Baicalin suppresses lung cancer growth by targeting PDZ-binding kinase/T-LAK cell-originated protein kinase

Baicalin is the main bioactive component extracted from the traditional Chinese medicine Baical Skullcap Root, and its anti-tumor activity has been studied in previous studies. PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK), a serine/threonine protein kinase, is highly expressed in many cancer cells and stimulates the tumorigenic properties, and so, it is a pivotal target for agent to cure cancers. We reported for the first time that baicalin suppressed PBK/TOPK activities by directly binding with PBK/TOPK in vitro and in vivo. Ex vivo studies showed that baicalin suppressed PBK/TOPK activity in JB6 Cl41 cells and H441 lung cancer cells. Moreover, knockdown of PBK/TOPK in H441 cells decreased their sensitivity to baicalin. In vivo study indicated that injection of baicalin in H441 tumor-bearing mice effectively suppressed cancer growth. The PBK/TOPK downstream signaling molecules Histone H3 and ERK2 in tumor tissues were also decreased after baicalin treatment. Taken together, baicalin can inhibit proliferation of lung cancer cells as a PBK/TOPK inhibitor both in vitro and in vivo.


Introduction
PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK), a serine-threonine mitogen-activated protein kinase, is a member of the MEK protein family [1,2]. It is involved in mitotic checkpoint of cell [3], DNA damage [4], tumor transformation and metastasis [5,6], and inflammation [7]. Previous studies showed that PBK/TOPK was highly expressed in multiple types of cancers and associated with poor prognosis, such as lymphoma, leukemia, melanoma, colorectal, breast and lung cancers, and cholangiocarcinoma [8][9][10][11][12][13][14]. In addition, it was reported that TOPK exhibits high expression levels in cancer tissues but low expression levels in normal tissues [15]. These suggest that TOPK might be a prominent drug target for cancer chemotherapy. However, there were few PBK/TOPK inhibitors reported on basic research. PBK/TOPK inhibitor HI-TOPK-032 [16], OTS964 [17] had brought some side-effects, which have been facing a critical challenge clinically [17]; thus we aimed to look for traditional medicine to inhibit PBK/TOPK activity.
Natural compounds have higher efficacy and lesser toxicity, and gain more and more interests to search for their potent phamaceutical values in chemoprevention and chemotherapy. Baicalin is a natural flavonoid glycoside, which has much perfect pharmacological efficacy to promote the human health in traditional medicine in the world, such as anti-diabetic [18], antioxidant [19] anti-inflammatory [20], and anti-cancer [21] functions. Significant anti-tumor effects of baicalin were observed in the lung cancer [22].
Although baicalin is a pleiotropic protein kinase enzymes inhibitor [23], the molecular mechanism of its pharmacological action is still incomplete, in lung cancer cells. Herein, we found a new target of baicalin. Baicalin can target PBK/TOPK protein kinase directly and inhibit the proliferation of lung cancer.

Cell culture
JB6 Cl41 was cultured at 37 • C in a 5% CO 2 incubator in MEM medium containing 5% fetal bovine serum (FBS). H441, H1975, and H1299 cells were cultured at 37 • C in a 10% CO 2 incubator in RPMI-1640 medium containing 10% FBS, respectively. The cells were starved 24 h before the addition of 20 ng/ml epidermal growth factor (EGF) in medium without serum.

MTS assay
In order to estimate cytotoxicity of baicalin, the cells were seeded (8 × 10 3 cells per well) in 96-well plates and cultured overnight. The cells were then fed fresh medium and treated with different doses of baicalin. After culturing for several times, the cytotoxicity of baicalin was detected by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay kit (Promega, Madison, WI). According to the instructions, the absorbance was read at 490 nm.

Soft agar assay
JB6 Cl41 cells (8 × 10 3 /ml) were treated by baicalin (0-100 μM) with exposure to EGF (20 ng/ml) in 1 ml of 0.33% Basal Media Eagle (BME) agar containing 10% FBS, 2 mM l-glutamine, and 25 μg/ml gentamicin. The cultures were maintained at 37 • C in 5% CO 2 incubator for 10 days and colonies were scored using a microscope Motic AE 20 (China) and the Motic Image Plus computer program (Media Cybernetics, Silver Spring, MD). Human lung cancer cells were treated as described above instead of exposing to EGF.

