JOJDC.MS.ID.555689

Abstract

Background: Human epidermoid carcinoma (A431) is an exceptionally resilient and malignant form of cancer, representing approximately 3% of all instances of malignant tumors. The main objective is to assess the potentiality of synthesized spiropyrrolidine in combating microbial infections caused by epidermoid skin disease pathogens and inhibiting a skin malignant cell line (A431).
Methods: This study was conducted to investigate apoptosis and evaluate the viability of A431 cell line utilizing Acridine Orange (AO) and Ethidium Bromide (EtBr). Flow cytometry was employed to quantify the fluorescence intensities of FITC-conjugated Annexin-V and PI in cellular samples.
Results: Spiropyrrolidine showed significant antibacterial and antifungal activity. It outperformed the standard antibiotic Amphotericin B in terms of efficacy. MTT assay on A431 showed significant inhibition of cell proliferation. Cancer cells treated with spiropyrrolidine showed a significant number of cells emitting red fluorescence, indicating they were in a late apoptotic or dead state. Cell cycle analysis revealed the cell cycle arrest at various stages in A431 cells. It also led to increased levels of DNA fragmentation and enhanced mitochondrial membrane permeability due to the activation of caspase-3 enzyme.
Conclusion: These results obtained suggests that the spiropyrrolidine heterocyclic hybrid has the potential as an effective therapeutic agent for treating malignant tumors.

Keywords:Human epidermoid carcinoma cell line; Antifungal activity; Apoptosis; Flow cytometry; Spiropyrrolidine heterocyclic hybrid

Introduction

Cancer is an intricate disease that results in uncontrolled cell division and proliferation by disrupting normal cellular processes. Cancer mortality is rising and is anticipated to exceed cardiovascular mortality soon [1,2]. According to the 2020 WHO global cancer report, 19.3 million new cases and 10 million deaths have been reported. Nearly 30.2 million new cancer cases and 16.3 million deaths are expected by 2040 [3]. To combat this disease, medicinal chemists must design and develop new anticancer agents with high specificity. Because of their three-dimensional nature, spiro cores play a key role in the structural composition of diverse natural products and biologically active synthetic analogs and can be expected to interact more proficiently with binding pockets of target protein in biological system than flat aromatic ring system as ligand [4].

In addition, spirocyclic cores constitute the essential units of many pharmacological agents and alkaloids besides being valuable synthetic intermediates for the construction of diverse heterocycles [5-7]. Besides reducing lipid peroxidation, acenaphthenequinone derivatives possess promising free radical scavenging activity and strong antioxidant properties [8-10] and hence medicinal and synthetic chemists have devoted substantial consideration to this structural core because of its medicinal significance. 4-Piperidone structural unit gains prime importance due to its structural simplicity and their broader range of applications in diverse fields [11-14]. Perhaps, for these reasons, the synthesis of hybrid heterocycles that incorporate spiro cores in addition to the above-mentioned structural units could be very valuable in medicinal chemistry. These hybrid spiroheterocycles could be constructed through a 1,3-Dipolar cycloaddition strategy by using non-stabilized azomethine ylides with olefins [15,16].

Human epidermoid carcinoma (A431) is an exceptionally resilient and malignant form of cancer, representing approximately 3% of all instances of malignant tumors. The prevalence of this cancer type has grown at a more rapid rate than any other type of cancer in recent decades. This carcinoma accounts for merely 4% of all skin cancer cases. About 80% of all skin cancer deaths are caused by this most malignant type of disease [17,18]. If epidermoid carcinoma is detected early in its progression, it can be effectively treated through a straightforward surgical procedure known as wide surgical excision. However, current treatments are often ineffective at altering the progression of epidermoid carcinoma once it has metastasized [19]. Side effects of surgery and radiotherapy include appetite loss, hair loss, decreased infection resistance, weakness, and fatigue [20]. Traditional cytotoxic treatments target dividing cells, both normal and malignant, increasing morbidity and limiting clinical benefits. Thus, novel drugs with better efficacy and fewer side effects are needed [17].

