One of the 5-aminosalicylates drug, mesalamine as a drug repurposing lead against breast cancer

Breast cancer is the world's second leading cause of death in women. The problem of chemoresistance in breast cancer is proving to be a challenge for researchers and several oncologists all around the world. Current treatment modalities are associated with severe toxicities and lower efficiency. Hence, there is an unmet need for the development of novel drugs that can be used as adjuvants in breast chemotherapy. One of the strategies used to overcome this problem and that has received scientific coverage over the years is ‘Drug Repurposing’. For this purpose, a list of 5-aminosalicylates drugs were evaluated for their drug repurposing potential in breast cancer. Mesalamine, sulfasalazine, balsalazide, and olsalazine were docked with high expression signatures in cancer cells such as EGFR (epidermal growth factor receptor), ERα (Estrogen Receptor alpha), Aromatase, mTOR (mammalian target of rapamycin), ALOX5 (Arachidonate 5-lipoxygenase), and Topoisomerase II. Docking analysis revealed that the selected ligands (drug) exhibited good binding affinity for all receptors. Based on the specificity with receptors, mesalamine was further selected for in vitro functional validation in a breast cancer cell line. Cell-based cytotoxicity assay in MCF-7 (Michigan Cancer Foundation-7) cells demonstrated the anticancer potential of mesalamine in breast cancer with IC-50 (Inhibitory Concentration) of 6.358 µM. Significant morphological alterations were observed in breast cells treated with mesalamine. Further studies are warranted to explore the anticancer effect of mesalamine in breast cancer and its role in combination therapies to be used as an adjuvant in chemotherapy.


Background
Cancer has proved to be one of the major causes of death throughout the world, comprising around 7.9 million deaths, which accounts for 13% of total deaths Sung et al. 2021). Breast cancer constitutes around 10.4% of total cancer cases and is in the fifth rank for causing maximum mortalities (Bhattacharyya et al. 2020). A number of side effects like fatigue, nausea, vomiting sensation, drastic hair loss, drying of the mouth, and gastric issues generally accompany chemotherapy. In certain cases, the cancer cells develop resistance against chemotherapeutic drugs and even inactivate them through metabolic pathways (Wang et al. 2019). The risk of recurrence is also seen in patients undergoing chemotherapy. The inability of drugs to suppress or inhibit the proteins involved in disease progression leads to higher rates of recurrence and ultimately mortality (Mahvi et al. 2018;Beckwitt et al. 2018).
Drug repurposing (also known as drug repositioning or drug reprofiling) is a new, interesting, simple-yet-effective strategy, which involves the identification of new therapeutic uses of a drug, beyond its prescribed indication by the food and drug administration (FDA) (Oliveira and Lang 2018). It has several impressive advantages, such as reusing drugs, therefore overcoming the obstacle of drug shortage and saving huge time, cost and effort; its already determined pharmacokinetic, pharmacodynamic, bioavailability, and toxicity profiles make it easier to enter further stages of clinical trials. In the last few years, this approach is known to have gained momentum, especially in the field of oncology (Zhang et al. 2020).
Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most well-known and widely used medicines, specifically for inflammation and pain relief. In addition to their antipyretic, antianalgesic, and anti-inflammatory properties, they have also been reported to show efficacy in treating deadly diseases like cancer and heart-related condition (Bindu et al. 2020;Wongrakpanich et al. 2018). One of the most common examples of drug repurposing of NSAIDs is the use of aspirin. It is reported to have shown anticancer and chemopreventive properties in colorectal cancer (CRC). Apart from this, several other anti-inflammatory drugs repurposed are Naproxen, Sulindac, Celecoxib, Licofelone, and Diclofenac for treating CRC (Pantziarka et al. 2016;Mohammed et al. 2018).
Aminosalicylates are one of the different classes of NSAIDs, most popularly prescribed for inflammatory bowel disease (IBD) and ulcerative colitis (UC). They are used to transport its active ingredient-'mesalamine' (5-aminosalicylic acid) to the sites targeted (Veloso et al. 2021;Lim et al. 2016;Ye and Langenberg 2015). The structure of mesalamine "mimics" that of salicylic acid, but differing in containing an amino group at the 5th position on the ring. It is even considered a first-line therapy used for treating mild to moderate UC and IBD, along with controlling their remission (Perrotta et al. 2015). Recent experimental and epidemiological evidence has demonstrated the inhibitory potential of mesalamine in CRC. It hampers and inhibits the growth of CRC cells by inhibiting the wnt/β-catenin pathway and activating the epidermal growth factor receptor (EGFR), two of the most important targets for CRC cells (Dixon et al. 2021;Stolfi et al. 2013). This study aims to delineate the drug repurposing potential of mesalamine in breast cancer. It starts with in silico screening of the potent drug candidates in order to predict and assess the binding affinities of ligands within the binding site of target receptors and developing initial repositioning hypotheses. This is followed by functional validation of repurposed drug in an in vitro cancer model.

