Cytotoxic effect and apoptotic induction of tricyclohexyltin p-methoxycinnamate on HT-29 colorectal cancer cells: Implications for anticancer therapeutics

Abstract

Colorectal cancer’s escalating prevalence in Malaysia prompts the exploration of innovative anticancer agents; amidst this backdrop, tricyclohexyltin p-methoxycinnamate emerges as a synthesized organotin complex with unique bioactive properties. Notably, the novelty of this research lies in its groundbreaking investigation into the hitherto unexplored anticancer potential and mode of cell death induced by tricyclohexyltin p-methoxycinnamate on colon cancer (human colorectal adenocarcinoma cell line (HT-29)) cell lines. This study pioneers the assessment of tricyclohexyltin p-methoxycinnamate’s cytotoxic effects through the “(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay,” revealing a dose- and time-dependent cytotoxicity with IC₅₀ values of 1.2 × 10-6M for 24 h, 1.0 × 10-6M for 48 h, and 5.0 × 10-7M for 72 h. The mode of cell death through “AO/PI” staining alongside cell cycle analysis, highlighting apoptosis as the predominant mode of cell death in the HT-29 cell line, accompanied by substantial cell cycle arrest at the sub-“G0” phase. The tricyclohexyltin p-methoxycinnamate’s shown potential antiproliferative properties, cell cycle arrest, and apoptosis in HT-29 cancer cells. This novel insight into the compound’s mode of action positions it as a promising candidate for future anticancer therapeutics. The study underscores the urgency of investigating innovative approaches amidst the rising colorectal cancer rates, emphasizing the compound’s potential through further in-depth studies and preclinical trials.

INTRODUCTION

The “organotin” compound was first synthesized by Lowiq in “1852,” and the series of the compounds was studied by Franklin in 1853.[1] The organotin compound was composed of tin and hydrocarbon substituents and was part of organometallic chemistry.[2] Organotin compounds have been used as “bactericides,” “fungicides,” “insecticides,” “herbicides,” and “wood preservatives,” demonstrating the biologically active properties of organotin compounds.[1] In 1929, scientists found that organotin complexes had shown antitumor activity against cancer cells,[3] and this caused increasing interest in organotin compounds due to their possibility of being used as an antitumor agent.[4] The antiproliferative effect of the organotin complex was due to its ability to interact with organometallic compounds with the DNA because it could recognize specific DNA sequences. This caused “modification” of the “DNA structure” or sequence changes. Variation of DNA structure or sequence caused affection of the gene expression effect because of the inability to access the “activator” or “repressor” protein.[5] The coordination number and types of groups linked to the central tin atom were necessary for DNA binding in organotin complexes. Anchoring to the phosphate group in the DNA sugar backbone was unnecessary because of the solid binding for nitrogen in the DNA bases. Therefore, thanks to this interaction, the tin core was maintained and was octahedrally stable.[3] Apoptosis held a pivotal role in upholding equilibrium between cellular demise and proliferation. Evasion of apoptosis disrupted this balance, drove unregulated cell proliferation, and contributed to various ailments, including cancer.[6] The p53 tumor suppressor, “TRAIL” receptor, caspases pathway, and “Bcl-2” family of proteins were examples of the apoptotic pathway, and some metal compounds could activate the apoptotic process through those pathways.[3] The organotin complex might have initiated the apoptotic pathway by releasing the metal compounds, which caused the metal-induced reactive oxygen species to be induced. This reactive oxygen species would thus cause metal-induced intracellular apoptosis.[7] For example, organotin complexes such as “di-n-butyltin” and “tri-n-butyltin” could induce apoptosis in rat thymocytes by inhibiting DNA synthesis. These organotin complexes could cause the release of Ca² + ions and then the release of cytochrome c from the mitochondrion. This would lead to caspase pathway activation and then cause DNA fragmentation. DNA fragmentation would cause the cells to undergo the apoptotic pathway.[8] Our current investigation showed the antiproliferative activity, mode of cell death, and cell cycle study of the organotin compounds, tricyclohexyltin p-methoxycinnamate, on the HT-29 cell line.

