The novel pterostilbene derivative ANK-199 induces autophagic cell death through regulating PI3 kinase class III/beclin 1/Atg‑related proteins in cisplatin‑resistant CAR human oral cancer cells

  • Authors:
    • Min-Tsang Hsieh
    • Hao-Ping Chen
    • Chi-Cheng Lu
    • Jo-Hua Chiang
    • Tian-Shung Wu
    • Daih-Huang Kuo
    • Li-Jiau Huang
    • Sheng-Chu Kuo
    • Jai-Sing Yang
  • View Affiliations

  • Published online on: June 2, 2014     https://doi.org/10.3892/ijo.2014.2478
  • Pages: 782-794
Metrics: HTML 0 views | PDF 0 views     Cited By (CrossRef): 0 citations

Abstract

Pterostilbene is an effective chemopreventive agent against multiple types of cancer cells. A novel pterostilbene derivative, ANK-199, was designed and synthesized by our group. Its antitumor activity and mechanism in cisplatin-resistant CAR human oral cancer cells were investigated in this study. Our results show that ANK-199 has an extremely low toxicity in normal oral cell lines. The formation of autophagic vacuoles and acidic vesicular organelles (AVOs) was observed in the ANK-199-treated CAR cells by monodansylcadaverine (MDC) and acridine orange (AO) staining, suggesting that ANK-199 is able to induce autophagic cell death in CAR cells. Neither DNA fragmentation nor DNA condensation was observed, which means that ANK-199-induced cell death is not triggered by apoptosis. In accordance with morphological observation, 3-MA, a specific inhibitor of PI3K kinase class III, can inhibit the autophagic vesicle formation induced by ANK-199. In addition, ANK-199 is also able to enhance the protein levels of autophagic proteins, Atg complex, beclin 1, PI3K class III and LC3-II, and mRNA expression of autophagic genes Atg7, Atg12, beclin 1 and LC3-II in the ANK-199-treated CAR cells. A molecular signaling pathway induced by ANK-199 was therefore summarized. Results presented in this study show that ANK-199 may become a novel therapeutic reagent for the treatment of oral cancer in the near future (patent pending).

Introduction

Pterostilbene, a natural stilbenoid compound of phenolic phytoalexin analogue, is found in narra tree, grape and blueberries (Fig. 1A) (14). It possesses many different pharmacological and biologic activities, such as anticancer activity with low intrinsic toxicity (46), anti-inflammatory properties (79), anti-oxidative effect (2), regulation of neutrophil function (10,11) and protection against free radical-mediated oxidative damage (1214). The anticancer activity of pterostilbene has drawn the most attention among of them so far (1,46). As reported in previous studies, pro-apoptosis (4,15,16), pro-autophagy (1719), telomerase inhibition (20), DNA damage (12,13,15), anti-angiogenesis (21), anti-metastasis (4,21) and immuno-stimulatory effects (10,11) are possible mechanisms responsible for its anticancer activity.

Pterostilbene is able to induce apoptosis in many different cancer cell lines, such as pancreatic cancer cells (22,23), breast cancer MCF-7 cells (20,24,25), docetaxel-induced multiple drug resistance (MDR) lung cancer cells (26), osteosarcoma cells (27), prostate cancer PC-3 and LNCaP cells (28,29), leukemia K562 cells (30,31), MDR and BCR-ABL-expressing leukemia cells (30,31), colon cancer cells (3234), hepatocellular carcinoma cells (35,36) and gastric carcinoma cells (7,15). On the other hand, it is also reported that autophagic death can be triggered by pterostilbene in leukemia HL60 (17) and MOLT4 cells (37), lung cancer cells (18,32,38), colon cancer HT29 cells (32), breast cancer MCF-7 cells (39), bladder cancer cells (17,40) and vascular endothelial cells (41). In addition, pterostilbene is capable of inhibiting tumorigenesis and metastasis with minor toxicity in vivo (4,22,38). It is safe in doses up to 250 mg/day in human clinical trial, and deserves further investigation as a potential anticancer agent (42). A novel pterostilbene derivative, ANK-199, was therefore designed and synthesized by our group (Fig. 1B).

