Discovery of a novel protein kinase C activator from Croton tiglium for inhibition of non-small cell lung cancer

Phytomedicine 65 (2019) 153100

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Original Article

Discovery of a novel protein kinase C activator from Croton tiglium for inhibition of non-small cell lung cancer

T

Wang Yuweia,1, Tang Chunpingb,1, Yao Shengb, Lai Huanlinga, Li Runzea, Xu Jiahuia, ⁎ ⁎ Wang Qianqiana, Fan Xing Xinga, Wu Qi Biaoa, Leung Elaine Lai-Hana,c,d, , Ye Yangb, , ⁎ Yao Xiaojuna, a

State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau (SAR), China State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academic of Sciences, Shanghai, China c Department of Thoracic Surgery, Guangzhou Institute of Respiratory Health and State Key Laboratory of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China d Respiratory Medicine Department, Taihe Hospital, Hubei University of Medicine, Hubei, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Non-small cell lung cancer Natural product Croton tiglium Protein kinase C Molecular modelling

Background: The incidence of non-small cell lung cancer (NSCLC) accounts for approximately 85–90% of lung cancer, which has been shown to be challenging for treatment owing to poorly understanding of pathological mechanisms. Natural products serve as a source of almost all pharmaceutical preparations or offer guidance for those chemicals that have entered clinical trials, especially in NSCLC. Purpose: We investigated the effect of B10G5, a natural products isolated from the Croton tiglium, in human nonsmall cell lung canceras as a protein kinase C (PKC) activator. Methods: The cell viability assay was evaluated by the MTT assay. The apoptosis and cell cycle distribution were assessed by flow cytometry. Reactive oxygen species (ROS) production was determined by using the fluorescent probe DCFDA. Cell migration ability of H1975 cells was analyzed by using the wound healing assay. The inhibiting effect of B10G5 against the phosphorylation level of the substrate by PKCs was assessed by using homogeneous time-resolved fluorescence (HTRF) technology. The correlation between PKCs and overall survival (OS) of Lung Adenocarcinoma (LUAD) patients was analysis by TCGA portal. The binding mode between B10G5 and the PKC isoforms was explored by molecular docking. Protein expression was detected by western blotting analysis. Results: B10G5 suppressed cell proliferation and colony formation, as well as migration ability of NSCLC cells, without significant toxic effect on normal lung cells. B10G5 induced the cell apoptosis through the development of PARP cleavage, which is evidenced by means of the production of mitochondrial ROS. In addition, the B10G5 inhibitory effect was also related to the cell cycle arrest at G2/M phase. Mechanistically, molecular modelling technology suggested that the potential target of B10G5 was associated with PKC family. In vitro PKC kinase assay indicated that B10G5 effectively activated the PKC activity. Western blotting data revealed that B10G5 upregulated PKC to activate PKC-mediated RAF/MEK/ERK pathway. Conclusion: Our results showed that B10G5, a naturally occurring phorbol ester, considered to be a potential and a valuable therapeutic chemical in the treatment of NSCLC.

Introduction Lung carcinoma is the leading cause of cancer‑related death in world‑wide. According to histological features, approximately 20% of

lung cancer cases are SCLC, and the other 80%, including adenocarcinoma, squamous cell carcinoma, large‑cell carcinoma and bronchoalveolar cell carcinoma, are classified as NSCLC, which has proven to be intractable to treat owing to insufficient understanding of

Abbreviations: ERK, extracellular signal–regulated protein kinases; HTRF, homogeneous time-resolved fluorescence; LUAD, Lung Adenocarcinoma; MAP, mitogenactivated protein; NC, nitrocellulose; NSCLC, non-small cell lung cancer; OS, overall survival; PKC, protein kinase C; ROS, reactive oxygen species; XP, extra precision ⁎ Corresponding authors. E-mail addresses: [email protected] (E.L.-H. Leung), [email protected] (Y. Ye), [email protected] (X. Yao). 1 These authors contribute equally https://doi.org/10.1016/j.phymed.2019.153100 Received 11 June 2019; Received in revised form 24 September 2019; Accepted 25 September 2019 0944-7113/ © 2019 Elsevier GmbH. All rights reserved.

