molecules Article A Methanol Extract of Scabiosa atropurpurea Enhances Doxorubicin Cytotoxicity against Resistant Colorectal Cancer Cells In Vitro Imene Ben Toumia 1,2,3,* , Mansour Sobeh 2,4, Marco Ponassi 3, Barbara Banelli 3, Anas Dameriha 2 , Michael Wink 2 , Leila Chekir Ghedira 1 and Camillo Rosano 3,* 1 Unit of Bioactive Natural Substances and Biotechnology UR17ES47, Faculty of Dental Medicine of Monastir, University of Monastir, Avicenne Street, 5000 Monastir, Tunisia; [email protected] 2 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany; [email protected] (M.S.); [email protected] (A.D.); [email protected] (M.W.) 3 IRCCS Policlinico San Martino, Largo R. Benzi 10, I-16132 Genova, Italy; [email protected] (M.P.); [email protected] (B.B.) 4 AgroBioSciences Research, Mohammed VI Polytechnic University, Lot 660–Hay MoulayRachid, 43150 Ben-Guerir, Morocco * Correspondence: [email protected] (I.B.T.); [email protected] (C.R.) Academic Editors: Bruno Botta, Cinzia Ingallina, Andrea Calcaterra and Deborah Quaglio Received: 30 September 2020; Accepted: 10 November 2020; Published: 12 November 2020 Abstract: Colorectal cancer is a malignancy with a high incidence. Currently, the drugs used in chemotherapy are often accompanied by strong side effects. Natural secondary metabolites can interfere with chemotherapeutic drugs and intensify their cytotoxic effects. This study aimed to profile the secondary metabolites from the methanol extract of Scabiosa atropurpurea and investigate their in vitro activities, alone or in combination with the chemotherapeutic agent doxorubicin. MTT assay was used to determine the cytotoxic activities. Annexin-V/PI double-staining analysis was employed to evaluate the apoptotic concentration. Multicaspase assay, quantitative reverse transcription real-time PCR (RT-qPCR), and ABC transporter activities were also performed. LC-MS analysis revealed 31 compounds including phenolic acids, flavonoids, and saponins. S. atropurpurea extract intensified doxorubicin anti-proliferative effects against resistant tumor cells and enhanced the cytotoxic effects towards Caco-2 cells after 48 h. The mRNA expression levels of Bax, caspase-3, and p21 were increased significantly whereas Bcl-2 expression level was decreased. Furthermore, the methanol extract reversed P-glycoprotein or multidrug resistance-associated protein in Caco-2 cells. In conclusion, S. atropurpurea improved chemosensitivity and modulated multidrug resistance in Caco-2 cells which makes it a good candidate for further research in order to develop a new potential cancer treatment. Keywords: Scabiosa atropurpurea; doxorubicin; Caco-2; multidrug resistance 1. Introduction Cancer is a world threat with nearly 9 million cancer-related deaths globally. Colorectal cancer (CRC) is diagnosed as the third most common cancer worldwide and the fourth leading cause of death related to malignancies. Globally, CRC is predicted to increase to 60% by 2030 [1]. This malignant disease is multifactorial and affected by several risk factors such as genetic, environmental factors, and lifestyle [2]. Chemotherapy is the most frequently applied therapy that demonstrated deleterious effects including 0.2–8.7% of chronic heart failure and 4–36% of cardiomyopathy among the patients [3]. Molecules 2020, 25, 5265; doi:10.3390/molecules25225265 www.mdpi.com/journal/molecules Molecules 2020, 25, 5265 2 of 12 During chemotherapy, drug resistance can be induced. Accordingly, higher doses are needed to achieve similar efficacy which leads to stronger side effects [4]. Drug combinations can be used as a way to synergisticallyMolecules 2020 potentiate, 25, x FOR PEER the REVIEW chemotherapeutic efficacy [5]. Generally, combining multiple2 of 13 drugs allows for the use of lower doses of conventional chemotherapeutical drugs, which may lead to the deleterious effects including 0.2–8.7% of chronic heart failure and 4–36% of cardiomyopathy among same or even higher efficacy due to synergism and lower resistance [5,6]. the patients [3]. During chemotherapy, drug resistance can be induced. Accordingly, higher doses Plantsare needed and their to achieve diverse similar secondary efficacy metabolites, which leads to among stronger them side polyphenols, effects [4]. Drug are combinations an interesting can source for the studybe used and as a developmentway to synergistically of drugs potentiate to treat the cancerchemotherapeutic [7]. The honeysuckleefficacy [5]. Generally, family, combining Caprifoliaceae, containsmultiple 42 genera, drugs among allows themfor the theuse genusof lowerScabiosa doses of[ 8conventional]. Scabiosa atropurpureachemotherapeuticalhas been drugs, demonstrated which as a valuablemay lead source to