Targeting LIMK1 with luteolin inhibits the growth of lung cancer in vitro and in vivo

Abstract Lung cancer is the leading cause of cancer‐related deaths. LIM domain kinase (LIMK) 1 is a member of serine/threonine kinase family and highly expressed in various cancers. Luteolin, a polyphenolic plant flavonoid, has been reported to suppress tumour proliferation through inducing apoptosis and autophagy via MAPK activation in glioma. However, the mechanism of luteolin on suppressing lung cancer growth is still unclear. We found that luteolin targeted LIMK1 from the in silico screening and significantly inhibited the LIMK1 kinase activity, which was confirmed with pull‐down binding assay and computational docking models. Treatment with luteolin inhibited lung cancer cells anchorage‐independent colony growth and induced apoptosis and cell cycle arrest at G1 phase. Luteolin also decreased the expression of cyclin D1 and increased the levels of cleaved caspase‐3 by down‐regulating LIMK1 signalling related targets, including p‐LIMK and p‐cofilin. Furthermore, luteolin suppressed the lung cancer patient‐derived xenograft tumour growth by decreasing Ki‐67, p‐LIMK and p‐cofilin expression in vivo. Taken together, these results provide insight into the mechanism that underlies the anticancer effects of luteolin on lung cancer, which involved in down‐regulation of LIMK1 and its interaction with cofilin. It also provides valuable evidence for translation towards lung cancer clinical trials with luteolin.


| INTRODUC TI ON
Lung cancer is the most leading cause of both men and women cancer death, and new cases in the worldwide. 1,2 While there is clearly an established risk for lung cancer associated with cigarette smoking, recent data indicated that increased risk of lung cancer in never smokers, especially in women. 2 Non-small cell lung cancer (NSCLC) is a major type (~85%) of lung cancer, and its targeted therapy is tried such as epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs); however, the treatment still limited due to acquired resistance and deficient efficacy. 3  Luteolin, a dietary flavone derived from vegetables, fruits and herbs, traditionally used in Chinese medicine. 5,6 It has beneficial effects including anti-inflammatory, anti-allergic, anticancer and antioxidant, to prevent the diseases. [5][6][7][8][9] Compared with traditional chemotherapy drug, luteolin showed less toxicity as a natural compound. 10,11 It inhibits critical events associated with carcinogenesis, including cell invasion, metastasis, transformation and angiogenesis, by inhibiting transcription factors, kinase modification and cell cycle arrest and inducing apoptosis. 5,7,9,10 Zao et al demonstrated that luteolin epigenetically activated the Nrf2 pathway by down-regulating DNA methyltransferase (DNMT) and histone deacetylase (HDAC) expression. 11 Kang et al demonstrated that luteolin promoted apoptotic human colon cancer cells death by up-regulation of Nrf2 expression through inhibition of DNA demethylase and the interaction of Nrf2 with p53. 5 The anticancer effect of luteolin has been investigated in various cancers including cervical, gastric, lung, colon and breast cancer. 12 Phospho-LIMK-1/2 (Thr 508/505)-R (catalog# sc-28409-R), LIMK-1(catalog# sc-28370), cofilin (catalog# sc-33779) and phosphocofilin (mSer3)-R (catalog# sc-21867-R) were purchased from Santa Cruz Technology.

| Cell cultures
The human normal lung epithelial cell (NL-20) and lung cancer cell lines (NCI-H1975 and NCI-H1650) were purchased from American Type Culture Collection (ATCC). Normal lung cell NL-20 was cultured in Ham's F12 medium with 1.5 g/L sodium bicarbonate, 2.7 g/L glucose, 2.0 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, 0.005 mg/ml insulin, 10 ng/mL epidermal growth factor, 0.001 mg/mL transferrin, 500 ng/mL hydrocortisone and 4% foetal bovine serum. NCI-H1975 and NCI-H1650 cells were cultured in RPMI-1640 containing penicillin (100 units/mL), streptomycin (100 μg/mL) and 10% foetal bovine serum (Biological Industries, Israel) and grown at 37°C in a humidified incubator containing 5% CO 2 . All cells were cytogenetically tested and authenticated before being frozen. Each vial of frozen cells was thawed and maintained in culture for a maximum of 2 months.

| Anchorage-independent cell growth assay
NCI-H1975 and NCI-H1650 cells were suspended in complete growth media and cell concentration were adjusted to 8,000 per millilitre, and then mixed with 0.3% agar contained different doses of luteolin was added in a top layer up a base layer of 0.5% agar in 6 well plates. The colonies were grown at 37°C in a humidified incubator containing 5% CO 2 . Two or three weeks later, the cell colonies were counted and photographed under a microscope using the Image-Pro Plus software (v.6.0) program (MediaCybernetics).

