Synergetic and antagonistic molecular effects mediated by the

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Synergetic and antagonistic molecular effects mediated by the feedback loop of p53 and JNK between Saikosaponin D and SP600125 on lung cancer A549 cells Xiaoman CHEN, Chenglin LIU, Ruilin ZHAO, Ping ZHAO, Ju WU, Nanjin ZHOU, and MUYING YING Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00595 • Publication Date (Web): 12 Sep 2018 Downloaded from http://pubs.acs.org on September 13, 2018

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Molecular Pharmaceutics

Synergetic and antagonistic molecular effects mediated by the feedback loop of p53 and JNK between Saikosaponin D and SP600125 on lung cancer A549 cells

Xiaoman Chen1#, Chenglin Liu1#, Ruilin Zhao1#, Ping Zhao1, Ju Wu1, Nanjin Zhou2 and Muying Ying1, 2*

1

Department of Molecular Biology and Biochemistry, Basic Medical College of

Nanchang University, Nanchang, PR China; 2Institute of Molecular Medicine, Jiangxi Academy of Medical Sciences, Nanchang, PR China.

#

These authors contributed equally to this work.

*Correspondence to: Muying Ying, Email: [email protected] Address: Bayi Road 603, Nanchang, Jiangxi Province, PR China, 330006 Tel: 86-0791-85788326; Fax: 86-0791-85788326

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Table of contents graphic: Ingenuity pathway analysis of these cell-cycle regulatory molecules in A549 cells treated with Ssd and SP600125


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Abstract We jointly analysed the changes in cell cycle arrest and distribution, the accumulation of sub-phase cells, apoptosis and proliferation in A549 cells treated with Saikosaponin D (Ssd) and JNK inhibitor SP600125 alone or in combination. Our results indicated that cell cycle arrest at G0/G1, S and G2/M phases was coupled with the accumulation of subG1, subS and subG2 cells, corresponding to early apoptosis, DNA endoreplication and later inhibitory proliferation, respectively. Analysing the expression of 18 cell cycle regulatory genes and JNK and phosphorylated JNK (pJNK) levels revealed an enhancement in these factors by Ssd. Additional SP600125 weakened or eliminated the Ssd-induced increase of these factors except that p53/p21 and Rassfia levels were further improved. Ingenuity Pathway Analysis (IPA) of the interactions of these factors revealed a negative synergistic effect on apoptosis while a positive synergistic effect on proliferative inhibition of the two drugs: 1) Ssd induced apoptosis via the activation of two axes: TGFα-JNK-p53 and TGFα-Rassfia-Mst1. By eliminating the Ssd-induced increase of JNK/pJNK, additional SP600125 weakened Ssd-induced apoptotic axis of TGFα-JNK-p53 and simultaneously abolished Ssd-induced apoptosis; 2) Ssd inhibited proliferation by the activation of two axes: TGFβ-p53/p21/p27/p15/p16 and TGFα-Rassfia-cyclin D1. By improving the Ssd-induced increase of p53/p21 and Rassfia, additional SP600125 enhanced the two axes of Ssd-induced inhibitory proliferation. Analysing JNK/pJNK, p53, phospho-p53 and TNF-α levels revealed an opposite association of JNK/pJNK with p53 while consistence with phospho-p53 and TNF-α, which supported the proposals that



JNK/pJNK negatively regulated p53 level while mediated p53 phosphorylation to transcriptionally activate TNF-α expression of apoptotic gene and trigger apoptosis. With the multiple roles, JNK/pJNK form a synergetic and antagonistic feedback loop with phospho-p53/p53. Within the feedback loop: 1) Ssd-induced apoptosis depended on JNK/pJNK activities mediating phospho-p53 that activated TNF-α expression; 2) By weakening the negative regulation of JNK/pJNK in p53, SP600125 enhanced p53 level and the Ssd-induced inhibitory proliferation axes of TGFβ-p53/p21/p27/p15/p16. The results indicated the central coordinating roles of the feedback loop in the synergistic and antagonistic effects of the two drugs in A549 cells and provided a rationale for the combination of Ssd with SP600125 in the treatment of lung cancer.

Key words: Saikosaponin D; SP600125 of JNK inhibitor; Cell cycle regulatory gene; Ingenuity Pathway Analysis; Feedback loop involving phospho-p53/p53 and JNK/pJNK; Lung cancer A549 cells.

Abbreviations: Saikosaponin D, Ssd; c-Jun N-terminal kinase, JNK; phosphorylation of JNK, pJNK; Human Umbilical Vein Endothelial Cells, HUVEC; Ingenuity Pathway Analysis, IPA; Kyoto Encyclopedia of Genes and Genomes, KEGG.