Microscale thermophoresis
PBK/TOPK protein was labeled with the Monolith NT™ Protein Labeling Kit RED (Cat# L001) according to the supplied labeling protocol. The PBK/TOPK protein were diluted in 20 mM HEPES (pH 7.4) and 0.05 (v/v) % Tween-20 to 50 nM. The baicalin stock was dissolved in ddH 2 O at a concentration of 5 mM; 5 mM baicalin was used as the highest concentration for the serial dilution. After 10-min incubation at room temperature the samples were loaded into Monolith™ standard-treated capillaries and the thermophoresis was measured at 25 • C after 30-min incubation on a Monolith NT.115 instrument (NanoTemper Technologies, München, Germany). Laser power was set to 40% using 30 s on-time. The LED power was set to 100%. The equilibrium dissociation constant K d values were fitted by using the NTAnalysis software (NanoTemper Technologies, München, Germany) [24].

Western blot
Different cell lines (5 × 10 8 ) were cultured in 6-cm diameter dishes, and the cells were harvested and disrupted in 300 μl of RIPA buffer. The samples were sonicated at 15 s for three times and centrifuged at 14000 rpm for 10 min. The quantity of protein was detected by the BCA method. The samples (30-50 μg protein) with 5× SDS loading buffer were heated at 95 • C for 10 min, and then cooled on ice. Next, the samples were separated on 10% sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) and subsequently transferred on to polyvinylidene fluoride membrane (PVDF), which were blocked with 5% non-fat milk and then incubated with a specific primary antibody at 4 • C overnight. The proteins were determined by chemiluminescence after hybridization with a horseradish peroxidase-conjugated secondary antibody. All the experiments were performed in triplicate, and band density was quantitated using the ImageJ 12.0 software.

In vitro kinase assay
Inactive Histone H3 proteins were used as the substrate for an in vitro kinase assay with active PBK/TOPK. Active PBK/TOPK was incubated with baicalin (10, 20, and 50 μM) and 100 μM ATP in 1× kinase buffer (25 mM Tris/HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ) at 32 • C for 90 min. Reactions were stopped and proteins were detected by Western blot. All the experiments were performed in triplicate.

Xenograft mouse model
Athymic nude mice (6-9 weeks) were obtained from Beijing HFK Bioscience Co., Ltd (Beijing, China). The animals were maintained at the Laboratory Animal Center, The Fourth Military Medical University, China. The animals were divided into two groups, vehicle group, and baicalin-treated group (n=10 of each group). H441 lung cancer cells (4 × 10 6 /0.1 ml) were injected subcutaneously into the right flank of each mouse anesthetized with pentobarbital. Treatment was started when the tumors reached a mean volume of 100 mm 3 . For the baicalin group, 2.5 mg baicalin formulated in 200 μl physiological saline, was administered to each mouse three times a week for 21 days by intraperitoneal (i.p.) injection. For the vehicle group, 200 μl physiological saline was administered to each mouse three times a week for 21 days by i.p. injection. Tumor volumes and body weights were measured. The mice were monitored until tumors reached 1 cm 3 total volume, at which time the mice were killed and the tumors were extracted. The tumors were embedded in a paraffin block and subjected to immunohistochemistry (IHC) or Hematoxylin and Eosin (H&E) staining. All animal experiments were performed following the protocols approved by the Laboratory Animal Center of the Fourth Military Medical University.

Statistical analysis
All quantitative data were expressed as mean values + − standard deviation, and significant differences were determined by Student's t test or by one-way ANOVA. A probability value of P<0.05 was used as the criterion for statistical significance.

Baicalin binds with PBK/TOPK by microscale thermophoresis and in vitro binding assay
The in vitro beads binding assay was detected by the binding between baicalin and PBK/TOPK in H441 cell lysates, which have high expression of PBK/TOPK. A strong band was seen in baicalin-conjugated beads group, whereas no obvious band representing PBK/TOPK was observed in beads without baicalin group ( Figure 1A).
To validate the veracity of in vitro beads binding assay, we employed microscale thermophoresis (MST) method to detect the binding affinity between the anti-tumor compounds and PBK/TOPK. This technology can quantitate protein and small molecule interactions with high sensitivity and low sample cost by detecting fluorescent changes in molecules during thermophoresis. Amongst four compounds assayed, baicalin exhibited the lowest K d of 23.2 + − 5.4 μM ( Figure 1B and Table 1), which meant the strongest binding between the baicalin and PBK/TOPK.

