BR Biomedical Reports 2049-9434 2049-9442 D.A. Spandidos BR-21-4-01831 10.3892/br.2024.1831 Articles Treatment with autophagic inhibitors enhances oligonol‑induced apoptotic effects in nasopharyngeal carcinoma cells WuYen-Ting 1 2 3 LinCheng-Han 4 ChiuWen-Chin 5 HsiehTsung-Jen 5 ChangSue-Joan 6 ChangYun-Ching 5 LanYu-Yan 5 Department of Pathology, Golden Hospital, Pingtung 90049, Taiwan, R.O.C. Department of Physical Therapy, Shu-Zen Junior College of Medicine and Management, Kaohsiung 82144, Taiwan, R.O.C. Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan, R.O.C. School of Medicine, College of Medicine, China Medical University, Taichung 40402, Taiwan, R.O.C. School of Medicine, College of Medicine, I-Shou University, Kaohsiung 82445, Taiwan, R.O.C. Department of Life Sciences, National Cheng Kung University, Tainan 70101, Taiwan, R.O.C. Correspondence to: Professor Yun-Ching Chang or Professor Yu-Yan Lan, School of Medicine, College of Medicine, I-Shou University, 8 Yida Road, Jiaosu, Yanchao, Kaohsiung 82445, Taiwan, R.O.C. ychang014@isu.edu.tw yyinmed@isu.edu.tw 10 2024 05 08 2024 21 4 143 24 04 2024 16 07 2024 Copyright: © 2024 Wu et al. 2024 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Although the combination of chemotherapy and radiotherapy has increased the survival rate of patients with nasopharyngeal carcinoma (NPC), certain patients do not respond well to the treatment and have a poor prognosis. Therefore, novel therapeutic drugs and strategies to improve prognosis of patients with NPC are required. As certain plant extracts can suppress the viability of cancer cells, the present study investigated whether oligonol, a polyphenolic compound primarily found in lychee fruit, exerts anticancer activities in NPC cells. MTT, ELISA and immunoblotting were performed to investigate cell survival, cytokeratin-18 fragment release, and the expression of apoptosis and autophagy markers, respectively. Oligonol decreased the viability of NPC-TW01 and NPC/HK1NPC cell lines. Oligonol increased the protein expression of several apoptosis markers, including cleaved caspase-8 and -3, cleaved PARP and cytokeratin 18 fragment. Moreover, it also increased expression of autophagy markers Beclin 1 and LC3-II, as well as LC3-II/LC3-I ratio in both NPC cell lines. Furthermore, treatment with autophagy inhibitors 3-methyladenine or LY294002 significantly increased oligonol-induced viability inhibition in NPC-TW01 cells. Combined treatment of oligonol + LY294002 reduced LC3-II expression and the LC3II/LC3I ratio while increasing cleaved caspase-8 and -3, cleaved PARP and cytokeratin 18 fragment expression in NPC-TW01 cells. These findings indicated autophagy inhibitors could enhance viability inhibition and apoptotic effects induced by oligonol in NPC cells.

 oligonol nasopharyngeal carcinoma viability autophagy apoptosis Funding: The present study was supported by the National Science and Technology Council (grant no. MOST-107-2320-B-471-001), Pingtung Veterans General Hospital (grant nos. PTVGH-E-11305 and PTVGH-11301) and I-Shou University (grant no. ISU111-S-06), Taiwan. Introduction

Nasopharyngeal carcinoma (NPC) is a highly metastatic malignant tumor originating from the epithelium of the nasopharynx (1). Genetic mutations, Epstein-Barr virus infection, tobacco smoking and consumption of alcohol and salted fish are risk factors for NPC (1). In 2019, age-standardized incidence rate (ASIR) of NPC was 2.12 globally. However, certain regions have higher ASIRs of NPC than the global average, including China, Singapore and Taiwan, with rates of 5.7, 10.81 and 7.14, respectively (2,3). The survival rate of patients with NPC has been improved significantly by combining chemotherapy and radiotherapy (4). However, ~30% of patients still have a poor prognosis due to distant metastases (5), suggesting that certain patients do not receive adequate benefits from the current treatment. Thus, improving the current treatments or developing new therapeutic drugs is necessary to improve prognosis of patients with NPC.