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S1). Other chemicals and reagents were purchased from Sigma-Aldrich unless stated otherwise. All chemicals were dissolved in dimethyl sulfoxide.

pathological mechanisms (Oyewumi et al., 2014; Siegel et al., 2017). Among its carcinogen signaling pathways, the protein kinase C (PKC) family plays an important role. PKC belongs to the phospholipiddependent serine/threonine kinases family and is divided into three subgroups based on their distinct regulation: classical (α, βI, βII, and γ), novel (δ, ε, η, and θ), and atypical (ζ and ιi). PKC isozymes are equipped with a commonly conserved N-terminal regulatory region, which consists of the C1 domain, C2 domain, as well as a C-terminal catalytic region responsible for ATP binding and phosphotransferase activity. Among them, the C1-domain is the binding site of phorbol ester and diacylglycerol (DAG) in cPKCs and nPKCs, where it is duplicated in tandem (C1a and C1b). The C2-domain in cPKCs binds to calcium, whereas in nPKCs it is primarily a calcium-unresponsive phospho-tyrosine binding motif. However, aPKCs does not bind phorbol ester/DAG or calcium (Mochly-Rosen et al., 2012). The activation of classical enzymes (cPKC) is mainly dependent on the concentration of Ca2+ and DAG, while the novel enzymes (nPKC) are mainly activated by means of diacylglycerol (DAG), and the activation of atypical enzyme (aPKC) is independent of calcium or DAG. PKC isozymes are referred to the activation of key cellular processes, containing cell proliferation, differentiation, and apoptosis (Berridge and Irvine, 1984; Nishizuka, 1992), which is regarded as therapeutic targets for several human diseases (Mochly-Rosen et al., 2012). For example, Hill et al. reported that PKCα plays a key role in K-Ras-mediated lung tumorigenesis, which evidenced the loss of expression of PKCα in primary human NSCLC tumors.(Hill et al., 2014) Expression of PKCβII in NSCLC has significant variability in tumor cells and stroma (Chang et al., 2011). PKCβ inhibition impairs proliferation and anchorage-independent growth of human NSCLC cells (Bae et al., 2007; Caino et al., 2012). It is elevated of PKCι expression in NSCLC, which is required for the transformed phenotype of NSCLC cells carrying oncogenic K-ras mutations (Frederick et al., 2008; Regala et al., 2009, 2005a, 2005b). PKC isozymes have been generally considered as an oncoprotein, however, several researchers have demonstrated that PKCs activity is often lost in human cancers (e.g., colorectal cancers, lung squamous cell carcinomas) due to the mutation causing loss of function, which supports a role of PKCs as tumor suppressors instead of tumor promoters (Antal et al., 2015; Newton, 2018). The increasing evidences demonstrated that the activation of PKC serves as a vital role in inducing the regression of human cancers. In particular, the activation of specific PKCs will lead to inhibition of proliferation and induction of apoptosis in cancer cells (Clavijo et al., 2007; DeVries-Seimon et al., 2007; Griner and Kazanietz, 2007). These results led scientists to believe that the treatment of cancer related to PKC should focus primarily on restoring their activity. Natural products were identified as a crucial source of therapeutically effective drugs, and they play a vital role in the prevention and treatment of cancer. Therefore, activating PKC by natural products may be a valuable therapeutic option for the treatment of NSCLC and may help overcome the emergence of drug resistance. So far, we have demonstrated that several natural products and their analogues can suppress the growth of NSCLC cells and induce their apoptosis through various mechanisms (Fan et al., 2017, 2015; Li et al., 2018, 2017). In present study, we identified a novel PKC activator with promising targeted anticancer activity in NSCLC. We revealed that B10G5 inhibits the proliferation of NSCLC cells by interacting with PKC to directly target and activate it, which will further result in abnormal activation of extracellular signal–regulated protein kinases (ERK) 1 and 2. Our results suggested that B10G5 is a potent agent for understanding the potential pathophysiological properties of PKC in lung cancer and further evaluating potential targeted therapies for NSCLC.