Baicalin inhibits EGF-induced anchorage-independent growth of JB6 Cl41 cells
The structural formula of baicalin is shown in Figure 2A. In order to detect the cytotoxicity of baicalin, different doses of the compound were used to treat JB6 Cl41 cells for 24 h. Cytotoxicity was measured by MTS assay and the results showed that baicalin had no effect on JB6 Cl41 cells viability up to 100 μM at 24 h ( Figure 2B). Furthermore, we detected whether baicalin had an effect on anchorage-independent growth of JB6 Cl41 cells treated with only EGF. The results demonstrated that JB6 Cl41 cells treated with different doses of baicalin formed fewer colonies compared with the control group ( Figure 2C). For example, colony formation was inhibited by more than 30% after treatment  Docking score/interaction potential of compounds with PBK/TOPK (kcal/mol). 2 The K d value is automatically calculated by the curve fitting, and presented as means + − S.D. 3 n.i. is no inhibition detected in the experiments. with baicalin at a concentration of 50 μM, and almost 60% colonies were inhibited at 100 μM baicalin ( Figure 2C). These results showed that baicalin could suppress EGF-induced anchorage-independent JB6 Cl41 cell growth.

Baicalin inhibits PBK/TOPK activity in vitro and ex vivo
The above data showed that baicalin directly binds with PBK/TOPK, implying that baicalin might inhibit the TOPK activity. To confirm this hypothesis, we performed an in vitro kinase assay with Histone H3 and ERK2 as the substrate with active PBK/TOPK in the presence of 25, 50, 100 μM of baicalin. The results indicated that the phosphorylation level of Histone H3 and ERK2 were substantially decreased in a dose-dependent manner after treatment with baicalin ( Figure 3A,B). HI-TOPK-032, a novel PBK/TOPK inhibitor, was used as a positive control [11]. Next, we detected whether baicalin could inhibit PBK/TOPK activities in JB6 Cl41 cells. Data indicated that the expression of phospho-Histone H3 and phospho-ERKs were attenuated by treatment with baicalin in a time- (Figure 3C,D) and dose-dependent manner ( Figure 3E,F).

Baicalin inhibits anchorage-independent growth of lung cancer cells
Previous studies revealed that PBK/TOPK is highly expressed in human lung cancer [25][26]. We attempted to determine whether baicalin could affect anchorage-independent growth of lung cancer cells. We used three lung cancer cell lines H441, H1975, and H1299 with high, middle, and low expression level of PBK/TOPK, respectively ( Figure  4A). First, we determined the cytotoxicity of baicalin by MTS assay. Different concentrations of the drug were used to treat lung cancer cell lines H441, H1975, and H1299 for 48 h, respectively. The results indicated that baicalin had different cytotoxicity toward different lung cancer cells. H441 cells with high PBK/TOPK expression were more sensitive to baicalin ( Figure 4B). The colony numbers of the cells were counted after culturing for 7 days using different concentrations of baicalin. The results showed that baicalin at 25, 50, and 100 μM inhibited colony formation of H441 cells on 27, 49, and 83%; H1975 cells on 25, 37, and 62% and H1299 on 5, 12, and 24%, respectively, compared with the non-treated cells ( Figure 4C-E). Overall, our results suggested that inhibitory effect of baicalin on colony formation was significant in H441 cells with a high expression level of PBK/TOPK.

Knocking down PBK/TOPK in H441 cells decreased the sensitivity of baicalin
To investigate whether the effects of baicalin are mediated directly through PBK/TOPK, first, we determined the efficiency of shPBK/TOPK, as well as the effect of shPBK/TOPK transfection on anchorage-independent growth. The expression of PBK/TOPK was obviously decreased after shPBK/TOPK ( Figure 5A). Moreover, baicalin suppressed anchorage-independent growth in shMOCK cells but had less effects in shPBK/TOPK cells ( Figure 5B). Next, Western blot results indicated that the phosphorylation level of Histone H3 and ERKs was substantially decreased with baicalin treatment in a time-dependent manner ( Figure 5C,D). The above results showed that PBK/TOPK is a direct target for baicalin to inhibit lung cancer cells growth.

