Role of Natural Bioactive Compounds in the Rise and Fall of Cancers Printed Edition of the Special Issue Published in Cancers www.mdpi.com/journal/cancers Claudio Luparello Edited by Volume 1 Role of Natural Bioactive Compounds in the Rise and Fall of Cancers Role of Natural Bioactive Compounds in the Rise and Fall of Cancers Volume 1 Editor Claudio Luparello MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editor Claudio Luparello Universit` a di Palermo Italy Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Cancers (ISSN 2072-6694) (available at: https://www.mdpi.com/journal/cancers/special issues/ cancers-NBC). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. Volume 1 ISBN 978-3-03943-254-7 ( H bk) ISBN 978-3-03943-255-4 (PDF) Volume 1-2 ISBN 978-3-03943- 294-3 ( H bk) ISBN 978-3-03943- 295-0 (PDF) c © 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Claudio Luparello Role of Natural Bioactive Compounds in the Rise and Fall of Cancers Reprinted from: Cancers 2020 , 12 , 2499, doi:10.3390/cancers12092499 . . . . . . . . . . . . . . . . 1 Emma Polonio-Alcal ́ a, S ` onia Palomeras, Daniel Torres-Oteros, Joana Relat, Marta Planas, Lidia Feliu, Joaquim Ciurana, Santiago Ruiz-Mart ́ ınez and Teresa Puig Fatty Acid Synthase Inhibitor G28 Shows Anticancer Activity in EGFR Tyrosine Kinase Inhibitor Resistant Lung Adenocarcinoma Models Reprinted from: Cancers 2020 , 12 , 1283, doi:10.3390/cancers12051283 . . . . . . . . . . . . . . . . 7 Ran Wei, Robert M. Hackman, Yuefei Wang and Gerardo G. Mackenzie Targeting Glycolysis with Epigallocatechin-3-Gallate Enhances the Efficacy of Chemotherapeutics in Pancreatic Cancer Cells and Xenografts Reprinted from: Cancers 2019 , 11 , 1496, doi:10.3390/cancers11101496 . . . . . . . . . . . . . . . . 25 Ammad Ahmad Farooqi, Marina Pinheiro, Andreia Granja, Fulvia Farabegoli, Salette Reis, Rukset Attar, Uteuliyev Yerzhan Sabitaliyevich, Baojun Xu and Aamir Ahmad EGCG Mediated Targeting of Deregulated Signaling Pathways and Non-Coding RNAs in Different Cancers: Focus on JAK/STAT, Wnt/ β -Catenin, TGF/SMAD, NOTCH, SHH/GLI, and TRAIL Mediated Signaling Pathways Reprinted from: Cancers 2020 , 12 , 951, doi:10.3390/cancers12040951 . . . . . . . . . . . . . . . . . 45 Dong Young Kang, Nipin Sp, Eun Seong Jo, Alexis Rugamba, Dae Young Hong, Hong Ghi Lee, Ji-Seung Yoo, Qing Liu, Kyoung-Jin Jang and Young Mok Yang The Inhibitory Mechanisms of Tumor PD-L1 Expression by Natural Bioactive Gallic Acid in Non-Small-Cell Lung Cancer (NSCLC) Cells Reprinted from: Cancers 2020 , 12 , 727, doi:10.3390/cancers12030727 . . . . . . . . . . . . . . . . . 67 Jessica Ruzzolini, Silvia Peppicelli, Francesca Bianchini, Elena Andreucci, Silvia Urciuoli, Annalisa Romani, Katia Tortora, Giovanna Caderni, Chiara Nediani and Lido Calorini Cancer Glycolytic Dependence as a New Target of Olive Leaf Extract Reprinted from: Cancers 2020 , 12 , 317, doi:10.3390/cancers12020317 . . . . . . . . . . . . . . . . . 83 Giada Juli, Manuela Oliverio, Dina Bellizzi, Maria Eugenia Gallo Cantafio, Katia Grillone, Giuseppe Passarino, Carmela Colica, Monica Nardi, Marco Rossi, Antonio Procopio, Pierosandro Tagliaferri, Pierfrancesco Tassone and Nicola Amodio Anti-tumor Activity and Epigenetic Impact of the Polyphenol Oleacein in Multiple Myeloma Reprinted from: Cancers 2019 , 11 , 990, doi:10.3390/cancers11070990 . . . . . . . . . . . . . . . . . 97 Emiliano Montalesi, Manuela Cipolletti, Patrizio Cracco, Marco Fiocchetti and Maria Marino Divergent Effects of Daidzein and Its Metabolites on Estrogen-Induced Survival of Breast Cancer Cells Reprinted from: Cancers 2020 , 12 , 167, doi:10.3390/cancers12010167 . . . . . . . . . . . . . . . . . 111 Nattanan Losuwannarak, Arnatchai Maiuthed, Nakarin Kitkumthorn, Asada Leelahavanichkul, Sittiruk Roytrakul and Pithi Chanvorachote Gigantol Targets Cancer Stem Cells and Destabilizes Tumors via the Suppression of the PI3K/AKT and JAK/STAT Pathways in Ectopic Lung Cancer Xenografts Reprinted from: Cancers 2019 , 11 , 2032, doi:10.3390/cancers11122032 . . . . . . . . . . . . . . . . 125 v Danilo Predes, Luiz F. S. Oliveira, La ́ ıs S. S. Ferreira, Lorena A. Maia, Jo ̃ ao M. A. Delou, Anderson Faletti, Igor Oliveira, Nathalia G. Amado, Alice H. Reis, Carlos A. M. Fraga, Ricardo Kuster, Fabio A. Mendes, Helena L. Borges and Jose G. Abreu The Chalcone Lonchocarpin Inhibits Wnt/ β -Catenin Signaling and Suppresses Colorectal Cancer Proliferation Reprinted from: Cancers 2019 , 11 , 1968, doi:10.3390/cancers11121968 . . . . . . . . . . . . . . . . 