’Atri, S. AKT is activated in an ataxia-telangiectasia and Rad3-related-dependent manner in response to Temozolomide and confers protection against drug-induced cell growth inhibition. Molecular pharmacology 2008, 74 (1), 173−83. (17) De Salvo, M.; Maresca, G.; D’Agnano, I.; Marchese, R.; Stigliano, A.; Gagliassi, R.; Brunetti, E.; Raza, G. H.; De Paula, U.; Bucci, B. Temozolomide induced c-Myc-mediated apoptosis via Akt

date, the synergistic effect of cordycepin on the cytotoxicity of TMZ has not been well studied in glioma cells. Therefore, we demonstrated that cordycepin inhibited AKT activation and enhanced activation of the AMPK signaling pathway in glioma cells. Moreover, we found that cordycepin augmented TMZ sensitivity in human glioma cells. Furthermore, the feasibility of the combined use of this drug and TMZ indeed requires a comprehensive evaluation in future investigations. In conclusion, we propose a novel model for the synergistic effect of the combination therapy of both TMZ and cordycepin on cytotoxicity, mainly through upregulation of AMPK and downregulation of the AKT signaling pathway in glioma cells. This synergy has been well demonstrated in this study using both in vitro and in vivo methods. Therefore, cordycepin, in combination with TMZ, may become a novel option to improve the therapeutic outcomes of patients with TMZresistant gliomas.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.8b00551.

■

MTT assay results and a Kaplan−Meier plot (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yong Chen: 0000-0003-4054-3395 Author Contributions

G.Z., Y.C., and Y.B. designed the study. Y.B., H.L., and D.Y. performed most of the experiments. Y.S. and Y.B. performed statistical analyses. S.Z. and Y.S. provided helpful suggestions relating to the manuscript. Experiments were performed under the supervision of G.Z. and Y.C. Y.B. wrote the manuscript. All authors read and approved the final manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China (Nos. 81672505 and 81772684), the S&T Development Planning Program of Jilin Province (Nos.20160101086JC, 20160312017ZG, 20180101152JC and 20150414024GH), the Jilin Provincial Education Department “13th Five-Year” Science and Technology Project (JJKH20180191KJ) and the Interdisciplinary Innovation Project of The First Hospital of Jilin University (JDYYJC001).

■

REFERENCES

(1) Louis, D. N.; Ohgaki, H.; Wiestler, O. D.; Cavenee, W. K.; Burger, P. C.; Jouvet, A.; Scheithauer, B. W.; Kleihues, P. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007, 114 (2), 97−109. (2) Stupp, R.; Mason, W. P.; van den Bent, M. J.; Weller, M.; Fisher, B.; Taphoorn, M. J.; Belanger, K.; Brandes, A. A.; Marosi, C.; Bogdahn, U.; Curschmann, J.; Janzer, R. C.; Ludwin, S. K.; Gorlia, T.; Allgeier, A.; Lacombe, D.; Cairncross, J. G.; Eisenhauer, E.; Mirimanoff, R. O. Radiotherapy plus concomitant and adjuvant M


