Telmisartan anti‐cancer activities mechanism through targeting N‐cadherin by mimicking ADH‐1 function

Abstract This study aimed to investigate if Telmisartan as a novel N‐cadherin antagonist, can overcome cell migration of cancer cells. We investigated the mechanism and influence of Docetaxel and Telmisartan (as an analogous to ADH‐1, which is a well‐known N‐cadherin antagonist) on cancer cells. The effect of ADH‐1 and Telmisartan on cell attachment in PC3, DU145, MDA‐MB‐468 cell lines using recombinant human N‐cadherin was studied. Cell viability assay was performed to examine the anti‐proliferative effects of Telmisartan, ADH‐1 and Docetaxel. Migration was examined via wound healing assay, and apoptosis was determined by flow cytometry. The expression of AKT‐1 as a downstream gene of N‐cadherin signalling pathway was assayed by real‐time PCR. Treatment of PC3, MDA‐MB‐468 and DU145 cells with Telmisartan (0.1 µM) and ADH‐1 (40 µM) resulted in 50%, 58% and approximately 20% reduction in cell attachment to N‐cadherin coated plate respectively. It shows reduction of cell attachment in PC3 and MDA‐MB‐468 cell lines appeared to be more sensitive than that of DU145 cells to the Telmisartan and ADH‐1 treatments. Telmisartan (0.1 µM) and Docetaxel (0.01 nM) significantly reduced cell migration in PC3 and MDA‐MB‐468 cell lines compared with the control group. Using Real‐time PCR, we found that Telmisartan, Docetaxel and ADH‐1 had significant influence on the AKT‐1 mRNA level. The results of the current study for the first time suggest that, Telmisartan, exerts anti‐proliferation and anti‐migration effects by targeting antagonistically N‐cadherin. Also, these data suggest that Telmisartan as a less expensive alternative to ADH‐1 could potentiate Docetaxel anticancer effects.