| Cell cycle and apoptosis analysis
Cells were seeded in 60-mm plates and cultured overnight at

| Computational docking model
In order to confirm whether luteolin can bind with LIMK1 kinase, we performed in silico docking method by using the Schrödinger Suite 2016 software programs (Schrödinger, 2016). 19 The LIMK1 structure was built with Prime followed by refining and minimizing loops in the binding site. The structure was prepared under the standard methods of the Protein Preparation Wizard (Schrödinger Suite 2016). Hydrogen atoms were added maintained pH to 7, and all water molecules were discarded. The LIMK1 ATP-binding site based receptor grid was generated for docking. Hence, we can get luteolin computational docking site. When average tumour volume was reached about 1000 mm 3 , mice were euthanized and tumours were extracted to further analysis.

| Immunohistochemistry (IHC) assay
Paraffin-embedded tumour tissues were prepared for H&E staining and IHC analysis. When antigen unmask finished, the tumour tissues were blocked with 5% goat serum and incubated at 4°C overnight with antibodies to detect some protein markers, such as Ki-67, p-LIMK (Thr508/505) and p-cofilin. After incubation with a rabbit secondary antibody, DAB (3,3'-diaminobenzidine) staining was used to visualize the protein targets according to the manufacturer's instructions.
Sectioned tissues were counterstained with haematoxylin, dehydrated through a graded series of alcohol into xylene and mounted under glass coverslips. After then, photographed under a microscope and analysed using the Image-Pro Plus software (v.6.0) program (MediaCybernetics).

| Statistical analysis
Data illustrated with error bars are the mean ± SD. Statistically significant differences were determined using the Student's t test or one-way ANOVA. For all analysis, a p value less than 0.05 was considered statistically significant.

| LIMK is a potential target in lung cancer cells
To targeting the LIMK1 kinase, we performed in silico virtual screening and found luteolin as a candidate. Then, we implemented an

| Luteolin inhibits the proliferation of lung cancer cells
The potency of luteolin on the cytotoxicity and proliferation of normal lung cells and lung cancer cells were determined by MTT assay (Figure 2). Luteolin also attenuated anchorage-independent cell growth of these two lung cancer cell lines in a concentration-dependent manner compared with DMSO treated control ( Figure 2C,D). Representative images of colonies illustrate the number of colonies ( Figure 2D).

| Luteolin directly inhibits LIMK1 activity
To confirm the role of luteolin in inhibiting LIMK activity, we performed

| Luteolin induces cell cycle arrest and apoptosis of lung cancer cells
To examine whether the cell growth inhibition by luteolin was from the regulation of cell cycle and apoptosis, we analysed cell cycle contribution and annexin V staining cells (Figure 4). The results revealed that luteolin-induced cell cycle arrest at G1 phase in NCI-H1975 and NCI-H1650 ( Figure 4A,B). To apoptosis analysis, NCI-H1975 and NCI-H1650 cells were treated with DMSO control and luteolin at the concentration of 5, 10, 20 or 40 μmol/L for 72 hours. Luteolin significantly induced apoptosis in a dose-dependent manner ( Figure 5A,B).

F I G U R E 3 LIMK1 is a potential target of luteolin. (A) The expression of LIMK1 in NCI-H1975 cells after knocking-down LIMK by Western blot. (B)
Proliferation of cells expression shmock and shLIMK #2 and 4 were examined by MTT. The asterisks (*P < .05, **P < .01, ***P < .001) indicate a significant decrease in proliferation compared with corresponding control. (C) Anchorageindependent growth was assessed after treatment with serially doses of luteolin in NCI-H1975 cells expressing shmock and shLIMK #2 and 4. The asterisks (*P < .05, **P < .01, ***P < .001) indicate a significant decrease in colony number compared with corresponding control. Data are shown as mean ± SD of values from triplicate samples Based on the effects induced by luteolin, we examined the expression of proteins associated with the G1 phase of cell cycle and apoptosis by Western blot (Figure 4C,D). Treatment of lung cancer cells with luteolin decreased the expression of cyclin D1 and cyclin D3, cell cycle markers compared with control ( Figure 4C,D). Furthermore, luteolininduced apoptosis markers, Bax, cleaved caspase 3, cleaved caspase-7 and cleaved PARP expression while reduced total form of caspase-3 and caspase-7 expression compared with DMSO controls, respectively ( Figure 5C,D).

| Luteolin down-regulates LIMK1 signalling pathways
To identify the effect of luteolin on LIMK1 signalling pathways, we treated the NCI-H1975 and NCI-H1650 cells with luteolin and detected their expressions ( Figure 6). Luteolin decreased the levels of phosphorylated p-LIMK1/2 and p-cofilin compared with total form of LIMK1, cofilin or DMSO treated controls, whereas ROCK1 and ROCK2 not changed ( Figure 6A,B).

