TQFL12, a novel synthetic derivative of TQ, inhibits triple‐negative breast cancer metastasis and invasion through activating AMPK/ACC pathway

Abstract Thymoquinone (TQ) has been reported as an anti‐tumour drug widely studied in various tumours, and its mechanism and effect of which has become a focus of current research. However, previous studies from our laboratory and other groups found that TQ showed weak anti‐tumour effects in many cancer cell lines and animal models. Therefore, it is necessary to modify and optimize the structure of TQ to obtain new chemical entities with high efficiency and low toxicity as candidates for development of new drugs in treating cancer. Therefore, we designed and synthesized several TQ derivatives. Systematic analysis, including in vitro and in vivo, was conducted on a panel of triple‐negative breast cancer (TNBC) cells and mouse model to demonstrate whether TQFL12, a new TQ derivative, is more efficient than TQ. We found that the anti‐proliferative effect of TQFL12 against TNBC cells is significantly stronger than TQ. We also demonstrated TQFL12 affects different aspects in breast cancer development including cell proliferation, migration, invasion and apoptosis. Moreover, TQFL12 inhibited tumour growth and metastasis in cancer cell–derived xenograft mouse model, with less toxicity compared with TQ. Finally, mechanism research indicated that TQFL12 increased AMPK/ACC activity by stabilizing AMPKα, while molecular docking supported the direct interaction between TQFL12 and AMPKα. Taken together, our findings suggest that TQFL12, as a novel chemical entity, possesses a better inhibitory effect on TNBC cells and less toxicity in both in vitro and in vivo studies. As such, TQFL12 could serve as a potential therapeutic agent for breast cancer.


| INTRODUC TI ON
Malignant tumours are the main cause of death of cancer patients, and the mechanism and therapeutics of tumour metastasis has become the most widely researched topic recently. Breast cancer is a worldwide malignant disease that seriously threatens mental and physical health of patients. [1][2][3][4] Triple-negative breast cancer (TNBC) and metastatic breast cancer (mBC) are aggressive and highly heterogeneous subtypes of breast cancer with poor prognosis, which continue to be a leading cause of cancer-related death in women. 4,5 Even though mortality rates have reduced over the recent years, the 5-year survival rate of advanced TNBC is still very low. 6,7 Scientists are attempting to tackle this challenge and to develop new therapies for those breast cancer patients.
Among various tumour treatment options, targeting therapy using small molecule drugs is so far the most effective treatment strategy. 8 Bioactive agents derived from natural products have gained substantial attentions and have long been used as therapeutic drug owing to their anti-cancer, anti-inflammatory, neuroprotective and other properties. [9][10][11] One of the most successful natural agents is artemisinin (Qinghaosu), which is considered as a gift in old Chinese medicine discovered by Youyou Tu. 12 However, lots of compounds from traditional Chinese medicine (TCM) have not been fully accepted, mainly due to their limited effects, poorly defined molecular mechanisms and high costs.
Moreover, malignant tumours are prone to developing resistance or inducing side effects in the host in response to traditional chemotherapy. Thus, finding a natural drug that is highly effective with low toxicity has become an urgent issue. Interestingly, thymoquinone (TQ), the main active compound isolated from black seed oil (Nigella sativa), has been reported to be a potential treatment option for a variety of diseases including cancer. 13,14 TQ can inhibit cancer cell metastases in various malignant tumours including prostate cancer, 15 breast cancer, 16,17 bladder cancer 18 and lung cancer. 19,20 With these promising therapeutic effects, TQ has a potential to being a new clinical treatment option. [21][22][23] While it does have the prospect, there are limitations in using TQ with its original form. For instance, the effective drug concentration of TQ is reportedly high and the latest studies reported that the IC 50 of TQ is higher than165µM in some breast cancer cells. 24 On the other side, derivatives of TQ were rarely studied, raising the research question of whether TQ derivatives could be new avenues for cancer treatment. As such, additional studies are necessary to assay TQ derivatives for their efficacy, toxicity and antitumour properties. In this study, we obtained new chemical entities via chemical modification and improved the bioactivity of TQ. A new compound, (E)-3-((4-chlorobenzylidene)amino)-5-isopropyl-2methylcyclohexa-2, 5-diene-1,4-dione (TQFL12, molecular formula: C 17 H 16 ClNO 2 ), was synthesized and its in vitro and in vivo antitumour activities against triple-negative breast cancer (TNBC) were evaluated. Moreover, we also investigated its molecular mechanism. better inhibitory effect on TNBC cells and less toxicity in both in vitro and in vivo studies. As such, TQFL12 could serve as a potential therapeutic agent for breast cancer.

