Background:Colorectal cancer (CRC) is the third most common cancer in Qatar and a major health concern for the Qatari population. Qatar has the highest rate of colon cancer compared to other countries in the eastern Mediterranean, West Asia and North Africa. Colon cancer is the most common cancer among Qatar's male population and according to the world age standard rates, around 20.8% of male Qataris have this cancer. Although progress in diagnosis and treatment has helped to extend and save the lives of many colorectal cancer patients, it still remains as one of the most prevalent human cancers. Many drugs such as 5-fluorouracil (5-FU) are being used to treat colorectal cancer, but patient(s) response to these drugs vary widely in terms of efficacy and toxicity. Moreover, it is observed that in colon cancer patients the tumor starts to develop resistance against these drugs over the course of treatment. The harmful side effects exhibited by the drugs used in cancer therapy as well as the increasing frequency of resistance to drugs have become the most challenging issues in the treatment of colorectal cancer. Hence, there exists an immediate need to discover better targeted and reliable drugs that could act as therapeutic agents which can prevent colon cancer progression and control distant metastasis as well as cure the disease with minimal side effects. Chemotherapeutic agents obtained from natural sources (plants) holds promising potential and have gained significant recognition in the field of cancer therapy. Pristimerin is a triterpenoid quinine methide present in various plant species of Cleastraceae and Hippocrateaceae families. Pristimerin has been shown to inhibit the proliferation of glioma, leukemia, myeloma, breast, lung, prostate and pancreatic cancer cell lines. Recent studies shows that pristimerin is a potent inhibitor of NF-κB. Induction of apoptosis by pristimerin was found to involve activation of caspases, mitochondrial dysfunction and inhibition of Akt signaling pathways. Pristimerin was reported to induce apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl.Material and MethodsReagentsAntibodies against Caspase 3, caspase-9, cleaved caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyadenosine 5’-diphosphate ribose polymerase (PARP) antibody was purchased from Cell Signaling Technologies (Beverly, MA). BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit with BD GolgiPlug, Propidium Iodide Staining Solution, Annexin V Binding Buffer, Mitochondrial Membrane Potential Detection (JC-1) Kit, Stain Buffer (FBS), Annexin V-FITC antibody, H2AX (pS139)-Alexa Fluor 647 antibody, Rabbit Anti- Active Caspase-3- BV605 antibody and PARP Cleaved Form-AF700 antibody were obtained from BD Biosciences (NJ, USA). CellROX Deep Red Reagent, MitoSOX Red Mitochondrial Superoxide Indicator, SYTOX Blue Nucleic Acid Stain and Hoechst 33342 solution were obtained from Molecular Probes, Life Technologies (CA, USA). Pristimerin, Cell Counting Kit-8 (CCK-8), DAPI, Glutathione-Reduced (GSH) and N-Acetyl-L-Cysteine (NAC) were obtained from Sigma-Aldrich (MO, USA). Human Apoptosis Antibody Array and Phospho-Kinase Antibody Array were purchased from R&D systems (MN, USA). Cell Death Detection ELISAPLUS kit was purchased from Roche Diagnostics (Mannheim, Germany).MethodsCell culture: Human colorectal cancer cell line HCT116 was cultured in DMEM, OXCO1 and SW48 cells were cultured in RPMI 1640 medium. Both culture medium were supplemented with 10% Heat Inactivated fetal bovine serum (FBS), 100 U/ml Penicillin and 100 U/ml Streptomycin. Cells were cultured at 37°C under a humidified 95%:5% (v/v) mixture of air and CO2.Pristimerin was dissolved in dimethylsulfoxide as a 10 mM stock solution and stored at 4°C for the in vitro experiments. Further dilution was done in cell culture medium as required.Cell Viability: Effect on the viability of CRC cell lines, HCT116, OXCO1 and SW48 were determined following treatment with various doses of Pristimerin for 24, 48 and 72 hours using WST-8 kit.Apoptosis: HCT116/OXCO1 cells were treated with various doses of Pristimerin for 24 hours. After incubation, cells were harvested, washed with PBS and stained with Annexin V-FITC/PI for 20 minutes at room temperature and apoptosis was measured by flow cytometry using BD LSRFortessa analyzer (BD Biosciences, USA).H2AX, active caspase-3 and cleaved PARP were quantified by flow cytometry. After treatment with Pristimerin, HCT116 cells were fixed and permeabilized using BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit, as per protocol from the manufacturer. 0.5 × 106 cells in Stain Buffer (FBS) were stained with 3 μL each of H2AX (pS139)-Alexa Fluor 647, Rabbit Anti- Active Caspase-3- BV605 and PARP Cleaved Form-AF700 antibodies for 30 minutes at room temperature. The cells were washed with Stain Buffer (FBS) and then analyzed by flow cytometry.Cell cycle analysis: 0.5 × 106 cells (HCT116/OXCO1) were briefly stained with Hoechst 33342 solution (10 μg/mL) and then analyzed by flow cytometry.Mitochondrial membrane potential: 0.5 × 106 cells (HCT116/OXCO1) were briefly stained with JC-1 stain for 15 minutes at 37°C as per instructions from the kit manufacturer. The cells were washed twice with 1x assay buffer and then analyzed by flow cytometry.ROS Production: CellROX Deep Red Oxidative Stress Reagent is a fluorogenic probe designed to reliably measure reactive oxygen species (ROS) in live cells. The signals from CellROX Deep Red Reagent is localized in the cytoplasm. The production of superoxide by mitochondria was quantitated using the MitoSOX Red reagent. It is rapidly oxidized by superoxide but not by other reactive oxygen species and reactive nitrogen species. HCT116/OXCO1 cells were treated with Pristimerin (0, 1, 2.5, 5 μM) for 24 h and finally analyzed by flow cytometry for quantification of ROS and superoxide.HCT116 cells were preincubated with NAC (2.5 mM) or GSH (5 mM) for 30 minutes before addition of Pristimerin (5 μM) in studies to confirm the role of ROS in induction of apoptosis.Western blot: Following treatment with various doses/combinations of Pristimerin for 24 hours, HCT116 cells were lysed with RIPA buffer and proteins were isolated. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies. Target proteins were detected using an enhanced chemiluminescence (Bio-Rad ChemiDoc MP imaging system).Statistics: Two-tailed Student's t tests was performed using the Origin Pro software to compare means of different treatment groups and a P value below 0.05 was considered statistically significant.ResultsThe results show that Pristimerin causes a dose dependent inhibition of cell proliferation in various CRC cell lines, HCT116, OXCO1 and SW48. The inhibition of proliferation correlated with the induction of apoptosis in HCT116 and OXCO1 cell lines. Treatment with Pristimerin leads to activation of Bid, loss of mitochondrial membrane potential, as determined by JC1 staining, activation of caspase-9, subsequent activation of caspase-3 followed by polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage and DNA double strand breaks (H2AX staining). Pristimerin was found to increase the oxidative stress in CRC cells in a dose dependent manner. Pretreatment of HCT116 cells with NAC/GSH, was found to inhibit Pristimerin mediated induction of apoptosis, confirming the role of ROS.ConclusionAltogether, these data confirm the role of ROS in the inhibition of cell proliferation and the induction of apoptosis in CRC cell lines by Pristimerin, and thus provide a strong rationale for pursuing the detailed mechanism of action and confirming the anti cancer activity using in vivo models and future clinical studies.KeywordsColorectal cancer, Pristimerin, oxidative stress, apoptosis.