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Introduction Cancer growth is characterized by an aberrant cell cycle, which is directly regulated by three key classes of regulatory molecules, namely, cyclins, cyclin-dependent kinases (Cdks) and CDK inhibitors (CKIs), which function as regulatory, catalytic and inhibitory subunits, respectively, in heterodimers of cyclin-Cdk or CKI-Cdk complexes. The fluctuating cyclin-Cdk complex plays a broad role in cell cycle control1. Whether Cdks are activated or inhibited depends on their partners, cyclins and CKIs. Only proper activation of phase-specific Cdks can guarantee the sequential transitions of the cell cycle and maintain apoptosis and proliferation with normal efficiency2. As a medicinal plant used to prolong life, Bupleurum falcatum L. (Umbelliferae) has been widely applied in health protection and disease treatment among people in Asia. Eleven types of saikosaponins have been isolated from its root, of which Saikosaponin D (Ssd) has the most pharmacological activities followed by Ssa3. Unlike Ssa, which has been shown to increase the levels of p16/15 but not other cell cycle regulators in HepG2 cells4, Ssd up-regulated the levels of all the 18 cell cycle regulatory genes in lung cancer A549 cells in this study. Ssd functions in anti-inflammation, cytoprotection of normal cells, liver disease and systemic lupus erythematosus through different signalling pathways5-6. By directly inhibiting an endoplasmic reticulum Ca2+ ATPase pump and the mammalian target of rapamycin (mTOR), Ssd induced autophagy in various cancer cells7, modulated the PKC pathway through PKCθ, JNK and NF-κB transcription factors, suppressed T cell activation by reducing CD69, CD71 and IL-26, abolished TNF-induced cancer cell



invasion and angiogenesis in HUVEC cells and induced apoptosis via enhancing the loss of mitochondrial membrane potential in HeLa cells8. As a suggested target for the treatment of inflammatory disease, apoptotic cell death and cancer9, increased c-Jun N-terminal kinase (JNK) activity has been widely reported in scenarios of cell proliferation and apoptosis. To understand the molecular events of Ssd drug activities and the possible cross-talk with the JNK pathway, Ssd and SP600125 (JNK inhibitor) were combined in A549 cells to investigate their effects on cell cycle progression, migration, apoptosis and proliferation, with a stress on molecular events analysed by changes in the expression of 18 key cell cycle regulatory genes and three apoptotic related genes (FAS, ARAF1 and TNF-α), the levels of JNK/pJNK (phosphorylated JNK), phospho-p53 (Ser15, Thr18 and Thr81) and phospho-p21 (Thr145). To gain insights into the regulatory mechanisms of the two drugs in cell cycle progression, apoptosis and proliferation of A549 cells, we analysed the interaction network of these molecules with the Ingenuity Pathway Analysis (IPA) database.

Materials and Methods Reagents, cells and cell culture Ssd (>99% purity, HPLC) and SP600125 (an anthrapyrazolone inhibitor of Jun N-terminal kinase) were purchased from Shanghai Yuanye Biological Technology (Shanghai, China). Stock solutions of Ssd and SP600125 were prepared in DMSO (Sigma, St Louis, MO, USA) and an equal volume of DMSO was added to the controls. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO,


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USA) unless otherwise stated. FastQuant RT kit was purchased from TianGen Biotech (Beijing, China). SYBR Green was purchased from ComWin Biotech (Beijing, China). A549 cells (American Type Culture Collection [ATCC] CCL185) were obtained from the Chinese Academy of Medical Sciences and Peking Union Medical College and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco/BRL, U.S.A) and penicillin/streptomycin (Thermo Fisher, USA) in a humidified incubator at 37 ℃ and 5% CO2. Cells were treated with the appropriate volume of DMSO as a control group.

Wound healing assay for cell migration To assess the effects of Ssd and SP600125 on cell migration, A549 cells (2.5 × 105/ml) were seeded into 6-well plates, cultured in DMEM with 5% FBS and incubated at 37℃ in an atmosphere of 5% CO2 overnight until confluent. An artificial wound was created along the centre of each monolayer using a sterile pipette tip. Wells were then washed twice with PBS to remove detached cells. The wounded monolayers of A549 cells were immediately treated with Ssd and SP600125 (0 µM Ssd/2 µl DMSO, 0.5 µM, 2 µM, 10 µM SP600125, 0.5 µM + 10 µM SP600125, 2 µM + 10 µM SP600125) and incubated for a further 12 h, 24 h, 36 h or 48 h. The denuded areas were captured and analysed using Java’s ImageJ software. For each well, at least five pictures were taken at a magnification of 100x after scraping. The cell migration distances were determined with the ratios of wound closure. The migration ratio of cells treated with 0 µM Ssd was set as 1.0, and the ratio of migrated cells was calculated as a percentage



of the control.

