INTRODUCTION:

Cancer is a major health concern in both developed and developing countries, second leading cause of mortality worldwide. Among the different types of cancer, breast cancer is the most common cancer found in females worldwide. In Indonesia, the number of breast cancer incidence remains the highest until 2018.¹

The current cancer treatment involves chemotherapies, radiation and hormone therapies.^2,3 On conventional chemotherapies, systemic adjuvants are used to target cancer cells through several mechanisms. One of them is the use of tamoxifen which is an estrogen antagonist.⁴ However, this adjuvant only targets breast cancer cells with positive estrogen receptors, therefore there are high chance it can develop resistance and causes side effects. For example, the high linkage between Tamoxifen and estrogenic activity in endometrial tissue is known to increase the risk of both endometrial tumors and cancers.⁵

Other adjuvants used are also known to have side effects such as hair loss, fatigue, and nausea, especially during the early treatment.⁶ There are also side effects occurring later on the treatment regiment, such as neurocognitive dysfunction, fertility, cardiomyopathy, even second cancers.^7,8

As adjuvant chemotherapy is still considered one of the most preferable choice for treatment, there has been developing research on finding new adjuvants that would cause less side effects but better efficacy for cancer. Recent researches have proposed the use of salicylic acid in treating cancers, and It has shown positive results. Especially, 5-aminosalicylic acid (5-ASA)–a derivative of salicylic acid, as treatment for colorectal cancer. It has been reported that 5-ASA that has been used as treatment for inflammatory bowel disease, has anticancer activity with less irritable side effects.^9–11

Substantial evidence have been made about the correlation of COX-2 over-expression and growth of both colorectal cancer cells and breast cancer cells (found in 40% of aggressive breast cancer cells). Expression of COX-2 in mammary cells show more vascularization, which is a hallmark of cancer, than mammary cells with knocked-out-COX-2.^12,13

Previous research has shown that alkylizing salicylic acid increases the bioenergy of mitochondria in breast cancer cells resulting in decreased respiratory control ratio on MCF-7, observed through oxygraphy.¹⁴ Compared to non-alkylated MCF-7, T47D has lower respiratory control ratio.¹⁵ Since the characteristics between these cells are similar, logically alkylated salicylic acid should be able to lower the respiratory control ratio of T47D as well, resulting in more effective treatment. Moreover, recent research on salicylic acid efficacy for cancer treatment was done to lung adenocarcinoma A549, and it was shown that salicylic acid decreases cell viability with a concentration-dependent manner.¹⁶

Conventional drug design is very complicated and requires a lot of time and money, therefore the presence of computational techniques including the Quantitative Structure-Activity Relationship (QSAR) method is one solution that can reduce time and costs as well as the trial and error factor in drug design.^17–19 In this study, the QSAR analysis of the alkylated salicylates was carried out so that the theoretical activity of the alkylated salicylates could be predicted.

Therefore, as more prominent cancer adjuvant with less side effect is needed and with growing interest on salicylic acid, this research aims to conduct in vitro cytotoxicity evaluation of alkyl derivatives of salicylic acid on breast T47D cancer cells. Alkyl derivatives of salicylic acid are expected to have higher anticancer activity on breast T47D cancer cells.

MATERIALS AND METHODS:

Material:

Tools used in this research include laminar air flow, incubator CO2, vortex, ELISA reader, 96-well plate, pipette tips (0,2-1000mL), filter unit 0,22um Millex GV (Millipore), and confocal microscope. Also melting point apparatus Innotech DMP 100.

Ingredients used in this research are alkylated salicylic acid derivatives, carcinoma cell line T47D, RPMI media which contains HEPES and GlutaMAX powder (Invitrogen Gibco BRL), MTT (Sigma), DMSO (Sigma), Fetal Bovine Serum (Gibco), NaHCO3 (Sigma), penicillin-streptomisin solution (Gibco/BTGIB), Phosphate Buffer Saline, 96-well, and tryptan blue stain 0,25% (Gibco).

Methods:

Research design used to know the cytotoxicity of salicylic acid derivatives is experimental and laboratory work research.