In recent years, increasing evidence (6-9) has indicated that bioactive compounds from plants have promising anticancer properties. For example, cordycepin and fucoidan are natural compounds extracted from Cordyceps sinensis and brown seaweed cell wall matrix, respectively (10,11). These compounds can induce apoptosis in various cancer cell lines, including esophageal, colorectal, liver, and non-small-cell lung cancer (12-15). Similarly, curcumin extracted from Curcuma longa inhibits the survival of non-small-cell lung, papillary thyroid, and bone cancer cells by inducing apoptosis (16-18) in vitro. It also suppresses bone cancer cell growth in vivo (18). Additionally, curcumin can inhibit oral cancer cell survival by inducing autophagy (19). Furthermore, paclitaxel, an anti-microtubule agent extracted from Taxus brevifolia, is used clinically to treat multiple cancers, including ovarian, lung, and breast (20).

Apoptosis is a programmed cell death characterized by specific biological features, such as plasma membrane blebbing, apoptotic body formation, cytokeratin 18 fragment release, DNA fragmentation and poly ADP-ribose polymerase (PARP) cleavage (21-23). In addition, caspases, a family of endoproteases, are divided into initiator and effector caspases (23); they are involved in executing apoptosis. Upon activation, initiator caspases such as caspase-8 can activate effector caspases, such as caspase-3. This leads to the cleavage of PARP, a DNA repair enzyme, ultimately contributing to apoptosis (23).

Autophagy, a regulated self-digestion mechanism, is characterized by formation of double-membraned vesicles called autophagosomes that engulf cytoplasmic contents and fuse with lysosomes for degradation (24). The formation of autophagosomes is regulated by autophagy-related proteins, such as Beclin 1, LC3, and Atg12-Atg5 conjugate (25,26). Beclin 1 initiates autophagy, while LC3-II contributes to autophagosome formation, a key feature of autophagy (25-27). Additionally, a previous study showed that the knockdown of Beclin 1 or LC3 decreases autophagic activity (28), indicating that Beclin 1 and LC3 serve essential roles in activating autophagy.

Certain anticancer substances, such as cisplatin and curcumin, can simultaneously induce apoptosis and autophagy in bladder and gastric cancers, respectively (29,30). Studies show that inhibiting autophagy can enhance cisplatin or curcumin-induced apoptosis (29,30), suggesting that autophagy may play a protective role in cancer cell survival.

Oligonol, a polyphenolic compound found in lychee fruit (31), has antioxidant and anti-inflammatory properties and can alleviate sarcopenia (32-34). Additionally, previous studies have suggested that oligonol may have anticancer activity, as it induces apoptosis in breast and ovarian cancer cells (35,36). Therefore, the present study evaluated the potential anticancer effects of oligonol in NPC cells.

 Materials and methods <sec> <title>Materials

Oligonol was provided by Toong Yeuan Enterprise Co., Ltd. DMSO, 3-methyladenine (3-MA), LY294002 and MTT were purchased from Sigma-Aldrich (Merck KGaA). Fetal bovine serum (FBS), PBS and RPMI-1640 medium were purchased from HyClone (Cytiva).

 Cell culture

NPC-TW01 cells were provided by Dr Chin-Hwa Tsai from the National Taiwan University in Taipei, Taiwan. These cells were established from moderately differentiated NPC tissue (37). The second cell line, NPC/HK1, was purchased from Quantum Biotechnology, Inc. and established from well-differentiated NPC tissues (38). Both cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS at 37˚C in a 5% CO2 incubator.

 MTT assay

NPC cells were seeded into a 6-well plate at a density of 3x105 cells/well for 24 h. Once the cells reached 80% confluence, they were treated with oligonol at concentrations of 3.125, 6.25, 12.5, 25, and 50 µg/ml for 48 h at 37˚C with 5% CO2. Cells not treated with oligonol were used as the control. Supernatant was removed, and 2 ml MTT reagent (0.5 mg/ml in PBS) was added to each well. After incubation at 37˚C and 5% CO2 for 4 h, supernatant was removed, and 1 ml DMSO was added to each well to dissolve the crystals. Next, 100 µl DMSO lysate was transferred from each well to a 96-well plate. ELISA reader (BMG LABTECH) measured the optical density at 570 nm. All experiments were carried out ≥3 times independently.

 ELISA

The concentration of cytokeratin 18 fragment levels in the cell culture supernatant was measured using the SimpleStep ELISA kit (cat. no. ab254515; Abcam) according to the manufacturer's instructions. Each experiment was conducted independently ≥3 times.