K E Y W O R D S
AMPK, anti-cancer, metastasis, thymoquinone derivative, triple-negative breast cancer recorded using an Agilent 6550 iFunnel Q-TOF LC/MS system. HPLC analysis was used to determine the purity (>98%) of the compound with a YMC Pack ODS-A (5 μm, 250 × 4.6 mm; YMC Co. Ltd) column.

| Cell Counting Kit-8 assays
Cells were plated in a 96-well plate with 3000-5000 cells/well and treated with various concentrations of TQ or TQFL12 for 16, 24 or 48 h. After the treatment, 10 µl of CCK8 reagent was added to each well and incubated at 37°C for 1 h. After incubation, the absorbance at 450 nm was measured using microplate reader. 25 Each experiment was repeated three times.  [26][27][28] Each experiment was repeated three times.

| Mouse xenograft assays
Animal experiments of mice were in compliance with institutional animal care guidelines and followed the university committeeapproved protocols. 16 To establish the breast cancer xenograft model, cells of mouse triple-negative breast cancer (4T1) were injected into the mammary fat pads of female BALB/c mice and the size of the tumours were measured every 5 days. 26 Four days after injection of the cells, the mice were randomly divided into seven groups with six in each group and treated with 0, 3.75 mg/kg, 7.5 mg/kg and 15 mg/kg of TQFL12, with 0, 3.75 mg/kg, 7.5 mg/kg and 15 mg/kg of TQ. The tumour sizes were continuously monitored during the treatment. At the end of the 27-day treatment (30-day of 4T1 cell injection), the mice were killed, the tumour tissues were dissected, and the weight of the tumour tissues was measured. To estimate the TQFL12 and TQ effects on tumour cell migration/invasion, the lungs of the animals were dissected out at the end of the treatment and the number of colonies formed in the lungs was counted.

| Histology
Tumour tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and sliced in 5 µm thickness. After dewaxing in a xylene series, the slides were dehydrated in alcohol and stained with H&E (haematoxylin and eosin). 26  BT549 cells were seeded in 6-well plate and incubated for 24 h and then treated with different concentrations (0, 2.5, 5, 10 μM) of TQFL12 for 12 h. Total RNA was extracted and reverse-transcribed into cDNA. Semi-quantitative RT-PCR for the PRKAA1 gene was performed using primers (forward primer: 5'-ggagccttgatgtggtagga-3', reverse primer: 5'-tttcatccagccttccattc-3'). GAPDH was served as internal control. Each experiment was repeated three times.

| Assays for the treatments of cycloheximide and TQFL12
4T1 or BT549 cells were treated with or without TQFL12 (5 μM) and were cultured for 24 h before 0.1 mg/ml (20 µM) of cycloheximide (CHX) was added. Cells were harvested and proteins in the lysate were used for Western blotting. Band intensities were semiquantified by densitometry and analysed using imaging scanner.

| Molecular docking
The file of AMPKα (NO.4CFH) from PDB (Protein Data Bank) was selected, and docking was conducted by Glide program in Schrodinger software. 25 TQFL12 was docked into the binding site of the AMPKα with the standard precision scoring mode. 25

| Statistical analysis
Three individual experiments were performed, and all data are presented as the mean ± standard deviation SD. The statistical differences were performed by one-way ANOVA using GraphPad Prism 6. p value <0.05 was considered significantly different. *p < 0.05, 0.01< **p < 0.05, ***p < 0.01, ****p < 0.001 are indicated as differences with p values.

| Design and synthesis of TQFL12, a novel TQ derivative
With TQ as the starting compound, we decided to add different chemically groups to TQ. Two steps were used to synthesize TQFL12 After heating and stirring at 80°C for 6 h, the reaction mixture was cooled to room temperature (RT). TLC verified that TQ had been reacted completely. The reaction mixture was run on a silica gel to obtain the pure product (NTQ) (Figure 1). NTQ shows red solid, yield  302.0948). The purity of TQFL12 is 98.2% ( Figure S6).

| The cytotoxic sensitivity of TQFL12 is higher than TQ on different breast cancer cells
To  Figure 2A~D and  Figure 2E &F and the IC 50 (Table 1 and Table 2) for MCF10A is significantly higher (>100µM) than that of all TNBC cell lines (<100µM). The IC 50 of TQ12 against TNBC cells 4T1 was as low as 20.24 μM. These results indicated that the cytotoxic effect of TQFL12 is more sensitive than TQ.