Phytochemical analysis:

Physical properties such as melting point, molecular mass, and solubility are also obtained. Melting point was obtained by using melting point apparatus Innotech DMP 100. Solubility was obtained from solubility curve and/or physical observation. Molecular mass was also obtained from physical observation while nMR of each compounds were obtained from available database.

Cell line preparation:

Breast cancer cell T47D culture is taken from the stock kept inside liquid tank in the locator with temperature of -196^oC. Cell culture is liquedify in water approximately 37,7C. Then it is grown in a couple tissue culture flasks with complete media (RMPI, 1% trypsin and 10% FBS), incubated in 37C with 5% CO₂ and 95% O₂ flows. After 24 hours, media is replaced and cells are grown until the amount is enough to be researched. After the culture is confluent (around 70%) and has minimum amount of cells, media is replaced by RPMI 1640 as much as 5ml. Cells are taken in the amount of 3 x 10⁴ cells/100 l medium –counted using haemocytometer.^20,21

MTT Assay:

T47D suspension with the amount of 100 µl and media with consistency of 3x10⁴ cells/100µl is distributed inside the 96-well and incubated in 24 hours. After incubated, 100µl tested suspension is added with varying concentrations. As a positive control, 100 doxorubicin was added to various concentration in the wells. As cell control, 100µl culture medium into the wells. As solvent control, DMSO is added (100µl) into the wells as well with adjusted delusion that matched the concentration of tested suspension. The wells are incubated in 24 hours inside the incubator with 5% CO2 and 95% O₂ flow. At the end of incubation, culture media is thrown out and then 10µl MTT is added (5 mg/mL PBS), then the cells are incubated as long as 3-4 hours. MTT reaction is stopped by adding stopper reagent SDS (100µl). Microplate filled with cell suspension was seized. Around 5 minutes then wrapped by aluminium foil, incubated in 1 night with room temperature. Alive cells will react to MTT forming purple color. Result of the test is read by ELISA reader with 492nm wavelength.^22,23

The result of absorbance reading by ELISA Reader in cytotoxicity of the compound and its derivatives towards T47D is then used as a tool to count the percentage of cell death with the formula:

C - T

% death = ------- x100 %

C stands for average absorbance rate from control cells and T stands for the average absorbance rate for cells with treatment. Analysis for growth inhibition is done using the 24^th version of IBM SPSS Statistics software and using Microsoft Office Excel 2016.

QSAR Analysis:

QSAR Structural modelling of alkylated salicylate derivatives conducted using HyperChem. Optimized the geometry using the ab initio method. Then the descriptors are calculated using MOE 2020.10. The descriptors calculated are AM1_E, AM1_Eele, AM1_HOMO, VSA, vol, AM1_LUMO, AM1_dipole, AM1_HF, log P (o/w), glob, ASA_H, mr. Statistical calculations with multilinear regression analysis to determine R2, CV LOO (cross-validation leave one out) the value of q², R² the relationship between experimental IC50 and IC50 prediction, and the highest significance of Pearson correlation.

The QSAR model's preparation for 10 starts with dividing the data into two groups, namely training sets and test sets. The distribution of training sets (7 compounds) and test sets (3 compounds) was carried out randomly (Table 1). A number of compounds in the training set, analyzed using Multiple Linear Regression (MLR) with the backward method run on the statistical program (SPSS® Release 16.0.0) to obtain several models of the relationship between MIC logs with electronic descriptors or for generating QSAR models and a test set for validating the quality of the models.

Table 1. Test Set and Training set of Alkyl derivatives of salicylic acid

No	R1	R2	IC50	Training/Test set
1	H	H	64	Test
2	H	CH₃-	15	Test
3	H	C2H₅-	17	Test
4	H	C4H₉-	1	Test
5	H	C₅H₁₀ - (isoamyl)	2	Test
6	H	C₆H₁₃-	25	Test
7	H	C₈H₁₅-	35	Test
8	H	C₉H₁₇-	38	Training
9	H	C₆H₆-	18	Training
10	H	Cl-	36	Training

RESULTS:

Physical and Chemical Properties:

Phytochemical analysis results and properties of salicylic acid derivatives shown in Table 2.