Human lung normal and cancer cell lines and culture conditions The human normal lung bronchial epithelial (BEAS-2B) and human lung cancer (A549 and H1975) cell lines were purchased from ATCC. Herein, human lung cancer A549 and H1975 cell lines were cultured in RPMI-1640 medium, and medium was supplemented with 10% fetal bovine serum (FBS; Gibco) with 100 U/ml penicillin and 100 μg/ml streptomycin. BEAS-2B lung cells were cultured in complete bronchial epithelial growth medium (Lonza, Walkersville, MD, USA) supplemented with insulin (5 μg/ml), epinephrine (0.5 μg/ml), human EGF (0.5 ng/ml), hydrocortisone (0.5 μg/ml), transferrin (10 μg/ml), gentamycin (50 μg/ml), retinoic acid (0.1 ng/ml), triiodothyronine (6.5 ng/ ml) and bovine pituitary extract (52 μg/ml). All cells were cultured in an environment at 37 °C, 5% CO2. Cell proliferation and viability assay Cell proliferation was demonstrated by using the standard 3-(4, 5dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide MTT assay. Firstly, well-grown cells were seeded in 96-well plates at a cell density of 3.0 × 103 cells/well and allowed to adhere for 24 h. Next, cells were exposed to the indicated concentration of B10G5 for 72 h (DMSO acted as a blank control). 10 μl MTT was added into each well and incubated for 4 h, then 100 μl of the resolved buffer (99% DMSO) was added to dissolve the dark blue crystals. The absorbance value at 570 nm was measured with a microplate reader (Tecan, Morrisville, NC, USA). Colony formation assay Briefly, H1975 cells were planted in 6-well plate at a cell density of 1 × 103 cells/well for 24 h and then exposed to the indicated concentration of B10G5 or DMSO. The medium was changed every three days was formed the visible colonies. Following three time of washes with ice-cold PBS, visible colonies were fixed with 4% paraformaldehyde (PFA) for 15 min and then stained with 0.5% crystal violet (0.5% crystal violet, 1% PFA, and 20% methanol in ddH2O) for 20 min. Finally, the colonies were photographed. Annexin V and PI staining assay Briefly, H1975 cells were exposed to the B10G5 or vehicle in the indicated concentrations for 48 h, then washed with ice-cold PBS and resuspended with 1 × binding buffer (100 μl). The cells were stained with 1 μl of dissolved propidium iodide (PI) (50 μg/ml) and 1 μl of annexin V-fluorescein isothiocyanate solution (2.5 μg/ml) for 15 min in darkness. Afterwards, 400 μl of pre-cooled 1 × binding buffer was added and gently mixed. The percentage of apoptotic cells was analyzed by using a BD Aria III Flow Cytometer (FACSAriaIII, BD Biosciences). The obtained data was processed by means of the Flowjo software (version 7.6). Cell cycle analysis For the cell cycle analysis, cells were seeded in 6-well plate at a cell density of 1 × 103 cells/well and cultured for 24 h. Then, cells were exposed to the indicated concentrations of B10G5 or vehicle and incubated for 48 h at 37 °C. The cells were collected after trypsinization, then washed twice with ice-cold PBS and immobilized in pre-cooling 70% methanol for 2 h in darkness. Subsequently, the cells were collected by centrifugation, and then mixed with RNaseA and stained with PI for 1 h in darkness after washing twice with PBS, followed by a BD FACSAria III Flow Cytometer (FACSAriaIII, BD Biosciences). The data

Material and methods Chemical and regents B10G5 was isolated from Croton tiglium (Purity > 99%, see Figure 2

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Table 1 The structure and IC50 values of identified compound from natural product library. No.

