Anticancer activity of a novel small molecule tubulin inhibitor STK899704

We have identified the small molecule STK899704 as a structurally novel tubulin inhibitor. STK899704 suppressed the proliferation of cancer cell lines from various origins with IC50 values ranging from 0.2 to 1.0 μM. STK899704 prevented the polymerization of purified tubulin in vitro and also depolymerized microtubule in cultured cells leading to mitotic arrest, associated with increased Cdc25C phosphorylation and the accumulation of both cyclin B1 and polo-like kinase 1 (Plk1), and apoptosis. Unlike many anticancer drugs such as Taxol and doxorubicin, STK899704 effectively displayed antiproliferative activity against multidrug-resistant cancer cell lines. The proposed binding mode of STK899704 is at the interface between αβ-tubulin heterodimer overlapping with the colchicine-binding site. Our in vivo carcinogenesis model further showed that STK 899704 is potent in both the prevention and regression of tumors, remarkably reducing the number and volume of skin tumor by STK899704 treatment. Moreover, it was significant to note that the efficacy of STK899704 was surprisingly comparable to 5-fluorouracil, a widely used anticancer therapeutic. Thus, our results demonstrate the potential of STK899704 to be developed as an anticancer chemotherapeutic and an alternative candidate for existing therapies.


Introduction
Microtubules, a major component of the cytoskeleton, are polymers of α-and β-tubulin heterodimers which play important roles in a variety of cellular processes including cellular a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 trafficking, maintaining cell polarity, cell signaling, cell migration, and cell proliferation [1]. During mitosis, microtubules form highly dynamic mitotic spindles, which are critical for the proper orientation and segregation of chromosomes [2]. The impairment of mitotic spindles leads to mitotic arrest and consequently apoptosis [3,4]. The critical role of microtubules in cell division and other cellular functions makes them an attractive target for cancer chemotherapy.
Microtubule-targeting agents are usually classified into two main groups, stabilizers and destabilizers, based on their mechanisms of action [5][6][7]. Microtubule-stabilizing agents, including paclitaxel (Taxol) and docetaxel, inhibit depolymerization and enhance microtubule polymerization. Most microtubule-stabilizing agents bind to the taxane-binding site or an overlapping site on β-tubulin. Microtubule-depolymerizing agents such as colchicine and vinca alkaloids, inhibit microtubule polymerization and usually bind to either the colchicineor vinca-binding site. Both stabilizers and destabilizers affect microtubule dynamics at lower concentrations than those that affect microtubule-polymer mass [5], and arrest cells at mitosis. Although microtubule-targeting agents especially paclitaxel and vinca alkaloids are widely used and in clinical success, both intrinsic and acquired drug resistances in cancer cells are significant limitations to clinical efficacy [8][9][10]. Resistance to microtubule-targeting agents is often related to the expression of multidrug resistance proteins such as the drug efflux pump P-glycoprotein (P-gp), resulting in the exportation of the agents from cancer cells preventing the intracellular accumulation of the active drug. Resistance can also arise from mutations in and/or alteration of tubulin isotype levels [11,12]. In addition to drug resistance, neurotoxicity is a common side effect, which leads to a dose limitation of microtubule targeting drugs in clinical use [13,14]. Therefore, in recent years, there has been great interest in the identification of novel tubulin-targeting drugs with lowered neurotoxicity and insensitivity to chemoresistance providing significant clinical benefits to cancer patients.
In our screening for antiproliferative agents from a small-molecule library, we identified STK899704 as a structurally novel antimitotic agent. STK899704 binds tubulin and inhibits its polymerization, leading to cell cycle arrest at mitosis and cell death. Molecular docking studies demonstrated that the binding site of STK899704 on tubulin overlaps with the colchicinebinding site. In addition, STK899704 exhibited antiproliferative activity against a broad range of cancer cell types regardless of multidrug-resistance phenotypes. The preclinical evaluation of novel compounds STK899704 revealed the effect on skin carcinogenesis model in vivo, demonstrating its chemopreventive and antitumor activities. As far as we know, this is the first study indicating that STK899704 has a potent therapeutic efficacy. Thus, our data suggest that STK899704 is a novel tubulin-depolymerizing agent with the potential to be further developed as an anticancer agent.