Cell proliferation assay Cell proliferation was evaluated using the Cell Counting kit-8 (CCK-8; Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). In brief, A549 cells were seeded into 96-well plates at a density of 5000 cells/well and cultured overnight for cell adherence. Then, A549 cells were treated with Ssd and SP600125 (0 µM Ssd/2 µl DMSO, 0.5 µM Ssd, 2 µM Ssd, 10 µM SP600125, 0.5 µM Ssd + 10 µM SP600125, 2 µM Ssd + 10 µM SP600125) for 12 h, 24 h, 36 h, 48 h and 60 h. Then, 100 µl of CCK-8 was added at different time points and incubated in the dark at 37°C for 3 h. The quantification of cell viability was determined by CCK-8 assay. Absorbance was measured at a wavelength of 450 nm using a spectrophotometer at different time points using the Multiskan Ascent microplate reader (ELx800; Bio-Tek Instruments, Inc., Winooski, VT, USA) The inhibition of cell growth was calculated as follows: inhibitory ratio % = (Control OD - Test OD)/Control OD × 100.

Annexin V-FITC and propidium iodide staining for the cell apoptosis assay After incubating with various concentrations of Ssd and SP600125 (0 µM Ssd/2 µl DMSO, 2 µM Ssd, 4 µM Ssd, 6 µM Ssd, 20 µM SP600125, 2 µM + 20 µM SP600125, 4 µM + 20 µM SP600125, or 6 µM + 20 µM SP600125) for 12 h, A549 cells were detached with trypsin and collected by centrifugation at 2,000 rpm for 5 min. The cell pellet was suspended in 195 µl binding buffer according to the manufacturer’s


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instructions of Annexin V-FITC/PI kit (Beyotime, China), stained with 5 µl annexin V-FITC and 10 µl propidium iodide (PI), and incubated with annexin V-FITC and PI in the dark for 30 min at room temperature. The cells were transferred into a BD Falcon® tube, and the percentage of apoptotic cells in each sample was analysed via FACSCalibur MT flow cytometer (Becton Dickinson Technologies, USA).

Cell cycle distribution A549 cells at the logarithmic growth phase were randomly seeded in 6-well culture plates (Corning, USA). After reaching 50% confluence, cells were cultured in serum-free medium for 24 h to induce cell quiescence. Cells were seeded at a density of 1.0 × 106 cells per well and treated with various concentrations of Ssd and SP600125 (0 µM Ssd/2 µl DMSO, 2 µM Ssd, 4 µM Ssd, 6 µM Ssd, 20 µM SP600125; 2 µM + 20 µM SP600125; 4 µM Ssd + 20 µM SP600125; 6 µM Ssd + 20 µM SP600125) for 24 h. Both floating and adherent cells were collected, washed with phosphate-buffered saline (PBS), and fixed in ice-cold 70% ethanol at 4°C overnight. Then, cells were stained with 500 µl PI solution [10 µg/ml PI (Beyotime, China) and 20 µl RNase A (Beyotime, China)] and incubated in a 37℃ water bath for 30 min. Data acquisition was performed on a FACScan (Becton-Dickinson, Franklin Lakes, NJ, USA) within 30 min and subsequently analysed using Lysis II software (Becton-Dickinson). For cell sorting experiments, cells containing 2N or 4N DNA were separated based on PI fluorescence using a FACSVantage SETM (Becton-Dickinson, Franklin Lakes, NJ, USA) and prepared for western blotting.



Western blotting Protein lysates were prepared from A549 cells and quantified by BCA assay following the instructions. Antibodies against cyclin B1 (1:800, 55004-1-AP), Aurora A (1:800, 10297-1-AP), p53 (1:2000, 10442-1-AP), p21 (1:1000, 10355-1-AP), p27 (1:1000, 25614-1-AP), p16 (1:600, 10883-1-AP), p15 (1:1000, 23652-1-AP), RB (1:500, 17218-1-AP), RBL1 (1:500, 13354-1-AP), PCNA (1:1000, 10205-2-AP), RBL2 (1:500, 27251-1-AP), Cdk1 (1:1000, 19532-1-AP), Cdk2 (1:600, 10122-1-AP), Cdk4 (1:1000, 11026-1-AP), Cdk6 (1:1000, 14052-1-AP), E2F1 (1:500, 12171-1-AP), GAPDH (1:5000, 60004-1-Ig), cyclin D1 (1:1000, 60186-1-Ig), ASAF1 (1:1000, 21710-1-AP), and goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Proteintech Group Inc., Wuhan Sanying (Wuhan, China). Antibodies against FAS (1:500, A2639, ABclonal), JNK1/JNK2/JNK3 (1:500, A11119, ABclonal), Phospho-MAPK8-T183 (1:1000, AP0276, ABclonal), Phospho-p53-S15 (1:1000, AP0083, ABclonal), Phospho-TP53-T18 (1:1000, AP0464, ABclonal), TNF-α (1:1000, A0277, ABclonal) were purchased from ABclonal Company. Antibodies against phospho-p21-Thr145 (1:1000, YP0200, Immunoway, China), phospho-p53-Thr81 (#2676, CST, Inc) and Rassf1 (1:2000, sc-58470, Santa Cruz Biotech., Inc) were also used. Western blotting was performed as previously published10. GAPDH was used as an internal control for western blotting. The levels of proteins were normalized and relatively quantified according to GAPDH levels in three independent experiments. Immunoreactive bands were detected using the enhanced chemiluminescence (ECL) detection system from Amersham Pharmacia Biotech (Arlington Heights, IL USA)


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according to the manufacturer’s instructions.

