Cytotoxicity Evaluation of Chalcone-Coumarin Conjugates Against A549 Cell Line

 

Raj Keshwar Prasad*, Kavita R. Loksh

Faculty of Pharmacy, Oriental University, Indore, Madhya Pradesh-453555.

 

ABSTRACT:

Coumarins and chalcones have been molecules of considerable interest amongst medicinal chemists owing to their vivid pharmacological potentials including anticancer. A few coumarin-chalcone conjugates (C1-C5) with good antioxidant potential were evaluated for in vitro cytotoxicity against A549 cell lines using MTT assay. The IC₅₀ of the conjugates was calculated after 24 h of treatment. The compounds C3 and C4 with higher mesomeric effects exhibited by the substituents were found to be possessing significant cytotoxic potential with IC₅₀ values of 22.90 ± 2.1, 26.73 ± 6.6 µM respectively.

 

 

INTRODUCTION:

Cancer is any malignant growth caused by abnormal and uncontrolled cell division.¹ It is the prime cause of death over the world with an estimated number of 11.4 million deaths by the year 2030.² Irrespective of the several therapeutic options, the treatment of cancer presents several hindrances and the persistent investigations to develop new, less toxic, and highly potent anticancer molecules is being conducted worldwide.^3-6It has been reported that most of the anticancer molecules are non-selective due to direct interaction with various cellular components. The α,β-unsaturated Michael acceptors have been molecules of wide interest as they can be modified to attain specific interaction with target nucleophiles in the cell.⁷ Chalcones, are α,β-unsaturated systems, found widely in plants and have shown a wide array of biological actions such as antiiflammatory⁸, antibacterial⁹, antimalarial¹⁰, antifungal¹⁰, antioxidant^11, and anticancer¹¹. Chalcones are also reported to be potential inducers of cellular apoptosis.¹² Coumarin, is also a class of compounds extensively found in plants, bacteria, and fungi. They are also known to possess several pharmacological and biological actions like antibacterial¹³, anti-inflammatory¹⁴, antioxidant¹⁵, antimutagenic^16, and anticancer¹⁷.

 

To assess for preliminary anticancer activity in terms of cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) in vitro cytotoxicity assays are considered two of the most economic, reliable and convenient methods.¹⁸

 

In our previous investigation, we have reported the synthesis and antioxidant potential of a few novel chalcone-coumarin conjugates. Owing to the antioxidant potential in the conjugates; in the present investigation, we have reported the preliminary cytotoxic action of the chalcone-coumarin conjugates against human lung cancer cell line.

 

MATERIAL AND METHODS:

Chemicals:

Dulbecco’s Modified Eagle’s Medium (DMEM), antibiotics, Amphotericin B, and Fetal Bovine Serum (FBS) were purchased from Sigma Aldrich. All other chemicals used were of analytical grade and used as obtained.

 

Cell lines and Culture:

The human lung cancer cell line (A549) was purchased from National Centre for Cell Science (NCCS, Pune, India). The cell line was maintained in the laboratory and the cells were grown using DMEM. The culture media was supplemented with 10% FBS and antibiotics (Penicillin G (100 U/mL), Gentamycin sulfate (50 µg/mL), Streptomycin (100 µg/mL) and Amphotericin B (2.5 µg/mL)). The maintenance of the cell line was done in humidified conditions with 5% CO₂ at 37°C in a CO₂ incubator.

 

MTT Assay:

The cytotoxic potential of the chalcone-coumarin conjugates was evaluated using an MTT reduction assay. The cells were seeded into 96-well plates at a density of 1 × 10 cells/well and incubated for 24 h at 37 °C. Then, three different concentrations of synthesized compounds (10, 25, and 50 µM), 5-fluorouracil (as positive control) were added in triplicate and the plates were incubated at 37 °C for another 72 h. After the incubation period, the media was removed; then, MTT solution in PBS at a concentration of 5 mg/mL was added for MTT assay. After 4 h of incubation, formazan crystals formed. Then, 60 μL DMSO was added to dissolve the crystals and, finally, absorbance was measured at a wavelength of 570 nm with background correction at 650 nm using a microplate reader (Bio-Rad, Japan). IC (concentration that results in 50 % inhibition of cell viability) for each compound was calculated.