 Immunoblotting assay

Extraction of total protein from cells and immunoblotting were performed as described previously (29). Primary antibodies for detecting PARP (cat. no. 9532; 1:1,000), cleaved PARP (cat. no. 9541; 1:1,000), caspase-8 (cat. no. 9746; 1:1,000), cleaved caspase-8 (cat. no. 9429; 1:1,000), caspase-3 (cat. no. 9662; 1:1,000), cleaved caspase-3 (cat. no. 9661; 1:1,000), Beclin 1 (cat. no. 3738; 1:1,000), LC3-I/II (cat. no. 4108; 1:1,000) and GAPDH (cat. no. 97166; 1:5,000) were purchased from Cell Signaling Technology, Inc. Horseradish peroxidase-conjugated secondary antibodies, including goat anti-rabbit (cat. no. 111-035-144; 1:5,000) and anti-mouse IgG (cat. no. 111-035-146; 1:5,000), were purchased from Jackson ImmunoResearch, Inc. Protein bands were detected using an enhanced chemiluminescence detection kit (SuperSignal™ West Pico PLUS Chemiluminescent Substrate; cat. no. 34580; Thermo Fisher Scientific, Inc.). All experiments were conducted independently ≥3 times. The intensities of protein bands were quantified using ImageJ software (version 1.43, National Institutes of Health).

 Statistical analysis

Data are expressed as the mean ± standard error of the mean of ≥3 independent experimental repeats. The statistical analysis was performed using SPSS software (version 17.0; SPSS, Inc.). An unpaired t-test was used to compare significant differences between two groups. One-way analysis of variance and Tukey's post hoc test were used to compare significant differences between >2 groups. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Oligonol inhibits viability of NPC cells

NPC cell lines NPC-TW01 and NPC/HK1 were treated with oligonol, and the cell viability was measured using an MTT assay. Oligonol could significantly inhibit the viability of both NPC cell lines at 12.5, 25.0 and 50.0 µg/ml compared with the control (Fig. 1A and B). In addition, the half-maximal inhibitory concentration of oligonol in both cell lines was 17 µg/ml.

 Oligonol induces apoptotic effects in NPC cells

Next, it was examined whether oligonol induces apoptotic effects in NPC cells. NPC-TW01 and NPC/HK1 cells were treated with 17 µg/ml oligonol. Immunoblotting assay was then performed to detect protein expression of apoptotic markers, such as cleaved caspase-8 and -3 and cleaved PARP (23) (Fig. 2A and F). Quantitative evaluation of immunoblots demonstrated that in both cell lines, oligonol significantly increased expression levels of cleaved caspase-8 (Fig. 2B and G), cleaved caspase-3 (Fig. 2C and H) and cleaved PARP (Fig. 2D and I) compared with the control. In addition, cytokeratin 18 is expressed in various types of epithelial cell (22). During apoptosis, it is cleaved by effector caspases and released from epithelial cells (22). Therefore, cytokeratin 18 fragments serve as a biological marker of apoptosis (22). ELISA indicated that oligonol significantly increased the release of cytokeratin 18 fragments from NPC cells compared with the control (Fig. 2E and J), suggesting that oligonol could induce apoptotic effects in NPC cells.

 Oligonol induces autophagic effects in NPC cells

As some natural extracts can simultaneously induce apoptosis and autophagy in cancer cells (39,40), it was investigated whether oligonol triggers autophagy in NPC cells. Beclin 1 and LC3-II were used as the autophagy markers in this study. Oligonol significantly induced protein expression of Beclin 1 and LC3-II (Fig. 3A and B). The conversion of LC3-I to LC3-II is a reliable marker for autophagy activation (41). LC3-II/LC3-I ratio was significantly increased by oligonol treatment compared with the control in both NPC cell lines (Fig. 3A and B), suggesting that oligonol also induced autophagic effects in NPC cells.