Quantitative real-time PCR Total RNA was isolated using Trizol (Invitrogen), and cDNAs was synthesized according to the standard protocol using FastQuant RT kit (TianGen, Beijing, China). qRT-PCR was performed by standard methods using SYBR (Kangwei, Beijing, China). Primers were designed and synthesised by Shanghai Songon Biotech Co., Ltd. (Table S1). Relative mRNA enrichment was calculated using the 2-∆∆CT method by normalising the quantity of GAPDH.

Statistical analysis All experiments were repeated in triplicate and results are reported as the mean ± SEM based on independent samples. Statistical analysis was performed using Student’s t test, and P < 0.05 was considered to be significant and represented by *. P < 0.01 was represented by **, P < 0.001 by ***. No significance was represented as ns in figures.

Bioinformatics analysis The data for the 18 cell cycle regulatory genes and JNK/pJNK were imported into the Ingenuity Pathway Analysis (IPA) database (Ingenuity H Systems, Redwood City, CA, USA; http://www.ingenuity.com) for human diseases in the molecular and cellular functions categories. Comparisons of the expression of these genes were performed



between the test and control groups. With the different networks of the Ingenuity Knowledge Base, biological processes were then algorithmically generated within the datasets. Pathway analysis was performed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg/mapper.html). By the integration of KEGG pathway-related cell cycle, apoptosis and proliferation information, the interaction networks of these cell cycle regulatory genes and JNK/pJNK signalling were built within the IPA database to analyse their possible regulatory mechanisms.

Results The effects of Ssd and SP600125 on migration and proliferation in A549 cells We investigated the effects of Ssd and SP600125 treatment in A549 cells, either alone or in combination, on cell migration and proliferation by wound scratch and CCK-8 assays. Quantitative evaluation indicated that the inhibitory rates of cell migration by wound scratch were 21.45% for 0.5 µM Ssd, 42.86% for 2 µM Ssd, 49.66% for 10 µM SP600125, 60.92% for 0.5 µM Ssd +10 µM SP600125 and 75.29% for 2 µM Ssd + 10 µM SP600125 after 48 h when compared to cell migration in the control groups (0 µM Ssd) (Figure 1A and B). The relative absorbance was determined by CCK-8 assay and showed that the inhibitory rate of cell proliferation was 4.06% for 0.5 µM Ssd, 17.58% for 2 µM Ssd, 22.18% for 20 µM SP600125, 30.62% for 0.5 µM Ssd +10 µM SP600125, and 36.71% for 2 µM Ssd + 10 µM SP600125 after 48 h when compared to cell proliferation in the control groups (0 µM Ssd) (Figure 1C). Evidently,


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the data indicated that the effects of SP600125 and Ssd on inhibitory proliferation and migration in A549 cells were positive synergistic, which was further confirmed by the following flow cytometry data.

The effects of Ssd and SP600125 on apoptosis and the cell cycle distribution Flow cytometry and Annexin V-PI staining assay were performed to determine the effects of Ssd and SP600125 on apoptosis and the cell cycle distribution, which indicated that: 1) Ssd alone blocked cell cycle transitions from G1 to S and from G2 to M phases, arrested cell cycle at both G0/G1 and G2/M phase and caused the concurrent accumulation of subG1 and subG2 cells, ranging from 56.17% with 0 µM Ssd to 68.23% with 4 µM Ssd and from 7.47% with 0 µM Ssd to 40.63% with 6 µM Ssd, respectively. Due to failure to leave G1 and enter the S phase with normal efficiency, the accumulated subG1 cells subsequently triggered early cell apoptosis, as indicated here, ranging from a rate of 3.66% with 0 µM to 30.09% with 6 µM Ssd (Figure 2A-D). Similarly, the failure to split one into two daughter cells, the accumulated subG2 cells accompanied the inhibitory proliferation, with an inhibitory rate of 16.86% with 2 µM Ssd after 36 h when compared to that of the DMSO control groups (Figure 1C); 2) With the accumulated tetraploid (4N) cells from G2 phase, SP600125 alone had no effect on apoptosis but inhibited proliferation when compared to DMSO control groups (Figure 2A). Analysing the distribution of cell cycle indicated that SP600125 continuously re-initiated DNA replication within S phase or DNA endoreplication and prevented the entry of cells into mitosis, which resulted in a