Chemical screening methods
All compounds from a small-molecule library obtained from Korea Chemicals Bank were evaluated for their antiproliferative activity on various cancer cell lines. The MTT assay was used to determine the cytotoxic effect of compounds and each IC 50 value was assessed by log-doseresponse curves. Also, the EZ-CyTox cell viability assay (Daeil Lab. Service, Korea) was performed according to the manufacturer's instructions and the absorbance was measured at 450 nm using VersaMax™ (Molecular Devices LLC, USA). Each IC 50 value was calculated using a nonlinear regression analysis using GraphPad Prism 6.0 program.
Considering the functions of microtubule in the maintenance of cellular morphology, further analyses that involved the disruption of cellular morphology was performed by microscopic examination and immunocytochemistry. The detailed methods including flow cytometric analysis are described in the following sections.

Flow cytometric analysis
Following compound treatment, cells were harvested and stained with propidium iodide (PI) according to the instruction of Cycletest Plus DNA Reagent Kit (BD Biosciences) or with anti-Annexin V-FITC (BD Biosciences) for 30 min to determine the percentage of cells with phosphatidylserine externalization. Flow cytometric analysis was performed using a FACSCalibur instrument (BD Biosciences).

Immunoblot and immunofluorescence staining
Cells were lysed with cold RIPA buffer and whole cell lysates were subjected to SDS-PAGE as previously reported [17]. To perform immunofluorescence staining, the cells were fixed and incubated with Alexa 488-conjugated α-Tubulin antibody. DNA was stained with Hoechst 33342 in PBS. Images were analyzed on a fluorescence microscope (Nikon Instruments Inc.).

Tubulin polymerization assay
The assay was performed according to the manufacturer's instructions (Cytoskeleton, Inc., USA). In brief, tubulin proteins (>97% pure) were suspended in G-PEM buffer (80 mM PIPES (pH 6.9), 2 mM MgCl 2 , 0.5 mM EDTA, and 1.0 mM GTP) to a final concentration of 4.0 mg/mL. The tubulin solution was then incubated with G-PEM buffer alone (control), and STK899704 (10 μM, final concentration) at 37˚C. Paclitaxel and vinblastine at final concentration of 5 μM were also used as positive enhancer and inhibitor controls, respectively. The polymerization of tubulin was measured by continuous monitoring of the turbidity change at 340 nm (VersaMax™).

Computer modeling study
To examine the binding mode of STK899704 with respect to impairing the activity of tubulin, we conducted docking simulations in the active site. Three dimensional atomic coordinates were extracted from the X-ray crystal structure of tubulin (PDB code: 1SA0) as the receptor model [18]. Gasteiger-Marsili atomic charges were determined for all the protein and ligand atoms to calculate the electrostatic interactions between tubulin and STK899704 [19]. Docking simulations to address the binding mode of STK899704 were then carried out with the modified version of AutoDock program whose outperformance had been well appreciated for various target proteins [20][21][22][23]. Of the twenty binding conformations of STK899704 generated with docking simulations, those differing by less than 1.5 Å in positional root-mean-square deviation were clustered together. The lowest-energy binding configuration in the top-ranked cluster was finally selected for further analysis.

Tubulin competitive binding SPA assay
The competitive binding SPA assay was performed as the manufacturer's instructions (Cytoskeleton, Inc.) using biotin-labeled tubulin, streptavidin-coated PVT SPA beads (Perkin Elmer), and Colchicine [ring C, methoxy-3 H] (1 mCi/mL, specific activity 85 Ci/mmol) (American Radiolabeled Chemicals, Inc.). Briefly, lyophilized biotin-labeled tubulin was incubated with streptavidin-coated PVT SPA beads for 30 min at 4˚C, and the premix beads were then incubated with