 Inhibition of autophagic effects promotes oligonol-induced viability inhibition and apoptotic effects in NPC cells

Because autophagy contributes to cancer cell survival or death (42-44), autophagy inhibitors 3-MA and LY294002(45) were used to investigate the effect of oligonol-induced autophagic effects on NPC cell viability. Treatment with 3-MA or LY294002 increased the viability inhibition induced by oligonol in NPC cells (Fig. 4A). Since previous studies indicate that blocking autophagy enhances the induction of apoptotic effects (29,46), the present study examined protein expression levels of LC3-II and apoptotic markers (cleaved caspase-8 and -3 and cleaved PARP) in NPC cells after combination treatment with oligonol and LY294002. LY294002 could suppress the oligonol-induced LC3-II expression (Fig. 4B) and increase in LC3-II/LC3-I ratio (Fig. 4C), indicating that the autophagy inhibitor, LY294002, inhibited oligonol-induced autophagy activation. Cotreatment of oligonol with LY294002 could significantly enhance the protein expression of cleaved caspase-8 (Fig. 4D), cleaved caspase-3 (Fig. 4E) and cleaved PARP (Fig. 4F) compared with oligonol alone. Additionally, cotreatment of oligonol with LY294002 significantly increased release of cytokeratin 18 fragments, another apoptotic marker, compared with oligonol alone (Fig. 4G). These findings suggested that inhibition of oligonol-induced autophagic effects by autophagy inhibitors enhanced viability inhibition and apoptotic effects in NPC cells.

 Discussion

Plant extracts have been reported to serve a key role in inhibiting the survival of cancer cells (12-15). Oligonol is a polyphenolic compound primarily extracted from lychee fruit that suppresses cancer cell viability (35,36). However, whether it has anticancer activity in NPC cells is still unknown. Here, oligonol effectively inhibited the viability of NPC cell lines NPC-TW01 and NPC/HK-1, which suggested that oligonol exerted anticancer effects in NPC cells.

As oligonol may induce apoptosis in cancer cells (35,36), the present study investigated its effects on NPC cells. Oligonol could stimulate the expression of apoptosis markers, including cleaved caspase-8 and -3 and cleaved PARP and induce the release of cytokeratin 18 fragments, suggesting that oligonol has the ability to induce apoptotic effects in NPC cells. Since caspase-8 can be cleaved (activated) through the apoptotic death receptor pathway (23), oligonol may also have the ability to activate the death receptor pathway. Previous studies have indicated that inhibiting Akt or inducing AMP-activated protein kinase (AMPK) activation promotes expression of Fas ligand (FasL) (47,48), an inducer of the death receptor pathway. As oligonol can inhibit Akt or promote AMPK activation (49,50), oligonol may induce expression of FasL by inhibiting Akt or inducing activation of AMPK in NPC cells. This could initiate the death receptor pathway and ultimately induce the activation of caspase-8. To determine if AMPK mediates oligonol-induced FasL expression in NPC cells, an AMPK inhibitor will be combined with oligonol to treat the NPC cells. The expression of FasL will then be assessed using an immunoblotting assay.

The present study examined whether oligonol induces autophagy in NPC cells, as certain natural extracts have been reported to simultaneously induce apoptosis and autophagy in cancer cells (39,40). Oligonol induced autophagic effects in NPC cells, as evidenced by increased expression of Beclin 1 and LC3-II, two autophagy markers. According to previous studies, inhibition of autophagy results in apoptosis in lung cancer cells treated with cisplatin and endometrial cancer cells treated with paclitaxel (29,44). The present study used two autophagy inhibitors (3-MA and LY294002) to confirm the role of autophagy in oligonol-induced viability inhibition and apoptotic effects. Autophagy inhibitors enhanced oligonol-induced viability inhibition. Expression of apoptosis markers, including cleaved caspase-8 and -3 and cleaved PARP and the release of cytokeratin 18 fragments significantly increased following cotreatment of oligonol with LY294002. These findings suggested that apoptotic effects were promoted by inhibiting oligonol-induced autophagic effects, leading to an increase in NPC cell viability inhibition. Therefore, oligonol-induced autophagy may serve a cytoprotective role in NPC cells.

It is unclear how the autophagic effects induced by oligonol inhibit the apoptotic effects in NPC cells. Induction of autophagy can cause mitochondria elongation (51,52), which results in inhibition of cytochrome c release (51,52), a pro-apoptotic factor associated with caspase-8 activation (51,53), preventing apoptosis and maintaining cell viability (51). Additionally, autophagosome formation proteins p62 and LC3-II interact with caspase-8, leading to its degradation when the mature autophagosome fuses with the lysosome (51,54). Therefore, oligonol-induced autophagy may decrease expression of cleaved caspase-8, which can inhibit the enhancement of apoptotic effects induced by oligonol in NPC cells. Further experiments should confirm these possible mechanisms in the future. For example, to investigate whether caspase-8 interacts with p62 under oligonol treatment in the NPC cells, protein-protein interaction techniques, such as co-immunoprecipitation and fluorescence resonance energy transfer, will be utilized.