| INTRODUC TI ON
Prostate cancer (PCa) is one of the most common solid tumour and globally the frequent cause of cancer-related death in men, after lung cancer. 1,2 Chemotherapy by docetaxel (DTX) is commonly used to treat a broad range of human malignancies, including lung cancer, breast, head and neck, stomach and PCa. 3 DTX belongs to the taxanes class and is the standard first-line treatment against metastatic PCa; however, its use for long term, results in many side effects and drug-resistance. Therefore, exploring new drugs with less side effects and potential to inhibit migration and metastasis of malignant cells is valuable. 4 PCa commonly metastasizes to organs such as liver, brain, lymph nodes and bone, which is one of the major causes of deaths related to this malignancy. 5 This process involves several steps, including cell-extracellular matrix and cell-cell attachments and detachments that allows the invasive cells disseminate from the primary tumour and migrate to the other organs. 6 In normal epithelium, cell-cell adhesion is maintained by many distinct junctions, such as tight junctions, which are composed of the complexes of cell adhesion molecules (CAMs), including selectin and cadherin proteins. 7 Epithelial to mesenchymal transition (EMT) is often associated with loss of E-cadherin expression level and increased the level of Ncadherin expression, leading to enhanced cell migration and invasiveness in various types of cancers. 7 Many studies have indicated that EMT plays a fundamental role in tumours development and progression, suggesting that new drugs targeting EMT and its mediators might be effective against different cancers. Many small molecules have been established for inhibiting EMT by targeting the mediators, such as the commercial drugs and nature-based compounds. Small molecules, which is composed about 75% of the anticancer FDAapproved drugs, have an extended history of using as drugs for the treatment of many diseases including diabetes, hypertension, infections, heart failure, rhinitis and cancers. 8,9 Previous study reported a novel peptide, ADH-1, that inhibit Ncadherin (a marker of EMT) function. 10 Yarom et al. 11 showed that ADH-1 was commonly well tolerated up to a dose of 1000 mg/m 2 when administered every three weeks in patients with incurable solid tumours. Other researchers demonstrated that significant reduction in tumour growth and lung metastasis by ADH-1 in a mouse model of pancreatic cancer. 12 Although ADH-1 has high specificity and tolerance, the oral administration of it, as the most convenient and comfortable way, is a difficult challenge for this drug. Based on the Lipinski's Rules, peptide and protein drugs are difficult to passing through the gastrointestinal mucosa and easily degraded by gastric acid, and resulting in poor bioavailability of oral delivery (less than 1%). Currently, most peptide and protein drugs are administered by invasive techniques such as subcutaneous or intravenous (IV) injection that require professional medical personnel. 13 Compared with peptides, the small molecules dominate the global drug market due to their advantages such as small size, oral availability, low cost, membrane-penetrating ability, ready synthesis and stability. 14 Therefore, the use of new drugs with less invasiveness administration route, controllability and high bioavailability is very efficient. 13 In previous in silico studies, after deep analysis of N-cadherin/ADH-1 interaction, we screened for FDA-approved small molecules which can act as potential inhibitors for N-cadherin protein. 15,16 Among seven screened candidate drugs, we decided to use one of these inhibitors, Telmisartan (Tel) in the current study. Our hypothesis was that Tel prevents cell migration and inhibits attachment of PCa and breast cancer cells to recombinant N-cadherin coated wells by suppressing N-cadherin. Tel is a specific and selective blocker for angiotensin II receptor, which is widely utilized as an antihypertensive drug and recently have been used for their anticancer effects. 17 Moreover, studies have demonstrated that Tel can inhibit the proliferation and growth of tumour cells. Moreover, it possess cytotoxic effects on different cancer cells when prescribed alone or in combination with other antitumor drugs. 18 To the best of our knowledge, no study has reported the effects of Tel on N-cadherin-mediated cell migration yet. Therefore, the aim of the present study was to investigate the potential anticancer effects of ADH-1, as a known N-cadherin antagonist, DTX, as the first line chemotherapy agent in PCa treatment, and Tel, as a novel small molecule, on cell viability, apoptosis, migration and cell attachment. In previous studies, the anticancer effects of Tel have been reported [18][19][20][21] ; so, in the current study, in addition to the N-cadherin antagonist function, we decided to investigate the anticancer effects of Tel in comparison to the first line chemotherapy drug for treatment of prostate cancer, DTX.
N-cadherin expression and homophilic ligation of N-cadherins between adjacent cells by activation of PI3K/AKT signalling pathway promotes growth regulatory events which was associated with angiogenesis, metastasis, programmed cell death and EMT in various cancers. 22,23 Therefore, the effect of aforementioned drugs on AKT-1 gene expression as a downstream gene of N-cadherin signalling was also investigated.
The two main types of prostate cancer cell lines (PC3 and DU145) and a breast cancer cell line (MDA-MB-468) were used for the present study. DU145 cell line lacks the N-cadherin expression, while PC3 and MDA-MB-468 cell lines highly express N-cadherin. 24 We evaluated the response of each cell lines to the aforementioned drugs.

| Materials
Recombinant human N-cadherin protein was obtained from R&D Systems. DTX, ADH-1 and Tel were obtained from MedChem Express. All drugs were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and diluted with media before each experiment.

| Cell culture
The PC3, DU145 (Prostate cancer) and MDA-MB-468 (Breast cancer) cell lines were obtained from the National Cell Bank of Iran (Pasteur Institute, Iran). The cells were cultured with RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin antibiotic solution and maintained in humidified incubator with 5% CO 2 at 37°C. All experiments were carried out at passage number 2-4 after reaching 70% confluency.
All concentrations were tested in quadruplicate wells and the experiment was repeated three times.