MTT Assay:

Based on MTT assay, IC50 value of alkylated salicylate towards T47D breast cancer cells are listed in table-3.

Table 2. Physical and chemical properties of each salicylic acid derivatives.^24–28

	Methyl Salicylate	Ethyl Salicylate	Butyl Salicylate	Isoamyl Salicylate	Octyl Salicylate
Formula	C₈H₈O₃	C₉H₁₀O₃	C₁₁H₁₄O₃	C₁₂H₁₆O₃	C₁₅H₂₂O₃
Melting Point	–8,6°C	1°C	–5,9°C	–75°C	25°C
Molecular mass	152,15 g/mol	166.17 g/mol	194,23 g/mol	208,25 g/mol	250,33 g/mol
Solubility in Water	Slightly soluble	Slightly soluble	Insoluble	Insoluble	Insoluble
Properties	Wintergreen-like odor, colorless yellowish or reddish liquid	Colorless to pale yellow liquid. Wintergreen-like and spicy odor	Trace of wintergreen for odor and colorless liquid	Slightly floral odor, colorless oily liquid.	Colorless oily liquid with a slight floral odor

Table 3. IC50 values of test samples against T47D breast cancer cells

Sample	IC50 Value (μg/mL)	IC50 Value (Mean ± SD μg/mL)	P-value
Methyl Salicylate	28,92787	14,79340 ± 11,4645	0,002
	6,1654
	19,92178
Ethyl Salicylate	21,9367	17,27127 ± 4,2654
	13,42195
	17,2265
Butyl Salicylate	2,0814	0,87320 ± 0,9117
	0,76929
	0,32881
Isoamyl Salicylate	0,51011	1,66443 ± 1,43668
	2,7879
	3,1659
Octyl Salicylate	11,95898	35,08997 ± 38,7909
	87,58544
	64,76183
Doxorubicin	0,013203	0,003815 ± 0,0066
	0,002274
	0,001292
Salicylic Acid	41,7211	63,67135 ± 30,0782599
	101,755
	75,0627

The table provides the IC50 values of each samples on three trials. Lower IC50 value suggests the best inhibitory activity of cancer growth. Hence Doxorubicin which has the lowest IC50 value, has the best inhibitory effect to the growth of T47D breast cancer cells (IC50 value = 0,003815±0,0066). Between the five salicylic acid derivatives, the lowest IC50 value belongs to butyl salicylate (IC50 value = 0,87320±0,9117). While the highest IC50 value belongs to salicylic acid.

Fig 1. Comparison of IC50 values between salicylic acid derivatives.

One-way ANOVA test shows significant correlation between IC50 groups with p-value 0,002 (<0,05). The data also fulfils Levene homogenous test with p-value >0,05. However, further post-hoc Tukey test reveals mostly insignificant results. Methyl salicylate shows significant results when compared to salicylic acid. Ethyl salicylate also shows significant result compared to salicylic acid. Butyl salicylate as well as isoamyl salicylate shows significant results when compared with octyl salicylate and salicylic acid. While doxorubicin shows significant results when compared to octyl salicylate and salicylic acid. Lastly, salicylic acid shows significant findings when compared to all salicylic acid derivatives except for octyl salicylate.

QSAR Analysis:

QSAR Analysis using MOE 2020.10 software, with log descriptors P, AM1_LUMO, mr and AVS on breast cancer cell line inhibitory activity. QSAR descriptors of 7 alkylated salicylate derivatives are shown in the Table 4.

