Finding a novel tetramethylated hydroxydiethylene of resveratrol analogue for potential anticancer agent

With the improvement and advance in cancer diagnosis and treatment, the cancer is still a major cause of morbidity and mortality throughout the world. Obviously, new breakthroughs in therapies remain be urgent needed. In this work, we designed and synthesized the compound 1-4, namely resveratrol analogues with methylation of hydroxy distyrene, to further explore its new anti-cancer potential. Encouragingly, compound 1 (( E )-4,4'-(ethene-1,2-diyl)bis(3,5-dimethylphenol)) exhibited cytotoxicity superior to resveratrol in MCF 7 cells. More importantly, the compound 1 showed greater toxicity to tumor cells than that to normal cells, which proved that it could selectively kill tumor cells. The favorable results encouraged us to explore the inhibitory mechanism of compound 1 on MCF 7 cells. The research finding indicated the compound 1 inhibited tumor cell proliferation by both arresting cell cycle in S phase and apoptosis via a prooxidant manner. In addition, the results further verified compound 1 caused cell cycle arrest in S phase and apoptosis by down-regulation of the cycling A1/cycling A2 expression and the rise of Bax/Bcl-2 ratio in a p21-dependant pathway in MCF 7 cells. Therefore, these results are helpful for the effective design of anticanceranticancer reagents and the better understanding of their mechanism of action.


Introduction
Cancer, as a global public health and social problem, is a major cause of morbidity and mortality, affecting all of humankind [1]. Due to multiple etiologies, complicated pathogenesis, and multi-stages, although great efforts have been made, many kinds of cancers still don't have efficient screening strategies, or just 3 have limited treatment options with high recurrence risks and/or adverse prognosis.
Obviously, there remains an urgent need for effective treatments and new breakthroughs in cancer therapy.
A large body of research has shown that carcinogenesis is associated with the cell cycle control, signal transduction or apoptosis leading to abnormal proliferation of cancer cells, which may provide the potential drug targets for cancer therapies [2][3][4][5]. Obviously, molecules with the potential of an anti-proliferation and proapoptotic effect on tumor cells, but not on normal cells, may be appropriate anticancer agents. ROS (Reactive oxygen species), as the intracellular second messengers, are closely involved in various physiology processes in normal cells.
However, once the redox homeostasis is disrupted, ROS will present heterogeneous effects depending on the different levels [6]. ROS may display the carcinogenic effect at low levels through the induction of cellular abnormal proliferation [7].
Interestingly, the basal ROS levels in cancer cells are higher than those in normal cells, which assign the negative role of ROS in tumorigenesis at high levels through prooxidant activity to induce apoptosis and/or cell cycle arrest by damage DNA [8][9].
Based on this, exploration of prooxidants strategies may be effective therapeutic measures.
Resveratrol (3,4′,5,-trihydroxy-trans-stilbene, RES), as a kind of natural polyphenolic compound in a wide variety of dietary, has attracted a large number of attention.
Because of its pharmacological properties, RES has been reported to be involved closely in various physiological processes, including anti/prooxidant, antiinflammation, cardio-and vascular-protection, neuroprotective activity, antimicrobial activity, as well as anti-carcinogenic properties [10]. A great deal of research has verified that RES worked via the following potential mechanisms: 4 acts as the antioxidant or prooxidant being responsible for cytotoxic, or as the antiproliferative and even pro-apoptotic molecular to induce apoptosis and/or cell cycle arrest against cancer [10,11]. However, due to the low bioavailability and rapid metabolism rate [12], many researches were focused on the structure modification to further exploit its potential [13][14][15]. Up to now, structure modifications of RES were focused mainly on the olefinic bridge and/or the benzene ring parts [14].
Among these structural modification strategies, methylation had attracted a great deal of attention because of the simple synthesis and magical effect for the improvement of medicine potency [14,[16][17]. Thus, to obtain anticancer drug molecules with excellent biological activity, we designed and synthesized resveratrol analogues 1-4 by methylation strategy in this work (Fig. 1). The cytotoxic activity of compounds and anticancer mechanism were further investigated subsequently. These results may provide an effective strategy for the design of anticancer reagents and the better understanding of anticancer mechanism.
The solution was heated and refluxed for 6 h under argon. The reaction liquid was cooled to room temperature, KF (500 mg) and ethyl acetate (5 mL) were added and stirred for 30 min, then extracted with ethyl acetate for three times. The organic phases were washed with a solution of saturated KF and water, dried over Na 2 SO 4 .
The solvent was evaporated and the residue was purified by column chromatography (silica gel; petroleum/ethyl acetate 5/1) to provide compound 1-4.
DMSO, as the effective solvent, was used to dissolve the compounds at first. The concentration of DMSO in cell suspension was less than 0.1 % (v/v).