Chewing the mixtures of betel leaf and areca nut is a popular custom in many South and Southeast Asia countries. It is a high risk factor for oral cavity carcinoma (43,44), and is the 4th most common cause of cancer death in Taiwanese males (45). Natural product with high anticancer activity and low toxicity, like pterostilbene, appears to be an ideal candidate to prevent or treat oral cancer, as it can directly contact with human oral mucosa without intravenous administration or surgery (22). The anti-oral cancer activity of pterostilbene derivative, ANK-199, was first investigated in this study. Both normal human oral cell lines and cisplatin-resistant CAR human oral cancer cell lines were used. Here, we report the cytotoxic effect and anticancer mechanism of ANK-199 in human oral cancer CAR cells.

Materials and methods

Chemicals and reagents

Dimethyl sulfoxide (DMSO), 3-methyladenine (3-MA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), monodansyl cadaverine (MDC), cisplatin, β-actin antibody, and Tween-20 were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), acridine orange (AO), and trypsin-EDTA were purchased from Life Technologies (Carlsbad, CA, USA). The primary antibodies (anti-Atg5, anti-Atg7, anti-Atg12, anti-Atg14, anti-Atg16L1, anti-beclin 1, anti-PI3K class III, anti-LC3-II, and anti-Rubicon) were obtained from Cell Signaling Technology (Danvers, MA, USA), and the horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit or mouse immunoglobulin for western blot analysis were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). ANK-199 [4-(3,5-dimethoxystyryl)phenyl 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate] was synthesized by Dr Sheng-Chu Kuo.

Cell culture

The human oral cancer cell line CAL 27 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). CAR, a cisplatin-resistant cell line, was established by clonal selection of CAL 27 using 10 cycles of 1 passage treatment with 10–80 μM of cisplatin followed by a recovery period of another passage. CAR cells were cultivated in DMEM supplemented with 10% FBS, 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine and 80 μM cisplatin. Human normal gingival fibroblasts cells (HGF) and human normal oral keratinocyte cells (OK) were kindly provided by Dr Tzong-Ming Shieh (Department of Dental Hygiene, China Medical University). HGF and OK cells were cultivated in DMEM as previously described for our study (45).

Cell viability and morphological examination

CAR cells (1×104 cells) in a 96-well plate were incubated with 0, 25, 50, 75 and 100 μM of ANK-199 for 24, 48 and 72 h. For incubation with the autophagy inhibitor, cells were pretreated with 3-MA (10 mM) for 1 h, followed by treatment with or without ANK-199 (50 and 75 μM) for 48 h. After washing the cells, DMEM containing MTT (0.5 mg/ml) of was added to detect viability as previously described (6). The cell viability was expressed as % of the control. Cell morphological examination of autophagic vacuoles was determined utilizing a phase-contrast microscope (46,47).

Observation of autophagic vacuoles by MDC and acidic vesicular organelles (AVO) with AO staining

CAR cells were seeded on sterile coverslips in tissue culture plates with a density of 5×104 cells/per coverslip. After 0, 50, 75 μM of ANK-199 treatment for 24 h, cells were stained with either 1 μg/ml AO or 0.1 mM MDC at 37°C for 10 min. The occurrece of autophagic vacuoles and AVO were immediately observed under fluorescence microscopy (Nikon, Melville, NY, USA) (4648).

Autophagy assay by LC3B-GFP imaging and nuclear stain

The induction of autophagy was detected with the Premo™ Autophagy Sensor (LC3B-GFP) BacMam 2.0 kit (Molecular Probes/Life Technologies). CAR cells were seeded on sterile coverslips in tissue culture plates with a density of 1×104 cells/per coverslip. After CAR cells were transfected with LC3B-GFP in accordance with the manufacturer’s protocol, cells were treated with 0, 50 and 75 μM of ANK-199 for 24 h. Cells were then fixed on ice with 4% paraformaldehyde, and the slides were mounted and analyzed by a fluorescence microscope. After treatments, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes/Life Technologies) and photographed using a fluorescence microscope (46,47,49).