In silico studies
The binding modes and the impact of drugs on the targets were investigated by molecular docking technique.

Library of compounds
A class of NSAIDs (aminosalicylates-mesalamine, sulfasalazine, balsalazide, and olsalazine) were retrieved from PubMed and docked with various general breast cancer targets to investigate and predict their binding energies. Prior to docking, they were prepared to show optimum results. Compounds belonging to the 'aminosalicylate' family of NSAIDs were retrieved from the PubChem database in Structure Data File (SDF) file format. Ligands were subjected to energy minimization by applying the 'MMFF94' force field. The structure files were converted to pdbqt format using open babel. The ligands were docked to the receptors using the multi docking approach. The compounds were selected considering the binding affinity (Kcal/mol), specificity, and molecular recognition during the procedure.

Therapeutic targets
3D X-ray structures of all receptors were retrieved and downloaded in Protein Data Bank (PDB) format from Protein Data Bank. The proteins used for docking are-EGFR (PDB ID: 3ERT), Aromatase (PDB ID: 3EQM), ERα (PDB ID: 1A52), ALOX 5 (PDB ID: 6N2W), mTOR (PDB ID: 4JSV), and Topoisomerase II (PDB ID: 1ZXM). The proteins were prepared by removing water molecules, docked ligand(s) and other heteroatoms (HETATM) (if any) using BIOVIA Discovery Studio (DS) visualizer and saving it as an individual compound This was followed by its energy minimization through the Swiss-PDB Viewer (SPDBV). The compound was uploaded onto AutoDock software version 4.2. Polar hydrogens were added and merged with non-polar hydrogens, along with applying Histidine hydrogens. Kollman charges were applied to the protein to determine the electron density and charge distribution. Lastly, the grid box (or grid map) was set appropriately for each receptor and calculated by AutoGrid. It contains a 3D lattice of regularly spaced points, surrounding either completely or partially, and centered on a region of interest of the compound under study, with a default grid spacing of 0.375 Å.

Property calculation of compounds
The compounds should possess a desirable biological property and toxicity. Hence, the selected compounds were further evaluated based on toxicity parameters using the SWISSADME program (Daina et al. 2017). The canonical SMILES of the compounds were used for identifying the properties which could be influenced in toxicity studies. These properties are essentially needed for understanding the structural alerts which can alter the functions later in biological studies.

Cell culture
The cytotoxic effect of mesalamine was further checked on MCF-7 (procured from NCCS), a breast cancer cell line. MCF-7 was maintained in DMEM supplemented with 10% FBS in 37 °C0 with 5% CO 2 in a humidified incubator supplemented with penicillin-streptomycin solution 100U/L.

Cell cytotoxicity assay
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was performed Approximately, 5000 cells per 200μL of sterile complete medium were seeded into each well of the 96-well microtiter plate and incubated for 24 h. After incubation, different drug concentrations (5 µM, 20 µM, 50 µM and 100 µM) were added to each lane except the control and again incubated for 24 h. The stock concentration of mesalamine was prepared in dimethyl sulfoxide (DMSO). Subsequent dilution of mesalamine was prepared in complete media. At the end of 24 h of drug treatment, MTT solution (5 mg/ ml) was freshly prepared, filtered, and added to each well and incubated for 4 h for the formation of formazan crystals. After incubation, the medium was discarded and 50 μl of DMSO was added to fully dissolve the crystals. The absorbance of each well was recorded using the Bio-Rad microplate reader at a wavelength of 570 nm. Relative cell viability was expressed as a percentage.

Phase-contrast microscopy
Approximately, 10,000 cells of MCF-7 were seeded in 500 µl of complete medium (DMEM along with 10% FBS) into each well of 24-well plates. Cells were further incubated with different concentrations (5 µM, 20 µM, 50 µM and 100 µM) of mesalamine for 24 h. Untreated cells were labeled as control. Phase-contrast microscopy of control and treated MCF-7 cells was performed using Nikon ECLIPSE TS100.