Skin carcinogenesis in vivo
A two-stages carcinogenesis was performed as previously described [24,25]. The dorsal skin area of the 6-7 week old mice was shaved 2 days before start of the experiment. Tumorigenesis was initiated by a single topical treatment with 100 μg of DMBA in 0.2 ml of acetone over a period of 1 week. Tumor promotion was then induced by treatment with 5 μg of TPA in 0.2 ml of acetone twice weekly. In order to measure the number and volume of skin tumors, mice were weighed and photographed every week starting from when first measurable tumors (1 mm 3 ) appeared. Tumor volume was calculated using the following formula: tumor volume 4π/ 3(l/2)(w/2)(h/2), where l is the length, w is the width, and h is the height [19]. At the end of the experiment, the mice were euthanized with CO 2 , and both tumors and skin were collected for histological and biochemical studies.
Within 30 minutes following the above dose of TPA application, STK899704 or 5-FU at 500 nM in 0.2 ml of acetone was treated twice a week over 15 weeks. The mice were divided into four groups and each group consisted of more than 20 mice. Two groups of mice were treated with acetone (vehicle) or TPA only, which served as negative or positive controls, respectively.

Test for adverse effects of STK899704 treatment
To test adverse effects of STK899704 treatment, the compound was applied onto the shaved area of dorsal skin in healthy group of mice twice weekly, 20 times in total. The effects were compared to control group treated with acetone only, and each group was consisted of more than 10 mice.
Collected samples of skin were fixed at 4˚C in 4% paraformaldehyde/PBS or 10% formalin solution and then sectioned for histological analyses [15]. The sections (10 μm thickness) were stained with hematoxylin/eosin and observed using research system microscope BX51 (Olympus).

Antiproliferative effect of STK899704 on various cancer cells and multidrug-resistant cell lines
In the course of screening for antiproliferative agents from a small-molecule library, we found that ethyl(2-methyl-3-((E)-((naphtha(2,1-b)furan-2-ylcarbonyl)hydrazono)methyl)-1Hindole-1-yl)acetate or STK899704 ( Fig 1A) displayed a potent antiproliferative effect against HeLa cervical cancer cells as shown in Fig 1B. STK899704 suppressed the growth of HeLa cells in a dose-dependent manner with an IC 50 of 350 nM ( Fig 1C). Furthermore, STK899704 inhibited the growth of a variety of human cancer cell lines including skin, bone, breast, colon, prostate, lung, stomach, brain, and liver cancers and leukemia with IC 50 values ranging from 0.35 to 1.54 μM (Fig 1C and S1 Table).
One major mechanism of acquired resistance to anticancer drug is mediated by overexpression of drug-efflux protein P-glycoprotein [8]. The antiproliferative activity of STK899704 was compared with doxorubicin and Taxol in K562, MCF7, and their respective P-glycoprotein overexpressing multidrug-resistance (MDR) cell lines, K562/ADR and MCF7/ADR [15,16]. The MDR cells were resistant to doxorubicin, Taxol, vinblastine, and colchicine with resistance factors (ratio of IC 50 of resistance cell line relative to its parental cell line) ranging from 7.6 to 582 fold (Table 1), whereas STK899704 exhibited a potent cytotoxic effect against these MDR cell lines as judged by resistance factors of 0.12 and 0.16 for K562/ADR and MCF7/ADR, respectively.