MATERIALS AND METHODS

The tricyclohexyltin p-methoxycinnamate was synthesized and provided by the “Department of Chemistry,” Faculty of Sciences, “University Malaya.” The HT-29 (colon cancer) cell line was acquired from the “American Type Culture Collection,” USA. The cell lines medium ‘”RPMI 1640” purchased from Nacalai Tesque, Kyoto, Japan. The “FBS” and trypsin purchased from PAA Laboratories, Austria. However, the phosphate buffer, RNAase A reagent, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) reagent solution, acridine orange (AO), and propidium iodide (PI) purchased from Sigma, USA. All reagents were of analytical grades.

Cytotoxic analysis

The cell culture was grown in “RPMI 1640” culture media complemented by 10% “FBS,” and “cell culture“ antibiotics. The analysis was performed through previously optimized and established method.[9] The cancer and normal cells were grown in a 5% CO₂ at 37°C. The cytotoxicity was evaluated through using MTT reagent. Ultimately, the absorbance was measured using an “enzyme-linked immunosorbent assay (ELISA)” 96-well plate reader at a wavelength of 490 nm.

Apoptosis analysis through using acridine orange/propidium iodide staining

The HT-29 cells were seeded into six-well plate and incubated at for 24 h in humidified environment. Subsequently, the old medium was removed from each well, and 2 mL of tricyclohexyltin p-methoxycinnamate at concentrations of 3.5 × 10-7M (IC₃₀), 1.2 × 10-6M (IC₅₀), and 2.25 × 10-6M (IC₇₀) was added into individual cells. The six-well plate was then incubated at 37°C with 5% CO₂ for an additional 24 h. The supernatant was removed, and the HT-29 cells were stained with 50 µL of AO (1 mg/mL) and 50 µL of PI (1 mg/mL). The stained cells were transferred onto a slide and examined using fluorescence microscope.

Cell cycle analysis through flow cytometry

Initially, 1 × 10^-6 cells were seeded into each well of a “six-well” plate, then incubated at 37°C with 5% CO₂ for 24 h. Following incubation, 2 mL of tricyclohexyltin p-methoxycinnamate with concentrations of 3.5 × 10-7M (IC₃₀), 1.2 × 10-6M (IC₅₀), and 2.25 × 10-6M (IC₇₀) were added to each well. Briefly, the cells were fixed in “70% ethanol” overnight at −20°C. Subsequently, 5 µL of RNAase A was added, followed by 50 µL of 1 mg/mL PI, and the mixture was incubated for 30 min. Finally, the stained samples were analyzed using a flow cytometer.

Statistical analysis

All the determinations were expressed in mean ± standard error of the mean. The dose–response relationship of tricyclohexyltin p-methoxycinnamate toward HT-29 cancer cell line was evaluated using the “Statistical Package for the Social Science” (SPSS) version 13.0 (SPSS Chicago (Ill., USA)). The different concentration of tricyclohexyltin p-methoxycinnamate in the AO/PI assay was analyzed through “one-way analysis of variance” and “Dunnett’s multiple-range” tests. A “P < 0.05” was considered significant in all the statistical analyses.

RESULTS AND DISCUSSIONS

The cytotoxicity assay revealed that escalating concentrations and prolonged treatment with tricyclohexyltin p-methoxycinnamate led to a reduction in the viability of the HT-29 cancer cell line [Figure 1]. Notably, tricyclohexyltin p-methoxycinnamate did not demonstrate significant toxicity toward Vero cells or healthy cells. The IC₅₀ values of tricyclohexyltin p-methoxycinnamate at 24, 48, and 72 h, along with those of the positive control, 5-fluorouracil, are detailed in Table 1.

Figure 1.

Graph percentage of HT-29 cancer cell line viability versus tricyclohexyltin p-methoxycinnamate concentration for specific screening after 24, 48, and 72 h of incubation

Table 1.