Western blot analysis

CAR cells (1×107 cells/75-T flask) were treated with ANK-199 (50 and 75 μM) for 48 h. At the end of incubation, the total proteins were prepared, and the protein concentration was measured by using a BCA assay kit (Pierce Chemical, Rockford, IL, USA). Equal amounts of cell lysates were run on 10% SDS-polyacrylamide gel electrophoresis and further employed by immunoblotting as described by Lin et al (46).

Real-time PCR analysis

CAR cells at a density of 5×106 in T75 flasks were incubated with or without 50 and 75 μM of ANK-199 for 24 h. Cells were collected, and total RNA was extracted by the Qiagen RNeasy mini kit (Qiagen Inc., Valencia, CA, USA). Each RNA sample was individually reverse-transcribed using the High Capacity cDNA Reverse Transcription kits (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was assessed for amplifications with 2X SYBR-Green PCR Master mix (Applied Biosystems), as well as forward and reverse primers for Atg7, Atg12, beclin 1 and LC3-II gene. (Human ATG7-F-CAGCAGTGACGATCGGATGA; human ATG7-R-GACGGGAAGGACATTATCAAACC; human ATG12-F-TGTGGCCTCAGAACAGTTGTTTA; human ATG12-R-CGCCTGAGACTTGCAGTAATGT; human BECN1-F-GGATGGTGTCTCTCGCAGATTC; human BECN1-R-GGTGCCGCCATCAGATG; human LC3-II-F-CCGACCGCTGTAAGGAGGTA; human LC3-II-R-AGGACGGGCAGCTGCTT) Applied Biosystems 7300 Real-Time PCR System was run in triplicate, and each value was expressed in the comparative threshold cycles (CT) method for the housekeeping gene GAPDH.

cDNA microarray analysis

CAR cells (5×106 per T75 flask) were incubated with or without 75 μM of ANK-199 for 24 h. Cells were scraped and collected by centrifugation. The total RNA was subsequently isolated as stated above, and the purity was assessed at 260 and 280 nm using a Nanodrop (ND-1000; Labtech International). Each sample (300 ng) was amplified and labeled using the GeneChip WT Sense Target Labeling and Control Reagents (900652) for Expression Analysis. Hybridization was performed against the Affymetrix GeneChip Human Gene 1.0 ST array. The arrays were hybridized for 17 h at 45°C and 60 rpm. Arrays were subsequently washed (Affymetrix Fluidics Station 450), stained with streptavidin-phycoerythrin (GeneChip Hybridization, Wash, and Stain Kit, 900720), and scanned on an Affymetrix GeneChip Scanner 3000. Resulting data were analyzed by using Expression Console software (Affymetrix) with default RMA parameters. Genes regulated by ANK-199 were determined with a 1.5-fold change. For detection of significantly over-represented GO biological processes, the DAVID functional annotation clustering tool (http://david.abcc.ncifcrf.gov) was used (DAVID Bioinformatics Resources 6.7). Enrichment was determined at DAVID calculated Benjamini value <0.05. Significance of overexpression of individual genes was determined (50).

Statistical analysis

All the statistical results were expressed as the mean ± SEM of triplicate samples. Statistical analyses of data were done using one-way ANOVA followed by Student’s t-test, and *p<0.05 and ***p<0.001 were considered significant.

Results

ANK-199 exhibits cytotoxicity and inhibits viable CAR cells

CAR cells were treated with different concentrations of ANK-199 for 24, 48 and 72 h. ANK-199 concentration- and time-dependently decreased cell viability of CAR cells (Fig. 2A). The half maximal inhibitory concentration (IC50) for a 24, 48 and 72-h treatment of ANK-199 in CAR cells were 106.21±3.21, 73.25±4.20 and 32.58±2.39 μM, respectively. To investigate whether the cell death was mediated through apoptosis by ANK-199, cells were treated with 50 and 75 μM ANK-199 for 48 h. The appearance of DNA fragmentation was not observed (data not shown), suggesting that ANK-199 was unable to induce apoptosis in CAR cells. As shown in Fig. 2B, ANK-199 was able to induce the formation of autophagic vacuoles in CAR cells in a time-dependent manner in the presence of 50 μM ANK-199 for 24, 48 and 72 h. This result implies that autophagic cell death plays a pivotal role in ANK-199-induced cell death. However, no viability impact and morphological trait change was observed in ANK-199-treated HGF and OK cells, suggesting that ANK-199 has an extremely low toxicity in normal oral cell lines. In accordance with this observation, the IC50 value of HGF and OK cells is greater than 100 μM (Fig. 3A and B). In short, ANK-199-induced cell death of CAR cells is mediated through autophagic death, rather than apoptosis.

