Novel Quinazoline-based Sulfonamide Derivative (3D) Induces Apoptosis in Colorectal Cancer by Inhibiting JAK2-STAT3 Pathway

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 species  

INTRODUCTION

Colorectal cancer (CRC) has the third highest incidence rate of malignant tumors worldwide. CRC is associated with increased morbidity and mortality over the years.¹ In the USA, it is estimated that 134,000 patients were diagnosed with CRC in 2016.² Surgery is the mainstay of treatment for early CRC. CRC patients are diagnosed with an advanced stage of disease, often with distant metastasis. Gastrointestinal epithelial cells acquire genetic and epigenetic mutations in oncogenes and tumor suppressor genes, which confer them for selective advantage on proliferation and self-renewal.^3,4Aberrant inflammation of gastrointestinal tract by bacterial infections also leads to CRC development.⁵Tumor growth and progression during tumor-associated inflammation involve multiple mechanisms including aberrant proliferation, antiapoptosis, angiogenesis, metastasis and tumor evasion.^6,7 During CRC development, normal epithelial cells become hyperproliferative that leads to benign adenoma, which further evolves into carcinoma leading to CRC metastasis.⁸

Many inflammatory signals promote tumorigenesis by involving activation of NF-κB and JAK/STAT signaling pathways. The JAK–STAT pathway is known be involved in tumor-associated inflammation, proliferation, invasion and migration. The STAT3 proteins are transcription factors that participate in cellular signaling using cytokine and growth factors from the cell membrane into nucleus. Constitutive activation of STAT3 has been reported in various human malignancies such as breast cancer, pancreatic cancer and glioblastoma.^9–12 Several previous reports have demonstrated that modulating JAK2–STAT3 pathway arrests growth of primary human cancer cells and promotes antitumor immunity.¹³ STAT3 as a drug target has been exploited in several cancers.¹⁴ Some studies have also reported that the inhibition of the JAK–STAT pathway induces apoptosis in CRC cells.¹⁵ During cancer pathogenesis, apoptosis deregulation has been widely recognized as a hallmark of cancer.¹⁶ Mainly, there are two major signaling pathways leading to cell death. The first is the death receptor-mediated extrinsic apoptotic pathway, which is activated from outside by proapoptotic ligands binding to the cell surface death receptor.¹⁷ The second is the mitochondrial apoptotic pathway (intrinsic apoptotic pathway), which is activated from inside the cell by Bcl2 family proteins.¹⁸ Both these pathways converge onto the activation of effector caspases, leading to apoptotic cell death program.

Recently, we published some derivatives of aminobenzenesulfonamide as potential antitumor agents and evaluated the mechanism of a novel antitumor agent (3c) and found that this particular derivative acts by inducing reactive oxygen species (ROS)-mediated apoptosis in CRC.^19,20 In the present study, we evaluated the inhibitory effect of a novel quinazoline-based sulfonamide derivative, 4-((2-(4-(dimethylamino)phenyl)quinazolin-4-yl)amino)benzenesulfonamide (3D), in CRC. We demonstrated that compound 3D inhibits proliferation of CRC cell lines by inducing ROS-mediated apoptosis. This finding further indicated that 3D induces p53, Bax and the intrinsic mitochondrial apoptotic pathway and inhibits Bcl2 family protein. Furthermore, 3D inhibits constitutive and IL-6-induced STAT3 phosphorylation and target genes.