Structure

IC50 Value (μM) H1975

A549

BEAS-2B

B10G5

0.11 ± 0.05

>20

>2

B10G6

0.16 ± 0.04

>20

>2

B10E10

0.55 ± 0.23

4.87 ± 1.23

1.55 ± 0.53

B16A8

0.0075 ± 0.0023

0.08 ± 0.03

>0.3

B23A7

0.19 ± 0.04

>5

3.65 ± 0.18

B23B8

0.97 ± 0.04

>5

1.49 ± 0.19

Cell migration assay

were processed via the Flowjo software (version 7.6).

Cell migration ability of H1975 cells was analyzed by using the wound healing assay. Briefly, H1975 cells were planted in 6-well plates at a cell density of 2 × 105 cells/well for 24 h, then cells with a wound were exposed to a range of B10G5 and PMA for 24 h. Finally, the migration of H1975 cells was photographed.

ROS measurement The ROS production was determined by using the fluorescent probe DCFDA. Briefly, H1975 cells were seeded in 6-well plate at a cell density of 2 × 105 cells/well and then serum-starved for 24 h. Next, the cells were firstly preprocessed with DCFDA for 0.5 h, and then dealt with the indicated concentration of B10G5 for 0.5 h. Subsequently, cells were harvested after trypsinization and centrifuged. The proportion of ROS-positive cells after washing twice with cold 1 × PBS was counted by using a BD FACSAria III flow cytometer.

In vitro PKC assay The inhibiting effect of B10G5 against the phosphorylation level of the substrate by PKCs was assessed by using homogeneous time-resolved fluorescence (HTRF) technology. The HTRF® KinEASE™ STK Kit 3

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Fig. 1. Predicted targets of B10G5. The predicted targets are ranked according to their scores. Green bars indicated the estimated probability that a specific protein will become a true target.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Cisbio, USA) and purified recombinant human PKC proteins, PKC α, PKC β and PKC δ (Carna, USA), were used. Briefly, 4 μl substrate and 2 μl kinase were added to a 384-well plate, and then indicated concentrations of B10G5 were added. Subsequently, the reaction was proceed with the addition of ATP for 30 min. For the detection of phosphorylated products, TK-antibody labeled with streptavidin-XL665 and Eu3+-cryptate were then added with EDTA for 2 h. Finally, the fluorescence value was measured at 665 nm (XL665) and 620 nm (cryptate) by using the TECAN Infinite 200 Pro-multilabel plate reader.

overall survival (OS) of Lung Adenocarcinoma (LUAD) patients. Survival analysis based on the Kaplan-Meier method was performed, and the log-rank test was also executed. p < 0.05 was selected as the cutoff value. Western blot In brief, H1975 cells were lysed in RIPA lysis buffer including protease inhibitor for 10 min on ice after treating with B10G5 and then boiled for 10 min in a 100 °C water bath. After cooling, Bio-Rad DCTM Protein Assay Kit (Bio-Rad, Hercules, CA, USA) was used to determine the concentration of total protein. Then, 30 μg protein lysate were loaded one by one and then separated by use of 10% SDS-polyacrylamide gel electrophoresis followed by transferring onto nitrocellulose (NC) membrane purchased from Millipore (Billerica, MA, USA). After blocking NC membranes with 5% skimmed milk powder dissolved in 1 X TBST, the NC membranes were incubated with different primary antibodies for 24 h at 4 °C. Antibodies are described in Supplementary Table S1. Next, the NC membranes were washed by the TBST buffer three times for 10 min, and then secondary fluorescent antibodies (anti-rabbit or anti-mouse) were added and incubated for 2 h at room temperature. After washing the membranes again by using TBST three times, the signal intensity of specific band in NC membranes was captured by using LI-COR Odessy scanner (Belfast, ME, USA).

Molecular docking The crystallographic structure of PKC α (PDB ID: 4RA4) (George et al., 2015), PKC β (PDB ID: 2I0E) (Grodsky et al., 2006), PKC δ (PDB ID: 1PTR) (Zhang et al., 1995) complexed with PRB was downloaded from protein data bank for validating the binding mode of B10G5. The structures of ligand were processed by using Schrödinger 2015 (Madhavi Sastry et al., 2013). The grid box of each complex was determined by centering on endogenous ligand. After drawing the structure of B10G5, B10G5 was prepared by using the LigPrep module with OPLS-2005 force field. The ionized state of ligand was assigned by use of Epik module (Shelley et al., 2007) at a pH of 7.0 ± 2.0. Subsequently, B10G5 was docked to the binding site of PKC isozymes by using the Glide (Friesner et al., 2006) extra precision (XP) mode. The best binding pose for B10G5 with PKC isozymes was preserved.

