Novel derivative of aminobenzenesulfonamide (3c) induces apoptosis in colorectal cancer cells through ROS generation and inhibits cell migration

Background: Colorectal cancer (CRC) is the 3rd most common type of cancer worldwide. New anti-cancer agents are needed for treating late stage colorectal cancer as most of the deaths occur due to cancer metastasis. A recently developed compound, 3c has shown to have potent antitumor effect; however the mechanism underlying the antitumor effect remains unknown. Methods: 3c-induced inhibition of proliferation was measured in the absence and presence NAC using MTT in HT-29 and SW620 cells and xCELLigence RTCA DP instrument. 3c-induced apoptotic studies were performed using flow cytometry. 3c-induced redox alterations were measured by ROS production using fluorescence plate reader and flow cytometry and mitochondrial membrane potential by flow cytometry; NADPH and GSH levels were determined by colorimetric assays. Bcl2 family protein expression and cytochrome c release and PARP activation was done by western blotting. Caspase activation was measured by ELISA. Cell migration assay was done using the real time xCELLigence RTCA DP system in SW620 cells and wound healing assay in HT-29. Results: Many anticancer therapeutics exert their effects by inducing reactive oxygen species (ROS). In this study, we demonstrate that 3c-induced inhibition of cell proliferation is reversed by the antioxidant, N-acetylcysteine, suggesting that 3c acts via increased production of ROS in HT-29 cells. This was confirmed by the direct measurement of ROS in 3c-treated colorectal cancer cells. Additionally, treatment with 3c resulted in decreased NADPH and glutathione levels in HT-29 cells. Further, investigation of the apoptotic pathway showed increased release of cytochrome c resulting in the activation of caspase-9, which in turn activated caspase-3 and −6. 3c also (i) increased p53 and Bax expression, (ii) decreased Bcl2 and BclxL expression and (iii) induced PARP cleavage in human colorectal cancer cells. Confirming our observations, NAC significantly inhibited induction of apoptosis, ROS production, cytochrome c release and PARP cleavage. The results further demonstrate that 3c inhibits cell migration by modulating EMT markers and inhibiting TGFβ-induced phosphorylation of Smad2 and Samd3. Conclusions: Our findings thus demonstrate that 3c disrupts redox balance in colorectal cancer cells and support the notion that this agent may be effective for the treatment of colorectal cancer

IL-17 and colorectal cancer risk in the Middle East: gene polymorphisms and expression

Omar A Al Obeed,1 Mansoor-Ali Vaali-Mohamed,1 Khayal A Alkhayal,1 Thamer A Bin Traiki,1 Ahmad M Zubaidi,1 Maha Arafah,2 Robert A Harris,3 Zahid Khan,4,* Maha-Hamadien Abdulla1,* 1Colorectal Research Chair, Department of Surgery, King Khalid University Hospital, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 2Department of Pathology, King Khalid University Hospital, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 3Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden; 4Genome Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia *These authors contributed equally to this work Background: IL-17 expressed by Th17 cells play a crucial role in tissue inflammation by induction of proinflammatory and neutrophil mobilizing cytokines, and IL-17 polymorphisms are associated with colorectal cancer (CRC).Objective: We investigated the expression of IL-17 and the association of IL-17 gene polymorphisms with CRC susceptibility in a Middle East population.Materials and methods: The study included 117 diagnosed CRC patients and 100 age- and gender-matched healthy controls. IL-17A rs2275913 (G197A) and IL-17F rs763780 (T7488C) single nucleotide polymorphisms, mRNA, and protein levels of IL-17A were assessed. Results: We observed significant association between rs2275913 in IL-17A and susceptibility to CRC (p = 0.016228). The AG and AA genotypes conferred 2-fold and 2.8-fold, respectively, higher risk of developing CRC compared with individuals having GG genotype. Stratification of the data based on gender and age revealed very strong association of CRC with IL17A rs2275913 only in males and “AG” genotype in patients ≤57 years of age at the time of disease diagnosis. The rs763780 in IL-17F was not linked with CRCs in our cohort. Furthermore, IL-17A mRNA expression in CRCs was significantly elevated compared to adjacent normal tissues, particularly in early stages of disease (p = 0.0005). Strong immunoreactivity to IL-17A protein was observed in 70% of early stage relative to 30% of late-stage tumors. Conclusion: The IL-17A G197A variant may be utilized as a genetic screening marker in assessing CRC risk, and its expression can be used as a biomarker for early detection of CRC in the Saudi population. Keywords: interleukin (IL), genetic polymorphism, colorectal cancer, Saudi populatio

Novel quinazoline-based sulfonamide derivative (3D) induces apoptosis in colorectal cancer by inhibiting JAK2–STAT3 pathway

Omar Al-Obeed,1 Mansoor-Ali Vaali-Mohammed,1 Wagdy M Eldehna,2 Khayal Al-Khayal,1 Amer Mahmood,3 Hatem A Abdel-Aziz,4 Ahmed Zubaidi,1 Ahmed Alafeefy,5 Maha Abdulla,1 Rehan Ahmad1 1Colorectal Research Chair, Department of Surgery, King Khaled University Hospital, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt; 3Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Saudi Arabia; 4Department of Applied Organic Chemistry, National Research Center, Cairo, Egypt; 5Department of Chemistry, Kulliyyah of Science, International Islamic University, Kuantan, Malaysia Introduction: Colorectal cancer (CRC) is a major worldwide health problem owing to its high prevalence and mortality rate. Developments in screening, prevention, biomarker, personalized therapies and chemotherapy have improved detection and treatment. However, despite these advances, many patients with advanced metastatic tumors still succumb to the disease. New anticancer agents are needed for treating advanced stage CRC as most of the deaths occur due to cancer metastasis. A recently developed novel sulfonamide derivative 4-((2-(4-(dimethylamino) phenyl)quinazolin-4-yl)amino)benzenesulfonamide (3D) has shown potent antitumor effect; however, the mechanism underlying the antitumor effect remains unknown. Materials and methods: 3D-mediated inhibition on cell viability was evaluated by MTT and real-time cell proliferation was measured by xCelligence RTDP instrument. Western blotting was used to measure pro-apoptotic, anti-apoptotic proteins and JAK2-STAT3 phosphorylation. Flow cytometry was used to measure ROS production and apoptosis. Results: Our study revealed that 3D treatment significantly reduced the viability of human CRC cells HT-29 and SW620. Furthermore, 3D treatment induced the generation of reactive oxygen species (ROS) in human CRC cells. Confirming our observation, N-acetylcysteine significantly inhibited apoptosis. This is further evidenced by the induction of p53 and Bax; release of cytochrome c; activation of caspase-9, caspase-7 and caspase-3; and cleavage of PARP in 3D-treated cells. This compound was found to have a significant effect on the inhibition of antiapoptotic proteins Bcl2 and BclxL. The results further demonstrate that 3D inhibits JAK2–STAT3 pathway by decreasing the constitutive and IL-6-induced phosphorylation of STAT3. 3D also decreases STAT3 target genes such as cyclin D1 and survivin. Furthermore, a combination study of 3D with doxorubicin (Dox) also showed more potent effects than single treatment of Dox in the inhibition of cell viability. Conclusion: Taken together, these findings indicate that 3D induces ROS-mediated apoptosis and inhibits JAK2–STAT3 signaling in CRC. Keywords: sulfonamide, apoptosis, colorectal cancer, STAT3 pathway, Bcl2 proteins, reactive oxygen specie