145 Cheol Park, Hee-Jae Cha, Eun Ok Choi, Hyesook Lee, Hyun Hwang-Bo, Seon Yeong Ji, Min Yeong Kim, So Young Kim, Su Hyun Hong, JaeHun Cheong, Gi-Young Kim, Seok Joong Yun, Hye Jin Hwang, Wun-Jae Kim and Yung Hyun Choi Isorhamnetin Induces Cell Cycle Arrest and Apoptosis Via Reactive Oxygen Species-Mediated AMP-Activated Protein Kinase Signaling Pathway Activation in Human Bladder Cancer Cells Reprinted from: Cancers 2019 , 11 , 1494, doi:10.3390/cancers11101494 . . . . . . . . . . . . . . . . 165 Diego Mu ̃ noz, Martina Brucoli, Silvia Zecchini, Adrian Sandoval-Hernandez, Gonzalo Arboleda, Fabian Lopez-Vallejo, Wilman Delgado, Matteo Giovarelli, Marco Coazzoli, Elisabetta Catalani, Clara De Palma, Cristiana Perrotta, Luis Cuca, Emilio Clementi and Davide Cervia XIAP as a Target of New Small Organic Natural Molecules Inducing Human Cancer Cell Death Reprinted from: Cancers 2019 , 11 , 1336, doi:10.3390/cancers11091336 . . . . . . . . . . . . . . . . 183 Shang-Tse Ho, Chi-Chen Lin, Yu-Tang Tung and Jyh-Horng Wu Molecular Mechanisms Underlying Yatein-Induced Cell-Cycle Arrest and Microtubule Destabilization in Human Lung Adenocarcinoma Cells Reprinted from: Cancers 2019 , 11 , 1384, doi:10.3390/cancers11091384 . . . . . . . . . . . . . . . . 213 Lorena Perrone, Simone Sampaolo and Mariarosa Anna Beatrice Melone Bioactive Phenolic Compounds in the Modulation of Central and Peripheral Nervous System Cancers: Facts and Misdeeds Reprinted from: Cancers 2020 , 12 , 454, doi:10.3390/cancers12020454 . . . . . . . . . . . . . . . . . 225 Ana M. Barbosa and F ́ atima Martel Targeting Glucose Transporters for Breast Cancer Therapy: The Effect of Natural and Synthetic Compounds Reprinted from: Cancers 2020 , 12 , 154, doi:10.3390/cancers12010154 . . . . . . . . . . . . . . . . . 243 Chon Phin Ong, Wai Leong Lee, Yin Quan Tang and Wei Hsum Yap Honokiol: A Review of Its Anticancer Potential and Mechanisms Reprinted from: Cancers 2020 , 12 , 48, doi:10.3390/cancers12010048 . . . . . . . . . . . . . . . . . 281 Qingyu Zhou, Hua Pan and Jing Li Molecular Insights into Potential Contributions of Natural Polyphenols to Lung Cancer Treatment Reprinted from: Cancers 2019 , 11 , 1565, doi:10.3390/cancers11101565 . . . . . . . . . . . . . . . . 325 Joanna Xuan Hui Goh, Loh Teng-Hern Tan, Joo Kheng Goh, Kok Gan Chan, Priyia Pusparajah, Learn-Han Lee and Bey-Hing Goh Nobiletin and Derivatives: Functional Compounds from Citrus Fruit Peel for Colon Cancer Chemoprevention Reprinted from: Cancers 2019 , 11 , 867, doi:10.3390/cancers11060867 . . . . . . . . . . . . . . . . . 359 vi About the Editor Claudio Luparello https://www.unipa.it/persone/docenti/l/claudio.luparello/en/. vii cancers Editorial Role of Natural Bioactive Compounds in the Rise and Fall of Cancers Claudio Luparello Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Viale delle Scienze, Edificio 16, 90128 Palermo, Italy; claudio.luparello@unipa.it; Tel.: + 39-091-238-97405 Received: 31 August 2020; Accepted: 2 September 2020; Published: 3 September 2020 Recent years have seen the idea of a close association between nutrition and the modulation of cancer development / progression reinforced. In fact, an increasing number of experimental and epidemiological evidence has been produced, supporting the concept that many di ff erent bioactive components of food (e.g., polyphenols, mono- and polyunsaturated fatty acids, methyl-group donors . . . ) may be implicated in either the promotion of or the protection against carcinogenesis. At the cellular level, such compounds can have an impact on di ff erent but sometimes intertwined processes, such as growth and di ff erentiation, DNA repair, programmed cell death, and oxidative stress. In addition, compelling evidence is starting to build up of the existence of primary epigenetic targets of dietary compounds, such as oncogenic / oncosuppressor miRNAs or DNA-modifying enzymes, which in turn impair gene expression and function. This editorial aims to summarize the themes of the 31 papers (20 original articles and 11 reviews) published in the Special Issue “Role of Natural Bioactive Compounds in the Rise and Fall of Cancers” presenting the