ANK-199 induces autophagic cell death in CAR cells

To further confirm the formation of autophagosome vesicles in ANK-199-treated CAR cells, the autophagic cell death caused by ANK-199 was monitored by using MDC staining, a popular fluorescent marker that preferentially accumulates in autophagic vacuoles (46,48). After cells were treated with 50 and 75 μM of ANK-199 for 48 h, autophagic vacuoles were easily observed under fluorescence microscopy (Fig. 4A). The intensity of MDC staining was directly proportional to the concentration of ANK-199. The ANK-199-triggered autophagic cell death was also examined by using AO staining. In Fig. 4B, AO staining of ANK-199-treated CAR cells clearly showed the presence of AVOs within the cytoplasm compared to control. Microtubule-associated protein 1 light-chain 3 (LC3) is an autophagic membrane marker for the detection of early autophagosome formation (46,49). The LC3 distribution in ANK-199-treated CAR cells was also investigated. A more punctate pattern of LC3B-GFP was observed in ANK-199 treated cells (Fig. 4C). The occurrence of DNA condensation was also investigated in the presence of 50 and 75 μM ANK-199 for 24 h. No significant change was observed in ANK-199-treated CAR cells under microscope, which means that ANK-199-induced cell death triggered by apoptosis is quite unlikely (Fig. 4D). Again, all of the above results support that ANK-199-induced cell death in CAR cells is mediated through the induction of autophagic death instead of apoptosis.

ANK-199 upregulates the autophagy-associated protein levels in CAR cells

The protein level of autophagy marker proteins, like Atg complex (Atg5, Atg7, Atg12, Atg14 and Atg16L1), beclin 1, PI3K class III, rubicon and LC3, was also investigated in ANK-199-treated CAR cells. As shown in Fig. 5, ANK-199 at 50 and 75 μM increased the protein levels of Atg5, Atg7, Atg12, Atg14 and Atg16L1, beclin 1, PI3K class III and LC3, but decreased the protein level of rubicon in CAR cells. Our results imply that ANK-199 induced autophagic cell death in CAR cells through interfering with the kinase class III/beclin 1/Atg-associated signal pathway.

ANK-199 stimulates the autophagy-associated mRNA levels in CAR cells

The mRNA level of autophagy-associated gene was also investigated in ANK-199-treated CAR cells. As shown in Fig. 6, ANK-199 is able to enhance the expression level of Atg7 gene (Fig. 6A), Atg12 gene (Fig. 6B), beclin 1 gene (Fig. 6C) and LC3-II gene (Fig. 6D) in CAR cells.

Protection effect of 3-MA against autophagy in ANK-199- treated CAR cells

3-MA, an inhibitor of PI3K kinase class III, has been shown to potently inhibit autophagy-dependent protein degradation and suppress the formation of autophagosomes. CAR cells were pretreated with 3-MA and then exposed to 50 or 75 μM of ANK-199. The formation of autophagic vacuoles and cell viability were then monitored under phase contrast microscopy. Our results showed that 3-MA can inhibit the formation of autophagic vacuoles (Fig. 7A) and enhance the viability of ANK-199-treated CAR cells (Fig. 7B), suggesting that ANK-199-induced autophagy in CAR cells is mediated through interference with the PI3K kinase class III.

Microarray analysis

The cDNA microarray experiments were carried out to examine the gene expression in ANK-199-treated CAR cells. The transcripts of 26 genes were upregulated, while these of 96 genes were downregulated in ANK-199-treated CAR cells (Table I). The important biological processes and Gene to Go Molecular Function test regulated by ANK-199 are listed in Table II and Table III. The formation of autophagosomes and autophagolysosome was observed during the course of ANK-199 induced autophagic cell death. In a good agreement with above results, membrane formation or reorganization is closely associated with following biological processes: cellular component biogenesis, actin cytoskeleton organization, regulation of actin filament-based process, regulation of cytoskeleton organization, regulation of actin polymerization or depolymerization, regulation of actin filament length.