 

RESULT AND DISCUSSION:

The MTT assay owing to its ease of use, accuracy, and rapid indication of toxicity along with sensitivity and specificity is the most widely preliminary method of assessment of cytotoxicity of natural as well as synthetic compounds.¹⁹ It is an in vitro whole-cell toxicity assay that employs colorimetric methods for determining the number of viable cells based on mitochondrial dehydrogenase activity measurement. In the MTT assay, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide is bio-reduced by dehydrogenase inside living cells to form a colored formazan dye. The number of viable cells is measured through colorimetry and works on the principle that the mitochondrial dehydrogenase enzymes which produce NADH or NADHP, reduce the colorless tetrazolium salt into a colored aqueous soluble formazan product by the mitochondrial activity of viable cells at 37 °C.¹⁸

 

The foundation of the present investigation is based on the antioxidant capacity of these compounds (C1-C5) against DPPH scavenging and hydroxyl radical scavenging assays.

 

 

C1                                                           C2

 

C3

 

C4                                                           C5

 

As per reports around 70-80% of the reported cancer cases are either breast cancer or lung cancer and hence in the present investigation, a non-small cell lung cancer cell line (A549) was used for cytotoxicity evaluation of the synthesized chalcone-coumarin conjugates. The compounds were tested at three different concentration levels (10, 25, and 50 µM) against the human lung cancer cell line and the results are depicted in table 1. Compounds C3 and C4 exhibited the lowest IC₅₀ values of 22.90 ± 2.1, 26.73 ± 6.6 µM amongst all the compounds.

 

Table 1 Cytotoxicity of the chalcone-coumarin conjugates against A549 cell line

Compound	IC50 (µM)
C1	39.48 ± 5.2
C2	35.09 ± 3.6
C3	22.90 ± 2.1
C4	26.73 ± 6.6
C5	31.17 ± 3.9
5-FU	6.39 ± 1.8

 

Figure 1 Comparative cytotoxicity of test compounds at different dose levels

 

The cytotoxicity exhibited by the chalcones could be attributed to its ability to cause molecular alterations like induction of apoptosis, or inhibition of tubulin polymerization and depolymerization or it may be owing to the capability of the chalcone nucleus to inhibit kinase enzymes.²⁰ The cytotoxic or cytostatic action of the natural and synthetic coumarin derivatives might be due to several mechanisms like telomerase inhibition, induction of caspase-9-mediated cellular apoptosis, inhibition of kinase activity or they may affect the p-gp of the cancer cells.²¹

 

It was found from the results that the presence of strong electron-donating groups (via mesomeric effect) in the ring substitution of the ring A of chalcone had a prominent effect on the IC₅₀ of the compounds against the cancer cell. Compound C3 and C4 with dual substitution of electron-donating substituents, OCH₃ and OH effects presented the highest cytotoxicity. The effect of the OH group alone on cytotoxicity was position-dependent. The cytotoxicity of the compounds decreased with substitution of the OH group on the 4-position (C1) with IC₅₀ of 39.48 ± 5.2 whereas when the OH group was on 2-position (C2) the IC₅₀ value was 35.09 ± 3.6.

 

A similar result was obtained when aromatic chalcones with pyrazoline were evaluated for anticancer action. The presence of the OH group on positions 2 and 3 was beneficial for the anticancer action as compared to the OH group on position 4.²² In general, chalcone derivatives are known to exhibit anticancer activity. We have not found any study in the literature on the synthesis and anticancer properties of chalcone compounds containing coumarin rings. A study however on chalcone containing benzofuran revealed that the presence of electron-withdrawing group on the ring B of chalcone.²³

 

CONCLUSION:

To conclude, an evaluation of the cytotoxicity of coumarin-chalcone conjugates against the A549 cell line was accomplished. On the basis of the MTT assay, compounds C3 and C4 were shown to be having good potential to inhibit the growth of the A549 cell line. The results displayed that the chalcone-coumarin conjugates may be useful for the development of newer anticancer molecules.

 

ACKNOWLEDGEMENT:

The authors are grateful to the Faculty of Pharmacy, Oriental University, Indore, M. P., India, for providing research support.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 27.01.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):5144-5147.