3D inhibits JAK2 and STAT3 phosphorylation

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Phosphorylation of STAT3 at Tyr-705 is critical for its dimerization and nuclear translocation. STAT3 is constitutively activated in HT-29 cells. Treatment of HT-29 cells with 3D resulted in the inhibition of STAT3 phosphorylation (Figure 5A). Janus kinases are upstream kinases that are known to phosphorylate STAT3. 3D treatment of HT-29 cells inhibited JAK2 phosphorylation in a dose-dependent manner (Figure 5A). STAT3 phosphorylation can be induced by IL-6. SW620 cells, which do not express phosphorylated STAT3, were used to determine if 3D is capable of inhibiting IL-6-induced STAT3 phosphorylation. We found that 3D inhibited IL-6-induced phosphorylation of STAT3 in SW620 cell line (Figure 5B, left). STAT3 together with NF-κB is known to be involved in inflammation-induced cancer progression. We also determined if 3D may also block NF-κB activation; however, 3D was not found to have any inhibitory effect on NF-κB activation (Figure 5B, right). Binding of STAT3 to the binding site present in the promoters of the target genes induces the transcription of several proliferation genes. Cyclin D1 is a STAT3 target gene for inducing cell proliferation, and another STAT3 target gene survivin is also known to inhibit caspase activation and thereby to inhibit apoptosis. The expression of STAT3 target genes such as cyclin D1 and survivin was decreased after treatment with 3D in a dose-dependent manner in HT-29 cells (Figure 5C). Similar results were obtained in SW620 cells (Figure 5D). AG490 is a known STAT3 inhibitor; we asked if 3D could have synergistic effect with AG490. Indeed, 3D inhibited STAT3 phosphorylation along with cyclin D1 and survivin synergistically with AG490 (Figure 5E). These findings thus indicate that 3D inhibited the JAK–STAT pathway and downstream target genes in CRC cells.

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3D sensitizes HT-29 to the Dox treatment

To compare the anticancer effect of 3D and potential synergy with a known standard drug, combination studies were performed with Dox. We investigated how 3D and Dox influence growth and proliferation of cancer cells in combination therapy. Treatment of HT-29 cells with Dox alone or in combination with 3D indicated that 3D potentiated the effect of Dox on inhibition of proliferation. Dox inhibited the cell viability of HT-29 in a dose-dependent manner; however, 3D significantly potentiated the effect of Dox (Figure 6A). Dox was found to be ineffective at lower concentration in inhibiting cell viability of metastatic CRC cell line SW620. However, in the combination approach, Dox with 3D significantly inhibited the cell viability (Figure 6B).

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3D has been reported to exert anticancer effect.19 However, the biochemical basis of the anticancer effect of 3D remains unknown. In the present study, we found that 3D inhibited the cell viability of human CRC cell lines in a dose- and time-dependent manner. 3D was found to have more sensitive effect on metastatic CRC cell SW620 as compared to adenocarcinoma CRC cell HT-29. Most of the inhibition of cell viability and proliferation occurs due to apoptosis induction. Most anticancer agents target apoptosis as a major route to eradicate cancer. This novel quinazoline-based derivative of sulfonamide (3D) significantly induced apoptosis as demonstrated by flow cytometry. Various anticancer agents have been shown to exhibit their anticancer effect by altering the levels of ROS.20–22ROS are byproducts of the oxygen metabolism during oxidative stress. ROS plays a vital role in the maintenance of homeostasis. 3D-induced apoptosis was found to be inhibited with addition of antioxidant NAC. These findings confirmed that 3D-induced apoptosis is ROS dependent. Excessive production of ROS leads to a loss of cellular integrity and cell death.23 Several therapeutic agents used to treat different malignant tumors have been shown to generate a high level of ROS.24 Our findings demonstrated that 3D robustly induces ROS generation in a dose-dependent manner in human CRC cells. These results thus demonstrate that the ROS production is an upstream event and is essential for the apoptosis induction in 3D-treated cells. These findings strongly support the idea that 3D-induced increase in ROS production may be an effective therapeutic strategy against CRC. Mitochondria are considered a prime location where cellular stress signals converge leading to the execution of apoptosis.25 Loss of mitochondrial membrane potential is an early event in the intrinsic apoptotic pathway. 3D treatment resulted in a significant loss of mitochondrial membrane potential.