Materials and Methods

Chemistry

In this research work, we used only commercially accessible reagents and solvents. No further purification was carried out of these materials. A thin-layer chromatography (TLC) on silica gel was used to monitor the reactions. A solvent mixture involving petroleum ether and ethyl acetate was used in the column chromatography. A Jeol 500 MHz NMR instrument was used to record the 1H and 13C NMR spectra in CDCl3 using TMS as an internal standard. Data acquisition and analysis were conducted using standard Jeol software. Chemical shifts are reported in parts per million (d-scale), whereas the coupling constant is given in Hertz. Perkin Elmer spectrum 100 FT-IR spectrometer was used to record the IR spectra.

Biology

All the chemicals, including Mueller-Hinton agar (MHA), yeast extract, peptone and soluble starch, were purchased from commercial suppliers such as Hi-Media, and Sigma-Aldrich.

Antimicrobial activity of spiropyrrolidine heterocyclic hybrid

The agar well diffusion method reported by Aritonang et al. [21] was used to test the antimicrobial activity of spiropyrrolidine heterocyclic hybrid against selected bacterial and yeast strains. S. flexineri (MTCC 1457) and E. coli (MTCC 40), B. subtilis (MTCC 6633) and S. aureus (MTCC 6908), and C. albicans (MTCC 227) and C. glabrata (MTCC 3019) were chosen for activity testing. A sterile cotton swab spread each culture evenly on nutrient agar plates. A gel-hole puncher created 6 mm wells in 4 mm agar plates. The wells were loaded with spiropyrrolidine heterocyclic hybrid at 25- 100 μg/μL concentration. For 24 h, the plates were incubated at 37 ℃. Positive controls were Streptomycin and Amphotericin-B. With a transparent ruler, the inhibition zone around each well was measured in millimetres after incubation.

MIC, MBC and MFC of spiropyrrolidine heterocyclic hybrid

The antimicrobial efficacy of the Spiropyrrolidine heterocyclic hybrid was assessed using the resazurin-based micro-broth dilution technique, according to the CLSI guidelines were followed for conducting the MIC. In a sterile 96-well plate with a flat bottom, each well received a 10 μL culture inoculum and 180 μL sterile nutrient broth media was added. Then, using a serial dilution method, 10 μL of 20x compounds were added to create a final concentration ranging from 0.5 to 64 μg/mL. Standard antibioticsampicillin (AMP) for Gram-positive bacteria (S. aureus, B. subtilis), ciprofloxacin (CIP) for Gram-negative bacteria (S. flexineri, E. coli) and fluconazole (FLC) for yeast (C. glabrata, C. albicans), respectively, following a 24 hr incubation period at 37 °C, 20 μL of 0.015 % a resazurin solution was added to each well, and the plates were left to stand for an hour to observe any color change. The lowest concentration prior to color change was considered as the MIC. To determine the MBC, MFC, Nutrient agar (NA) media plates were swabbed with bacteria, fungi sample solutions from each well and incubated for 24 hr at 37 °C. If the extract grows 8 times the MIC with an unknown MBC/MFC, it is considered static (ND). The lowest concentration of each chemical that resulted in either complete inhibition or ≥99% suppression of the growth was defined as the MBC/MFC [22,23].

In-vitro anticancer activity of spiropyrrolidine heterocyclic hybrid by MTT assay

The A431 cell line, derived from human epidermoid carcinoma, was obtained from NCCS in Pune, India. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) under standard conditions, incubated for 24 hours at 37 ℃ in a 5% CO2 atmosphere with 95% humidity, and supplemented with 10% foetal bovine serum (FBS) to enhance cell proliferation. Post-incubation, cells were seeded at 20,000/well in 96-well microtiter plates with 200 μL of culture medium. A431 cells were then treated with various concentrations of spiropyrrolidine heterocyclic hybrid compound (12.5-200 μg/mL).