latest findings on the intracellular pathways and mechanisms a ff ected by selected natural molecules influencing the fine-tuning of cancer phenotype. Plant polyphenols have been among the most studied natural compounds by the contributors to this issue. In the original article group, Polonio-Alcal à et al. [ 1 ] showed the additive and synergistic e ff ects of the flavonoids ( − )-epigallocatechin-3-gallate (EGCG) from green tea and its naphthalene derivative G28, which are fatty acid synthase inhibitors, in combination with epidermal growth factor receptor tyrosine kinase inhibitors on gefitinib-resistant lung adenocarcinoma models. Moreover, Wei et al. [ 2 ] examined the e ff ect and mechanism of action of EGCG alone and in combination with current chemotherapeutics on pancreatic cancer cell growth, demonstrated the impairment of cell proliferation via the phosphofructokinase inhibition-mediated suppression of glycolysis in a ROS-dependent manner, and the additive enhancement of the anticancer e ff ect of gemcitabine both in vitro and in pancreatic xenografts by the further inhibition of glycolysis and the impairment of cell kinetics. In the Review section, Farooqi et al. [ 3 ] analyzed the pleiotropic abilities of EGCG to regulate intracellular signalizations such as those related to JAK / STAT, Wnt / β -catenin, TGF / SMAD, SHH / GLI and NOTCH pathways, also commenting on the ability of EGCG to modulate non-coding RNAs and the methylation-associated machinery in di ff erent cancers. Other natural phenolic compounds whose activity is discussed in the original articles of this issue are: – Gallic acid (3,4,5-trihydroxybenzoic acid), widely distributed in natural plants, fruits, and green tea, whose tumor-suppressive e ff ect via the p53-mediated downregulation of the transmembrane protein PD-L1 was demonstrated by Kang et al. [4] in non-small-cell lung cancer models; – Oleuropein, the main bioactive phenolic component of Olea europaea L., whose presence in enriched extracts from olive leaves was proven to reduce the glycolytic rate of a wide range of solid and liquid tumor cells via the downregulation of the three key e ff ectors of the glycolytic Cancers 2020 , 12 , 2499; doi:10.3390 / cancers12092499 www.mdpi.com / journal / cancers 1 Cancers 2020 , 12 , 2499 pathway, GLUT-1, PKM2 and MCT4, likely resulting in a decreased glucose entrance and biomass production [5]; – Oleacein (3,4-dihydroxyphenylethanol), the main secoiridoid contained in extra virgin olive oil, able to elicit significant anti-tumor activity by promoting cell cycle arrest and apoptosis in multiple myeloma cells due to its histone deacetylase inhibitory properties [6]; – Dadzein (7,4 ′ -dihydroxyisoflavone), present in soybeans, whose 4-sulphate metabolite produced by gut microbiota was found to exert an anti-estrogenic e ff ect on ER α -positive breast cancer cells via the downregulation of the anti-apoptotic neuroglobin protein thus rendering cells more prone to the paclitaxel treatment [7]; – Gigantol, a bibenzyl compound from orchid species, whose ability to destabilize tumor integrity via the suppression of the PI3K / AKT / mTOR and JAK / STAT pathways was demonstrated by Losuwannarak et al. [8] in non-small-cell lung cancer models in vitro and in vivo; – Lonchocarpin, a chalcone isolated from Lonchocarpus sericeus , proven to be a powerful inhibitor of the Wnt / β -catenin pathway able to selectively suppress the migration and proliferation of a panel of colorectal cancer cell lines in vitro and in a preclinical colorectal cancer mouse model [9]; – Isorhamnetin, (3 ′ -methoxy-3,4 ′ ,5,7-tetrahydroxyflavone), a flavonol aglycone found in some medicinal plants, able to exert an anti-proliferative e ff ect on human bladder cancer cells via the induction of cell cycle arrest during the G2 / M phase and apoptosis, accompanied by the activation of the AMPK signaling pathway and ROS overproduction [10]; – Erioquinol, eriopodol A and gibbilimbol B, derived from Piper genus plants, whose ability to inhibit XIAP protein, involved in the regulation of caspase-dependent / independent cell death pathways, was reported by Muñoz et al. [11] in breast cancer cell lines; – Vatein, isolated from Calocedrus formosana Florin leaves extract, proven to interfere with cell cycle and microtubule dynamics in lung adenocarcinoma cells, also inhibiting