Baicalin inhibits tumor growth by suppressing PBK/TOPK activity in vivo
In order to detect the anti-tumor efficacy of baicalin in xenograft model, the left flank of 6-week old athymic nude mice was injected subcutaneously in H441 cells. The mice were divided into vehicle and baicalin treatment group when the tumors reached a mean volume of 100 mm 3 . The above data showed that tumors treated with 50 mg/kg baicalin grew remarkably more slowly and the size of tumors was smaller than the vehicle group ( Figure 6A). However, the weights of mice had not significantly changed between the baicalin-treated group and vehicle ( Figure 6B), which demonstrated that the concentration of baicalin used for the experiment had no toxicity to the mice. To further explore whether the anti-tumor effect of baicalin was associated with its inhibition of PBK/TOPK activities, tumor extracts from either group were analyzed for immunohistochemistry. The results indicated that the phosphorylation expression of Histone H3 and ERKs were substantially decreased in the baicalin-treated group compared with the vehicle group ( Figure 6C). Taken together, our results showed that baicalin inhibited tumor growth by suppressing PBK/TOPK activities in vivo.

Discussion
Lung cancer is a common disease of human malignancy, and its incidence in Asia is increasing gradually year by year [27]. There is a 5-year survival rate of only 15%. Despite routine chemotherapy in aggressive lung cancer, almost all patients ultimately develop resistance to tyrosine kinase inhibitor because of EGFR mutation [28][29][30]. Therefore, it is a good strategy to inhibit other signaling pathway to overcome TKIs resistance of lung cancer. Previous studies have shown that the therapeutic effect in lung cancer through inhibiting PBK/TOPK signaling pathway [31][32].
PBK/TOPK, a 322-amino serine/threonine kinase, is a member of MEK family [15]. PBK/TOPK is a regulator of mitosis, such as formation of spindle midzone and cytokinesis [33]. Previous studies indicated that PBK/TOPK is highly expressed in several malignancies, especially in lung cancer [25]. PBK/TOPK overexpression was significantly correlated with poor prognosis and served as a prognostic marker for lung cancer [26,34]. Because TOPK seems highly expressed in cancer cells and not in normal cells, in which TOPK expression is very low. Therefore, inhibitors of PBK/TOPK would be expected to be an excellent drug target for cancer chemotherapy treatment of lung cancer.
MST screening was performed as a novel way to screen a selective PBK/TOPK inhibitor from several potential anti-tumor natural compounds. We identified a natural compound, baicalin can inhibit the proliferation of lung cancer by blocking PBK/TOPK activity in vitro and in vivo.
Natural compounds from plants have brilliant efficacy and no toxicity, which make more and more various kinds of natural compounds widely used for chemoprevention and chemotherapy. Baicalin, a major flavonoid compound from Scutellaria baicalensis has been reported to possess anti-diabetes [35], antioxidative [36], anti-inflammation [37], anti-cancer [38], and neuroprotective [39] effects. Previous studies showed that baicalin inhibited lung cancer cells proliferation and metastasis [22]. Baicalin also can attenuate resistance to chemoradiotherapy of lung cancer [40][41]. Although baicalin is a pleiotropic protein kinase inhibitor, the detailed mechanism of its pharmacological function remains unclear in lung cancer cells. Herein, for the first time, we found that baicalin can target PBK/TOPK kinase directly and inhibit the proliferation of lung cancer.
In short, the result of present studies identified that PBK/TOPK was a direct and important target of baicalin for inhibition of lung cancer proliferation and transformation. Because the activation of PBK/TOPK signaling pathway is closely related to the occurrence, development, and biological behavior of lung cancer and targeting PBK/TOPK can affect the chemotherapy of lung cancer [42][43][44], our findings provided a better therapy or enhanced the efficacy of chemoradiotherapy for lung cancer by targeting PBK/TOPK with baicalin.