STK899704 induced mitotic arrest
The treatment of HeLa cells with STK899704 resulted in a dose-dependent accumulation of G 2 /M phase cells with 4N DNA content and, concomitant decrease in G 1 and S phase cells    (Fig 2A). The increased G 2 /M phase cells were accompanied by a marked increase in rounded, mitotic-like cell morphology, suggesting that STK899704 might induce mitotic arrest. To address this, we assessed the percentage of mitotic cells by quantification of the number of cells with highly condensed chromosomes, characteristic of particular mitotic chromosomes [26] and found that STK899704 treatment resulted in a dose-dependent increase in mitotic indices ( Fig 2B). Hence, these results indicate that STK899704 causes cell cycle arrest at M phase.
In addition, we evaluated the effect of STK899704 on cell cycle related proteins, including Cdc25C, histone H3, cyclin B1, and polo-like kinase (Plk1), in comparison with nocodazole, a tubulin depolymerizing agent and well-known mitotic blocker [6]. During mitosis, Cdc25C activity is hyperphosphorylated causing its slower migration on SDS-PAGE [27,28], whereas the phosphorylation of histone H3 at S10 is required for proper chromosome condensation and segregation [29,30]. Both Cyclin B1 and Plk1 expression oscillate throughout the cell cycle. Their levels are minimal in G 1 phase, begin to accumulate in S phase, and reach the maximum at G 2 /M boundary [31][32][33][34]. As expected, the treatment of HeLa cells with nocodazole, as well as STK899704, resulted in elevated Cdc25C and histone H3 phosphorylation levels and accumulated cyclin B1 and Plk1 levels (Fig 2C), confirming that STK899704 induces mitotic arrest.
To examine whether the antimitotic activity of STK899704 is reversible, HeLa cells were treated with STK899704 (1 and 5 μM) for 18 h. Nocodazole, a reversible inhibitor of microtubule assembly [35], was included as control. As shown in Fig 2D, HeLa cells were efficiently arrested at G 2 /M phase (>80%) upon treatment with nocodazole and STK899704 overnight. After the removal of nocodazole and STK899704, the G 2 /M-arrested HeLa cells were able to re-enter the cell cycle with a minimal delayed in cells treated with a high concentration of STK899704 (5.0 μM) (Fig 2D). These results indicated that STK899704, like nocodazole, acts in a reversible manner.

STK899704 interfered with tubulin polymerization and mitotic spindle organization
Given the fact that microtubules are the major structural component of a mitotic spindle, whose function is critical for chromosome segregation during mitosis [2], and that a large number of antimitotic agents interact with tubulin and thereby alter its polymerization and dynamics [5], we examined the effect of STK899704 on tubulin polymerization in vitro in comparison with known microtubule binding agents by tracking the change in turbidity over time.
In the absence of treatment, tubulin heterodimers self-assembled to form linear tubulin polymers in a time-dependent manner as shown in Fig 3A. Treatment with the microtubule-stabilizing agent paclitaxel resulted in enhanced tubulin polymerization, whereas the microtubuledepolymerizing agent vinblastine, as well as STK899704, interfered with tubulin polymerization (Fig 3A).
To further determine whether STK899704 could affect mitotic spindle organization in cells, HeLa cells were treated with DMSO control or test compounds, followed by formaldehyde fixation and subsequent staining to visualize α-tubulin, γ-tubulin, and chromosomes. As shown in Fig 3B, Taxol, a microtubule stabilizing agent, significantly induced multipolar spindles with highly condensed chromosomes resulting from enhanced tubulin polymerization.   (Fig 3B and 3C). In some instances, spindle appeared normal but chromosomes were not aligned at the metaphase plate. In others, multipolar spindles were observed. This abnormal spindle morphology suggests that nocodazole and STK899704 at low concentration interfere with spindle microtubule dynamics. However, cells treated with higher concentrations of nocodazole (0.3 μM) or STK899704 (5.0 μM) exhibited condensed chromosome with aggregated tubulin and disrupted microtubules. Taken together, these results suggest that STK899704, like nocodazole, is a microtubuledepolymerizing agent.
We next search for potential binding site of STK899704 on tubulin. Using computer modeling and X-ray structure PDB code 1SA0 [18], we determined the proposed binding site of STK899704 on tubulin (Fig 3D, red stick) is located between the α-and β-subunit of tubulin at the colchicine binding site (Fig 3D, green stick). Comparison of their proposed binding modes showed that colchicine binds to the β-tubulin subunit whereas the STK899704 ligand spans across the intra dimer interface suggesting that the binding site of STK899704 somehow overlaps with colchicine. We confirmed that STK899704 interacts directly with this binding site using a tubulin competitive binding SPA assay. In addition, we showed that STK899704 competitively inhibited [ 3 H]colchicine binding to biotinylated tubulin, similar to unlabeled colchicine, whereas vinblastine did not significantly influence the binding of [ 3 H]colchicine ( Fig  3E). Thus, these data indicate that STK899704 is a new class of tubulin inhibitor; and its antiproliferative activity is due to its binding to tubulin at the colchicine-binding site.