Cell viability assay
The cell lines (MCF-7, HeLa, H1299, MRC-5 and UC2 involved) viability was evaluated by the MTT assay [18][19]. The initial densities were 2.5×10 4 cells/mL. After cell culture for 24 h, the culture medium was replaced, and the cells were incubated with target compounds at gradient concentrations (1 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, four wells were used for each concentration, and repeat 3 times.) for 72 h in 96-well flat microtiter plates. Subsequently, 100 μL culture medium containing 10% MTT was added to each well. Incubation for another 4 h at 37 ℃ in the dark, remove the culture medium, and add 100 μL DMSO in each cell. The absorbance was determined at 570 nm by a microplate reader (Thermo Fisher 7 1510).
Then the culture medium was replaced by indicated concentrations of compound 1

Nuclear Staining with DAPI
The morphological changes of nucleus were observed by fluorescence microscopy with DAPI staining. Firstly, 8×10 4 /mL MCF-7 cells were planked in a 6-well plate with slide in each cell for 24 h. Later, the cells were treated with gradient concentration (control, 5μM, 10μM, 20μM) of compound 1 for another 24 h. Then the treated cells were fixed with 4 % paraformaldehyde overnight. DAPI staining for 10 min and the cells were determinated by fluorescence microscopy (Olympus BX41).

Measurement of ROS
DCFH-DA produces fluorescent signal after oxidation by ROS, which is used to reflect the intracellular level of ROS. MCF-7 cells were seeded in a 6-well plate for 24 h, and were treated with gradient concentration (control, 5μM, 10μM, 20μM, 30μM and 40μM) of compound 1 for another 6 h or 9 h. Discarded the culture medium, collected and washed the MCF-7 cells, which were resuspended in PBS (3μM DCFH-DA containing) for 0.5 h at 37 °C in the dark. Then washed the cells by PBS and detected the fluorescence intensity by flow cytometry (BD FACSCalibur) at once.

Western blot analysis
The lysates of treated MCF-7 cells with different concentration of compound 1 were extracted by RIPA Lysis Buffer containing PMSF (P0020, Solarbio, China) following the instructions. After centrifugation, the protein supernatants were collected, and the protein concentration was measured by BCA protein assay kit (R0020, Beyotime Institute of Biotechnology, China). Then the SDS-PAGE loading buffer (1:1 v/v) was added into the proteins sample, and boiled for 5min. The extracted proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane by a wet transfer method. After blocking with 5% nonfat milk in TBST for 1.5 h at room temperature, the PVDF membrane was incubated with primary antibodies (1:2000 against p53, p21, Bax, Bcl-2, Cyclin A1, Cyclin A2 and 1:10000 against GAPDH) overnight at 4 °C, followed by the incubation with secondary antibody at room temperature for another 1.5 h. The immunoblots were visualized with an Ultra ECL kit (LK-U1421, MultiSciences Biotech Co., Ltd, China).

9
All experiments were repeated at least three times. The results were shown as mean ± SD and the differences were analyzed by ANOVA using SPSS 22.0.

Synthesis of the compounds 1-4
The compounds 1-4 ( Fig. 1) were synthesized on basis of one step Heck and Stille series reaction (Scheme 1). Synthesis details and characterization obtained were described in Method section.

Cytotoxic activities of the resveratrol and compound 1-4
Firstly, we investigated the anti-proliferative activities of resveratrol and compound 1-4 by MTT assay in various cell lines. As shown in Table 1, compound 1 possessed the best cytotoxicity in MCF-7 cells compared to that of other compounds, which may be attributed to the change of the molecular plane structure with four methyl groups in aromatic rings of ortho-position of the double bond [14]. Moreover, the compound 1 exhibited more than ten times anti-proliferative activity compared to that of RES. Therefore, methylation is an effective strategy for improving biological activities of hydroxystyrene. More importantly, the cytotoxicity of the compound 1 in tumor cells (MCF-7) was about two times higher than that in normal cells (MRC-5 and UC2), which provided a foundation for the selectivity of anticancer. The excellent cytotoxicity and selectivity of compound 1 encouraged us to further research the mechanism of inhibitory effect on the tumor proliferation. a The results are presented as the mean ± SD for 4 replications in 3 separate trials after treatment with compounds for 72 h.