| Cell migration assay
Monolayer wound healing assay was used to determine cell migration according to described protocols. 26

| Cell attachment assay
Cell attachment assay was carried out in the adhesive 96-well plates as described previously. 27 Briefly, the wells were coated with 5 µg/mL of recombinant human N-cadherin, and then, 50 μL of cell suspension (4 × 10 5 /mL) were loaded to each coated well together with 50 μL of drugs diluted with cell culture medium and incubated in 37°C. In the next step, unbound cells were removed, and the adherent cells were fixed by glutaraldehyde.
The fixed cells were stained with crystal violet (0.1% (w/v) in methylethanesulphonic acid (MES)), and finally, the optical density of each well was measured with a microplate reader at 570 nm.

| Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from cells by the Tripure reagent, and concentration of RNA was determined by a Nanodrop 1000 spec-

| Apoptosis assay by Flow Cytometry
The status of cell death was measured by flow cytometry method.
Briefly, 4 × 10 5 cells were seeded in 6 well plates and incubated with drugs (treated cells) or cell culture medium (control cells) for 48 hours. In the next step, the cells were detached using cell dissociation buffer (non-enzymatic) and were washed twice with PBS.
Then, the cells were stained with Annexin-V conjugate (FITC) and propidium iodide (PI) (MabTag). After incubation for 20 minutes in the dark, the population of apoptotic cells was analysed by flow cytometry (BD FACS Calibrator, Biosiences Company). Each experiment was repeated by three independent experiments and the results analysed by FlowJo 7.6 software.  Table 1.

| Effects of DTX and Tel on cell migration in prostate and breast cancer cell lines
To

| Effects of Tel and ADH-1 on cell attachment in prostate and breast cancer cell lines
To

| Effects of DTX, Tel and ADH-1 on AKT-1 expression level in prostate and breast cancer cell lines
As shown in Figure 4,

| Effects of DTX, Tel and ADH-1 on induction of apoptosis in prostate and breast cancer cell lines
In order to find out if the Tel and ADH-1 treatment also induces cel-   Note: Data are expressed as the mean ± standard deviation of three independent experiments (n = 12).

| DISCUSS ION
In In fact N-cadherin contribution on generation of cell-cell junctions is less than that of E-cadherin, and its higher expression in PC3 cells is closely related to 'more mesenchymal phenotype' and increased cell invasion capacity of these cells compared with DU145 cells. 37 Based on our previous in silico studies, 15,16 we hypothesized that Tel could act as N-cadherin antagonist. Therefore, in contrast to DU145 of Tel on N-cadherin inhibition. 19,21,38,40 In the present study for the first time, a new mechanism is suggested for Tel by which exerts its functions.
Upon EMT, in which E-cadherin switches to N-cadherin, tumour cells tend to detach from their primary location. After invasion into blood vessels, the tumour cells avoid from anoikis process due to their aggregation and attachment to endothelial cells, which is mediated by adhesion molecules such as N-cadherin. 35  There are several mechanisms by which Thymoquinone could exert its anticancer activity including, inhibition of JAK/STAT signalling pathway, epidermal growth factor receptor, Wnt signalling pathway and activation of PTEN, P53, caspase-3,9 as well as induction of reactive oxygen species production that resulting in DNA damage.
In general, Thymoquinone via these actions can induce cancer cell apoptosis. 42 Zhang et al. 39 19,36,40,48 The fact that Tel not only inhibited cell attachment and cell migration but also suppression of AKT-1 expression, reduced cell proliferation and increased apoptosis in PC3 and MDA-MB-468 cell lines, indicates the potential of this small molecule to exert its multimodal antitumor effects in vitro.
In conclusion, based on our results it could be concluded that Tel, as a small molecule, is specifically interact with N-cadherin and it might be the mechanism by which Tel exerts its effects in addition to the previously reported mechanisms such as its partial agonist role for PPARγ. This study highly implicates N-cadherin as a valid target for treatment of human PCa, and suggests that N-cadherin antagonists such as ADH-1 and Tel that target its adhesive function should be developed for use in treatment of human PCa. Results of the present investigation could suggest Tel as a less expensive alternative to ADH-1 which potentiate DTX anticancer effects, although many functions and interactions of Tel inhibitory effects on N-cadherin needs to be explored in vivo in the future studies.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available within the article.