Half-maximal inhibitory concentration values of tricyclohexyltin p-methoxycinnamate and positive control (5-fluorouracil) on HT-29 colon cancer cell line after 24-, 48- and 72-h incubation

Duration of incubation (h)	Tricyclohexyltin p-methoxycinnamate, IC50 ± SEM	5-fluorouracil (IC50 ± SEM)
24	1.25×10−6±5.77×10−8 M	-
48	1.17×10−6±2.21×10−7 M	-
72	4.58×10−7±9.61×10−8 M	2.43×10−4±1.45×10−5 M

SEM: Standard error of mean, IC₅₀: Half-maximal inhibitory concentration

Inducing apoptosis in cancer cells represents an optimal strategy for cancer treatment, as it avoids causing damage to the surrounding tissue, unlike cell death through necrosis, which may lead to injury in the adjacent tissue.[10] Previous investigations on organotin complexes have provided evidence suggesting that these complexes can induce apoptosis in cells, with a study demonstrating p53-dependent mediation of apoptosis in tumor cells.[3] Our study treated the “HT-29” colon cancer cell line with varying concentrations of IC₃₀, IC₅₀, and IC₇₀ of tricyclohexyltin p-methoxycinnamate. Fluorescent images of HT-29 cells following exposure to tricyclohexyltin p-methoxycinnamate revealed a concentration-dependent impact, as illustrated in Figure 2. Observable changes included DNA fragmentation, membrane blebbing, and apoptotic body formation, indicative of apoptosis in the treated cells. The negative control (without treatment) exhibited a predominance of green viable cells compared to red apoptotic cells. Notably, IC₇₀, featuring a higher concentration of tricyclohexyltin p-methoxycinnamate, displayed more “red color” apoptotic cells than “green color” viable cells. Meanwhile, IC₅₀ demonstrated a nearly equal distribution of “red apoptotic” cells and “green viable” cells, and IC₃₀, with a lower concentration of tricyclohexyltin p-methoxycinnamate, exhibited a lower number of red apoptotic cells than green viable cells [Table 2]. Analysis of the treated cancer cell lines indicated a reduction in cell viability, with a higher abundance of red apoptotic cells compared to the control group. This substantiates the antiproliferative or apoptotic effect of tricyclohexyltin p-methoxycinnamate on cancer cells. Furthermore, the dose-dependent relationship was evident, as a higher dose of tricyclohexyltin p-methoxycinnamate resulted in an increased number of apoptotic cells. This confirmation supports the notion that tricyclohexyltin p-methoxycinnamate influences cell viability in a concentration-dependent manner. Statistical analysis using SPSS affirmed a significant difference in the dose–response relationship of tricyclohexyltin p-methoxycinnamate toward the HT-29 colon cancer cell line, as demonstrated by the AO/PI assay. This substantiates that tricyclohexyltin p-methoxycinnamate exerts its apoptotic effect on cells across different concentrations.

Figure 2.

Fluorescent images of acridine orange and propidium iodide staining. The arrows represent increase in IC value dependant change in cells. HT-29 Colorectal cancer cell line was treated with Tricyclohexyltin p-methoxycinnamate in a dose-dependent manner; (a) treatment of IC₃₀, (b) treatment of IC₅₀, (c) treatment of IC₇₀, and (d) untreated cells

Table 2.

Quantitative analysis mean number of viable and apoptotic cells observed with different concentrations of tricyclohexyltin p-methoxycinnamate under fluorescent microscope

	Apoptotic cells			Viable cells
	Mean	SD	SEM	Mean	SD	SEM
Control	0.00	0.00	0.00	108.00	15.72	9.07
IC30	51.00	5.57	3.21	59.67	5.69	3.28
IC50	38.33	1.53	0.88	31.67	0.58	0.33
IC70	54.67	13.01	7.51	24.33	3.06	1.76

SEM: Standard error of mean, SD: Standard deviation, IC: Inhibitory concentration