Statistical analysis TCGA verification and survival analysis of PKCs All of the experimental results were expressed as mean values ± standard error (mean ± S.D.). One-way ANOVA test followed by Bonferroni's post-tests was used to statistical analysis. Significance differences were accepted at the level of p < 0.05.

TCGA portal (http://tumorsurvival.org/index.html, accessed 23 March 2018), a tool for in-depth analyses of The Cancer Genome Atlas (TCGA) data, was used to determine the correlation between PKCs and 4

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Fig. 2. B10G5 activated the PKCs by targeting PKC kinase domain. (A-B) The effect of B10G5 on PKCs and its phosphorylation in H1975 for 1 h by western blot analysis. (C) In vitro kinase assay with recombinant PKCs for the effect of 40 μM B10G5 and 10 μM PMA (positive control). B10G5 has a significantly activation of PKCs enzyme at 40 μM. (D-F) Binding model of B10G5 into PKC α (D), PKC β (E), PKC δ (F), respectively. The key residues of PKCs interact with B10G5 was represented and shown as sticks. The hydrogen bonds are displayed through a dotted red line. (G-I) A two-dimensional interaction map of B10G5 and PKC α (G), PKC β (H), PKC δ (I), respectively. Data was represented as mean ± SD.

Result

All 2, 000 compounds were initially evaluated in all cell lines for 72 h at 20 μM, and then best promising compounds were reserved for determining IC50 value. Only 6 compounds showed IC50 less than 1 μM in H1975 cell line and their structures were listed in Table 1. These data implied that B10G5 and B10G6 have an approximate 200 fold selectivity for H1975 and A549, but B10G5 with lower cytotoxicity. Besides, we also addressed the cytotoxic effect of B10G5 on normal lung BEAS-2B cells. Interestingly, the results showed that the IC50 value of B10G5 in H1975 cells was approximate 20-fold lower than that of BEAS-2B cells, indicating that B10G5 has strong inhibitory selectivity for NSCLC cells, rather than normal lung cells..

Six compounds were identified from a natural product library based on cytotoxicity against human NSCLC cells and normal lung bronchial epithelial cells To discover potent anti-tumor agent from natural products, we screened a natural products library consisting of 2, 000 compounds (provided by Prof. Yang Ye, from Shanghai Institute of Materia Medica, CAS) by using standard MTT assay on human NSCLC cells (A549 and H1975 cells) and normal lung bronchial epithelial cells (BEAS-2B cells). 5

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Fig. 3. Kaplan-Meier survival curve for PKC α (A), PKC β (B) and PKC δ (C) expression levels in TCGA patients with lung adenocarcinoma. The log-rank test was carried out on the relevant results.

that B10G5 could be considered as a potential PKC activator. To gain more structural basis of the potential binding mode between B10G5 and the PKC isoforms, molecular docking experiments were employed to explore the structural basis (See Fig. 2D–F). The docking scores of B10G5 for PKC α, PKC β and PKC δ were −6.89 kcal/mol, −7.04 kcal/mol and −7.49 kcal/mol, respectively. Compared with the docking score between PKCs and B10G5, the complex of PKC δ and B10G5 was the most stable. Besides, hydrophobic interactions and hydrogen-bonding interactions stabilize the entire system (see Fig. 2G–I). These docking results supported the B10G5 is a PKC activator.