STK899704 induced cell death following prolonged mitotic arrest
We next examined whether STK899704 would eventually induce cell death after prolonged mitotic arrest by determining the subG 1 populations of HeLa cells. Similar to the effect of colchicine and nocodazole, treatment with STK899704 resulted in a marked increase in the subG 1 population at 48 h (Fig 4A). Since caspases are the key mediators of apoptosis [36], Z-VAD-FMK, a cell-permeable and irreversible pancaspase inhibitor, was used to examine whether caspases are involved in the increased subG 1 population after STK899704 treatment. As shown in Fig 4B, co-treatment with Z-VAD-FMK prevented the accumulation of the subG 1 population compared with STK899704 treatment alone. These results suggest that STK899704 treatment induces apoptosis in a time-dependent manner, associated with the increase in the subG 1 population.
In addition, the effects of STK899704, colchicine, and nocodazole on the cleavage of initiator caspases were analyzed. Caspase-3 and caspase-7 are the effector caspases that are activated by initiator caspases such as caspase-8 and caspase-9. As shown in Fig 4C, the cleaved caspases were clearly detectable at 48 h in a concentration-dependent manner. The level of PARP cleavage was also examined since activation of effector caspases such as caspase-3 leads to downstream cleavage of various substrates including poly (ADP-ribose) polymerase (PARP) during apoptosis [36,37]. Consistent with active caspase levels, PARP cleavage was also prominent at 48 h after treatment with STK899704, colchicine, and nocodazole (Fig 4C). Taken together, these results indicate that STK899704 induces prolonged mitotic arrest and consequently leads to apoptosis.

STK899704 demonstrated prominent antitumor activity in vivo
Due to the physicochemical properties of STK899704, which is very lipophilic (XlogP3 = 5.7, pubchem), and the efficient antiproliferative activity shown earlier, we concluded that STK899704 might be an effective local antitumor agent when directly applied to the skin. Therefore, we assessed the antitumor effect of STK899704 in a skin carcinogenesis model in vivo and used 5-fluorouracil (5-FU), a widely used therapeutics for skin cancer, as a comparison treatment in this study.
After tumor initiation by DMBA treatment, mice were treated with STK899704 or 5-FU was co-treated with TPA to group of mice twice a week for 15 weeks. As shown in Fig 5A, TPA treatment alone resulted in the formation of many tumors on the back of the mice. In contrast, both number and size of tumors were significantly decreased by STK899704 or 5-FU treatment, exhibiting an antitumor promotion effect of STK899704. For statistical analyses, changes in total number and volume of tumors were weekly monitored during treatment ( Fig  5B and 5C). Surprisingly, STK899704 treatment exhibited approximately 76% reduction in the average number of DMBA/TPA-induced skin tumors, while 5-FU treatments were found to decrease skin tumors by about 70% compared to the TPA treatment alone at 15 weeks ( Fig  5B). Similarly, STK899704 treatment reduced the tumor volume by approximately 80%, while 5-FU treatments decreased the tumor volume by about 76% at 15 weeks (Fig 5C). At 15 weeks, skin tumors from each group of mice were isolated, and their weight was measured as shown in the Fig 5D. The mean weight of the skin tumors was obviously decreased 9.4-fold in the STK899704-treated group. These results indicate that STK899704 exhibits potent both chemopreventive and antitumor effects, and the efficacy of STK899704 showed beyond that of 5-FU.
The effects of STK899704 treatment on normal skin were also evaluated in healthy group of mice. Visual inspection and histological examination of the skin sections revealed that there were no abnormal symptoms such as skin inflammation and hyperplasia (S2 Fig).