Induction of cell apoptosis in MCF-7 cells
Apoptosis, as programmed death, is an important way of cell death. Therefore, the effect of compound 1 on inducing apoptosis was detected by flow cytometry after Annexin V-FITC/PI double staining. The results showed that when the concentration of compound 1 increased to 20 μM for 24 h, the apoptosis was observed (Fig. 2a).
As the extending of incubation time (48 h), the apoptotic effect showed concentration-dependent characteristics (Fig. 2b). On the other hand, apoptosis is a distinct characterised process with different morphological and biochemical features, so the DAPI staining was carried out to investigate the morphological changes of nucleus characteristic. After different concentrations of compound 1 treatment for 24 h, the MCF-7 cells presented chromatin condensation and nuclear fragmentation (arrow) at the concentration of 20 μM (Fig. 3), which further conformed apoptosis induction by compound 1 in MCF-7. Therefore, compound 1 is able to kill cancer cells by apoptosis manner.

Induction of cell cycle arrest in MCF-7 cells
Cell cycle is another factor of affecting the proliferation. In the following, the effect of cell cycle arrest was investigated. The result showed that compound 1 displayed the significant inhibitory effect on proliferation above 10 μM after incubation for 24 h, and the cells were arrested in S phase (Fig. 4). Meanwhile the effect showed concentration-dependent characteristics (Fig. 4b). Obviously, these results highlighted compound 1 inhibited tumor cell proliferation through binary pathways of cycle arrest and apoptosis which had been identified ( Fig. 2 and Fig. 3).

Effect of compound 1 induced ROS level in MCF-7 cells
ROS, as the important second message, are responsible for regulating cellular activities. However, high levels of ROS may be toxic to cancer cells and suppress the tumor proliferation by induction of apoptosis and/or cell cycle arrest [8].
Therefore, the ROS level in MCF-7 cells was detected by flow cytometry. As shown in Figure 5A, the ROS level expressed a slight increase at 20 μM after compound 1 treatment for 6 h and 9 h (Fig. 5a), but showed a significant rise with the increasement of the concentration. Interestingly, when the ROS scavengers, including GSH and NAC, were added to the cells 1 hour earlier than compound 1, the cell survival rate was reversed to some extent (Fig. 5b). Thus, compound 1 may increase intracellular ROS level, which may further induce the apoptosis and cell cycle arrest signal pathway to inhibit cellular activity.

MCF-7 Cells
Whereafter, we further investigated the expression levels of apoptosis-related proteins to explore the anticancer mechanism of compound 1. Hence, the expression levels of apoptosis-associated proteins including p53, p21, Bax and Bcl-2 were tested after treatment for 24 h with compound 1 in MCF-7 cells. Exhilaratingly, the results showed that compound 1 could induce up-regulation of pro-apoptotic protein Bax and down-regulation of anti-apoptotic protein Bcl-2, which increased the Bax/Bcl-2 ratio (Fig. 6a) compared to untreated MCF-7 cells. More importantly, as the tumor suppressor and the upstream regulatory protein of apoptosis pathway, p21 showed a marked increase (Fig. 6a). However, there was little difference in p53 between treated and untreated groups.

MCF-7 Cells
Based on the effect of compound 1 on the cell cycle progression, cyclin A1 and cyclin A2 expression related to S phase were tested by western blot analysis. As