Article

Molecular Pharmaceutics signalling in MGMT expressing glioblastoma cells. Int. J. Radiat. Biol. 2011, 87 (5), 518−33. (18) Molina, J. R.; Hayashi, Y.; Stephens, C.; Georgescu, M. M. Invasive glioblastoma cells acquire stemness and increased Akt activation. Neoplasia (New York, N.Y.) 2010, 12 (6), 453−63. (19) Manning, B. D.; Cantley, L. C. AKT/PKB signaling: navigating downstream. Cell 2007, 129 (7), 1261−74. (20) Wu, W. D.; Hu, Z. M.; Shang, M. J.; Zhao, D. J.; Zhang, C. W.; Hong, D. F.; Huang, D. S. Cordycepin down-regulates multiple drug resistant (MDR)/HIF-1alpha through regulating AMPK/mTORC1 signaling in GBC-SD gallbladder cancer cells. Int. J. Mol. Sci. 2014, 15 (7), 12778−90. (21) Chen, Y.; Qu, K.; Zhao, C.; Wu, L.; Ren, J.; Wang, J.; Qu, X. Insights into the biomedical effects of carboxylated single-wall carbon nanotubes on telomerase and telomeres. Nat. Commun. 2012, 3, 1074. (22) Tao, X.; Ning, Y.; Zhao, X.; Pan, T. The effects of cordycepin on the cell proliferation, migration and apoptosis in human lung cancer cell lines A549 and NCI-H460. J. Pharm. Pharmacol. 2016, 68 (7), 901−11. (23) Wu, C.; Guo, Y.; Su, Y.; Zhang, X.; Luan, H.; Zhang, X.; Zhu, H.; He, H.; Wang, X.; Sun, G.; Sun, X.; Guo, P.; Zhu, P. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the gamma1 subunit. J. Cell Mol. Med. 2014, 18 (2), 293−304. (24) Grossman, S. A.; Ye, X.; Piantadosi, S.; Desideri, S.; Nabors, L. B.; Rosenfeld, M.; Fisher, J. Survival of patients with newly diagnosed glioblastoma treated with radiation and Temozolomide in research studies in the United States. Clin. Cancer Res. 2010, 16 (8), 2443−9. (25) Choi, S.; Lim, M. H.; Kim, K. M.; Jeon, B. H.; Song, W. O.; Kim, T. W. Cordycepin-induced apoptosis and autophagy in breast cancer cells are independent of the estrogen receptor. Toxicol. Appl. Pharmacol. 2011, 257 (2), 165−73. (26) Mihaylova, M. M.; Shaw, R. J. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat. Cell Biol. 2011, 13 (9), 1016−23. (27) West, K. A.; Castillo, S. S.; Dennis, P. A. Activation of the PI3K/Akt pathway and chemotherapeutic resistance. Drug Resist. Updates 2002, 5 (6), 234−48. (28) Bellacosa, A.; Kumar, C. C.; Di Cristofano, A.; Testa, J. R. Activation of AKT kinases in cancer: implications for therapeutic targeting. Adv. Cancer Res. 2005, 94, 29−86. (29) Choo, E. J.; Rhee, Y. H.; Jeong, S. J.; Lee, H. J.; Kim, H. S.; Ko, H. S.; Kim, J. H.; Kwon, T. R.; Jung, J. H.; Kim, J. H.; Lee, H. J.; Lee, E. O.; Kim, D. K.; Chen, C. Y.; Kim, S. H. Anethole exerts antimetatstaic activity via inhibition of matrix metalloproteinase 2/9 and AKT/mitogen-activated kinase/nuclear factor kappa B signaling pathways. Biol. Pharm. Bull. 2011, 34 (1), 41−6. (30) Yuan, J.; Wang, A.; He, Y.; Si, Z.; Xu, S.; Zhang, S.; Wang, K.; Wang, D.; Liu, Y. Cordycepin attenuates traumatic brain injuryinduced impairments of blood-brain barrier integrity in rats. Brain Res. Bull. 2016, 127, 171−176. (31) Yu, Z.; Zhao, G.; Xie, G.; Zhao, L.; Chen, Y.; Yu, H.; Zhang, Z.; Li, C.; Li, Y. Metformin and Temozolomide act synergistically to inhibit growth of glioma cells and glioma stem cells in vitro and in vivo. Oncotarget 2015, 6 (32), 32930−32943. (32) Wang, X. A.; Xiang, S. S.; Li, H. F.; Wu, X. S.; Li, M. L.; Shu, Y. J.; Zhang, F.; Cao, Y.; Ye, Y. Y.; Bao, R. F.; Weng, H.; Wu, W. G.; Mu, J. S.; Hu, Y. P.; Jiang, L.; Tan, Z. J.; Lu, W.; Wang, P.; Liu, Y. B. Cordycepin induces S phase arrest and apoptosis in human gallbladder cancer cells. Molecules 2014, 19 (8), 11350−65. (33) Chen, Y. H.; Hao, L. J.; Hung, C. P.; Chen, J. W.; Leu, S. F.; Huang, B. M. Apoptotic effect of cisplatin and cordycepin on OC3 human oral cancer cells. Chin. J. Integr. Med. 2014, 20 (8), 624−32. (34) Du, Y.; Yu, J.; Du, L.; Tang, J.; Feng, W. H. Cordycepin enhances Epstein-Barr virus lytic infection and Epstein-Barr viruspositive tumor treatment efficacy by doxorubicin. Cancer Lett. 2016, 376 (2), 240−8. (35) Chen, Y. H.; Wang, J. Y.; Pan, B. S.; Mu, Y. F.; Lai, M. S.; So, E. C.; Wong, T. S.; Huang, B. M. Cordycepin enhances cisplatin

apoptotic effect through caspase/MAPK pathways in human head and neck tumor cells. Onco Targets Ther 2013, 6, 983−998. (36) Kang, F. C.; Chen, P. J.; Pan, B. S.; Lai, M. S.; Chen, Y. C.; Huang, B. M. Apoptotic effect of cordycepin combined with cisplatin and/or paclitaxel on MA-10 mouse Leydig tumor cells. OncoTargets Ther. 2015, 8, 2345−60.

N


Cordycepin Augments the Chemosensitivity of Human Glioma Cells to

Recommend Documents