Biochemical and Molecular Investigation of In Vitro Antioxidant and Anticancer Activity Spectrum of Crude Extracts of Willow Leaves <i>Salix safsaf</i>
Mourad A. M. Aboul-Soud,
Abdelkader E. Ashour,
Jonathan K. Challis,
Atallah F. Ahmed,
Ashok Kumar,
Amr Nassrallah,
Tariq A. Alahmari,
Quaiser Saquib,
Maqsood A. Siddiqui,
Yazeed Al-Sheikh,
Hany A. El-Shemy,
Ahmed M. Aboul-Enein,
Khalid M. Alghamdi,
Paul D. Jones,
John P. Giesy
Affiliations
Mourad A. M. Aboul-Soud
Chair of Medical and Molecular Genetics Research, Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh 11433, Saudi Arabia
Abdelkader E. Ashour
Department of Basic Medical Sciences, Kulliyyah (College) of Medicine, International Islamic University Malaysia, Kuantan 25200, Pahang, Malaysia
Jonathan K. Challis
Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
Atallah F. Ahmed
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
Ashok Kumar
Vitiligo Research Chair, College of Medicine, King Saud University, Riyadh 11451, Saudi Arabia
Amr Nassrallah
Biochemistry Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt
Tariq A. Alahmari
Chair of Medical and Molecular Genetics Research, Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh 11433, Saudi Arabia
Quaiser Saquib
Zoology Department, College of Sciences, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
Maqsood A. Siddiqui
Zoology Department, College of Sciences, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
Yazeed Al-Sheikh
Chair of Medical and Molecular Genetics Research, Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh 11433, Saudi Arabia
Hany A. El-Shemy
Biochemistry Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt
Ahmed M. Aboul-Enein
Biochemistry Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt
Khalid M. Alghamdi
Vitiligo Research Chair, College of Medicine, King Saud University, Riyadh 11451, Saudi Arabia
Paul D. Jones
Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
John P. Giesy
Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
Organic fractions and extracts of willow (Salix safsaf) leaves, produced by sequential solvent extraction as well as infusion and decoction, exhibited anticancer potencies in four cancerous cell lines, including breast (MCF-7), colorectal (HCT-116), cervical (HeLa) and liver (HepG2). Results of the MTT assay revealed that chloroform (CHCl3) and ethyl acetate (EtOAc)-soluble fractions exhibited specific anticancer activities as marginal toxicities were observed against two non-cancerous control cell lines (BJ-1 and MCF-12). Ultra-high-resolution mass spectrometry Q-Exactive™ HF Hybrid Quadrupole-Orbitrap™ coupled with liquid chromatography (UHPLC) indicated that both extracts are enriched in features belonging to major phenolic and purine derivatives. Fluorescence-activated cell sorter analysis (FACS), employing annexin V-FITC/PI double staining indicated that the observed cytotoxic potency was mediated via apoptosis. FACS analysis, monitoring the increase in fluorescence signal, associated with oxidation of DCFH to DCF, indicated that the mechanism of apoptosis is independent of reactive oxygen species (ROS). Results of immunoblotting and RT-qPCR assays showed that treatment with organic fractions under investigation resulted in significant up-regulation of pro-apoptotic protein and mRNA markers for Caspase-3, p53 and Bax, whereas it resulted in a significant reduction in amounts of both protein and mRNA of the anti-apoptotic marker Bcl-2. FACS analysis also indicated that pre-treatment and co-treatment of human amniotic epithelial (WISH) cells exposed to the ROS H2O2 with EtOAc fraction provide a cytoprotective and antioxidant capacity against generated oxidative stress. In conclusion, our findings highlight the importance of natural phenolic and flavonoid compounds with unparalleled and unique antioxidant and anticancer properties.