tumor growth in a xenograft mouse model [12]. In the Review section, Perrone et al. [ 13 ] discussed the e ff ects of polyphenols in preventing the progression of central and peripheral nervous system tumors underlining the beneficial e ff ect of dietary compounds on the microbioma–intestine–brain axis. Barbosa and Martel [ 14 ] examined the role played by a wide variety of synthetic and natural substances, including polyphenols, on the impairment of glucose uptake by neoplastic breast cells thereby resulting in a tumor-restraining e ff ect. Ong et al. [ 15 ] reported the broad-range in vitro / in vivo anticancer properties of the Magnolia -derived polyphenol honokiol based upon its ability to impair cell cycle progression, inhibit epithelial–mesenchymal transition, and suppress cell motility, invasion, metastasis and angiogenesis. Zhou et al. [ 16 ] summarized the late preclinical studies on the applications of bioactive polyphenols in lung cancer therapy, focusing on the molecular mechanisms at the basis of their therapeutic e ff ects and also discussing the potential of the polyphenol-based combination therapy. Goh et al. [ 17 ] reviewed data on the anti-colon cancer properties of nobiletin, a polymethoxyflavone extracted from citrus peel, and its derivatives which are able to arrest the cell cycle, inhibit cell proliferation, induce apoptosis, prevent tumor formation, reduce inflammatory e ff ects and limit angiogenesis, also exploring better drug delivery strategies due to the low oral bioavailability of the compounds. Ong et al. [ 18 ] focused their review on the pharmacological properties and therapeutic potential of formononetin [7-hydroxy-3-(4-methoxyphenyl)-4H-1-benzopyran-4-one], one of the main bioactive components of red clover, which regulates various transcription factor- and growth factor-mediated oncogenic pathways attenuating metastasis and tumor growth in vivo in multiple cancer cell models and also alleviating the possible causes of chronic inflammation that are linked to the cancer survival of neoplastic cells and their resistance against chemotherapy. The other articles and reviews addressed further cancer-related issues relevant to types of compounds of a di ff erent nature, specifically: 2 Cancers 2020 , 12 , 2499 – The methanolic extract of Malva pseudolavatera leaves, which showed a promising selective anti-proliferative and pro-apoptotic e ff ect on acute myeloid leukemia cell lines, determining PARP cleavage, cytochrome-c release, Bax / Bcl-2 ratio increase and ROS overproduction [19]; – Eicosapentaenoic acid, an ω -3 polyunsaturated fatty acid, which played a protective role, both alone and in combination with angiotensin-converting enzyme inhibitors, in attenuating adipocyte-induced proinflammatory cytokine expression and the migration of breast cancer cells in an in vitro model of obesity-induced breast cancer [20]; – Fucoidan, a sulphated polysaccharide derived from brown seaweed, whose combination with gemcitabine determined an enhanced pro-apoptotic and cell cycle-inhibitory activity on selected uterine carcinosarcoma and stromal sarcoma cell lines [21]; – Nicotin, whose mechanisms underlying the promotion of melanoma cell proliferation and migration mediated through α 9-nAChR-initiated carcinogenic signaling and PD-L1 expression were reported by Nguyen et al. [22]; – The ethyl acetate fraction of the crude extract of Streptomyces sp. MUM256, isolated from mangrove soil in Malaysia, and the cyclic dipeptides contained whose ability to induce cell cycle arrest and apoptosis was demonstrated by Tan et al. [23] in colon cancer cells; – Manoalide, an antibiotic sesterterpenoid isolated from the marine sponge Lu ff ariella variabilis , which preferentially inhibits the proliferation of oral cancer cells inducing apoptosis and DNA damages via oxidative stresses, such as intracellular ROS and MitoSOX / MitoMP [24]; – λ -carrageenan, a family of linear sulfated polysaccharides, proven to enhance the e ff ect of radiotherapy by suppressing the survival and invasiveness of di ff erent cancer cell lines in vitro and in vivo through the Rac GTPase-activating protein 1 pathway [25]; – Ethanol, which was found to trigger a pro-survival autophagic response following the induction of oxidative and endoplasmic reticulum stress in colon cancer cells, and the activation of Nrf2 