 Acknowledgements

Not applicable.

 Availability of data and materials

The datasets generated in the present study may be requested from the corresponding author.

 Author's contributions

YTW, YCC and YYL designed the study. CHL, WCC, TJH and SJC performed experiments. YTW collected and analyzed the data. YTW and YYL wrote the manuscript. YCC and YYL revised the manuscript. YTW and YYL confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

 Ethics approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References Jicman StanDNiculetELunguMOnisorCRebegeaLVesaDBezmanLBujoreanuFCSarbuMIMihailovRNasopharyngeal carcinoma: A new synthesis of literature data (Review)Exp Ther Med2313620223506981710.3892/etm.2021.11059 BaiRSunJXuYSunZZhaoXIncidence and mortality trends of nasopharynx cancer from 1990 to 2019 in China: An age-period-cohort analysisBMC Public Health22135120223584096410.1186/s12889-022-13688-7 YuHYinXMaoYChenMTangQYanSThe global burden of nasopharyngeal carcinoma from 2009 to 2019: An observational study based on the Global Burden of Disease Study 2019Eur Arch Otorhinolaryngol2791519153320223414615010.1007/s00405-021-06922-2 BlanchardPLeeAMarguetSLeclercqJNgWTMaJChanATHuangPYBenhamouEZhuGChemotherapy and radiotherapy in nasopharyngeal carcinoma: An update of the MAC-NPC meta-analysisLancet Oncol1664565520152595771410.1016/S1470-2045(15)70126-9 WuJZhouQPanZWangYHuLChenGWangSLyuJDevelopment and validation of a nomogram for predicting long-term overall survival in nasopharyngeal carcinoma: A population-based studyMedicine (Baltimore)99e1897420203197791410.1097/MD.0000000000018974 ShrihastiniVMuthuramalingamPAdarshanSSujithaMChenJTShinHRameshMPlant derived bioactive compounds, their anti-cancer effects and in silico approaches as an alternative target treatment strategy for breast cancer: An updated overviewCancers (Basel)13622220213494484010.3390/cancers13246222 AsmaSTAcarozUImreKMorarAShahSRAHussainSZArslan-AcarozDDemirbasHHajrulai-MusliuZIstanbullugilFRNatural Products/Bioactive Compounds as a Source of Anticancer DrugsCancers (Basel)14620320223655168710.3390/cancers14246203 YuanMZhangGBaiWHanXLiCBianSThe role of bioactive compounds in natural products extracted from plants in cancer treatment and their mechanisms related to anticancer effectsOxid Med Cell Longev2022142986920223521124010.1155/2022/1429869 EsmeetaAAdhikarySDharshnaaVSwarnamughiPUmmul MaqsummiyaZBanerjeeAPathakSDuttaroyAKPlant-derived bioactive compounds in colon cancer treatment: An updated reviewBiomed Pharmacother15311338420223582031710.1016/j.biopha.2022.113384 van WeeldenGBobinskiMOklaKvan WeeldenWJRomanoAPijnenborgJMAFucoidan structure and activity in relation to anti-cancer mechanismsMar Drugs173220193062104510.3390/md17010032 SuNWWuSHChiCWTsaiTHChenYJCordycepin, isolated from medicinal fungus Cordyceps sinensis, enhances radiosensitivity of oral cancer associated with modulation of DNA damage repairFood Chem Toxicol12440041020193057671010.1016/j.fct.2018.12.025 XuJCZhouXPWangXAXuMDChenTChenTYZhouPHZhangYQCordycepin induces apoptosis and G2/M Phase Arrest through the ERK pathways in esophageal cancer cellsJ Cancer102415242420193125874610.7150/jca.32071 LiSZRenJWFeiJZhangXDDuRLCordycepin induces Bax-dependent apoptosis in colorectal cancer cellsMol Med Rep1990190820193053547910.3892/mmr.2018.9717 PAKALSMMKMAnticancer effect of fucoidan on cell proliferation, cell cycle progression, genetic damage and apoptotic cell death in HepG2 cancer cellsToxicol Rep655656320193124978910.1016/j.toxrep.2019.06.005 ChenXSunLWeiXLuHTanYSunZJiangJAntitumor effect and molecular mechanism of fucoidan in NSCLCBMC Complement Med Ther212520213343085410.1186/s12906-020-03191-0 EndoHInoueIMasunakaKTanakaMYanoMCurcumin induces apoptosis in lung cancer cells by 14-3-3 protein-mediated activation of BadBiosci Biotechnol Biochem842440244720203284158110.1080/09168451.2020.1808443 