P53 is a well-known tumor suppressor that plays important role in apoptosis and prevents cells from tumorigenic alterations.26 Our compound of interest induces p53 expression in a dose-dependent manner. The balance between the proapoptotic proteins (p53, Bax) and antiapoptotic proteins (Bcl2, BclxL) determines the cell fate. The induction of Bax expression is essential for apoptosis in human colon cancer cells.27 The overexpression of Bcl2 and BclxL inhibits apoptosis and promotes survival of cancer cells.28 The antiapoptotic Bcl2 protein is reported to be overexpressed in colon cancer. The Bcl2-mediated apoptosis inhibition restores the tumorigenicity of regressed colon cancer.29 In this study, we found that 3D induces the expression of proapoptotic proteins such as p53 and Bax; however, it inhibits the expression of Bcl2 and BclxL in human adenocarcinoma and metastatic CRC cells. Thus, the induction of Bax expression and the inhibition of Bcl2 and BclxL expression provide a mechanistic basis for 3D-induced apoptosis. Induction in Bax activation leads to opening of VDAC channel, thereby releasing cytochrome c. Cytochrome c release from mitochondria into cytosol resulted in activation of caspases. 3D was indeed found to enhance cytochrome c release from mitochondria into cytosol. 3D treatment also resulted in PARP cleavage. Caspase cascade begins with activation of initiator caspase-9, which in turn activated caspase-3 and -6, leading to mitochondria-mediated cell death pathway. Caspase-8 is involved in death receptor-mediated apoptosis pathway. 3D was found to activate caspase-9, -3 and -6 and to a little extent caspase-8 as well. These findings indicate that 3D induces mainly intrinsic apoptotic death pathway and to some extent extrinsic apoptotic pathway.

The essential role of STAT3 in cancer tumorigenesis makes STAT3 an important target for cancer therapeutics.15 We studied the effect of 3D on STAT3 signaling pathway in human CRC cell lines. STAT3 has been reported to be constitutively active in HT-29 cells. 3D was found to decrease phosphorylation of STAT3 at Tyr-705 in HT-29 cells. 3D also inhibited the IL-6-induced phosphorylation of STAT3 in metastatic CRC cell line SW620. Inhibition of STAT3 phosphorylation is an essential step in blocking STAT3 pathway as STAT3 phosphorylation leads to STAT3 dimerization, followed by translocation into nucleus and binding to the promoters of target genes. STAT3 is activated by the upstream kinases including JAK2.12 Our finding indicates that 3D inhibits JAK2 phosphorylation in human CRC cells. 3D-induced inhibition of JAK2 and STAT3 suggests that apoptosis induction by 3D is mediated by inhibition of JAK2-STAT3 signaling. 3D may affect other pathways as well. This was further demonstrated by 3D-induced inhibition of STAT3 target genes such as cyclin D1 and survivin. AG490 is known to inhibit JAK–STAT pathway; 3D treatment in combination with AG490 further inhibits the STAT3 signaling in CRC cells. The anticancer efficacies of current therapeutics are limited because of the high degree of cancer clonal heterogeneity and intratumor genetic variation. Therefore, use of combinations of molecular-targeted agents has been on the rise for better therapeutics. Our compound of interest 3D sensitizes the efficacy of standard anticancer drug Dox. Drug–drug interaction can pose serious side effects in patients. The interaction could be pharmacokinetic (PK) and pharmacodynamic (PD) in nature. The PK interactions result in altered distribution, absorption and elimination of the drug. Pharmacodynamic interactions cause alterations in the way a drug or compound affects a tissue or organ system. These interactions affect the action of a drug in a qualitative way by either enhancing (synergy or additive) or decreasing (antagonize) the therapeutic effect. The interaction between 3D and Dox may be synergistic or additive with resultant potentiation of cytotoxicity in combination therapy. Further studies are needed to study the interaction between 3D and other cytotoxic agents in combination therapy.