Cell viability was measured after incubation at 37 ℃ in a humid environment for the specified time. The assay used Camptothecin (20 μM) as a positive control and untreated cells as a negative control. Each well was incubated with 200 μL of freshly prepared MTT solution at 37 ℃ for 4 h, resulting in a purple precipitate. To dissolve formazan crystals, the MTT solution was aspirated, 100 μL of buffered DMSO was added to each well, and the plates were vigorously shaken. A microtiter plate reader (ELX-800, BioTek, USA) measured viable cell absorbance at 570 nm [24]. IC50 values were the final results. The following formulae calculated cell viability percentage:

Apoptotic assay of cancer cell line by dual staining method

A study was conducted to investigate apoptosis and evaluate the viability of the Human epidermoid carcinoma (A431) cell line utilizing Acridine Orange (AO) and Ethidium Bromide (EtBr) [25]. Cancer cells were cultured in 6-well plates and subjected to the IC50 concentration of the spiropyrrolidine heterocyclic hybrid. The cells that did not receive treatment with the spiropyrrolidine heterocyclic hybrid compound were referred to as the control group. Subsequently, the cells in each of the 6 wells were treated with 200 mL of AO and EtBr for a duration of 10 min. The resulting images were then captured and recorded using ZEN Blue software, and subsequently analyzed using ImageJ Software v1.48.

Apoptosis assay via flowcytometry

The Annexin V/FITC method measured apoptosis. Flow cytometry was employed to quantify the fluorescence intensities of FITC-conjugated Annexin-V and PI in cellular samples. A431 cells were cultured in 6-well plates at a density of 1 x 106 cells per well. Following an overnight incubation, the spiropyrrolidine heterocyclic hybrid was administered at the concentration that inhibits 50% of cell viability, as determined by the assay. After trypsinisation, cells spun at 1000 rpm for 10 min. They were gently mixed in 100 μL AnnexinV-FITC binding buffer and incubated at 25 ℃ for 10 min in the dark. The cells were centrifuged for 5 min at 2000 rpm. They were carefully re-suspended in 500 μL AnnexinVFITC binding buffer. Before conducting flow cytometry analysis, 5 μL of PI was added to the mixture in an ice bath. The Cell Quest software analysed apoptotic cells [26,27].

Cell cycle analysis

The A431 cells were seeded at 1x106/well in a 6-well plate. In serum-free media, the cells were exposed to spiropyrrolidine heterocyclic hybrid at IC50 concentrations after 12 h. They were incubated for 24 to 48 h. Following trypsinization, the cells were centrifuged at 1000 rpm for 10 min to obtain the pellet. The pellet was resuspended in PBS and fixed in 70% ethanol for two hours. The cells were subsequently centrifuged at 1000 rpm for 10 min. The pellet was resuspended in PBS. Subsequent to one minute, the cells were centrifuged once more, and the pellet was resuspended in a 1 μL propidium iodide staining solution. Cell Quest software was used for flow cytometric analysis after 15 min of PI incubation. The Excitation and emission wavelengths were 488 and 670 nm, respectively, for the analysis [13,28].

Results and Discussion

A literature-reported method was used to prepare prerequisite starting material 1 [29]. The schematic representation for the synthesis of required N-propargyl-3’,5’- bisarylmethylidenepiperidin-4-one 3 is shown in scheme 1 and was prepared by the reaction of propargyl bromide 2 with the N-unsubstituted-3’,5’-bis (4-phenylmethylidene) piperidin-4-one 1 in good yield. NMR data obtained for the structural elucidation of dipolarophile 3 was in accordance with the literature report [30,31]. The synthetic method for the efficient construction of spiropyrrolidine in the present study is based on a one-pot three-component approach involving N-propargyl-3’,5’-bis(4- phenylmethylidene) piperidin-4-one 3, acenaphthenequinone 4 and L-phenylalanine 5 as outlined in scheme 1.

Scheme 1. Synthesis of highly functionalized spiropyrrolidine heterocyclic hybrid 6

The spiropyrrolidine 6 was synthesized in a good yield by the 1,3-dipolarcycloaddition reaction of an equimolar mixture of 3, 4 and 5 under reflux in methanol. In the materials and methods section, detailed experimental conditions are described. A careful structural elucidation of the spiropyrrolidine 6 was performed with the help of FT-IR and NMR spectroscopic data (vide appendix). A reasonable mechanism to rationalize the formation of spiropyrrolidine involves the initial reaction of acenaphthenequinone and L-proline followed by dehydration and concomitant decarboxylation to furnish the azomethine ylide, then by the addition of nucleophilic carbon of azomethine ylide to the more electron-deficient β-carbon of 3 to afford the spiropyrrolidine 6. During the reaction, two C-C bonds and a C-N bond, along with four adjoining stereocenters were formed and were shown as thick bonds in scheme 1.