and HO-1 also leading to the acquisition of a more aggressive phenotype [26]; – Colchicine, an alkaloid present in the medicinal plant Colchicum autumnale , whose enhanced anticancer e ff ects and reduced cytotoxicity on colon cancer cells when delivered in the nanoformulated form was reported by AbouAitah et al. [27]. In the Review section, Del Corn ò et al. [ 28 ] discussed the modulatory e ff ects of dietary β -glucans, present in diverse edible mushrooms, baker’s yeast, cereals and seaweeds, on human innate immunity cells and their potential role in cancer control. Lee et al. [ 29 ] reviewed a large number of data on the role played by di ff erent cytokines, lipids and other natural molecules on the suppression of epithelial–mesenchymal transition in cancer progression. Ennour-Idrissi et al. [ 30 ] focused their attention on the bioaccumulation of persistent organic pollutants in the food chain and the association of exposure with breast cancer risk. Farooqi et al. [ 31 ] presented the current views about the ability of berberine, a natural alkaloid compound found in several medicinal plants, to target di ff erent signaling cascades in various cancers, also discussing the nanocarrier strategies developed to improve the delivery of the compound. The number of manuscripts published in this Special Issue indicates an active interest in research about the molecular / pharmacological mechanisms used by natural products exerting anti-tumoral e ff ects which deserve further and deeper studies. I wish to thank all the contributors of this issue for sharing with us their experimental or reviewed data which will surely attract readers’ attention and encourage the publication of other high-quality papers in this field. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. 3 Cancers 2020 , 12 , 2499 References 1. Polonio-Alcal á , E.; Palomeras, S.; Torres-Oteros, D.; Relat, J.; Planas, M.; Feliu, L.; Ciurana, J.; Ruiz-Mart í nez, S.; Puig, T. Fatty acid synthase inhibitor G28 shows anticancer activity in EGFR tyrosine kinase inhibitor resistant lung adenocarcinoma models. Cancers 2020 , 12 , 1283. [CrossRef] 2. 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Nobiletin and derivatives: Functional compounds from citrus fruit peel for colon cancer chemoprevention. Cancers 2019 , 11 , 867. [CrossRef] 18. Ong, S.K.L.; Shanmugam, M.K.; Fan, L.; Fraser, S.E.; Arfuso, F.; Ahn, K.S.; Sethi, G.; Bishayee, A. Focus on formononetin: Anticancer potential and molecular targets. Cancers 2019 , 11 , 611. [CrossRef] 4 Cancers 2020 , 12 , 2499 19. El Khoury, M.; Haykal, T.; Hodroj, M.H.; Najem, S.A.; Sarkis, R.; Taleb, R.I.; Rizk, S. Malva pseudolavatera leaf extract promotes ROS induction leading to apoptosis in acute myeloid leukemia cells in vitro Cancers 2020 , 12 , 435. [CrossRef] 20. Rasha, F.; Kahathuduwa, C.; Ramalingam, L.; Hernandez, A.; Moussa, H.; Moustaid-Moussa, N. Combined e ff ects of eicosapentaenoic acid and adipocyte renin–angiotensin system inhibition on breast cancer cell inflammation and migration. Cancers 2020 , 12 , 220. [CrossRef] 21. Bobi ́ nski, M.; Okła, K.; Łuszczki, J.; Bednarek, W.; Wawruszak, A.; Moreno-Bueno, G.; Dmoszy ́ nska-Graniczka, M.; Tarkowski, R.; Kotarski, J. Isobolographic analysis demonstrates the additive and synergistic e ff ects of gemcitabine combined with fucoidan in uterine sarcomas and carcinosarcoma cells. Cancers 2020 , 12 , 107. [CrossRef] [PubMed] 22. Nguyen, H.D.; Liao, Y.-C.; Ho, Y.-S.; Chen, L.-C.; Chang, H.-W.; Cheng, T.-C.; Liu, D.; Lee, W.-R.; Shen, S.-C.; Wu, C.-H.; et al. The α 9 nicotinic acetylcholine receptor mediates nicotine-induced PD-L1 expression and regulates melanoma cell proliferation and migration. Cancers 2019 , 11 , 1991. [CrossRef] [PubMed] 23. Tan, L.T.-H.; Chan, C.-K.; Chan, K.-G.; Pusparajah, P.; Khan, T.M.; Ser, H.-L.; Lee, L.-H.; Goh, B.-H. Streptomyces sp. MUM256: A source for apoptosis inducing and cell cycle-arresting bioactive compounds against colon cancer cells. Cancers 2019 , 11 , 1742. [CrossRef] [PubMed] 24. Wang, H.-R.; Tang, J.-Y.; Wang, Y.-Y.; Farooqi, A.A.; Yen, C.-Y.; Yuan, S.-S.F.; Huang, H.-W.; Chang, H.-W. Manoalide preferentially provides antiproliferation of oral cancer cells by oxidative stress-mediated apoptosis and DNA damage. Cancers 2019 , 11 , 1303. [CrossRef] [PubMed] 25. Wu, P.-H.; Onodera, Y.; Recuenco, F.C.; Giaccia, A.J.; Le, Q.