Table 4. Descriptors of alkylated salicylate derivatives against IC50 Breast cancer cell line

Molecule	MIC	logP(o/w)	AM1_LUMO	mr	$PRED
1 (Test)	64	1.2730	-0.5910	3.5745	63.2282
2 (Test)	15	1.5370	-0.4960	4.0857	16.7126
3 (Test)	17	1.8780	-0.4609	4.5537	12.1668
4 (Test)	1	2.9340	-0.4599	5.4936	-2.2693
8 (Training)	2	2.85	-0.4633	5.4598	13.1047
9 (Training)	25	3.818	-0.4571	6.4284	20.5761
10 (Training)	35	4.7020	-0.4483	7.3601	35.4810

The results of the analysis of 3 descriptors of 7 alkylated salicylate derivatives and their relationship with breast cancer cell line inhibition had R²: 0.94 with RMSE 4.08 and R² Cross-Validation (RCV) value of 0.90. The RCV value close to 1 indicates a good level of inhibition influenced by 3 important descriptors including log P with a significance of 0.885, AM1_LUMO with a less significant value of 0.178, and mr with a significance of 1. R as correlation coefficient and R²is the square correlation coefficient, which describe the relative measure of the quality of fit by the regression equations. The closer the values to 1, it fits the regression equation better.

The observations show that the first equation meets the best statistical criteria. The multilinear regression results show a good correlation, with experimental results (R² = 0.94) and cross-validation LOO (q² = 0.90). The q² value that is close to 1 shows the degree of deviation between the predicted IC50 activity value and the experimental IC50 value is relatively small. Then from the predicted IC50 and experimental IC50 relationship curves, the first equation has an R² value of 0.94, and the Pearson correlation output shows 3 significance which is higher than other equations. The equation obtained,

Log (1 / IC50) = -1236.21871 -236.42806 * logP (o / w) -1186.53810 * AM1_LUMO +251.55826 * mr ((n = 7 r = 0.94 RMSE = 4.08), shows that influential descriptors, namely: log P, mr, and AM_1LUMO has effect on the inhibitory activity of the breast cancer cell line.

DISCUSSION:

All alkylated salicylic acid derivatives have progressive pattern between concentration and inhibition activity. The higher the concentration is the better the inhibition activity against breast cancer T47D cells.

Butyl salicylate has the highest inhibition activity both at low and high concentration. Starting from concentration 1,5625μg/mL butyl salicylate already has the highest mean of percentage of inhibition compared to all compounds. Meanwhile, octyl salicylate has the lowest percentage of inhibition out of all compounds at both end of the concentration spectrum.

When compared to their structure and physical properties, there was no significant correlations with percentage of inhibition. Melting points can be an indicator of the length of the hydrocarbon chains. Compounds with higher melting points tend to have longer lengths.²⁹ However, the compounds tested in this research does not confer to this rule as their melting points are fluctuating. Hence, the longer the length of the chains do not determine the anticancer activity against T47D breast cancer cells between alkylated salicylic acid compounds.

According to Krippendorff, IC50 is categorized into three classes; IC50 below 1µM is considered high, IC50 between 1 and 10µM is considered medium, and IC50 above 10µM is considered low.³⁰ However, in a study conducted in Thailand, IC50 are categorized into four groups: 20g/ml as active, >20–100g/ml as moderately active, >100–1000g/ml as weakly active, and >1000g/ml as inactive.³¹

All the compounds tested are considered as active except for octyl salicylate and salicylic acid. Out of all, the best IC50 value comes from doxorubicin. Among the salicylic acid derivatives, the best is butyl salicylate (fig 1).

There is no significant correlation found whatsoever between structure and physical properties of alkylated salicylic acid derivatives and IC50 values. The parent chain, which is salicylic acid has the worst IC50 value but adding an alkyl branch to the parent chain increases the value of IC50 value. This supports the post-hoc Tukey test which have shown mostly insignificant findings. However, all salicylic acid derivatives have significant correlation with salicylic acid except for octyl salicylate (p>0,05). Therefore, the administration of alkyl branch enhances cytotoxicity activity against T47D breast cancer cells.

Between salicylic acid derivatives, most have insignificant relationship with each other. Except butyl salicylate and isoamyl salicylate who have significant relationship when compared with octyl salicylate. If we compare between these three compounds, there might be a possibility that the shorter length of alkyl chain has better cytotoxicity effect on T47D breast cancer cells. However, methyl salicylate and ethyl salicylate have lower cytotoxicity effect compared to butyl salicylate despite having shorter length. This leads to the possibility that the cytotoxicity effect becomes ineffective when the alkyl chain is too short or too long. The optimal length would be as long as butyl salicylate. As the relation between butyl salicylate and isoamyl salicylate is not significant (p>0,05), hence we cannot draw conclusion whether cytotoxicity effect is better on branched or straight alkyl chains.