KhanAQAhmedEIElareerNFathimaHPrabhuKSSiveenKSKulinskiMAziziFDermimeSAhmadACurcumin-Mediated apoptotic cell death in papillary thyroid cancer and cancer stem-like cells through targeting of the JAK/STAT3 signaling pathwayInt J Mol Sci2143820203193667510.3390/ijms21020438 SunYLiuLWangYHeAHuHZhangJHanMHuangYCurcumin inhibits the proliferation and invasion of MG-63 cells through inactivation of the p-JAK2/p-STAT3 pathwayOnco Targets Ther122011202120193093671810.2147/OTT.S172909 KimJYChoTJWooBHChoiKULeeCHRyuMHParkHRCurcumin-induced autophagy contributes to the decreased survival of oral cancer cellsArch Oral Biol571018102520122255499510.1016/j.archoralbio.2012.04.005 Sharifi-RadJQuispeCPatraJKSinghYDPandaMKDasGAdetunjiCOMichaelOSSytarOPolitoLPaclitaxel: Application in modern oncology and nanomedicine-based cancer therapyOxid Med Cell Longev2021368770020213470777610.1155/2021/3687700 ZhangYChenXGueydanCHanJPlasma membrane changes during programmed cell deathsCell Res2892120182907650010.1038/cr.2017.133 UlukayaEKaraagacEAriFOralAYAdimSBTokullugilAHEvrenselTChemotherapy increases caspase-cleaved cytokeratin 18 in the serum of breast cancer patientsRadiol Oncol4511612220112293394410.2478/v10019-011-0006-7 CarneiroBAEl-DeiryWSTargeting apoptosis in cancer therapyNat Rev Clin Oncol1739541720203220327710.1038/s41571-020-0341-y KhandiaRDadarMMunjalADhamaKKarthikKTiwariRYatooMIIqbalHMNSinghKPJoshiSKChaicumpaWA comprehensive review of autophagy and its various roles in infectious, non-infectious, and lifestyle diseases: Current knowledge and prospects for disease prevention, novel drug design, and therapyCells867420193127729110.3390/cells8070674 WirthMJoachimJToozeSAAutophagosome formation-the role of ULK1 and Beclin1-PI3KC3 complexes in setting the stageSemin Cancer Biol2330130920132372715710.1016/j.semcancer.2013.05.007 ZhangTYuJChengSZhangYZhouCHQinJLuoHResearch progress on the anticancer molecular mechanism of targets regulating cell autophagyPharmacology10822423720233688930110.1159/000529279 SunYYaoXZhangQJZhuMLiuZPCiBXieYCarlsonDRothermelBASunYBeclin-1-Dependent autophagy protects the heart during sepsisCirculation1382247226220182985351710.1161/CIRCULATIONAHA.117.032821 HamurcuZDelibasiNGeceneSŞenerEFDönmez-AltuntaşHÖzkulYCanatanHOzpolatBTargeting LC3 and Beclin-1 autophagy genes suppresses proliferation, survival, migration and invasion by inhibition of Cyclin-D1 and uPAR/Integrin β1/Src signaling in triple negative breast cancer cellsJ Cancer Res Clin Oncol14441543020182928836310.1007/s00432-017-2557-5 ChenJZhangLZhouHWangWLuoYYangHYiHInhibition of autophagy promotes cisplatin-induced apoptotic cell death through Atg5 and Beclin 1 in A549 human lung cancer cellsMol Med Rep176859686520182951276210.3892/mmr.2018.8686 LiWZhouYYangJLiHZhangHZhengPCurcumin induces apoptotic cell death and protective autophagy in human gastric cancer cellsOncol Rep373459346620172849843310.3892/or.2017.5637 FujiiHSunBNishiokaHHiroseAAruomaOIEvaluation of the safety and toxicity of the oligomerized polyphenol OligonolFood Chem Toxicol4537838720071708167110.1016/j.fct.2006.08.026 SakuraiTNishiokaHFujiiHNakanoNKizakiTRadakZIzawaTHagaSOhnoHAntioxidative effects of a new lychee fruit-derived polyphenol mixture, oligonol, converted into a low-molecular form in adipocytesBiosci Biotechnol Biochem7246347620081825648510.1271/bbb.70567 LeeCBakJYoonSMoonJOProtective effect of oligonol on dimethylnitrosamine-induced liver fibrosis in rats via the JNK/NF-κB and PI3K/Akt/Nrf2 signaling pathwaysAntioxidants (Basel)1036620213367102810.3390/antiox10030366 ChangYCChenYTLiuHWChanYCLiuMYHuSHTsengWTWuHLWangMFChangSJOligonol alleviates sarcopenia by regulation of signaling pathways involved in protein turnover and mitochondrial qualityMol Nutr Food Res63e180110220193079386710.1002/mnfr.201801102 JoEHLeeSJAhnNSParkJSHwangJWKimSHAruomaOILeeYSKangKSInduction of apoptosis in MCF-7 and MDA-MB-231 