Survival analysis
Publicly available level 3 TCGA RNASeq data for breast cancer (n = 1097) and CRC (n = 380) along with clinical information of patients were downloaded using UCSC Xena Functional Genomics Explorer. Data were log2(x + 1) transformed and RSEM (RNA-Seq by Expectation-Maximization) normalized. Known targets {arachidonate 5-lipoxygenase (ALOX5) and peroxisome proliferator-activated receptor gamma (PPARG)} of mesalamine were chosen from the DurgSurv database and probed into TCGA data. Patients were divided based on the expression median value for their respective gene and classified as High expression class and Low expression class. R software was used to do survival analysis using survival packages. Survminer R package was used to generate Kaplan Meier plot.

Statistical analysis
The experimental data were shown as mean ± SE. Oneway ANOVA followed by the Turkey HDS post hoc test was used to calculate the significance between control and treated groups. IC-50 was calculated by GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA, www. graph pad. com". Statistical significance was set at P ≤ 0.05.

Molecular Docking study
The docking analysis of aminosalicylates (mesalamine sulfasalazine olsalazine balsalazide) was done using AutoDock Vina software. The program generated 10 possible docking conformations of the ligand for each of the selected receptors. From the possible conformation, the best conformation was selected based on the binding energy and stability (number of hydrogen bonds, pi-pi, or van der Waals interactions). The grid box parameter used for docking was set as following  Table 1) revealed binding affinities, different amino acids, and the interaction between mesalamine and different receptors [ Fig. 1 (A,a-EGFR, B,b-ERα, C,c-Aromatase), Fig. 2 (A,a-mTOR, B,b-ALOX5, C,c-Topoisomerase II)]. Mesalamine showed moderate affinities for all. Olsalazine had the highest affinity for Topoisomerase II with binding energies of − 9.4 kcal/mol respectively (Table 1), whereas sulfasalazine and balsalazide had the highest affinities toward the receptor Aromatase with binding energies of − 9.1 and − 8.1 kcal/mol respectively (Table 1). Each docked ligand complex was stabilized with hydrogen bonds with pi-pi interactions where non-covalent interactions involving pi systems dispersed electron densities. On further analysis, 2D Ligplot showed mesalamine in mesalamine -Topoisomerase II complex, formed four hydrogen bonds with Gln 376, Thr 49, Tyr 165, and Tyr 50, one carbon-hydrogen also showed with His 42 along with Pi-Pi stacked interaction of Tyr 50. The complex was stabilized with many six van der Waals forces as well (Fig. 1f ). Olsalazine bound to Topoisomerase II with fourteen van der Waals interacting residues five conventional hydrogen bonds, Asn 120, Arg 98, Asn 150, Arg 162, Gly 164 and two pi-alkyl bonds with Ala 167, Ile 125 residues ( Table 2). Docking analysis of sulfasalazine revealed that the ligand formed eight van der Waals forces, and five hydrogen bonds with Gly 439, Thr 310, Leu 372, Met 374, Arg 115. There were two pi-sigma interactions observed in Val 370, Leu 152. Interestingly, the Aromatase receptor    (Table 2). However, Ala 306 and Phe 430 formed pi-sigma and pi-pi T-shaped interactions, respectively.

Property calculation
The properties of the selected compounds were carried out using a server called 'SWISS ADME' (Daina et al. 2017). It is a well-known web server, mainly used to determine ADMET (Adsorption, Distribution, Metabolism, Excretion, and Toxicity) and physiochemical features for studying the pharmacodynamics of drugs. Additional file 1: Tables S1 and S2 displays six important features that are usually determined prior to any pharmaceutical research. The partition coefficient between n-octanol and water (log Po/w) is defined as lipophilicity and is also the affinity of a drug toward lipids (non-polar solvent) and denoted as 'LogP' . Size is defined in terms of molecular weight in 'g/mol' . Polarity is stated as topological polar surface area (TPSA) and takes all polar atoms of the molecule into account. Solubility of a molecule is estimating the aqueous solubility of the compound and is defined as 'Log S' . The ratio of sp 3 hybridized carbons over the total carbon count of the molecule is taken and defined as 'Fraction csp3' for saturation. The flexibility estimates total number of rotatable bonds in the molecule that eventually determines affinity and pose upon binding to its receptor (Constantinescu et al. 2019;Sumalapao et al. 2020). It is clearly seen that the property of 'Instauration' does not fall in its ideal range for all four compounds, along with the property of 'Polarity' not being seen in majority of compounds.

The cytotoxic effect of mesalamine on MCF-7 cells
To examine the effect of mesalamine on the proliferation of breast cancer cells, a cell-based cytotoxicity assay was performed using MTT. MCF-7 cells were seeded in 96well plates (5000 cells/100 µL). The next day, cells were treated with increasing concentrations of mesalamine for 24 h. The MTT assay relies on mitochondrial dehydrogenases converting MTT to a purple formazan product in the cell. The purple color produced by this method is proportional to the viability of the cells. In our experimental setup, the growth of breast cancer cells was suppressed in a dose-dependent manner upon treatment with mesalamine. Upon further analysis, the IC50 value     of mesalamine was determined to be 6.358 µM (Fig. 3A). Figure 3B depicts the control for the rest of the MTT figures.