B10G5 is a PKC activator interacting with PKCs In order to query the molecular targets of B10G5, Swiss Target Prediction tool (Gfeller et al., 2014) was employed to predict the potential molecular target, which were shown in Fig. 1. The predicted targets included PKC isozymes, transient receptor potential cation channel subfamily V member 4, prostaglandin G/H synthase 1/2, quinone oxidoreductase, Microtubule-associated protein tau, glucocorticoid receptor and mineralocorticoid receptor, which were ranked based on their possibility score with respect to the B10G5 structure. These results showed that the targets of B10G5 may be associated with PKC isozymes. As a result, to better compare the effect of B10G5, we firstly characterized the dose dependence of B10G5 on PKC isoforms by western blotting. As shown in Fig. 2A and B, treatment with B10G5 caused a rapid shift of phosphorylated pan PKC and PKC δ. Among PKC isoforms, PKC α, PKC β and PKC δ were selected to further evaluate whether B10G5 increased PKCs activity by using an in vitro kinase assay. It can be seen from Fig. 2C that B10G5 significantly activated PKC α, PKC β and PKC δ in 40 μM, while PMA was used as blank control in 10 μM. In addition, B10G5 increased the basal activity of both PKC α and PKC β activity with similar potency. Compared with the activation activities of PKC α and PKC β, B10G5 markedly elevated the activity of PKC δ with nearly 1.7-fold. These results demonstrated

TCGA validation and the Kaplan-Meier plot To reveal the importance of PKCs for overall survival of lung cancer patients, TCGA data of LUAD patients were used via the TCGA portal. Kaplan-Meier curve for overall survival of TCGA patients with LUAD was retrieved on the basis of the low and high expression of PKCs. Our results indicated that the expression level of PKC α was found to be negatively correlated with overall survival in LUAD (Fig. 3A), while the expression level of PKC β and PKC δ protein was found to be significantly correlated with overall survival in LUAD (Fig. 3B and C). Importantly, the overall survival rates of those patients with PKC β (p = 0.00067) and PKC δ (p = 0.0002823) in the high expression group 6

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Fig. 4. B10G5 markedly induced cell apoptosis in H1975 cells. (A) The proportion of apoptotic cells after B10G5 treatment for 48 h. (B) Statistical analysis of apoptosis data. (C) PARP was cleaved after treatment with B10G5 for 48 h. (D) Statistical analysis of the densitometry of cleaved PARP. (E) The ROS levels after B10G5 treatment for 48 h in H1975 cells. (F) Statistical analysis of the percentage of ROS generation. (G) Colony formation assay for H1975 cells treated with B10G5. In B, D and F, data were presented as mean ± SD (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001).

treatment was quantitatively measured by flow cytometry analysis in combination with western blotting assay. It can be seen from Fig. 4A that the percentage of apoptotic cells dramatically increased after exposure to 100 nM B10G5 for 48 h compared with the control. From the flow cytometry results in Fig. 4B, a dose-dependent increase of the percentage of apoptotic cells was observed in H1975 cells. Moreover, PARP was significantly cleaved and activated by B10G5 (Fig. 4C and D). It suggested that B10G5-induced apoptosis in H1975 cells is mediated

were markedly higher than those in the low expression group. These results further demonstrated that activation of PKC β and PKC δ activity may be a valuable strategy in NSCLC treatment. B10G5 significantly induces apoptosis in H1975 cells To study how B10G5 induced cell apoptosis in H1975 cells, the proportion of apoptotic cells after B10G5 and PMA (positive control) 7

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Fig. 5. B10G5 induced cell cycle arrest in H1975 cells. (A) H1975 cells were treated with B10G5 for 48 h. (B) Statistical analysis of cell cycle after B10G5 treatment. (C) Western blot analysis of the expression levels of Cyclin B1 and Cyclin B1 after treatment with B10G5 for 48 h. (D-E) Statistical analysis of the densitometry of Cyclin B1 and Cyclin B1, respectively. (F) The wound healing of H1975 cells treated with B10G5 for 24 h was recorded. (G) Western blot analysis of the protein levels of E-cadherin after 24 h treatment. (H) Statistical analysis of the densitometry signals of E-cadherin. All data was presented as mean ± SD (n = 3, * p < 0.05, ** p < 0.01).