decrease in the total and G2/M cells but an increase in S phase cells, ranging from 31.36% (DMSO) to 38.25% with 20 µM SP600125 (Figure 2B-D). Additionally, similar observations have been reported in HCT116 cells11. SP600125 induced endoreduplication by activating p21 transcription, which inhibited cyclin E/Cdk2 activity but did not directly regulate cyclin B1/Cdc2 activity12. Consistently, the following results of western blotting and qPCR also indicated that SP600125 alone increased p21 and Cdk2 levels; 3) With Ssd and SP600125 in A549 cells, SP600125 synchronously abolished Ssd-induced subG1 accumulation (ranging from 68.23% with 4 µM Ssd to 47.61% with 4 µM Ssd + 20 µM SP600125) and apoptosis (ranging from 30.09% with 6 µM Ssd to 5.09% with 6 µM Ssd + 20 µM SP600125) (Figure 2A-D), while further enhanced Ssd-induced subG2 accumulation (ranging from 19.09% with 4 µM Ssd to 47.36% with 4 µM Ssd + 20 µM SP600125) and Ssd inhibitory proliferation (ranging from 17.58% with 2 µM Ssd to 36.71% with 2 µM Ssd + 10 µM SP600125 after 48 h) (Figure 1C and 2B-D). The additional SP600125 synchronously abolished Ssd-induced apoptosis while enhanced Ssd-inhibited proliferation, which indicated that: 1) Ssd-induced apoptosis depended on JNK activity; 2) the two drugs had a negative synergetic effect on apoptosis while a positive synergetic effect on proliferative inhibition in A549 cells.

Molecular events of A549 cells treated with Ssd and SP600125 The orderly transition of cell cycle phases depends on the completion of the previous stage and proper activation of the next one, which is monitored by phase-specific cell


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cycle regulatory molecules, particularly cyclins, Cdks and CKIs. To understand the molecular events of the pharmacological actions of Ssd and SP600125 in A549 cells, we examined the changes of 18 key cell cycle regulators and the total JNK/pJNK levels in A549 cells treated with the two drugs. The 18 cell cycle regulators can be broadly classified into positive (cyclin B1/D1, Cdk1/2/4/6, Aurora A, E2F1 and PCNA) and negative (CKIs: p53/21/27/16/15; RB family members: RB1, RBL1 and RBL2 and Rassfia) regulators. JNK and pJNK are two key components reflecting the activation of the JNK pathway. Our results indicated: 1) Somewhat surprisingly, A549 cells treated with Ssd alone exhibited increased mRNA and protein levels of all 18 cell cycle regulatory genes and JNK/pJNK levels when compared to the control groups (Figure 3A-C). JNK/pJNK negatively regulates p53 levels by inhibiting transcription of p53 mRNA13 and targeting p53-ubiquitinated degradation14. The concurrent Ssd-induced increase in JNK/pJNK levels partially abrogated the effects of Ssd on increasing p53 levels, which resulted in the observations that Ssd alone modestly increased p53 mRNA and protein levels in A549 cells (Figure 3A and B). The p53 protein experienced twice JNK/pJNK-mediated inhibition: transcriptional inhibition13 and targeting degradation14, while p53 mRNA experienced once JNK/pJNK-mediated transcriptional inhibition. Additionally, the opposite correlations of p53 with JNK/pJNK levels also indicated the negative regulation of JNK/pJNK in p53 level (Figure 3C); 2) Without significant effects on the expression of other regulators, SP600125 alone increased p53/p21, Cdk2, cyclin D1 and Rassfia while reduced JNK/pJNK levels when compared to control groups (3A-C); 3) Except for the



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duplicated effects on increasing p53/21 and Rassfia levels, Ssd and SP600125 in A549 cells mutually weakened or eliminated their effects on the levels of other members. Additionally, Ssd seemed to increase the phosphorylated levels of Cdk2/4, cyclin D1, RB1 and Auraro A, which were also weakened by SP600125 (Figure 3A-C).

Ingenuity pathway analysis of these cell cycle regulatory molecules Despite the fundamental difference in apoptosis, proliferation and cell cycle progression, increasing amounts of evidence have indicated that at some point, these processes use a common biochemical pathway while achieve different results15. To better understand the regulatory mechanisms of Ssd and SP600125 in the decision of cell fate, we analysed the expression of these molecules using the IPA database and identified 96 Ingenuity Canonical Pathways that may be associated with these cell cycle regulatory genes (Table S2). By integrating pathways related to cell cycle, apoptosis and proliferation in Table S2, we built an interaction network of these genes regulating cell cycle, apoptosis and proliferation in the API database (Figure 4). Our results indicated that Ssd alone induced apoptosis through TGFα-JNK-p53 and TGFα-Rassfia-Mst1 axes, both of which needed TGFα receptor (Figure 2-4). With Ssd and SP600125 in A549 cells, additional SP600125 eliminated the Ssd-induced increase of JNK/pJNK levels and simultaneously abolished Ssd-induced apoptosis. Conversely, the additional SP600125 further enhanced the Ssd-induced increase of p53/p21