Phase-contrast microscopy
Control and treated cells were observed under an inverted light microscope, using phase-contrast microscopy to spot morphological changes and microphotographs were documented. Cells treated with increasing concentrations of mesalamine displayed significant morphological alterations at 5, 20, and 50 µM (Fig. 3C, D, E). This change in morphology becomes more evident in cells treated with a higher concentration of mesalamine, i.e., > 100 µM (Fig. 4F). A higher concentration of mesalamine (670 µM) resulted in cell apoptosis and degradation in a few cells, while the others were observed to have lost their morphology and become round in nature, with large vacuoles (Additional file 2: Figure S1). The cells seem to have been arrested in the G1 phase.

Survival analysis
To extend our findings, the gene expression levels of different targets of mesalamine in breast and colorectal cancer were undertaken. Mesalamine has already been reported to have protective effects against colorectal cancer in inflammatory bowel disease (Dixon et al. 2021).
We did comparative studies based on the TCGA database (Atlas et al. 2019). We correlated the expression level of targets of mesalamine with the survival of breast and colorectal cancer patients. Known targets of mesalamine were retrieved from the DurgSurv database (Amelio et al. 2014). We examined the expression of ALOX5 (Arachidonate 5-lipoxygenase) and PPARG (peroxisome proliferator-activated receptor gamma), in 1097 tumor samples of invasive breast and 380 samples of colorectal cancer from the cancer genome atlas (TCGA). The log-rank test was used to examine the difference in survival outcomes between low and high expression patient groups for each gene. Mesalamine has been reported to inhibit ALOX 5 (Nielsen et al. 1987;El-Nagar et al. 2018). In both cancer types, poor survival of cancer patients was associated with high expression ALOX5 with (P value = 0.10) (Fig. 4A, C). Interestingly, Kaplan-Meier survival plots displayed that high expression of PPARG predicted shorter survival in breast cancer patients by TCGA database analysis but in the case of colorectal cancer high expression of PPARG in the samples was associated with better survival probability (P value = 0.4) (Fig. 4B, D).