after B10G5 and PMA (positive control) treatment by using flow cytometry analysis. Cells were exposed to B10G5 for 48 h and then DCFHDA was loaded to measure intracellular levels of ROS. As shown in Fig. 4E and F, we discovered that B10G5 induced a dramatically increase in DCF fluorescence with a dose-dependent manner. In addition, we also studied whether B10G5 can inhibit colony formation in H1975 cells. Colony formation assay was performed. It can be seen from in Fig. 4G that B10G5 markedly repressed the colony

by the activation of pro-apoptotic proteins PARP. B10G5 enhances ROS generation, but inhibits colony formation in H1975 cells As an early signal of apoptosis (Liou and Storz, 2010), reactive oxygen species (ROS) are the cause of several anti-tumor drugs that inhibit tumor progression. Intracellular ROS production was evaluated 8

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Fig. 6. The effect of B10G5 on the PKC-mediated RAF/MEK/ERK signaling pathway in H1975 cells after 1 h (A) or 24 h (B) treatment. The levels of the indicated cRAF/MEK/ERK and of GADPH as loading control were detected after treatment with B10G5 for 6 or 24 h. Table 2 Summary of reported PKC activators. Compounds

Structures

Proposed mechanism

References

Acadesine

PKC ε activator

(Robert et al., 2009)

Bryostatin

Non-selective PKC activator

(Kortmansky and Schwartz, 2003; Zhang et al., 1996)

Vibsanin A

Non-selective PKC activator

(Yu et al., 2016)

Roy-Bz

PKC δ activator

(Bessa et al., 2018)

B10G5 suppresses the cell migration ability in H1975 cell line

formation ability of H1975 cell line in a dose-dependent manner.

To investigate the effect of B10G5 on the migration of H1975 cells, a wound healing assay was employed. It can be seen from Fig. 5F that B10G5 caused significant migration inhibition. In addition, the effect of B10G5 on migration protein levels was determined by immunoblotting. From Fig. 5G and H, we found that the expression of E-cadherin increased with a dose-dependent manner. It suggested that B10G5 suppressed the cell migration of H1975 cell by upregulating the expression of E-cadherin.

B10G5 causes cell cycle arrest in H1975 cells In order to evaluate cell cycle profile of H1975 cells treated with B10G5, cell cycle analysis was profiled by using flow cytometry after H1975 cells were exposed to a range of B10G5 for 48 h. As shown in Fig. 5A and B, treatment with B10G5 resulted in cell cycle arrest at the G2/M phase with a dose-dependent manner. Furthermore, the effect of B10G5 on cycle-associated protein levels was determined by immunoblotting. It can be seen from Fig. 5C–E that the expression of Cyclin B1 and Cyclin D1 decreased with a dose-dependent manner. Together, these results indicated that B10G5 caused G2/M phase arrest leading to the decrease of the expression of Cyclin B1 and Cyclin D1.

B10G5 effects on the PKC-mediated RAF/MEK/ERK signaling pathway It has been well documented that PKC plays an important role in the activation of the mitogen-activated protein (MAP) kinase pathway, especially MEK/ERK/MAP kinase signaling pathway (Ueda et al., 9

