br Results br DSC targets TOPK but not MEK br
3-DSC targets TOPK, but not MEK
To determine whether 3-DSC aﬀects TOPK activity, we conducted a TOPK kinase assay (Fig. 1A). 3-DSC inhibited TOPK kinase activity, as compared to the vehicle control but did not aﬀect the kinase activity of MEK, which also belongs to the MAPKK family (Fig. 1A, B). Also, 3-DSC treatment did not aﬀect the autophosphorylation of TOPK (Fig. 1A). To examine whether direct binding between 3-DSC and TOPK occurred, ex vivo and in vitro pull-down assays were conducted (Fig. 1C and D). HCT-15 cell lysates or a human recombinant active TOPK were incubated with 3-DSC. Results confirmed that 3-DSC binds to TOPK, but not to MEK, in the HCT-15 cell lysate, which highly expresses TOPK (Fig. 1C). To determine whether 3-DSC binds to the ATP binding pocket of TOPK, we performed an ATP competitive pull-down assay using recombinant active TOPK. Binding between 3-DSC and TOPK was determined in the presence of increasing concentrations of ATP (0, 10, 100, or 1000 μM). The increasing ATP concentrations reduced binding between 3-DSC and TOPK (Fig. 1E), suggesting that the 3-DSC-TOPK binding may occur through the TOPK ATP-binding pocket. To further examine this inter-action, we conducted in silico analysis. We docked TOPK at the ATP binding pocket of TOPK and ran several protocols in the Schrödinger Suite 2016 (Fig. 1F). From the docking model, we found that 3-DSC bound to and fit into the ATP binding pocket of TOPK very well and that hydrogen bonds were formed between 3-DSC and TOPK at Thr42 and Asn172 amino E-64 sites. These results indicated that 3-DSC could be a potential inhibitor against TOPK activity.
3-DSC suppresses growth of colon cancers by targeting TOPK
To study the eﬀect of 3-DSC on colon cancer cell growth, we first conducted a 3-DSC toxicity assay on a normal colon cell line, CCD-18Co (Supplementary Fig. 2). Results indicated that 3-DSC did not aﬀect normal cell growth, even at the highest concentration tested, 20 μM. Crucially, 3-DSC significantly inhibited the growth of colon cancer cell lines, HCT-15, HCT-116, SW620, and DLD1 in a time- and dose-de-pendent manner (Fig. 2A). Also, in an anchorage-independent cell growth assay, 3-DSC retarded the growth of sensitive cell lines, HCT-15, HCT116, SW620, and DLD1 in a concentration-dependent manner (Fig. 2B and C).
3-DSC induces cell cycle arrest and apoptosis of colon cancer cells
3-DSC inhibits the TOPK signaling pathways
We have shown that 3-DSC inhibited cell growth and induced cell cycle arrest and apoptosis in colon cancer cell lines by targeting TOPK. Next, we determined whether 3-DSC also aﬀected signaling pathways downstream of TOPK. When we treated HCT-15 or HCT-116 cells with 3-DSC for 24 h, the expression levels of pTOPK, pERKs, pRSK and pc-Jun were suppressed in a dose dependent manner; however, the ex-pression levels of total TOPK, ERKs, RSK and c-Jun did not change (Fig. 5).
Abnormal signaling of TOPK promotes cancer development, in-cluding breast cancer (Park et al., 2006), colorectal cancer (Zhu et al., 2007), lung cancer (Shih et al., 2012), and hepatocellular carcinoma (He et al., 2010). TOPK has been studied as a potential therapeutic target against cancer (Fan et al., 2016; Vishchuk et al., 2016; Zeng et al., 2016) and inhibitors have been developed (Kim et al., 2012; Matsuo et al., 2014; Park et al., 2017). However, these inhibitors have therapeutic limitations, including significant side eﬀects, poor eﬃcacy, and low selectivity. To identify new drugs that target TOPK, we screened several compounds and identified 3-DSC from Caesalpinia sappan L. 3-DSC has been reported to exert activity against tuberculosis (Seo et al., 2017) and has anti-allergic (Yodsaoue et al., 2009) and anti-inflammatory activities (Kim et al., 2014). However, potential antic-ancer activity has not yet been studied. Thus, we investigated the ef-fects of 3-DSC against colon cancer cell growth and identified its me-chanism of action. First we found that 3-DSC inhibited TOPK, but not MEK, kinase activity (Fig. 1B). We also used kinase profiling assays to determine whether 3-DSC could target other kinases, such as the stress-activated serine/threonine-specific kinase 2 (SAPK2) α, c-Jun N-term-inal kinase 1 (JNK1) α, JNK2α, mitogen-activated protein kinase 1 (MAPK1), MAPK2, ribosomal S6 kinase 1 (RSK1), RSK2, protein kinase Bα (PKBα), PKBβ, and Src. We found that the only other kinase targeted by 3-DSC was Src. However, the level of inhibition of Src was much lower at about 30% (Supplementary Fig. 3). Thus 3-DSC might be a specific inhibitor against TOPK. The specificity of 3-DSC for TOPK is higher than that for the previously reported HI-TOPK-032 compound (Kim et al., 2012). By targeting TOPK, 3-DSC significantly inhibited cell growth and induced cell cycle arrest and apoptosis in various colon cancer cell lines (Figs. 2–4). 3-DSC did not exert toxicity against normal colon cells, even at the highest dose of 20 μM compared to HI-TOPK-032 (Supplementary Fig. 2, Supplementary Fig. 4A). However, HI-TOPK-032 significantly inhibited cell growth at low doses (Supple-mentary Fig. 4B and C). TOPK has been reported to interact with the DNA binding domain of the tumor suppressor p53 protein and modulate expression of the transcription target p21 (Hu et al., 2010). Indeed, when we treated colon cancer cell lines with 3-DSC, we observed sig-nificant apoptosis only in cells with wild-type p53 (HCT15 and HCT116), but not in cells with mutant p53 (SW260 and DLD1), sug-gesting that 3-DSC induced apoptosis in a p53-dependent manner (Fig. 4A, B). Because TOPK is activated in cancer cells, it transfers signals to down-stream eﬀectors, including extracellular signal–regu-lated kinases (ERKs), RSK, or c-Jun (Kim et al., 2012; Zhu et al., 2007). We found that 3-DSC interfered with downstream signaling and re-duced the expression of phosphorylated ERKs (pERKs), RSK (pRSK), and c-Jun (pc-Jun), but did not aﬀect the total expression of ERKs, RSK,