levels

and

the

Ssd-induced

inhibitory


proliferation

axis

of

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TGFβ-p53/p21/p27/p15/p16 (Figure 2-4). By increasing CKIs (p53/p21/p27/p16/p15) levels (Figure 3), Ssd alone activated the TGFβ-p53/p21/p27/p15/p16 axis to induce replicative senescence, block cell cycle progression, promote apoptosis and inhibit proliferation in A549 cells16 (Figure 4). As Cdks inhibitors, the p53/p21/p27/p16/p15 proteins inactivated Cdks in heterodimers of phase-specific cyclin-Cdk complexes, such as cyclin D1-Cdk4/6, cyclin E-Cdk2 and CDC25A at G1 phase, cyclin A/B-Cdk1 at G2 phase, cyclin A-Cdk2 at S phase and cyclin A-Cdk1/2 at M phase (Figure 4). The inactivation of Cdks resulted in the attenuation of Cdk-mediated phosphorylation of Rb members required for continued cell cycle transition17 (Figure 2-4). In contrast to the negative synergistic effect of the two drugs on apoptosis, SP600125 and Ssd showed positive synergistic effect on inhibitory proliferation. By enhancing the Ssd-induced increase in p53/p21 and Rassfia levels, SP600125 intensified the two Ssd-induced inhibitory proliferation axes of TGFβ-p53/p21/p27/p15/p16 and TGFα-Rassfia-cyclin D1.

The synergetic and antagonistic feedback loop involving JNK/pJNK and p53 IPA results indicated a synergetic and antagonistic feedback loop involving JNK/pJNK and phospho-p53/p53 of the two drugs in the decision of A549 cell fate (Figure 4). Ssd induced apoptosis via the activation of TGFα-JNK-p53 axis. The increased JNK/pJNK mediated phospho-p53 activation required for Ssd-induced apoptosis (Figure 4). SP600125 inhibited JNK/pJNK activities and simultaneously abolished Ssd-induced apoptosis. The p53 is well known to induce apoptosis,



particularly in response to cell stress events activating JNK18. SP600125 increased p53 level that failed to trigger apoptosis. The observations may be attributed to the simultaneous reduce of JNK/pJNK levels by SP600125 that resulted in the decreased level of JNK-mediating phospho-p53. The phospho-p53 activity is essential for transcriptional activation of apoptotic gene expression and the subsequent apoptosis18 (Figure 4). By weakening JNK/pJNK negative regulation in p53 level, SP600125 increased p53 level and further enhanced the Ssd-induced inhibitory proliferation axis of TGFβ-p53/p21/p27/p15/p16. Therefore, the two drugs showed additive effects on inhibitory proliferation in A549 cells.

To further get insights into the synergetic and antagonistic molecular effects, we examined the changes of phospho-p53 levels on three residues (Ser15,Thr18 and Thr81) and p21 on Thr145, and the levels of three apoptotic related molecules (APAF1: apoptotic peptidase activating factor; TNF-α: tumor necrosis factor α; and FAS: The fas receptor, first apoptosis signal) in A549 cells treated with the two drugs alone or in combination by western blotting (Figure 5). Consistently, the change of JNK/pJNK levels is consistent with phospho-p53 levels (S15, T18 and T81) and TNF-α while opposite to p53 level in A549 cells treated with the two drugs (Figure 3C and 5). On the one hand, Ssd increased JNK/pJNK levels accompanied with the subsequent increase of the JNK-mediated phospho-p53 (S15, T18 and T81) levels (Figure 3C and 5). Concomitant with the increased phospho-p53 is the transcriptional activation of p53 to induce the expression of apoptotic related genes and trigger


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apoptosis18. Among APAF1, TNF-α and FAS of three apoptotic related genes, the change of TNF-α expression was consistent with the levels of JNK/pJNK and the phospho-p53 (S15, T18 and T81) (Figure 5). Thereby, TNF-α may participate in the Ssd-induced apoptosis in A549 cells. With Ssd and SP600125 in A549 cells, SP600125 inhibited JNK/pJNK activities that accompanied with the reduced levels of phospho-p53 (S15, T18 and T81) and TNF-α, and abolished Ssd-induced apoptosis (Figure 2-5). On the other hand. JNK/pJNK negatively regulated p53 level by inhibiting transcription of p53 mRNA13 and targeting p53-ubiquitinated degradation14. SP600125 inhibited JNK/pJNK activities, and weakened the negative regulation of JNK/pJNK in p53 level. Therefore, p53 level was further increased in A549 cells treated with Ssd and additional SP600125 (Figure 3 and 5), which resulted in the enhancement

of

the

Ssd-induced

inhibitory

proliferation

axes

of

TGFβ-p53/p21/p27/p15/p16 (Figure 2-5). Structural algorithms predicted that JNK-mediated phospho-p53 at Thr81 promotes the expression of apoptotic target genes in response to JNK activation by cell stress in various cells18. Arguably, the most important phosphoepitope governing p53 activation is Ser15 as its phosphorylation is required for subsequent phosphorylation of other p53 residues19-20.