Antimicrobial activity of compound

Spiropyrrolidine heterocyclic hybrid was assessed for its antimicrobial activity using the agar well diffusion assay. Culture plates showed circular inhibition zones. The compound is treated Gram-positive, Gram-negative, and yeast strains. When treated with 100 μL of spiropyrrolidine, S. flexineri, B. subtilis, and C. glabrata showed the highest growth inhibition with zones measuring 21.3±0.2 mm, 17 ±0.3 mm, and 20.6 ±0.3 mm, respectively. E. coli, S. aureus, and C. albicans showed the lowest growth inhibition with zones measuring 18 ±0.5 mm, 16.8 ± 0.2 mm.

Figure 1(A-F) displayed zone of inhibition against tested pathogens in various concentrations and Figure 2 graphically depict the measured data. Furthermore, the spiropyrrolidine heterocyclic hybrid also significantly inhibited the growth of gram-negative bacteria. A well-inhibited zone is formed by spiropyrrolidine heterocyclic hybrid. Differences in cell wall composition and thickness may explain inhibition zone variation. This heterocyclic compound may disrupt the bacterial membrane surface, increasing cell permeability and altering respiration. Thus, enzymes degrade, DNA proteins are disrupted, and cells die [32].

Minimal Inhibitory Concentration of compound 6

The antimicrobial assessment of compound was evaluated using the resazurin-based micro-broth dilution technique and showed effective antimicrobial assessment activity against both Gram-positive and Gram-negative bacteria, as well as yeast. Compound effectiveness was comparable to conventional standard medications such as ampicillin (AMP), ciprofloxacin (CIP), and fluconazole (FLC) (Figure 3). The lowest MIC value was observed against C. glabrata (8 μg/mL), indicating strong antibacterial activity. The MBC values (32, 64 μg/mL) were generally higher than the MIC values, indicating that higher concentrations were required for bactericidal effects. The MFC values were not determined. The lowest concentration of each chemical that resulted in either complete inhibition or ≥99 % suppression of the growth was defined as the MBC/MFC (Table 1 and 2).

In-vitro anticancer activity of compound 6 by MTT assay

The colorimetric MTT assay was used to assess the in-vitro cytotoxic potential of spiropyrrolidine heterocyclic hybrid against Human epidermoid carcinoma (A431) cancer cell line at various concentrations. The results were measured in absorbance at 570 nm since this assay is a colorimetric measurement. The cells treated with spiropyrrolidine heterocyclic hybrid showed morphological changes in Figure 4 (A-G). This spiropyrrolidine heterocyclic hybrid exhibit significant anticancer activity, with 50% cell mortality at 134.36 μg/mL. At doses of 12.5, 25, 50, and 100 μg/mL of spiropyrrolidine heterocyclic hybrid, cell viability decreased by 69.70%, 59.78%, 52.21%, and 44.65%, respectively. At 200 μg/mL, cell viability decreased to 39.96%, Figure 4(H) depicts these percentages. The cancer cell line inhibitory mechanism of spiropyrrolidine heterocyclic hybrid is unknown. Spiropyrrolidine heterocyclic hybrid compound may inhibit abnormally increased signaling proteins or interact with functional groups of intracellular proteins and enzymes and DNA nitrogen bases, causing cell death [32,33].

Note: Decreasing the polarity of the extract increased the antibacterial efficacy against E. coli which may be due to increased permeability into fatty acid outer membrane in Gram-negative bacteria.

Apoptotic assay by dual staining method

The AO/EtBr staining technique is a reliable and comprehensive method for detecting apoptosis in carcinoma cells. The control cells in this experiment exhibited green fluorescence after being stained with AO, indicating the presence of viable cells. Apoptotic cells, on the other hand, were stained red with EtBr (Figure 5). However, only a limited number of viable cells were found in the spiropyrrolidine heterocyclic hybrid treated cancer cells. The majority of cells emitted red fluorescence, indicating their late apoptotic or dead state. The data showed that the IC50 concentration of spiropyrrolidine heterocyclic hybrid exhibited significant cytotoxicity against the human epidermoid carcinoma (A431) cancer cell line.