the ofsame pharmaceutical or even higher efficacy and health-promoting due to synergism and properties lower resistance because [5,6]. of its antioxidant, hepatoprotective,Plants and their hypoglycemic diverse secondary activities. metabolites, Chlorogenic among acidthem methylpolyphenols, ester, are luteolin, an interesting and luteolin source for the study and development of drugs to treat cancer [7]. The honeysuckle family, 7-O-glucosideCaprifoliaceae, have been contains isolated 42 genera, from among the total them ethanol the genus extract Scabiosa of aerial[8]. Scabiosa parts atropurpurea [9], whereby has luteolin been and chlorogenicdemonstrated acid were as reporteda valuable tosource show of pharmaceutical anticancer activity and health-promoting [10,11]. properties because of its Despiteantioxidant, the existence hepatoprotective, of several and andhypoglycemic valuable acti biologicalvities. Chlorogenic data about acid S.methyl atropurpurea ester, luteolin,properties, the chemicaland luteolin constituents 7-O-glucoside of its have methanol been isolated extract from remain the to uncharacterized.tal ethanol extract of Furthermore, aerial parts [9], the full anticancerwhereby potential luteolin of and the chlorogenic extract has acid not were yet repo beenrted exhaustively to show anticancer investigated. activity [10,11]. In the present study, Despite the existence of several and valuable biological data about S. atropurpurea properties, the we profiled the phytoconstituents of the methanol leaf extract utilizing LC-MS/MS. We determined its chemical constituents of its methanol extract remain uncharacterized. Furthermore, the full anticanceranticancer activities potential alone of and the as extract doxorubicin has not yet adjuvant been exhaustivelyin vitro against investigated. Caco-2 In cells, the present resistant study, colorectal cancer cells.we profiled Additionally, the phytoconstituents the ability of of the the extractmethanol to leaf modulate extract utilizing multidrug LC- resistance-associatedMS/MS. We determined protein 1 (MRP1)itsand anticancer P-glycoprotein activities alone (P-gp, and MDR1, as doxorubicin or ABCB1) adjuvant was explored.in vitro against Caco-2 cells, resistant colorectal cancer cells. Additionally, the ability of the extract to modulate multidrug resistance- 2. Resultsassociated protein 1 (MRP1) and p-glycoprotein (p-gp, MDR1, or ABCB1) was explored. 2.1. LC-MS2. Results Analysis of Scabiosa atropurpurea Leaves HPLC-MS2.1. LC-MS/MS Analysis in the of negative Scabiosa atropurpurea mode (ESI Leaves ( )) was used to characterize the secondary metabolites − of the methanolHPLC-MS/MS leaf extract in the fromnegativeS. mode atropurpurea (ESI (−)) .was We used tentatively to characterize identified the secondary 31 compounds metabolites based on theirof molecular the methanol weights leaf extract and from fragmentation S. atropurpurea. pattern We tentatively according identified to the 31 literature, compounds in-house based on library, and authentictheir molecular references. weights The and compoundsfragmentation belongpattern according to several to the classes: literature, phenolic in-houseacids, library,flavonoids, and and saponins.authentic The references. LC-MS profileThe compounds of the leaf belong extract to isseveral shown classes: in Figure phenolic1 and acids, the chemical flavonoids, composition and is presentedsaponins. in Table The LC-MS1. profile of the leaf extract is shown in Figure 1 and the chemical composition is presented in Table 1. Figure 1. LC-MS profile of the methanol extract from S. atropurpurea leaves. Molecules 2020, 25, 5265 3 of 12 Table 1. Polyphenolics in a methanol extract of S. atropurpurea leaves. No. Rt M H MS/MS Tentatively Identified Compound − 1 1.43 191 127, 173 Quinic acid a 2 7.71 315 153 Protocatechuic acid 3-glucoside a 3 10.35 325 119, 163 p-Coumaric acid 3-glucoside a 4 11.28 353 179, 191 Neochlorogenic acid 5 12.46 353 179, 191 Cryptochlorogenic acid 6 13.53 353 179, 191 Chlorogenic acid a,b 7 14.28 373 149, 193 Ferrulic acid derivative 8 15.82 625 301, 463 Quercetin diglucoside 9 16.07 337 119, 163, 191 p-Coumeroylquinic acid a 10 17.12 609 285, 327, 447 Luteolin-7,3-diglucoside a 11 17.52 625 179, 255, 301, 463 Quercetin 3,4’-diglucoside a 12 17.62 367 127, 173, 191 3-O-Caffeoylquinic acid methyl ester 13 19.16 609 285, 327, 447 Luteolin-7,3’-diglucoside a 14 22.95 515 353 3,4-Dicaffeoylquinic acid 15 23.35 579 285, 447 Luteolin-pentosyl-glucoside 16 23.89 579 285, 447 Luteolin-pentosyl-glucoside 17 24.43 515 179, 353 3,5-Dicaffeoylquinic acid 18 25.05 593 285, 447 Luteolin 7-rutinoside a 19 25.71 447 285 Luteolin 7-O-β-d-glucoside b 20 26.88 447 179, 285 Luteolin 3’-glucoside