Table I

The genes with more than 1.5-fold changes in mRNA levels in CAR cells after ANK-199 (50 μM) 24-h treatment identified by DNA microarray.

Table I

The genes with more than 1.5-fold changes in mRNA levels in CAR cells after ANK-199 (50 μM) 24-h treatment identified by DNA microarray.

AccessionGeneFC
XR_042379LOC401875: hypothetical LOC4018757.34
NM_198581ZC3H6: zinc finger CCCH-type containing 65.68
NM_004755RPS6KA5: ribosomal protein S6 kinase, 90 kDa, polypeptide 53.27
NM_006472TXNIP: thioredoxin interacting protein3.00
NM_001506GPR32: G protein-coupled receptor 322.15
NM_018387STRBP: spermatid perinuclear RNA binding protein2.04
BC007928C21orf119: chromosome 21 open reading frame 1191.93
BC108718LOC389765: similar to KIF27C1.88
NM_153335LYK5: protein kinase LYK51.86
ENST00000021776CCT8L1: chaperonin containing TCP1, subunit 8 (theta)-like 11.81
NM_018434RNF130: ring finger protein 1301.75
NM_181684KRTAP12-2: keratin associated protein 12-21.72
NM_201266NRP2: neuropilin 21.71
NM_181351NCAM1: neural cell adhesion molecule 11.63
NM_004064CDKN1B: cyclin-dependent kinase inhibitor 1B (p27, Kip1)1.63
NM_007051FAF1: Fas (TNFRSF6) associated factor 11.62
NM_001039752SLC22A10: solute carrier family 22, member 101.62
NM_198993STAC2: SH3 and cysteine rich domain 21.62
NM_018257PCMTD2: protein-L-isoaspartate (D-aspartate) O-methyltransferase domain containing 21.62
NM_000901NR3C2: nuclear receptor subfamily 3, group C, member 21.62
BC093665FAM92B: family with sequence similarity 92, member B1.56
NM_016609SLC22A17: solute carrier family 22, member 171.55
NM_181605KRTAP6-3: keratin associated protein 6-31.55
XM_938903LOC649839: similar to large subunit ribosomal protein L36a1.53
BC021739LOC554201: hypothetical LOC5542011.53
ENST00000329244LOC100132169: similar to hCG17428521.50
NM_021109TMSB4X: thymosin beta 4, X-linked−1.50
NM_019896POLE4: polymerase (DNA-directed), epsilon 4 (p12 subunit)−1.51
NM_015475FAM98A: family with sequence similarity 98, member A−1.51
NM_006136CAPZA2: capping protein (actin filament) muscle Z-line, alpha 2−1.51
NM_001128619LUZP6: leucine zipper protein 6−1.51
NM_014248RBX1: ring-box 1−1.52
NM_024755SLTM: SAFB-like, transcription modulator−1.53
NM_003168SUPT4H1: suppressor of Ty 4 homolog 1 (S. cerevisiae)−1.53
NM_017892PRPF40A: PRP40 pre-mRNA processing factor 40 homolog A (S. cerevisiae)−1.53
NM_004776B4GALT5: UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 5−1.53
NM_002090CXCL3: chemokine (C-X-C motif) ligand 3−1.53
NM_001005333MAGED1: melanoma antigen family D, 1−1.53
NM_000937POLR2A: polymerase (RNA) II (DNA directed) polypeptide A, 220 kDa−1.54
NM_001349DARS: aspartyl-tRNA synthetase−1.55
NM_003348UBE2N: ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast)−1.56
NM_001614ACTG1: actin, gamma 1−1.56
NM_053024PFN2: profilin 2−1.56
NM_003010MAP2K4: mitogen-activated protein kinase kinase 4−1.57
NM_001099771A26C1B: ANKRD26-like family C, member 1B−1.57
NM_006000TUBA4A: tubulin, alpha 4a−1.58
NM_133494NEK7: NIMA (never in mitosis gene a)-related kinase 7−1.59
NM_173647RNF149: ring finger protein 149−1.59
NM_182917EIF4G1: eukaryotic translation initiation factor 4 gamma, 1−1.59
NM_002599PDE2A: phosphodiesterase 2A, cGMP-stimulated−1.62
NM_005066SFPQ: splicing factor proline/glutamine-rich (polypyrimidine tract binding protein associated)−1.64
NM_001039479KIAA0317: KIAA0317−1.64
NM_001127649PEX26: peroxisomal biogenesis factor 26−1.64
NM_015153PHF3: PHD finger protein 3−1.64
NM_007189ABCF2: ATP-binding cassette, sub-family F (GCN20), member 2−1.64
NM_007126VCP: valosin-containing protein−1.64
NM_012234RYBP: RING1 and YY1 binding protein−1.65
NR_004845LOC644936: cytoplasmic beta-actin pseudogene−1.65
NM_014795ZEB2: zinc finger E-box binding homeobox 2−1.66
NM_005998CCT3: chaperonin containing TCP1, subunit 3 (gamma)−1.67
NM_001099692EIF5AL1: eukaryotic translation initiation factor 5A-like 1−1.67