Consistent with these observations, SP600125 has been shown to trigger polyploidization that cannot be sustained by TP53-/- cells, resulting in the activation of

mitotic

catastrophe,

an

oncosuppressive

mechanism

for

eradicating

mitosis-incompetent cells21. With decreased JNK activity, JNK1 null fibroblasts



derived from c-jun-/- mouse embryos exhibit a severe cell cycle and apoptosis defect13. Primary Jun-/- fibroblasts, with elevated levels of p53/p21 and reduced Jun activity, have proliferation- and stress-induced apoptotic defects, which can be reverted fully by simultaneous deletion of p5313, 22.

Discussion This study investigated the pharmacological activities of Ssd and its possible molecular mechanism by combination treatment of Ssd with SP600126 in A549 cells. Ssd alone prevented the transition of subG1 to S and subG2 to M, arrested the cell cycle at both G1 and G2 phases with the concomitant accumulation of subG1 and subG2 cells. Due to failure to properly complete the previous phase and activate next one, the accumulated subG1 and subG2 cells could promote early apoptosis and inhibit late cell proliferation, respectively. SP600125 treatment alone in A549 cells decreased the total number of cells and cells in G2/M, while continuously re-initiating DNA replication within S phase, resulting in the accumulation of subS cells. With Ssd and SP600125 treatment in A549 cells, the two drugs demonstrated negative synergistic effects on apoptosis but positive synergistic effects on proliferation inhibition. Ssd-induced G0/G1 arrest, subG1 accumulation and apoptosis were concomitantly abolished by the additional SP600125. Conversely, Ssd-induced G2/M arrest, subG2 accumulation and inhibitory proliferation were synchronously enhanced by the additional SP600125. By jointly analysing the changes in cell cycle arrest and distribution, the accumulation of sub-phase cells, apoptosis and proliferation, our


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results indicated reciprocal coupling of cell cycle arrest at G1/G0 with accumulated subG1 cells and cell apoptosis, accumulated S cells with DNA endoreplication, and cell cycle arrest at G2/M with accumulated subG2 cells and inhibitory proliferation in A549 cells treated with Ssd and SP600125, respectively.

During cell cycle progression, regulatory proteins are precisely synthesised and degraded to assure orderly transition through each phase of cell cycle. Ssd in A549 cells increased the levels of both the 18 positive and negative regulators (Figure 3). The Ssd-induced increase of positive regulators failed to induce transition from subG1 to S and subG2 to M with normal efficiency in A549 cells, which may suggest the defects of Cdk activities, which is essential for cell cycle transition. As an emergency braking system for cell cycle, the strong and simultaneous increase of negative cell cycle regulators of CKIs (p53/p21/p27/p16/p15), RB family members (RB1, RBL1 and RBL2) and Rassfia may be responsible for the defects (Figure 6). By binding to its specific cyclin/Cdk (such as p21/cyclin D1, A, E/Cdk2, 423; p27/cyclin A, D2, E/Cdk2, 424; p16/cyclin D/Cdk4, 6 and p15/cyclin D/Cdk4, 625), increased CKIs attenuate Cdk-mediated phosphorylation of Rb members and control cell cycle transition at both G1/S phase and G2/M phase26. p21 and p27 can interact with all cyclin/Cdks while p16 selectively inhibits Cdk4/6-mediated RB1 phosphorylation26 (Figure 6). In addition, by binding to and sequestering the activation of E2Fs, the increased RB1/L1/L2 decreases the efficiency of E2F-mediated transcription of target genes necessary for G1/S and G2/M transition27. Through different temporal profiles



of interaction with different E2F/DP1 complexes , RB1/L1/L2 mediated growth arrest in different ways with similar functional consequences of growth suppression28. Additionally, through blocking DNA synthesis and controlling microtubule stability and mitosis, Rassfia arrests cell cycle transition and inhibits proliferation in cancer cells29 (Figure 6). Therefore, these positive regulators seem to not be sufficient to maintain cell cycle progression with normal efficiency when CKIs are present at high levels.

Ssd and SP600125 showed a negative synergetic effect on apoptosis while a positive synergetic effect on inhibitory proliferation in A549 cells (Figure 2). With the multiple roles, JNK/pJNK formed a feedback loop with phospho-p53/p53, which coordinated the synergistic and antagonistic molecular effects of the two drugs in A549 cells. Within the feedback loop, on the one hand, Ssd-induced apoptosis depended on JNK/pJNK activities mediating phospho-p53. The phospho-p53 activation increased the expression of TNF-α and triggered apoptosis18 (Figure 2-7). SP600125 inhibited JNK/pJNK activities accompanied with the reduced levels of phospho-p53 and TNF-α, and the synchronous abolishment of Ssd-induced apoptosis. On the other hand JNK/pJNK negatively regulated p53 level by inhibiting p53 transcription13 and targeting p53-ubiquitinated degradation14. By weakening the JNK/pJNK negative regulation in p53 level, SP600125 improved the Ssd-induced increase of p53 and Rassfia levels, and enhanced the Ssd-induced inhibitory proliferation axes of TGFβ-p53/p21/p27/p15/p16 and TGFα-Rassfia-cyclin D1. Phosphorylation often