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activity of PKC activity, especially PKC δ. As we know, classical PKC ligands such as PMA and prostaglandins bind to the C1 domain of PKC in the form of structural analogs of DAG. Considering the similarity of the structure between PMA and B10G5, we speculated that B10G5 interacts specifically with the phorbol binding C1-domain of PKC. In accordance with this hypothesis, molecular docking results indicated that B10G5 interacts with the C1 domains of PKC. Although the detailed structural basis for the binding of B10G5 to PKCs remains to be studied, our results identify B10G5 as an effective PKC activator with satisfying binding affinity. Although some small molecules have been reported in the literature as direct PKC activators (Table 2), gefitinib-resistant cells have not been used as research objects. Compare with these four activators, the chemical structure of the identified compound B10G5 is simpler and easier to synthesize. Thus, the structural optimization based on B10G5 compound to improve the activity is practicable. After identifying PKC as a target of B10G5, we further analyzed the molecular mechanism of B10G5 on NSCLC cells. Our results showed that B10G5 remarkably increased the percentage of apoptosis, with the upregulation of cleaved PARP. Besides, as a promising target, regulation of cell cycle checkpoints significantly contributed to the treatment of cancer. (Takashi et al., 2001) Accordingly, we discovered that B10G5 significantly increased the proportion of NSCLC cells at G2/M phase. Activation of PKC was also related with the generation of ROS, which was observed in other models (Inoguchi et al., 2003; Stein et al., 2017; Yang et al., 2018), and may play an important role in the development of cardiac mitochondrial dysfunction. Supporting this PKC-mediated ROS generation, we found that B10G promoted the ROS generation with a dose-dependent manner. Taken together, our study indicated that B10G5 inhibited the cell growth by inducing cell apoptosis and cell cycle arrest at G2/M phase by upregulating cleaved PARP and downregulating Cyclin B1 and Cyclin D1 (see Fig. 7). In conclusion, we have identified a PKC activator B10G5 from natural products library which inhibits cell proliferation and induces cell apoptosis of NSCLC cells. B10G5 exerts the anticancer action by activating PKC-mediated RAF/MEK/ERK signaling pathways. In summary, our study indicated that B10G5 is a novel PKC activator, which has a great potential for treatment of NSCLC. In the future, we will further chemically-modified this compound into several derivates and compare their in vitro and in vivo therapeutic effect.

Fig. 7. Proposed mechanism of action mechanism of B10G5. B10G5 induced the activation of PKCs, resulting in cleaved PARP, ROS generation, G2/M phase arrest, upregulated E-cadherin and PKC-mediated RAF/MEK/ERK pathway, which lead to the apoptosis of NSCLC cells.

1996). To address the effects of B10G5 on downstream signaling pathways, we investigated RAF/MEK/ERK pathway by immunoblotting after treatment for 1 h to evaluate the duration of signal activation. As shown in Fig. 6A, B10G5 induced the phosphorylation of cRaf, MEK, and ERK in a dose-dependent manner (Fig. 6A). In addition, downregulation after ligand binding is a potential feedback mechanism for PKC, which reduces the activation of PKC signaling pathways (Christine and Alexandra, 2008; Gao et al., 2008; Leontieva and Black, 2004; Parker et al., 1995). At the same time, down-regulation after B10G5 treatment for 24 h is also observed at 24 h in H1975 cells (Fig. 6B). Discussion PKC isozymes play an important role in human diseases, including NSCLC. The PKC family consists of nine genes that are involved in many targets that regulate a variety of cellular functions, including cell proliferation, differentiation, migration, apoptosis, and survival. (Dempsey et al., 2000) However, PKC was regarded as a tricky target in cancer treatment because of contradictory evidence as to whether they serve as tumor suppressors or as oncogenes. Recently, a meta-analysis of PKC inhibitors in combination with chemotherapy showed that PKC inhibitors significantly reduced the response rate of non-small cell lung cancer.(Zhang et al., 2015) Subsequently, several scientists have revealed that PKC activity is usually lost in certain cancer attributed to the loss-of-function mutation, which supports the potential role of PKC isozymes as tumor suppressors rather than tumor promoters. (Antal et al., 2015) This phenomenon led scientists to believe that cancer treatment strategy should concentrate on activating PKC activity instead of inhibiting their activity. Additionally, the expression level of PKC β and PKC δ protein were found to be significantly correlated with overall survival in lung adenocarcinoma by using a Kaplan-Meier plot for survival analysis. Our results identified that the overall survival of patients with high expression of PKCβ and PKCδ was significantly higher than those in the low expression group. Therefore, developing PKC activator may be a potentially valuable strategy in cancer therapy. In present study, we screened a natural products library, and identified a novel PKC activator B10G5 by combining molecular modelling, biochemical, structural, and cellular analysis. Herein, for the first time, we present evidence that B10G5 exhibits potent anticancer activity against NSCLC cells by activating PKC-mediated signaling pathway. After obtaining the potential target is associated with PKC family, our in vitro kinase profile of B10G5 has shown that B10G5 increased the basal

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