-T.; Shimizu, S.; Shirato, H.; Nam, J.-M. Lambda-carrageenan enhances the e ff ects of radiation therapy in cancer treatment by suppressing cancer cell invasion and metastasis through Racgap1 inhibition. Cancers 2019 , 11 , 1192. [CrossRef] 26. Cernigliaro, C.; D’Anneo, A.; Carlisi, D.; Giuliano, M.; Marino Gammazza, A.; Barone, R.; Longhitano, L.; Cappello, F.; Emanuele, S.; Distefano, A.; et al. Ethanol-mediated stress promotes autophagic survival and aggressiveness of colon cancer cells via activation of Nrf2 / HO-1 pathway. Cancers 2019 , 11 , 505. [CrossRef] 27. AbouAitah, K.; Hassan, H.A.; Swiderska-Sroda, A.; Gohar, L.; Shaker, O.G.; Wojnarowicz, J.; Opalinska, A.; Smalc-Koziorowska, J.; Gierlotka, S.; Lojkowski, W. Targeted nano-drug delivery of colchicine against colon cancer cells by means of mesoporous silica nanoparticles. Cancers 2020 , 12 , 144. [CrossRef] 28. Del Corn ò , M.; Gessani, S.; Conti, L. Shaping the innate immune response by dietary glucans: Any role in the control of cancer? Cancers 2020 , 12 , 155. [CrossRef] 29. Lee, C.H. Reversal of epithelial–mesenchymal transition by natural anti-inflammatory and pro-resolving lipids. Cancers 2019 , 11 , 1841. [CrossRef] 30. Ennour-Idrissi, K.; Ayotte, P.; Diorio, C. Persistent Organic pollutants and breast cancer: A systematic review and critical appraisal of the literature. Cancers 2019 , 11 , 1063. [CrossRef] 31. Farooqi, A.A.; Qureshi, M.Z.; Khalid, S.; Attar, R.; Martinelli, C.; Sabitaliyevich, U.Y.; Nurmurzayevich, S.B.; Taverna, S.; Poltronieri, P.; Xu, B. Regulation of cell signaling pathways by berberine in di ff erent cancers: Searching for missing pieces of an incomplete jig-saw puzzle for an e ff ective cancer therapy. Cancers 2019 , 11 , 478. [CrossRef] [PubMed] © 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http: // creativecommons.org / licenses / by / 4.0 / ). 5 cancers Article Fatty Acid Synthase Inhibitor G28 Shows Anticancer Activity in EGFR Tyrosine Kinase Inhibitor Resistant Lung Adenocarcinoma Models Emma Polonio-Alcal á 1,2, † , S ò nia Palomeras 1, † , Daniel Torres-Oteros 3 , Joana Relat 3,4,5 , Marta Planas 6 , Lidia Feliu 6 , Joaquim Ciurana 2 , Santiago Ruiz-Mart í nez 1, * and Teresa Puig 1, * 1 New Therapeutic Targets Laboratory (TargetsLab)-Oncology Unit, Department of Medical Sciences, Faculty of Medicine, University of Girona, 17003 Girona, Spain; emma.polonio@udg.edu (E.P.-A.); sonia.palomeras@udg.edu (S.P.) 2 Product, Process and Production Engineering Research Group (GREP), Department of Mechanical Engineering and Industrial Construction, University of Girona, 17003 Girona, Spain; quim.ciurana@udg.edu 3 Department of Nutrition, Food Sciences and Gastronomy, School of Pharmacy and Food Sciences, Food Torribera Campus, University of Barcelona, E-08921 Santa Coloma de Gramanet, Spain; danytoot@hotmail.com (D.T.-O.); jrelat@ub.edu (J.R.) 4 Institute of Nutrition and Food Safety of the University of Barcelona (INSA-UB), E-08921 Santa Coloma de Gramenet, Spain 5 CIBER Physiopathology of Obesity and Nutrition (CIBER-OBN), Instituto de Salud Carlos III, E-28029 Madrid, Spain 6 LIPPSO, Department of Chemistry, University of Girona, 17003 Girona, Spain; marta.planas@udg.edu (M.P.); lidia.feliu@udg.edu (L.F.) * Correspondence: santiago.ruiz@udg.edu (S.R.-M.); teresa.puig@udg.edu (T.P.); Tel.: + 34-972-419-548 (S.R.-M.); + 34-972-419-628 (T.P.) † These authors contributed equally to this work. Received: 26 March 2020; Accepted: 16 May 2020; Published: 19 May 2020 Abstract: Epidermal growth factor receptor (EGFR) tyrosine kinases inhibitors (TKIs) are e ff ective therapies for non-small cell lung cancer (NSCLC) patients whose tumors harbor an EGFR activating mutation. However, this treatment is not curative due to primary and secondary resistance such as T790M mutation in exon 20. Recently, activation of transducer and activator of transcription 3 (STAT3) in NSCLC appeared as an alternative resistance mechanism allowing cancer cells to elude the EGFR signaling. Overexpression of fatty acid synthase (FASN), a multifunctional enzyme essential for endogenous lipogenesis, has been related to resistance and the regulation of the EGFR / Jak2 / STAT signaling pathways. Using EGFR mutated (EGFRm) NSCLC sensitive and EGFR TKIs’ resistant models (Gefitinib Resistant, GR) we studied the