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serves as a marker that triggers subsequent ubiquitination, in particular where ubiquitination leads to degradation14, 30. The p53-phosphorylated activation may be accompanied with the subsequent p53-ubiquitinated degradation. The multiple roles of JNK/pJNK in negatively regulating p53 transcription, mediating phospho-p53 activation and triggering p53-ubiquitinated degradation constitute the dynamic interactions within the feedback loop. Maybe, only the perfect combination can maintain p53 proper activity in normal cell cycle progression.

Consistently, SP600125 decreased JNK/pJNK level with the concurrent increase of p53 level in adult mouse brains without inducting apoptosis31. In addition, SP600125 activated p21 transcription and induced the phosphorylation of p21 at Thr 14512. The increased phosphorylated p21 located in cytoplasm have been indicated to be anti-apoptotic12, 32. Cyclin D1 was efficiently induced in G1 progression by treatments that increase JNK activity33. The TGFα-Rassfia-Mst1/Cyclin D1 axis may directly link Ssd-induced apoptosis to cell cycle arrest at the G1/G0 phase and inhibition of proliferation. By assembling a translational inhibitory complex on cyclin D1 mRNA, Rassfia negatively regulates cyclin D1 level and blocks cyclin D1 accumulation and G1/S phase transition of cell cycle34.

Traditional herbal formulae and herbal plant extracts have been recognized as attractive sources for novel multi-targeted therapy of cancer with minimal side effects. By increasing protein synthesis, including positive and negative regulators of cell



cycle, Ssd may benefit the overall metabolic balance of cells, which in turn increases Ssd-based therapeutic effects and decreases side effects in cancer treatment. The dual roles of Ssd in initiating apoptosis and inhibiting proliferation come together to ensure correct concurrence of Ssd activities in minimizing survival rate of A549 cells. Our study unveils previously uncharacterized synergistic and antagonistic actions and its molecular effects of Ssd and SP600125 on apoptosis and proliferation in A549 cells, and provides a rationale for the combination of Ssd with SP600125 in the treatment of lung cancer.

Acknowledgment This work was supported by grants from the National Nature Science Foundation of China (31160233), the Science and Technology Foundation of Jiangxi Province (20142BAB204013) and Graduate Student Innovation Special Foundation of Jiangxi Province (YC2018-S087).

Conflict of Interest Disclosure The authors declare that they have no competing interests.

Supporting Information Table S1 Primers designed for quantitative real time PCR. Table S2 Ingenuity Canonical Pathways that these molecular may participate in.


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Authors' contributions M.Y. was responsible for experimental design, data analysis and manuscript writing. C. X., L. C. and Z. L. prepared figures 1, 2, 3 4 and 5. Z. P., W.J. and Z. N. prepared supplementary tables 1 and 2. All authors read and approved the final manuscript.

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Figure Captions Figure 1 The inhibitory effects of Ssd and SP600125 alone or in combination on A549 cell migration and proliferation. A. A549 cell migration was assessed with scratch wound assay and expressed by a wound healing area. B. The picture were expanded 100. C. The effect of Ssd on A549 cell proliferation was determined by CCK-8 assay. Data (Mean ± SD) are expressed as the percentage of the controls from three independent experiments that yielded similar results, ***p < 0.001.

Figure 2 Effects of Ssd and SP600125 alone or in combination on A549 cell cycle distribution, apoptosis and proliferation. A. Apoptosis levels were evaluated by Annexin V-PI staining after A549 cells treated with the two drugs as indicated. The



plots show PI staining at the x-axis (DNA content) and cell counts at the y-axis. B. Flow cytometric analysis of apoptosis in A549 cells treated with the two drugs as indicated. C. Relative percentage of cells in various cell-cycle phases over time of drug treatment were analyzed using propidium iodide (PI) staining and evaluated by flow cytometric analysis. D. Cell percentages of different cell-cycle phase.

Figure 3 Expression change of 18 cell-cycle regulatory genes in A549 cells treated with Ssd and SP600125 alone or in combination. A. Western blotting analysis of the 18 cell-cycle regulatory genes. Ssd alone increased mRNA and protein levels of all these 18 cell-cycle regulatory genes. With Ssd and SP600125 in A549 cells, except for the duplicated effects on increasing p53/21 and Rassfia levels, the two drugs mutually weakened or eliminated their effects on the levels of other members. B. Real-time PCR analyzed mRNA levels of the 18 cell-cycle regulators in A549 cells treated with Ssd and SP600125, either alone or in combination after 36 hours. Data presented as mean ± SD (n=3). **p < 0.05, ***p < 0.001. P

Synergetic and antagonistic molecular effects mediated by the

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