Ziziphus Nummularia Attenuates the Malignant Phenotype of Human Pancreatic Cancer Cells: Role of ROS

molecules Article Ziziphus nummularia Attenuates the Malignant Phenotype of Human Pancreatic Cancer Cells: Role of ROS Joelle Mesmar 1,†, Manal M. Fardoun 1,†, Rola Abdallah 1,†, Yusra Al Dhaheri 2, Hadi M. Yassine 3 , Rabah Iratni 2 , Adnan Badran 4, Ali H. Eid 5,6,* and Elias Baydoun 1,* 1 Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut P.O. Box 11-0236, Lebanon; [email protected] (J.M.); [email protected] (M.M.F.); [email protected] (R.A.) 2 Department of Biology, College of Science, United Arab Emirates University, Al-Ain P.O. Box 15551, United Arab Emirates; [email protected] (Y.A.D.); [email protected] (R.I.) 3 Biomedical Research Center, Qatar University, Doha P.O. Box 2713, Qatar; [email protected] 4 Department of Basic Sciences, University of Petra, Amman P.O. Box 961343, Jordan; [email protected] 5 Department of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, Doha P.O. Box 2713, Qatar 6 Biomedical and Pharmaceutical Research Unit, QU Health, Qatar University, Doha P.O. Box 2713, Qatar * Correspondence: [email protected] (A.H.E.); [email protected] (E.B.) † These authors contributed equally to this work. Abstract: Pancreatic cancer (PC) is the fourth leading cause of all cancer-related deaths. Despite major improvements in treating PC, low survival rate remains a major challenge, indicating the need Citation: Mesmar, J.; Fardoun, M.M.; for alternative approaches, including herbal medicine. Among medicinal plants is Ziziphus nummu- Abdallah, R.; Al Dhaheri, Y.; Yassine, laria (family Rhamnaceae), which is a thorny shrub rich in bioactive molecules. Leaves of Ziziphus H.M.; Iratni, R.; Badran, A.; Eid, A.H.; nummularia have been used to treat many pathological conditions, including cancer. However, their Baydoun, E. Ziziphus nummularia effects on human PC are still unknown. Here, we show that the treatment of human pancreatic ductal Attenuates the Malignant Phenotype adenocarcinoma cells (Capan-2) with Ziziphus nummularia ethanolic extract (ZNE) (100–300 µg/mL) of Human Pancreatic Cancer Cells: Role of ROS. Molecules 2021, 26, 4295. attenuated cell proliferation in a time- and concentration-dependent manner. Pretreatment with https://doi.org/10.3390/ N-acetylcysteine, an ROS scavenger, attenuated the anti-proliferative effect of ZNE. In addition, molecules26144295 ZNE significantly decreased the migratory and invasive capacity of Capan-2 with a concomitant downregulation of integrin α2 and increased cell–cell aggregation. In addition, ZNE inhibited in ovo Academic Editors: angiogenesis as well as reduced VEGF and nitric oxide levels. Furthermore, ZNE downregulated Alessandro Attanzio and the ERK1/2 and NF-κB signaling pathways, which are known to drive tumorigenic and metastatic Luisa Tesoriere events. Taken together, our results suggest that ZNE can attenuate the malignant phenotype of Capan-2 by inhibiting hallmarks of PC. Our data also provide evidence for the potential anticancer Received: 14 April 2021 effect of Ziziphus nummularia, which may represent a new resource of novel anticancer compounds, Accepted: 8 July 2021 especially ones that can be utilized for the management of PC. Published: 15 July 2021 Keywords: herbal medicine; pancreatic cancer; malignant phenotype; ROS; Ziziphus nummularia Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Cancer is the second leading cause of death worldwide, accounting for 13% of global mortalities [1]. In 2018, 9.6 million people died from cancer [1], with pancreatic cancer (PC) being the fourth leading cause of cancer-related deaths [2]. Despite great advancements Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. in its management and treatment, PC continues to exhibit a low survival rate with 95% This article is an open access article of cases being incurable [3,4]. This low rate is largely due to delayed diagnosis as well distributed under the terms and as resistance to therapies [5,6]. This prompts the need for alternative approaches such conditions of the Creative Commons as herbal medicine. Relevantly, the use of herbs and plants for the treatment of several Attribution (CC BY) license (https:// diseases including cancer is receiving increasing interest due to its low cost, somewhat creativecommons.org/licenses/by/ more tolerable side effects, and more acceptance among patients, in addition to evidence 4.0/). from folk medicine and accumulated wisdom of previous generations [7]. Molecules 2021, 26, 4295. https://doi.org/10.3390/molecules26144295 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 4295 2 of 23 Ziziphus nummularia, commonly known as Sidr, is a branched thorny shrub belonging to the family Rhamnaceae [8]. It grows largely in arid and dry areas [9], and it has been used in folk medicine [10,11]. Since it is bountiful in bioactive molecules such as tannins, flavonoids, steroids, glycosides, and alkaloids, Ziziphus nummularia has been used to treat a wide spectrum of pathological