Discussion
Carcinogenesis is a complex and heterogeneous process, with typical characteristic, namely, uncontrolled cells division. Obviously, multiple mechanisms inhibiting cell proliferation, which were especially associated with apoptotic signal pathway and cell cycle regulation, become the preferred option of anticancer targets [3,[18][19], and the therapeutic strategies targeting intracellular ROS levels, which were influenced by the natural products and their structural analogues act as prooxidants, were the research hotspots [8,22]. In this study, tetramethylated hydroxystyrene of resveratrol analogues, namely, compound 1-4, were designed 13 and synthesized based on the resveratrol skeleton units to improve the selectivity of the anticancer activity and to exploit its potency.
Encouragingly, compound 1 showed a higher anti-proliferative activity and selectivity of anti-cancer compared with that of resveratrol and other compounds (Table 1). According to the subsequent studies, compound 1 expressed markedly inhibitory effect in the MCF-7 cells by both cell cycle arrest and pro-apoptosis activity. This result showed that the introduction of four methyl groups into the double bond ortho sites of aromatic rings significantly improved its cytotoxicity. For that reason, the distortion of distyrene space structure with introduction of methyl group may bond the protein more closely.
As we known, apoptosis is a key factor in leading to the cell death, which may be induced by various stresses such as therapeutic agents through induction ROS by triggering oxidative stress [23][24]. In this study, ROS levels were markedly elevated at a higher concentration, and the cell viability was also enhanced after ROS scavenger combined with compound 1 treatment (Fig. 5). Obviously, these results supported compound 1 upregulated the ROS levels to mediate the cell apoptosis and inhibit the proliferation of cancer cells. The main mechanisms may be referred to the imbalance of pro-apoptotic and anti-apoptotic proteins or/and the activation of caspase pathway [5]. Interestingly, our studies showed that the pro-apoptotic protein Bax (up-regulation) and anti-apoptotic protein Bcl-2 (down-regulation) promoted a shift in the Bax/Bcl-2 ratio toward apoptosis (Fig. 6a). Meanwhile, as a master apoptosis modulator in response to the stimuli and the upstream regulation protein of apoptosis pathway by both p53-dependent and p53-independent pathways [23], p21 showed an obvious up-regulation in a dosage dependent manner after compound 1 treatment. However, the expression of p53, as a tumor suppressor and a key regulator in apoptosis induction [26], was not obvious change in the present study, which indicated that the increased expression of p21 caused by compound 1 in p53-independent may be an important reason for the apoptosis of tumor cells. However, due to the caspase-3 activity deficiency in MCF-7 cells [27], compound 1 didn't induce caspase-3 activity at high concentrations in this study (result not show). Therefore, the realization of apoptosis induced by compound 1 mainly depends on p21 rather than upstream protein p53.
On the other hand, cycle arrest is another important manner for affecting the cell activities. The cell cycle traverses the four distinct phases, including G1, S, G2, and finally M phase, which are guarded by G1/S, intra-S, and G2/M checkpoints in response to DNA damage or cellular perturbations [28]. Numerous researches suggested that ROS, as key signaling intermediates, could damage the DNA and disturb the cell cycle progression by phosphorylation or ubiquitination of cell cycle regulatory proteins including cyclins and cyclin-dependent kinase (CDKs). [24, [29][30]. In this study, the cell cycle analysis result verified that the MCF-7 cells were arrested in S phase after compound 1 treatment (Figure 4), which indicated that compound 1 might inhibit the proliferation activity underlying the ROS-induced DNA damage, and then arrest cell cycle in S phase. In addition, S phase progression is regulated by the cycling A/CDK2 complexs [21]. Obviously, downregulated expression of cycling A1 and cycling A2 in MCF-7 cells after compound 1 treatment in the study provided a proper explanation of blocking cell cycle in S phase (Fig. 6b). Moreover, p21, as a vital cell cycle inhibitor, serves a role by inhibiting cyclin-CDK complexes [31]. The expression of p21 showed obvious upregulation in a dosage dependent manner accompany with ROS levels after compound 1 treatment, which indicated compound 1 might induce cell cycle arrest 15 by upregulation p21 interacting with cycling A/CDK2 complexs which are required for S phase progression to inhibit cellular proliferation. p53, as a key regulator in cell cycle regulation, plays an important in regulating the cell proliferation [5,26].
However, the expression level of p53 was not obvious change in the present study, which suggested the cell cycle arrest might be p53-independent and chiefly regulated by induction of DNA damage via ROS in a p21-dependent manner , which reached a similar conclusion just like apoptosis study.

Conclusion
The study indicated that the tetramethylated hydroxy distyrene of resveratrol analogue, compound 1 showed significant and selective anticancer activities by both apoptosis induction and cell cycle arrest in S phase involving p21-dependant pathway in a prooxidant manner. However, the exact mechanism and potential therapeutic application of compound 1 is being continuing studied in this team. Declarations YB2018002; No. QN2018008; No. QN2018012).

Availability of data and materials
Data supporting our findings is contained within the manuscript. Additional data will be shared upon request to the corresponding author.

Authors' contributions
Kang The nuclear chromatin condensation and the formation of apoptotic bodies with DAPI staining  The expression of apoptosis-related proteins and cycle-regulatory proteins of S phase was ev

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download. Scheme 1.tif