NM_138689PPP1R14B: protein phosphatase 1, regulatory (inhibitor) subunit 14B−1.67
NM_015665AAAS: achalasia, adrenocortical insufficiency, alacrimia (Allgrove, triple-A)−1.67
NM_001099692EIF5AL1: eukaryotic translation initiation factor 5A-like 1−1.68
NM_001099692EIF5AL1: eukaryotic translation initiation factor 5A-like 1−1.68
NM_002154HSPA4: heat shock 70 kDa protein 4−1.68
NM_013451FER1L3: fer-1-like 3, myoferlin (C. elegans)−1.70
NM_000303PMM2: phosphomannomutase 2−1.71
NM_002795PSMB3: proteasome (prosome, macropain) subunit, beta type, 3−1.71
NM_001363DKC1: dyskeratosis congenita 1, dyskerin−1.71
NM_001102ACTN1: actinin, alpha 1−1.73
NM_004299ABCB7: ATP-binding cassette, sub-family B (MDR/TAP), member 7−1.73
NM_005857ZMPSTE24: zinc metallopeptidase (STE24 homolog, S. cerevisiae)−1.74
NM_152265BTF3L4: basic transcription factor 3-like 4−1.74
NM_020409MRPL47: mitochondrial ribosomal protein L47−1.74
NM_006148LASP1: LIM and SH3 protein 1−1.76
BC065192C2orf12: chromosome 2 open reading frame 12−1.76
NM_001414EIF2B1: eukaryotic translation initiation factor 2B, subunit 1 alpha, 26 kDa−1.78
NM_003222TFAP2C: transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma)−1.78
NM_007350PHLDA1: pleckstrin homology-like domain, family A, member 1−1.79
ENST00000242577DYNLL1: dynein, light chain, LC8-type 1−1.79
NM_032830CIRH1A: cirrhosis, autosomal recessive 1A (cirhin)−1.79
NM_152265BTF3L4: basic transcription factor 3-like 4−1.80
NM_176816CCDC125: coiled-coil domain containing 125−1.80
NM_001039690CTF8: chromosome transmission fidelity factor 8 homolog (S. cerevisiae)−1.83
NM_001127257SLC39A10: solute carrier family 39 (zinc transporter), member 10−1.84
XM_001716411LOC128322: hypothetical LOC128322−1.86
NM_002370MAGOH: mago-nashi homolog, proliferation-associated (Drosophila)−1.90
NM_138578BCL2L1: BCL2-like 1−1.91
NM_003580NSMAF: neutral sphingomyelinase (N-SMase) activation associated factor−1.92
NM_015922NSDHL: NAD(P) dependent steroid dehydrogenase-like−1.92
NM_001797CDH11: cadherin 11, type 2, OB-cadherin (osteoblast)−1.93
NM_021242MID1IP1: MID1 interacting protein 1 [gastrulation specific G12 homolog (zebrafish)]−1.93
NM_005968HNRNPM: heterogeneous nuclear ribonucleoprotein M−1.95
NM_012338TSPAN12: tetraspanin 12−1.97
NM_014953DIS3: DIS3 mitotic control homolog (S. cerevisiae)−2.02
NM_003130SRI: sorcin−2.11
NM_018993RIN2: Ras and Rab interactor 2−2.12
NM_004093EFNB2: ephrin-B2−2.13
NM_032256TMEM117: transmembrane protein 117−2.14
NM_005415SLC20A1: solute carrier family 20 (phosphate transporter), member 1−2.15
NM_017872THG1L: tRNA-histidine guanylyltransferase 1-like (S. cerevisiae)−2.20
NM_014624S100A6: S100 calcium binding protein A6−2.24
NM_153618SEMA6D: sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D−2.27
NM_014604TAX1BP3: Tax1 (human T-cell leukemia virus type I) binding protein 3−2.27
NM_033505SELI: selenoprotein I−2.30
NM_003666BLZF1: basic leucine zipper nuclear factor 1−2.35
NM_002714PPP1R10: protein phosphatase 1, regulatory (inhibitor) subunit 10−2.50
NM_002714PPP1R10: protein phosphatase 1, regulatory (inhibitor) subunit 10−2.50
NM_002714PPP1R10: protein phosphatase 1, regulatory (inhibitor) subunit 10−2.50
NR_003003SCARNA17: small Cajal body-specific RNA 17−2.51
NR_002738SNORD57: small nucleolar RNA, C/D box 57−2.57
NM_006080SEMA3A: sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A−2.57
NM_009587LGALS9: lectin, galactoside-binding, soluble, 9−2.65
NM_003234TFRC: transferrin receptor (p90, CD71)−2.85
NM_138966NETO1: neuropilin (NRP) and tolloid (TLL)-like 1−2.97
NM_001098272HMGCS1: 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble)−2.97
NM_006350FST: follistatin−4.21
NM_024090ELOVL6: ELOVL family member 6, elongation of long chain fatty acids(FEN1/Elo2, SUR4/Elo3-like, yeast)−4.46
NM_005328HAS2: hyaluronan synthase 2−5.00
NM_001753CAV1: caveolin 1, caveolae protein, 22 kDa−5.04
NM_033439IL33: interleukin 33−5.96