breast cancer cells by Oligonol is mediated by Bcl-2 family regulation and MEK/ERK signalingEur J Cancer Prev1634234720071755420710.1097/01.cej.0000236247.86360.db KimMParkWHLeeSSuhDHKimKNoJHKimYBOligonol, a low molecular weight polyphenol, enhances apoptotic cell death in ovarian cancer cells via suppressing NF-κB activationNutr Cancer7114114820193063358710.1080/01635581.2018.1557215 AimjongjunSMahmudZJiramongkolYAlasiriGYaoSYagüeEJanvilisriTLamEWLapatinib sensitivity in nasopharyngeal carcinoma is modulated by SIRT2-mediated FOXO3 deacetylationBMC Cancer19110620193172700610.1186/s12885-019-6308-7 HuangDPHoJHPoonYFChewECSawDLuiMLiCLMakLSLaiSHLauWHEstablishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynxInt J Cancer261271321980625906410.1002/ijc.2910260202 LiYLiRZhuSZhouRWangLDUJWangYZhouBMaiLCordycepin induces apoptosis and autophagy in human neuroblastoma SK-N-SH and BE(2)-M17 cellsOncol Lett92541254720152613710310.3892/ol.2015.3066 GongXJiangLLiWLiangQLiZCurcumin induces apoptosis and autophagy inhuman renal cell carcinoma cells via Akt/mTOR suppressionBioengineered125017502720213440271810.1080/21655979.2021.1960765 FangSWanXZouXSunSHaoXLiangCZhangZZhangFSunBLiHYuBArsenic trioxide induces macrophage autophagy and atheroprotection by regulating ROS-dependent TFEB nuclear translocation and AKT/mTOR pathwayCell Death Dis128820213346218210.1038/s41419-020-03357-1 WangCYTsaiSWChienHHChenTYSheuSYSoECHuangBMCordycepin inhibits human gestational choriocarcinoma cell growth by disrupting centrosome homeostasisDrug Des Devel Ther142987300020203280163910.2147/DDDT.S252401 JiangYJiFLiuYHeMZhangZYangJWangNZhongCJinQYeXChenTCisplatin-induced autophagy protects breast cancer cells from apoptosis by regulating yes-associated proteinOncol Rep383668367620172903961610.3892/or.2017.6035 LiuSLiXAutophagy inhibition enhances sensitivity of endometrial carcinoma cells to paclitaxelInt J Oncol462399240820152582508810.3892/ijo.2015.2937 PasquierBAutophagy inhibitorsCell Mol Life Sci73985100120162665891410.1007/s00018-015-2104-y PanXZhangXSunHZhangJYanMZhangHAutophagy inhibition promotes 5-fluorouraci-induced apoptosis by stimulating ROS formation in human non-small cell lung cancer A549 cellsPLoS One8e5667920132344121210.1371/journal.pone.0056679 SayedDHeMHongCGaoSRaneSYangZAbdellatifMMicroRNA-21 is a downstream effector of AKT that mediates its antiapoptotic effects via suppression of Fas ligandJ Biol Chem285202812029020102040434810.1074/jbc.M110.109207 TsaiMCChenCCTsengTHChangYCLinYJTsaiINWangCCWangCJHibiscus anthocyanins extracts induce apoptosis by activating AMP-Activated protein kinase in human colorectal cancer cellsNutrients15397220233776475610.3390/nu15183972 ParkJYKimYImJAYouSLeeHInhibition of adipogenesis by oligonol through Akt-mTOR inhibition in 3T3-L1 adipocytesEvid Based Complement Alternat Med201489527220142529506910.1155/2014/895272 ParkSKSeongRKKimJASonSJKimYYokozawaTShinOSOligonol promotes anti-aging pathways via modulation of SIRT1-AMPK-Autophagy pathwayNutr Res Pract1031020162686591010.4162/nrp.2016.10.1.3 BataNCosfordNDPCell survival and cell death at the intersection of autophagy and apoptosis: Implications for current and future cancer therapeuticsACS Pharmacol Transl Sci41728174620213492700710.1021/acsptsci.1c00130 GomesLCDi BenedettoGScorranoLDuring autophagy mitochondria elongate, are spared from degradation and sustain cell viabilityNat Cell Biol1358959820112147885710.1038/ncb2220 PanJXuGYeungSCCytochrome c release is upstream to activation of caspase-9, caspase-8, and caspase-3 in the enhanced apoptosis of anaplastic thyroid cancer cells induced by manumycin and paclitaxelJ Clin Endocrinol Metab864731474020011160053310.1210/jcem.86.10.7860 HouWHanJLuCGoldsteinLARabinowichHAutophagic degradation of active caspase-8: A crosstalk mechanism between autophagy and apoptosisAutophagy689190020102072483110.4161/auto.6.7.13038