Apoptosis assay via flowcytometry

An apoptotic assay was performed by using Annexin V/FITC in order to determine the ability of spiropyrrolidine heterocyclic hybrid to induce apoptosis. Analysis of the fluorescence intensities of FITC-conjugated annexin-V and PI in cells was done using flow cytometry. After being exposed to spiropyrrolidine heterocyclic hybrid at an IC50 value of 134.36 μg/mL, a significant portion of the cells (61.1%) exhibited early apoptosis, while a smaller percentage (7.99%) displayed late apoptosis. The findings of this study demonstrate that the spiropyrrolidine heterocyclic hybrid has the ability to trigger apoptosis in A431 cell line, as shown in Figure 6 (A-C). Comparably, late apoptosis was 14.33% in cells treated with CTP and 46.32% in cells treated with compounds. Notably, prior studies on the anticancer effects of medications such as paclitaxel or cisplatin have shown that these drugs induce two distinct pathways of apoptosis, necrosis, and cell death, in varying degrees [13].

Based on the fluorescence intensities of cells shown in Figure 7(A-C), it can be observed that the spiropyrrolidine heterocyclic hybrid exhibits slightly moderate activity compared to the positive control (Doxorubicin, 1 μM). Based on the figure, M1 and M2 represent the fluorescence intensities of viable and apoptotic dying cells, respectively, induced by Annexin V-FITC. In the untreated cells [Figure 7A], the percentage of viable cells is approximately 99.83%. However, in the positive control group treated with Doxorubicin (Figure 7B), this percentage decreases to 4.76%. Similarly, when the cells are treated with the spiropyrrolidine heterocyclic hybrid, the percentage of viable cells further decreases to 22.03% (Figure 7C) after incubation. Thus, it can be inferred that the spiropyrrolidine heterocyclic hybrid exhibits a moderate level of activity, indicating its potential as a therapeutic agent against A431 cancer cells when compared to the standard compound, Doxorubicin (1μM). Moreover, the analysis conducted in this study is preliminary; thus, we are limited to discussing the elevated therapeutic values of certain derivatives. Consequently, it may be premature to assess the apoptosis-inducing capacity of spiropyrrolidine heterocyclic hybrid. In the absence of highly reactive or aggressive groups within the moiety, necrosis may not be evident, prompting the observation of apoptosis instead. In order to entirely realize this apoptotic pathway, one must trace the chemical pathways by which a series of groups sequentially oxidise intracellular proteins and kill the cells [28].

Conclusion

The spiropyrrolidine synthesized in the study showed significant antibacterial activity against a variety of pathogens as well as potent antifungal properties against Candida albicans. It outperformed the standard antibiotic Amphotericin B in terms of efficacy. MTT assay of spiropyrrolidine on human epidermoid carcinoma (A431), a malignant skin cell line, showed significant inhibition of cell proliferation. The hybrid molecule exhibited enhanced effectiveness when administered at higher doses. Furthermore, cancer cells treated with spiropyrrolidine showed a significant number of cells emitting red fluorescence, indicating they were in a late apoptotic or dead state. It was observed that the concentration of spiropyrrolidine, referred to as the IC50, had a notable impact on the cytotoxicity of the A431 cancer cell line. At an IC50 value of 134.36 μg/mL, a considerable proportion of cells (61.1%) showed early apoptosis, while a smaller fraction (7.99%) exhibited late apoptosis. Cell cycle analysis revealed that spiropyrrolidine induced cell cycle arrest at various stages in A431 cells. Additionally, it led to increased levels of DNA fragmentation and enhanced mitochondrial membrane permeability due to the activation of the caspase-3 enzyme. Hence, it is worthwhile to expand future research on this heterocyclic hybrid to encompass in vivo models and drug safety trials as studies like this will contribute to the development of new active agents for the antimicrobial and anticancer drug pipeline.

Acknowledgements

The authors are thankful to the Central Lab, College of Science, King Saud University for spectroscopic analysis. The authors extend their appreciation to P.G. Department of Studies in Botany, Karnatak University, Dharwad and Department of Biomedical Science, SDM Research Institute for Biomedical Sciences, Shri Dharmasthala Manjunatheshwara University, Sattur, Dharwad for antimicrobial studies and Scientific & Industrial Research Centre, Bangalore for anticancer studies. The authors are also grateful to Applied Organic Synthesis research group, Department of Chemistry, College of Science, and King Saud University for lab facilities.

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