Discussion
In our effort to discover novel antiproliferative agents, we identified STK899704 as a novel tubulin inhibitor. STK899704 has an acylhydrazone moiety known as a useful scaffold for drug development, especially for anticancer therapy due to its antimitotic activity [38][39][40][41][42]. Further analyses showed that STK899704 strongly induces the accumulation of mitotic cells in a concentration-dependent and reversible manner (Fig 2A, 2B and 2D). These results suggest that STK899704 induces cell cycle arrest in mitosis. In addition, the antimitotic effect of STK899704 was confirmed by increased phosphorylation of Cdc25C and accumulation of cyclin B1 and Plk1 (Fig 2C), which are associated with mitosis [18,27,28,[31][32][33][34][35][36][37]. Moreover, prolonged mitotic arrest leads to a time-dependent increase in subG 1 population ( Fig 4A) and activation of caspases ( Fig 4C). The effector caspases are implicated in the late, irreversible phase of the apoptotic pathway and carry out the proteolytic degradation of a broad range of cellular targets, such as PARP which plays an important role in diverse cellular processes, including DNA damage response [36,37]. Therefore, increased DNA fragmentation and altered levels of active caspases suggest that prolonged mitotic arrest induced by STK899704 eventually trigger programmed cell death.
We also found that the antimitotic activity of STK899704 is ascribed to its ability to prevent tubulin polymerization in vitro and disrupt microtubules in cells (Fig 3A and 3B), indicating that STK899704 is a microtubule-destabilizing agent. Computer modeling studies reveal that STK899704 possibly binds to the colchicine-binding site (Fig 3D). The colchicine ligand lies in β-tubulin subunit whereas STK899704 ligand traverses across the interface between αβ-tubulin heterodimer. Additionally, the results from Figs 2D and 3D suggest that STK899704 does not covalently bind to tubulin, allowing for the full reversibility of its intracellular activity.
One of the major limitations of anticancer drugs in clinical use is drug resistance, acquired or intrinsic, especially resistance due to the expression of the drug-efflux protein P-gp [8]. Unlike Taxol and the Topoisomeras II inhibitor doxorubicin, STK899704 exhibits equal or better potency in both parental and multidrug-resistant cells overexpressing P-glycoprotein. Therefore, STK899704 may overcome the resistance mediated by the multidrug resistance protein P-gp.
In this study, the efficacy of STK899704 was investigated using a DMBA/TPA-induced skin carcinogenesis model (Fig 5). Skin cancer is a generic term for malignant tumor occurring on the skin and its incidence rate has been generally on the rise [43,44]. The overall prevalence of skin cancer has been reported to be higher in Caucasians and becomes higher with age, in part due to the accumulative exposure to UV light [45][46][47]. Thus, skin cancer is emerging as a global issue in terms of disease prevention ranging from Europe and the United States whose major races are Caucasians, to Asia where the population age is steadily rising. Therefore, development of a therapy with convenient applicability and low side effect would be in high demand. Here we reported that topically applied STK899704 exhibited efficient antitumor effect. In response to STK899704 treatment, approximately 80% of the total number of skin tumors dramatically decreased (Fig 5B). Compared to the TPA-treated control group, tumors size was also reduced to approximately 80% by STK899704 treatments (Fig 5C). Thus, the prominent antitumor activity of STK899704 has been proved in vivo animal model.
Except surgical removal of the lesion, 5-FU and Imiquimod are the most widely used topical therapeutics for skin cancer [48,49]. However, 5-FU is occasionally accompanied by many side effects including local pain, itchiness, burning, stinging, and dermatitis. Imiquimod acts as immune response modifier that can stimulate the immune system to primarily produce interferon-α (INF-α) leading to nonspecific inflammation or dermatitis. Since the antitumor effect of STK899704 was surprisingly comparable to 5-FU ( Fig 5) and did not induce abnormal symptoms or alteration in skin conditions (S2 Fig), STK899704 is a promising alternative to 5-FU or Imiquimod for treatment of skin cancer.
Taken together, our data indicate that the antiproliferative effect of STK899704 is ascribed to its microtubule destabilizing activity, resulting in mitotic arrest and apoptosis. In addition, the antiproliferative activity of STK899704 seems to be effective regardless of any multidrugresistance phenotype. Our study suggests that STK899704 is a promising candidate for further development as a chemotherapeutic agent, particularly for the treatment of skin cancer as well as MDR cancer.  (6) was obtained in 86% yield in step 5, by N-substitution of 2-methyl-1H-indole-3-carbaldehyde (5) by ethyl bromoacetate using sodium hydride as a base in THF solvent and it was confirmed by the absence of indole-NH signal at δ 11.962 ppm instead appeared new signals at δ 4.208-4.155 ppm, quartet for-CH 2 and δ 1.241-1.205 ppm triplet for-CH 3