| TQFL12 inhibits breast cancer cell growth, migration and invasion by altering cell apoptosis but slightly affects cell cycle
EMT (epithelial-to-mesenchymal transition) is correlated with cancer metastasis. Inhibition of EMT might be a valuable approach in cancer therapy. It was reported that TQ could inhibit breast cancer cell growth, migration and invasion. 16 To determine the specific effects of TQFL12, we analysed its effect on cancer cell

| Effects of TQFL12 on breast cancer cellderived xenograft tumours in vivo
To demonstrate that TQFL12 is capable of repressing breast cancer  Figure 4A showed the per cent survival of mice in the TQ group. High-concentration TQ (15 mg/kg) was very toxic, and the mice began to die when the experiment was performed at the 7th day and all died at the 21st day. Figure 4B showed that the TQ treatment reduced tumour size. Meanwhile, the body weights of the animals showed that the toxic effect were more dramatic in the treatment of TQ than that in TQFL12 treatment ( Figure 4C). As shown in Figure 4E-H that compared to control, the sizes, weights and tissue morphology of the tumours were significantly reduced by TQFL12 and TQ treatment in a dose-dependent manner. Importantly, TQFL12 is less toxic compared with TQ ( Figure 4E-F). The statistical analysis for comparing between the groups of TQFL12 (7.5mg/kg) and TQ (7.5mg/kg) are presented in Figure S8.   the TQFL12-treated mice. Furthermore, Figure 5B showed that the average number of colonies per mice, which represents cancer cell migration and invasion, reduced in a dose-dependent manner when the mice were treated with TQ. Meanwhile, compared to TQFL12treated group, there are more and larger colonies in the lungs of the TQ-treated group on the same concentration ( Figure 5CandD).

| TQFL12 inhibits breast cancer cell invasion and migration better than TQ in vivo
Interestingly, images of in situ tumours treated with TQFL12 showed more air bubbles compared to those of the control group ( Figure 6E, right panel via left panel). These in vivo data unambiguously demonstrated that TQFL12 is better capable at inhibiting breast cancer cell growth, migration and invasion.

| TQFL12 affects AMPK signalling and stabilizes itself in triple-negative breast cancer cells
We next sought to explore the underlying signalling pathways that are affected by which TQFL12 controls tumour progression.
Therefore, we performed Western blot using different cancer cell lines treated with TQFL12. Results showed that, similar to TQ, 29 TQFL12 affects AMPKα (5'-adenosine monophosphate-activated protein kinase alpha) total and phosphorylated protein levels in both 4T1 cells ( Figure 6A, left panel) and BT549 ( Figure 6B, right panel).
Then, timing of TQFL12 treatment was executed and found that, comparing to the 0 hour (no TQFL12 treatment), total and phosphorylated AMPKα protein levels are gradually increased from 1 h, and at 8 h, reaches a peak ( Figure 6B). Accordingly, p-ACC was also gradually increased from 1 h and, at 12 h, reaches a peak ( Figure 6B). These results indicated that TQFL12 may directly affect AMPKα protein stability.
Then, we tested whether TQFL12 stabilizes AMPK. To do so, BT549 cells were treated with or without TQFL12 for 8h, and CHX, an inhibitor protein synthesis, was added with different time points.
Cells were harvested and proteins in the lysate were used for Western blotting and band intensities were semi-quantified. Figure 6C shows the TQFL12 treatments significantly increase AMPKα protein levels ( Figure 6C, left panel, Western blots; right panel, quantitative curves).
But the mRNA levels for PRKAA1 showed no difference ( Figure 6D), indicating that TQFL12 increase AMPKα protein levels is not due to the increase of mRNA transcription. Altogether, we confirmed that TQFL12 affects AMPK signalling by directly stabilizing AMPKα.

| TQFL12 interacts with hydrophobic surface of AMPKα
To further determine whether TQFL12 interacts with AMPKα, molecular docking experiment was conducted. We found that molecular docking score of TQFL12 and AMPKα is −5.08 kcal/mol. TQFL12 can form a strong hydrogen bond with AMPKα's side chain hydroxy group residue Val24 with the distance of 3.2 Å ( Figure 7A). In addition, the benzene ring of TQFL12 can form significant hydrophobic interactions with residue Leu22 ( Figure 7B). Furthermore, 2D modelling showed interaction between TQFL12 and AMPKα ( Figure 7C), whereas Figure 7C revealed hydrophobic surface of TQFL12 on AMPKα.

| DISCUSS ION
TQ is known as an anti-tumour candidate compound that is characterized by its small molecular size, and recent studies have touched on its biological function and mechanism of action. 20 showed the compounds with unsaturated side chains conferred a greater activity than those compounds with equally long saturated chains, but the in vivo efficacy was not evaluated. Other than these, no additional chemical modification of TQ has been reported. 33,36 Previous studies showed that some synthetic compounds with chlorophenyl exhibited good anti-cancer activity and it also possessed the potential to improve the bioavailability. We conduct a study of structural modification of TQ by chloromethylation to determine whether there is any benefit of chlorobenzyl modification on TQ. Taken together, our discovery of TQFL12's effect on the AMPK pathway in cancer is significant and could have great clinical potential.

CO N FLI C T O F I NTE R E S T
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DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.