Oligonol inhibits viability of nasopharyngeal carcinoma cells. (A) NPC-TW01 and (B) NPC/HK1 cells were treated with oligonol for 48 h. The MTT assay was used to measure the cell viability. *P<0.05 vs. Ctrl. Ctrl, control.

Oligonol induces apoptotic effects in nasopharyngeal carcinoma cells. NPC-TW01 were treated with 17 µg/ml oligonol for 48 h. (A) Protein expression of caspase-8, cleaved caspase-8, caspase-3, cleaved caspase-3, PARP, cleaved PARP and GAPDH was detected using an immunoblotting assay. The protein expression levels of cleaved caspase-8 (B) cleaved caspase-3 (C) and cleaved PARP (D) were quantified. (E) Concentration of cytokeratin 18 fragment in the cell culture supernatants was measured by ELISA. NPC/HK1 cells were treated with 17 µg/ml oligonol for 48 h. (F) Protein expression of caspase-8, cleaved caspase-8, caspase-3, cleaved caspase-3, PARP, cleaved PARP and GAPDH was detected using an immunoblotting assay. The protein expression levels of cleaved caspase-8 (G) cleaved caspase-3 (H) and cleaved PARP (I) were quantified. (J) Concentration of cytokeratin 18 fragment in the cell culture supernatants was measured by ELISA. *P<0.05 vs. Ctrl. Ctrl, control.

Oligonol induces autophagic effects in nasopharyngeal carcinoma cells. (A) NPC-TW01 and (B) NPC/HK1 cells were treated with 17 µg/ml oligonol for 48 h. Protein expression of Beclin 1, LC3-I, LC3-II and GAPDH was detected using an immunoblotting assay. *P<0.05 vs. Ctrl. Ctrl, control.

Inhibition of autophagic effects promotes oligonol-induced viability inhibition and apoptotic effects in nasopharyngeal cells. (A) NPC-TW01 cells were treated with 17 µg/ml Oligo and DMSO, 3-MA or LY. Cell viability was measured using MTT assay. (B) NPC-TW01 cells were treated with 17 µg/ml Oligo and DMSO or LY. Protein expression of LC3-I, LC3-II, C8, cleaved C8, C3, cleaved C3, PARP, cleaved PARP and GAPDH was examined by an immunoblotting assay. (C) Quantitative analysis of LC3-II levels normalized using LC3-I. The protein expression levels of cleaved C (D) 8 and (E) 3 and (F) cleaved PARP were quantified using ImageJ software. (G) Concentration of cytokeratin 18 fragment in cell culture supernatant was measured by ELISA. *P<0.05 vs. Ctrl + DMSO; #P<0.05 vs. oligo + DMSO. Ctrl, control; oligo, oligonol; 3-MA, 3-Methyladenine; LY, LY294002; C, caspase.