| Luteolin suppresses LIMK1-mediated tumour growth in patient-derived xenograft mice
The anticancer activity of luteolin was then evaluated in lung cancer patient-derived xenograft (PDX) mice experiments (Figure 7 and supplementary Figure S3). Everyday administration of luteolin (100 mg/kg) for 59 days retarded the tumour growth and weight without changing mouse body weight compared with vehicletreated control ( Figure 7A-C and supplementary Figure 3A-B). To determine whether the correlation between luteolin efficacy and Ki-67, p-Limk1/2 and p-cofilin expression that we observed in vitro could be recapitulated in vivo, we performed immunohistochemistry (IHC) analysis in the tumour samples ( Figure 7D,E). The results showed that proliferation marker Ki-67 and signalling marker F I G U R E 5 Luteolin induces cell apoptosis of lung cancer cells. Effects of luteolin on cell apoptosis were in NCI-H1975 and NCI-H1650 lung cancer cells (A). Cells were treated with DMSO or 5, 10, 20 and 40 μmol/L of luteolin and then incubated for 72 h (annexin-V staining assay, apoptosis marker expression). Representative plots of flow cytometry analysis of cell apoptosis of NCI-H1975 and NCI-H1650 (B). Data were shown compared with DMSO treated group. *P < .05; **P < .01; ***P < .001 compared with controls. The effects of luteolin on the expression of biomarkers associated with cell apoptosis (Bax, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7 and cleaved PARP) (C) are shown by Western blot. Quantitative analysis results of three bathes of biomarkers associated with cell apoptosis (Bax, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7 and cleaved PARP) (D) p-Limk1/2 and p-cofilin expression were significantly decreased in luteolin-treated tissues compared with vehicle treated ( Figure 7D,E and supplementary Figure S3C). Therefore, we reinforced the notion that p-Limk1/2 expression is essential for the anticancer activity of luteolin.

| D ISCUSS I ON
Natural compounds from plants have drawn more and more attention due to their function in protecting and suppressing the growth of different human cancers. These food-based products, F I G U R E 6 Luteolin down-regulates LIMK1-related signalling pathways. NCI-H1975 and NCI-H1650 cells were treated with increasing doses of luteolin (5, 10, 20 and 40 μmol/L). The expression and /or phosphorylation of the indicated proteins, ROCK1, ROCK2, p-LIMK, LIMK1, p-cofilin and cofilin were assessed by Western blot (A). β-Actin was used for the internal control to verify equal protein loading. Quantitative analysis results of three bathes of indicated proteins associated with LIMK1-related signalling pathways, ROCK1, ROCK2, p-LIMK, LIMK1, p-cofilin and cofilin (B). Data were shown compared with DMSO treated group. *P < .05; **P < .01; ***P < .001 compared with controls being chemo-preventive agents, are considered to be safer and more effectual against proliferation of cancer. 22 Luteolin is a flavonoid found in different plants such as vegetables, medicinal herbs and fruits. 5,6 It acts as an anticancer agent against various types of human malignancies such as lung, breast, prostate, colon and pancreatic cancer through apoptosis induction, cell cycle arrest and proliferation inhibition as well as metastasis down-regulation. 8,9 However, the molecular mechanisms of its anticancer activities are still unclear.
In this study, we found that luteolin has the potential to treat lung cancer due to its ability to regulate a new target LIMK1/cofilin signalling pathway. As activation of LIMK/cofilin signalling induces cancer development, invasion and metastasis, it is certain that intervening expression and activity can retard cancer cell proliferation, migration and invasion through modulation the target gene expression. [23][24][25][26] Herein, we investigated LIMK inhibitor from the Chinese medicine library by in silico virtual screening and selected luteolin as a candidate. Then, we confirmed the binding between luteolin and LIMK1 and inhibition of kinase activity by luteolin (Figure 1), whereas luteolin did not affect the mRNA level of limk (Supplementary Figure   S1). Byun et al reported that luteolin targeted to protein kinase Cε (PKCε) and Src kinase activities and inhibited UVB-induced skin carcinogenesis and its signalling pathways. 27 The inhibitory activity of luteolin on PKCε and Src kinases was similar against LIMK kinase activity at 20 µmol/L ( Figure 1A). In this study, we have used LIMK highly expressed lung cancer cells, NCI-H1975 and NCI-H1650.
When we knockdown of LIMK expression in Figure 3, the anchorageindependent colony number was significantly decreased and luteolin could not do further inhibition. It means LIMK is the major target Cucurbitacin I and E can inhibit kinetic activity of LIMK to phosphorylate cofilin and then retarded proliferation and migration of HeLa cells and Caco-2 human epithelial colorectal adenocarcinoma. 34,35 Though so many LIMK1 inhibitors exist, few inhibitors act on nonsmall cell lung adenocarcinoma cells.
Natural compound, diallyl disulfide from garlic extract also reduced the colorectal cancer cell migration and invasion. 35,36 However, still it needs to expand more animal experiments and pharmacokinetics analysis for applying the cancer patient treatment.
In summary, our study identified luteolin as a LIMK inhibitor that suppressed tumour growth by inhibiting LIMK kinase activity and related signalling pathways.

ACK N OWLED G M ENTS
We wish to thank Ran Yang, Sen Yang, Bingbing Lu and Fangfang Liu in the China-US (Henan) Hormel Cancer Institute for supporting experiments.

CO N FLI C T O F I NTE R E S T
No potential conflicts of interest were disclosed.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.