Discussion
In recent years, a plethora of studies have shed light on mechanisms that sustain cancer cell growth and survival and have pointed out the problem of cancer resistance to chemotherapy and other treatment options currently available (Wang et al. 2019;Cree and Charlton 2017). Problems such as recurrence, metastasis, chemo, and radio-resistance have promoted drug repurposing as the go-to solution to find better candidates with fewer adverse side effects (Kirtonia et al. 2021). The repurposing or repositioning approach has since been repeatedly adopted and sought after as it drastically brings down the time and cost in comparison to de novo drug discovery methods. Traditional methods involve numerous trial and error experiments. Plethora of novel drugs show promising results in early stages but only few of them reach to phase 3 clinical trials and still it remains one of the biggest hurdles in cancer therapeutics (Parasrampuria et al. 2018). The current investigation deals with understanding the drug repurposing potential of 5-aminosalicylates in cancer via molecular screening followed by in vitro study. Drugs that are potential candidates for repurposing should ideally have similar characteristic traits like market availability, acclaimed drugs and toxicity as that of conventionally used drugs. They need to exhibit a distinctive mode of action against the targeted condition and sufficient efficiency at the dosage administered (Pushpakom et al. 2019). Various drugs already being utilized for different conditions have shown immense potential in cancer therapeutics. Many off-patent pharmaceuticals have been proved to show properties against cancer by 16% of relevant clinical studies and 50% data from humans (Verbaanderd et al. 2020). Specifically, clinical trials have proven the efficacy of drugs like chloroquine, all-trans retinoic acid, verapamil, itraconazole and aspirin, against cancer (Correia et al. 2021). Along with these, mesalamine is another pharmaceutical showing activity against colorectal cancer and research is also being conducted to study its exact mechanism of action. These studies have indicated that strong doses of mesalamine hinder the development of colon cancer, and this obstruction is seen to be enhanced further when compounds derived from mesalamine are administered (Dixon et al. 2021).
Mesalamine is the bioactive moiety of aminoacylates which are known to be dual inhibitors of COX2 and 5LOX receptors involved in the anti-inflammatory pathways and their role in cancer has been highlighted by many studies (Harris et al. 2020). Affinity toward other targets also points to the multi-target inhibition ability of mesalamine being a small molecule with a structure almost similar to that of aspirin. The target receptors selected for this study are well known to play a huge role in the development and progression of breast cancer. The proteins namely, EGFR, Aromatase, ALOX5, and mTOR are usually overexpressed in breast cancer  (Larionov 2018;Fu et al. 2020). The overexpression of EGFR results in the large size of the tumor, poor differentiation as well as clinical outcomes; Aromatase is responsible for regulating hyperplasia when circulating estrogen is not present; ALOX5 is known to play a critical role in inflammation tied to cancer such as breast and colorectal. mTOR gives a great advantage to the growth of tumors, activates protein synthesis, and suppresses autophagy (Harris et al. 2020;Sood et al. 2021;Hare and Harvey 2017;Sigismund et al. 2018). mTOR plays a central role on PI3K/Akt/mTOR pathway which is one of the critical pathway affecting cell survival and metabolism in normal and pathophysiological situations, especially in cancer (Mossmann et al. 2018). ER α regulate the proliferation of breast cancer cells owing to their respective hormone ligands and by expressing oncogenic proteins (such as cyclin D1 and c-Myc) (Trabert et al. 2020;Xue et al. 2019). Topoisomerase II is responsible for DNA replication in the cells and has been a target for multiple chemotherapeutic agents (Arthi et al. 2021;You and Gao 2019). PPARs have already been reported to play a significant role in cancer cell proliferation and tumor growth (Gou et al. 2017). The role of EGFR is mostly in ER+ positive carcinoma and is a target for hormone and chemotherapy. In the present investigation, mesalamine along with sulfasalazine, balsalazide, and olsalazine was docked with different receptors and mesalamine showed moderate to strong affinity for all the receptors and was comparable for all the targets for which docking was performed as compared to other ligands.
Although sulfasalazine displayed a better binding affinity for all receptors, we still have chosen mesalamine for further functional validation in an in vitro model. Sulfasalazine, olsalazine, and balsalazide are the prodrug as compared to mesalamine (Sandborn and Hanauer 2003). Oral mesalamine has been shown to be safe and effective for both short and long-term use. Although sulfasalazine has been used for the therapeutics of ulcerative colitis, to avoid its side effects, direct application of mesalamine has been under investigation.
Cell-based cytotoxicity assay in MCF-7 demonstrated the anticancer potential of mesalamine. The IC50 of mesalamine on MCF-7 cells was calculated to be 6.358 µM, with R-value of 0.9298, which implies a strong positive correlation between the increasing concentrations of mesalamine and its inhibitory effect on the MCF-7 cells. The same trend was observed while investigating morphological alterations and cytotoxic effects under phase-contrast microscopy. For a drug, the values of a potent inhibitor can lie anywhere between the range of nano-molar to milli-molar concentrations of the substance. For mesalamine, the anticancer and cytotoxicity effects could only be observed more evidently at higher concentrations, where cells were arrested in the initial stages of the cell cycle and/or were undergoing apoptosis. The plausible mechanism of action could be in accordance with that in colorectal cancer as the pathways contributing to the development and progression of cancer are alike in both disease conditions. Next, we performed an indirect correlation analysis between the expression of target genes of mesalamine in breast and colorectal cancer with the survival of patients. Higher expression of ALOX5 was correlated with poor survival of both breast and colorectal cancer patients. Mesalamine is known to inhibit ALOX5 (Nielsen et al. 1987;El-Nagar et al. 2018). Therefore, if mesalamine is administered to breast cancer and colorectal cancer patients where expression of ALOX5 is elevated, it will lead to downregulation of ALOX5 and hence lead to better outcomes in breast and colorectal cancer, which fits, into our hypothesis of drug repurposing of mesalamine in breast cancer. A similar survival analysis was performed for PPARG in breast and colorectal cancer patients. There have been reports of the involvement of PPARG in mesalamine -mediated inhibition of colon cancer cells (Schwab et al. 2008). Survival analysis demonstrated high PPARG expression was correlated to shorter survival in breast cancer patients.
However, the opposite scenario existed for colorectal cancer patients. Mesalamine, being an agonist of PPARG will result in upregulation of its expression (Bertin et al. 2013). Therefore, when administered to colorectal cancer patients, it will elevate PPARG expression, which can lead to better survival outcomes. Breast cancer analysis resulted in contrasting observations. However, these all are indirect correlations and many more functional studies are warranted to comment on the efficacy of mesalamine in breast cancer.

Conclusions
This explorative study aimed to address the anticancer potential of mesalamine, one of the 5-aminosalicylates drugs for breast cancer. Text mining and knowledge-based repurposing approach was employed, and we were able to identify a potential candidate mesalamine that could be