role of the natural polyphenolic anti-FASN compound ( − )-epigallocatechin-3-gallate (EGCG), and its derivative G28 to overcome EGFR TKIs’ resistance. We show that G28’s cytotoxicity is independent of TKIs’ resistance mechanisms displaying synergistic e ff ects in combination with gefitinib and osimertinib in the resistant T790M negative (T790M − ) model and showing a reduction of activated EGFR and STAT3 in T790M positive (T790M + ) models. Our results provide the bases for further investigation of G28 in combination with TKIs to overcome the EGFR TKI resistance in NSCLC. Keywords: NSCLC; EGFR TKI; FASN inhibitors; resistance; STAT3; EGCG 1. Introduction Lung cancer is the most incident and the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) subtype is the most common and it represents 80–85% of lung cancers diagnosed. Cancers 2020 , 12 , 1283; doi:10.3390 / cancers12051283 www.mdpi.com / journal / cancers 7 Cancers 2020 , 12 , 1283 Early-stage NSCLC patients have long-term survival after surgery. However, approximately 75% of patients are diagnosed in advanced stages [1,2]. Gefitinib is a first generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). It was approved in 2003 by the Food and Drug Administration (FDA) for the treatment of patients whose tumors harbor an EGFR sensitizing / activating mutation (EGFRm) i.e., exon 19 deletion ( Δ E746-A750) or the point mutation in exon 21 that leads to the amino acid substitution L858R [ 3 – 5 ]. Despite this therapy represents a breakthrough in the treatment of EGFRm NSCLC patients, in a median time of 9–16 months nearly all patients do not achieve a complete response. One of the most common resistance mechanisms described is the EGFR point mutation in exon 20 that leads to the replacement of threonine 790 by methionine (T790M), which normally derives to lethal disease progression [ 6 ]. Osimertinib is an irreversible third generation TKI e ff ective in EGFRm T790M positive (T790M + ) patients. However, the point mutation C797S in exon 20 has appeared as the main resistance mechanism to the latest FDA-approved TKI [ 6 ]. Other mechanisms for EGFR TKI resistance include Met amplification, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3KCA) mutations, appearance of stem-like properties as evidenced by increase in epithelial–mesenchymal transition (EMT) and histological transformation, epidermal growth factor receptor type 2 (ErbB2) gene amplification, increase of insulin-like growth factor 1 receptor (IGF-1R) signaling pathway through the loss of inhibitory insulin-like growth factor-binding protein (IGF-BP) and loss or reduction of phosphatase and tensin homolog (PTEN), activation of AXL tyrosine kinase receptor or B-Raf proto-oncogene, and serine / threonine kinase (BRAF) mutations [6–12]. Recently, the activation of a signal transducer and activator of transcription 3 (STAT3) has been described as an alternative resistance mechanism allowing cancer cells to escape the EGFR signaling or the TKI suppression [ 13 ]. STAT3 is involved in the transcription of many genes related to cell di ff erentiation, proliferation, resistance to apoptosis, angiogenesis, metastasis, and immune response [ 14 – 16 ]. Besides being phosphorylated by EGFR [ 17 ], STAT3 can also be activated in response to di ff erent cytokines and growth factors such as interleukin 6 (IL-6), interferon-alpha (IFN- α ) or epidermal growth factor (EGF), among others [ 18 ]. Approximately 60% of patients show STAT3 activation, which correlates with poorly di ff erentiated tumors, the presence of metastasis, and the late clinical stage [ 19 , 20 ]. STAT3 phosphorylation has been related to disease progression in a small cohort of patients after EGFR TKI treatment [ 21 ]. Additionally, neither gefitinib nor osimertinib are able to inhibit STAT3 activation [22,23]. Energy metabolism deregulation has been described as a hallmark of cancer, allowing cell growth and proliferation [ 24 ]. Fatty acid synthase (FASN) is an essential enzyme for the de novo synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and NADPH [ 25 ]. Unlike most normal cells, highly-proliferative cancer cells overexpress this lipogenic enzyme, being an interesting target in cancer therapy [ 26 , 27 ]. FASN is strongly associated to poor prognosis and resistance to treatment in di ff erent human tumors such as