[i] FC, fold change.

Table II

Gene to GO Biological Process test for over-representation (ANK-199 to control).

Table II

Gene to GO Biological Process test for over-representation (ANK-199 to control).

TermCount%p-value
GO:0044087 - regulation of cellular component biogenesis96.3829798.59E-06
GO:0030036 - actin cytoskeleton organization107.0921993.75E-05
GO:0032956 - regulation of actin cytoskeleton organization74.9645394.43E-05
GO:0043254 - regulation of protein complex assembly74.9645394.72E-05
GO:0032970 - regulation of actin filament-based process74.9645395.34E-05
GO:0051493 - regulation of cytoskeleton organization85.6737595.75E-05
GO:0030029 - actin filament-based process107.0921996.17E-05
GO:0007010 - cytoskeleton organization139.2198587.13E-05
GO:0008064 - regulation of actin polymerization or depolymerization64.2553197.77E-05
GO:0030832 - regulation of actin filament length64.2553199.07E-05

Table III

Gene to GO Molecular Function test for over-representation (ANK-199 to control).

Table III

Gene to GO Molecular Function test for over-representation (ANK-199 to control).

TermCount%p-value
GO:0003723 - RNA binding2114.893621.54E-07
GO:0000166 - nucleotide binding3323.404266.28E-05
GO:0008092 - cytoskeletal protein binding117.8014180.00346
GO:0032553 - ribonucleotide binding2417.021280.005183
GO:0032555 - purine ribonucleotide binding2417.021280.005183
GO:0017076 - purine nucleotide binding2417.021280.008794
GO:0003779 - actin binding85.6737590.009366
GO:0019900 - kinase binding64.2553190.009636
GO:0047485 - protein N-terminus binding42.8368790.016454
GO:0003924 - GTPase activity64.2553190.018492