Overall, the result shows positive outcome on the inhibition of COX-2 by salicylic acid derivatives. As have been mentioned before, COX-2 inhibition can reduce the growth of breast cancer cells.^32–34 This is because overexpression of COX-2 pathway plays a role in activating pro-tumorigenic agents.^12,35

The administration of alkyl groups to salicylic acid has been proven to increase the IC50 value as seen in Table 3. This corresponds to previous studies indicating that alkylation improves the affinity between the compound and the substance (T47D cancer cells) through Van der Waals interaction.^12,36,37 Another study also found that PGE2, the product of COX-2, overexpression induces syalyltransferase activity to increase as has been seen in breast cancer.³⁸ Therefore, there is a link between breast cancer pathology and COX-2 upregulation as the mechanism pathway of cancer growth.^39,40 COX-2 inhibitor can serve as a novel breast cancer treatment. And as has been shown through this research results, alkyl salicylic acid has cytotoxicity effect towards T47D breast cancer cells.

From the QSAR equation model, the activity of the alkylated salicylate derivative compounds will increase with the addition of alkyl substituents that have high AM1_LUMO energy, large log P, and mr. It can be proposed that the substituent which has a large mr that is and has an alkyl branch, the substituent with the lower total energy, the higher the dipole moment value, i.e. the more polar the substituent. Substituents that have a large log P (logarithm of solubility in octanol/water) such as hydroxyl groups (-OH), carboxyl (COOH).¹⁸ Modifying the steric bulk or the chain length of alkyl substituents can alter selectivity and/or activity. The compound example with modify with alkylation is isoprenaline, it has greater selectivity for the ß-adrenoceptors over the a-adrenoceptors. The selectivity is attributed to the bulky isopropyl group which can fit in a hydrophobic pocket present in the ß-adrenoceptors.

Log P is a key physicochemical property that plays a crucial role in determining ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties and the overall suitability of drug candidates. There is increasing evidence to suggest that control of physicochemical properties such as lipophilicity, within a defined optimal range, can improve compound quality and the likelihood of therapeutic success.

CONCLUSION:

The higher the concentration of the compound, the more cytotoxicity activity found against T47D breast cancer cells and the better the IC50 value. Alkylating salicylic acid improves cytotoxicity effect against T47D breast cancer cells. Butyl salicylate has the most significant cytotoxicity effect against T47D breast cancer cells among all salicylic acid derivatives with mean IC50 value 0,87320 (SD±0,9117). The substituent that has a large mr is I (13.8741), the substituent with the lower total energy, the higher the dipole moment value, which is the more polar one. Substituents that have a large log P (logarithm of solubility in octanol/water) such as hydroxyl groups (-OH), carboxyl (COOH) will increase the inhibitory activity of the compound. Substituents that have a large mr, substituents with low total energy, high dipole moment values are those that are more polar. Substituents that have a large log P (logarithm of solubility in octanol/water) such as hydroxyl groups (-OH), carboxyl (COOH) will increase the inhibitory activity of the compound.

RECOMMENDATIONS:

Further research needs to be done to validate the effectivity of salicylic acid derivatives against T47D breast cancer cells, preferably using in vivo method and nMR should be done.

ACKNOWLEDGEMENT:

We thank Directorate for higher education, Ministry of Research and Technology, Republic of Indonesia and University of Indonesia for the PUTI research grant.

CONFLICT OF INTEREST:

Authors declare no conflict interests

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Received on 26.02.2021 Modified on 27.12.2021

Research J. Pharm. and Tech 2022; 15(10):4607-4613.

DOI: 10.52711/0974-360X.2022.00773

QSAR and Anticancer effect of Alkyl salicylate on Breast cancer T47D Cells Lines