breast [28], bladder [29], pancreatic [30], or lung cancer [31]. Moreover, FASN overexpression has also been proposed as a multidrug resistance mechanism, protecting cells from drug-induced apoptosis through the overproduction of palmitic acid [ 32 ]. The natural compound ( − )-epigallocatechin-3-gallate (EGCG) is a polyphenolic flavonoid from green tea that has been broadly studied for its cardiovascular, neuroprotective, anticancer, and antimicrobial properties [ 33 , 34 ]. EGCG has been reported to compete with NADPH to bind the β -ketoacyl reductase domain of FASN [ 35 ]. The ability of several FASN inhibitors to regulate the canonical EGFR / Jak2 / STAT pathway has also been stated in the literature [ 36 , 37 ]. We and others have shown that FASN inhibition is mainly related to EGFR / HER2 signaling pathways, leading to cytotoxic e ff ects in vitro and in vivo in a wide range of carcinomas, including breast and lung [ 38 – 42 ]. To date, many EGCG derivatives have been developed to improve e ffi cacy and increase stability in physiological conditions. Among them, the naphthalene derivative G28 has shown interesting antiproliferative features against sensitive and resistant breast cancer cells [38,43,44]. 8 Cancers 2020 , 12 , 1283 The purpose of this work was to study the role of FASN inhibitors (EGCG and G28) to overcome TKI resistance in NSCLC. FASN inhibitors were tested alone and in combination with EGFR TKIs (gefitinib and osimertinib) in EGFRm NSCLC models resistant to EGFR TKIs (Gefitinib Resistant, GR). In addition, we also evaluated gene and protein expression changes of FASN, EGFR, and STAT3 resulting from treatments with FASN inhibitors and EGFR TKIs alone or in combination. We show that FASN inhibitor G28 cytotoxicity is independent of EGFR TKI resistance mechanisms. Interestingly, G28 compound exhibited a cytotoxic e ff ect in combination with gefitinib showing changes in EGFR / STAT3 pathway in T790M positive (T790M + ) GR models and strong synergism in combination with gefitinib or osimertinib in T790M negative (T790M − ) GR model. 2. Results 2.1. EGFRm GR NSCLC Models Are Sensitive to FASN Inhibition In order to study the role of FASN in the acquisition of EGFR TKI resistance in NSCLC, we used the sensitive PC9 cell line carrying the EGFR exon 19 deletion (ELREA) and three GR models, two T790M + models (PC9-GR1 and PC9-GR4), and one T790M − model (PC9-GR3) [45]. 2.1.1. EGFRm NSCLC Models Overexpress FASN Firstly, we determined whether EGFRm NSCLC models express FASN enzyme. Hence, FASN protein (Figure 1a) and mRNA expression levels (Figure 1b) were analyzed by immunoblotting and quantitative real time PCR (qRT-PCR), respectively. Figure 1. FASN protein and mRNA expression levels in sensitive (PC9) and Gefitinib Resistant (GR) models (PC9-GR1, PC9-GR3, and PC9-GR4). ( a ) Western blot analysis (quantification in upper panel and bands in lower panel) of FASN protein expression. GAPDH was used as a loading control. Results shown are representative from at least three independent experiments. ( b ) FASN endogenous mRNA levels were obtained by qRT-PCR and normalized against the GAPDH gene. FASN expression in the sensitive cells was normalized to 1 an expressed as a fold change, to which all other conditions were compared. Results shown are mean ± SE from three independent experiments. * p < 0.050, *** p < 0.001 indicate levels of statistically significance. 9 Cancers 2020 , 12 , 1283 All models showed FASN protein and mRNA expression. Despite no di ff erences in mRNA, GR models presented significantly higher protein expression levels (PC9-GR1 p = 8.710 × 10 − 4 ; PC9-GR3 p = 3.160 × 10 − 4 , and PC9-GR4 p = 0.049) in comparison to PC9. 2.1.2. PC9-GR3 Model Is Resistant to Gefitinib and Osimertinib We confirmed the resistance to EGFR TKIs in PC9 and GR models. For that, we measured the cytotoxic e ff ect of gefitinib and osimertinib on all models by determining the half-maximal inhibitory concentration (IC 50 ) using the MTT assay (Figure 2). Figure 2. Cell proliferation inhibition of EGFR TKIs (gefitinib and osimertinib) in parental and Gefitinib Resistant (GR) models. Sensitive (PC9) and GR models (PC9-GR1, PC9-GR3, and PC9-GR4) were treated with increasing concentrations of ( a ) gefitinib (from 2.5 × 10 − 3 to 1 μ M for PC9 and 1–40 μ M for GR models) and ( b ) osimertinib (0.02