Discussion

Apoptosis, autophagy and necrosis are three major routes that lead to cell death (51). Both apoptosis and autophagy belong to the form of cell programmed death, but necrosis does not (5154). Autophagic death can promote cell survival or cell death when cells experience stress, such as damage, nutrient starvation, aging and pathogen infection (5557). Indeed, induction of autophagic death for cancer cells is thought to be one of the best strategies in chemotherapy (5860). Not only many autophagy-related proteins (Atgs) are involved in this process, but also a specific morphological and biochemical modification can be observed (56,60,61). Autophagy is first triggered by membrane nucleation, which is mediated by phosphatidylinositol 3-kinase (PI3K) class III, beclin 1 (the mammalian ortholog of yeast ATG6), rubicon and Atg14 (6264). The cytoplasm and phagophore of various organelles are then sequestered by a membrane to form an autophagosomes. Atg16L1-Atg12-Atg7-Atg5 complex and microtubule-associated protein 1 light chain 3 type II (LC3-II) (membrane-bound form) are absolutely required for autophagosome formation (55,6567). The autophagosome fuses with the lysosome then forming autophagolysosome, eventually resulting in the degradation of the captured proteins or organelles by lysosomal enzymes (55,68,69). Once cells undergo autophagic cell death, an autophagosomal marker LC3-II increases from the conversion of LC3-I (70,71).

Our results demonstrated that the ANK-199 can induce the formation of autophagic vesicle (Fig. 4A) and acidic vesicular organelles (Fig. 4B). It also simultaneously enhances the protein level of autophagic proteins, Atg complex, beclin 1, PI3K class III and LC3-II (Fig. 5), and mRNA expression of autophagic genes Atg7, Atg12, beclin 1 and LC3-II (Fig. 6). More importantly, 3-MA, a specific inhibitor of PI3K kinase class III, can inhibit the autophagic vesicle formation induced by ANK-199 (Fig. 7). All of the above results support that ANK-199-induced cell death in CAR cells is mediated through the induction of autophagic death. This molecular signaling pathway induced by ANK-199 is summarized in Fig. 8.

However, ANK-199 treatment duration for CAR cells is 72 h. We cannot completely rule out the possibility that apoptotic cell death or other signaling pathways can be induced by ANK-199 for longer treatment time. A series of pterostilbene derivatives have been synthesized as less toxic anticancer candidates (30,40,72,73). ANK-199 is less toxic than pterostilbene (unpublished data). More importantly, ANK-199 has much less cytotoxicity in the normal oral cells (HGF and OK) than that in CAR cells (Fig. 3). This is a novel finding regarding that ANK-199 represents a promising candidate as an anti-oral cancer drug with low toxicity to normal cells. Results presented in this study show that ANK-199 may become a novel therapeutic reagent for the treatment of oral cancer in the near future (patent pending).

Acknowledgements

This study was supported by AnnCare Bio-Tech Center Inc. and a research grant from the Ministry of Health and Welfare, Executive Yuan, Taiwan (DOH101-TD-C-111-005) awarded to S.-C.K.

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August 2014
Volume 45 Issue 2

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APA
Hsieh, M., Chen, H., Lu, C., Chiang, J., Wu, T., Kuo, D. ... Yang, J. (2014). The novel pterostilbene derivative ANK-199 induces autophagic cell death through regulating PI3 kinase class III/beclin 1/Atg‑related proteins in cisplatin‑resistant CAR human oral cancer cells. International Journal of Oncology, 45, 782-794. https://doi.org/10.3892/ijo.2014.2478
MLA
Hsieh, M., Chen, H., Lu, C., Chiang, J., Wu, T., Kuo, D., Huang, L., Kuo, S., Yang, J."The novel pterostilbene derivative ANK-199 induces autophagic cell death through regulating PI3 kinase class III/beclin 1/Atg‑related proteins in cisplatin‑resistant CAR human oral cancer cells". International Journal of Oncology 45.2 (2014): 782-794.
Chicago
Hsieh, M., Chen, H., Lu, C., Chiang, J., Wu, T., Kuo, D., Huang, L., Kuo, S., Yang, J."The novel pterostilbene derivative ANK-199 induces autophagic cell death through regulating PI3 kinase class III/beclin 1/Atg‑related proteins in cisplatin‑resistant CAR human oral cancer cells". International Journal of Oncology 45, no. 2 (2014): 782-794. https://doi.org/10.3892/ijo.2014.2478