conditions including cold, diarrhea, anemia, bronchitis, indigestion, and inflammation [12]. In addition, its extracts are well known to inhibit the malignant phenotype of several cancer types, such as human breast cancer (MCF-7), leukemia (K-562), ovarian cancer (OVCAR-3), human colon adenocarcinoma (HT-29), and human kidney carcinoma (A-498) [13,14]. In vivo, a chemical extracted from Z. nummularia decreased the tumor volume of Ehrlich ascites carcinoma-bearing mice, and more importantly, it increased their survival [13]. These studies suggest that Z. nummularia may have a beneficial effect in the fight against cancer, which prompted us to test the effect of this shrub on PC, which is a rather formidable and unpreventable type of cancer [15]. In the present study, we examined the effect of the Z. nummularia ethanolic extract (ZNE) on the malignant phenotype of human pancreatic ductal adenocarcinoma cells (Capan-2). We tested the effects of ZNE on the proliferation, migration, adhesion, aggregation, and invasion of Capan-2. In addition, we assessed whether ZNE modulates in ovo angiogenesis using a chorioallantoic membrane assay. To mechanistically determine the basis of ZNE effects, we also assessed the phosphorylation of ERK1/2 activity, α2 integrin expression, and VEGF levels. Moreover, the roles of ROS and NF-κB in the ZNE-modulated proliferation of Capan-2 cells were tested. 2. Results 2.1. ZNE Inhibits the Proliferation of Capan-2 Pancreatic Cancer Cells To examine the anti-proliferative activity of Ziziphus nummularia ethanolic extract (ZNE) on PC cells, the effect of various concentrations (0, 100, 200, 300, and 600 µg/mL) on the viability of the Capan-2 cell line was assessed (Figure1a). The results show that ZNE treatment decreased cell viability in a concentration-dependent manner. For instance, cell viability was 92.9 ± 2.8, 54.9 ± 7.8, 31.3 ± 6.5, or 1.8 ± 2.8% that of control after treatment with 100, 200, 300, or 600 µg/mL of ZNE, respectively. The IC50 (half maximal inhibitory concentration) was 251, 217, and 209 µg/mL at 24, 48, and 72 h, respectively. Morphological observation of ZNE-treated cells not only showed a concentration- dependent decrease in the number of cells per microscopic field but also that ZNE-treated cells exhibited features characteristic of apoptosis (Figure1b). Indeed, at higher magnifications, cells showed cytoplasmic shrinkage, rounded shape, loss of epithelial morphology, and appearance of membrane blebbing and apoptotic bodies (Figure1c,d). 2.2. ZNE Induced Caspase-3-Dependent Apoptosis in Capan-2 Cells Having shown that ZNE induces morphological changes characteristic of apoptosis, we next sought to confirm that apoptosis is indeed activated in Capan-2 cells. Toward this, we first examined the protein level of procaspase-3, which upon activation of apoptosis becomes cleaved into a lower molecular weight active caspase-2. Western blotting analysis showed that the level of procaspase-3 in ZNE-treated capan-2 cells was significantly reduced (1 versus 0.6 ± 0.07-fold reduction). This suggests that ZNE likely induces apoptosis in these cells (Figure2a). To further confirm apoptosis in ZNE-treated PC cells, we measured the activity of active caspase 3/7. Our results shown clearly show a significant activation of caspase 3/7 in ZNE-treated compared to vehicle-treated cells (2.67 ± 0.35-fold increase; Figure2b ). To- gether, these results strongly suggest that ZNE mediates its anti-pancreatic cancer activities, at least through the activation of the type I programmed cell death (PCD1). Molecules 2021, 26, 4295 3 of 23 Molecules 2021, 26, x FOR PEER REVIEW 3 of 23 Figure 1. InhibitionFigure of cellular 1. Inhibition viability ofby cellularZiziphus viability nummularia by Ziziphus ethanolic nummularia extract. (a) Capanethanolic-2 cancer extract. cells were(a) Capan-2 treated with and without cancerthe indicated cells were concentrations treated with of Ziziphus and without nummularia the indicated ethanolic concentrations extract (ZNE) for of Ziziphus24, 48, and nummularia 72 h. Cell viability was carriedethanolic using the extract metabolic (ZNE)-dye for- 24,based 48, MTT and 72assay h. Cell, as viabilitydescribed was in Materi carriedals using and Methods. the metabolic-dye-based Data represent the mean of three independentMTT assay, asexperiments described inperformed Materials inand triplicate. Methods. Morphological Data represent changes the were mean observed of three independentat magnifications of 4× (b), 10×experiments (c), or 40× (d performed). Arrows show in triplicate. (1) cell shrinkage, Morphological (2) membrane changes blebbing, were observed (3) apoptotic at magnifications bodies, and (4) of echinoid spikes. * denotes p < 0.05, and ** p < 0.01. 4× (b), 10× (c), or 40× (d). Arrows show (1) cell shrinkage, (2) membrane blebbing, (3) apoptotic bodies, and (4) echinoid spikes. * denotes p < 0.05, and ** p < 0.01. Molecules 2021, 26, x FOR PEER REVIEW 4 of 23 2.2.

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