Cell cycle phase distribution analysis using RNAase A/propidium iodide assay

To further validate the apoptogenic effect on HT-29 cells, an RNAase/PI assay was conducted. The RNAase A/PI assay is a valuable and sensitive technique for assessing cell cycle progression or DNA content within cells. The “propidium iodide” PI is a “DNA-intercalating” fluorescent dye capable of permeating through a permeabilized membrane and intercalating into DNA. The intensity of the PI signal correlates directly with the “DNA” content.[11] RNAase A introduced to degrade RNA, as PI also binds to RNA, thereby preventing false-positive results. In the RNAase A/PI assay [Figure 3], the untreated group exhibited 55.56% of cells accumulated in the “sub-G0” phase. Conversely, the IC₃₀ treated group demonstrated an accumulation of 63.41% of cells in the “sub-G0” phase, the IC₅₀ treated group displayed 66.00%, and the IC₇₀-treated group showed 71.58% in the “sub-G0” phase. The increased percentage of cells in the “sub-G0” phase from IC₃₀ to IC₇₀ signifies a higher incidence of apoptosis at elevated concentrations of tricyclohexyltin p-methoxycinnamate. A higher percentage of cells in the “sub-G0” phase indicates DNA cleavage and apoptosis during programmed cell death. Cellular endonucleases degrade DNA during apoptosis, resulting in apoptotic cell nuclei containing less DNA than healthy cell nuclei. Consequently, the treatment-inducing apoptosis led to fewer cells in the G0/G1 phase, with evident sub-G0 peaks in the fluorescence histograms. These sub-G0 peaks in the histogram facilitate quantifying apoptotic cells in the samples. Organotin (IV) compounds have garnered significant attention for their anticancer properties, comparable to other metallodrugs.[12] Diorganotin (IV) and triorganotin (IV) compounds have been extensively studied for their in vitro antitumor properties against various cancers. The quantity and type of alkyl or aryl substituents, along with the number of Sn-C bonds in an organotin molecule, may influence the biological effects of the product.[13,14,15] These substances exhibit fewer adverse effects, superior excretion qualities, and heightened antiproliferative activity at low concentrations than other platinum-based medications.[16]

Figure 3.

Cell cycle analysis of treated HT-29 colon cancer cell line. Untreated and Tricyclohexyltin p-methoxycinnamate with IC₇₀, IC₅₀, and IC₃₀ values

The two prominent mechanisms through which cancer cells undergo cell death are apoptosis and necrosis. Initiating apoptosis, characterized by programmed cell death, or inducing cell lysis leading to inflammation – a characteristic feature of necrosis, are significant avenues in cancer treatment.[17,18,19] Our research underscores the inhibitory effect of tricyclohexyltin p-methoxycinnamate on the growth of the HT-29 colon cancer cell line, resulting in cell death by inducing cell arrest. This cell arrest may activate specific cell death pathways, potentially leading to programmed cell death. Notably, our findings strongly indicate that apoptosis is the primary cause of cell death. However, verification through caspase investigations is crucial to determine whether the process is intrinsic or extrinsic. The process of apoptosis is well-defined, involving a cascade of enzymatic processes within cells. According to Jan and Chaudhry,[6] phosphatidylserine (PS) translocates from the cytoplasm to the cell’s surface during the initial phases of apoptosis induction. The distinctive characteristic of cytotoxic agents lies in their ability to induce apoptosis while maintaining membrane integrity. Further research is necessary to fully validate apoptosis by delineating each step of cell death, from DNA fragmentation to PS exposure, triggered during this cellular event. Future research investigations may delve into the activation of caspases, the mitochondrial route, and the role of death receptors. These factors will strengthen the conclusion regarding the apoptotic process of tricyclohexyltin p-methoxycinnamate in HT-29 colon cancer cells and contribute to advancing our understanding of the apoptotic process. Considering these results, research into the cellular and molecular impacts of tricyclohexyltin p-methoxycinnamate is warranted to ensure its potential as a therapeutic agent.

CONCLUSION

Overall, the rising incidence of colorectal cancer in Malaysia highlights the critical need to address this worldwide health issue. Due to its diverse bioactive properties, tricyclohexyltin p-methoxycinnamate is promising as a potential therapeutic agent. This work investigated the cytotoxic effects and mechanisms of cell death induced by tricyclohexyltin p-methoxycinnamate on colon cancer (HT-29) cell lines. This study emphasizes tricyclohexyltin p-methoxycinnamate’s antiproliferative qualities and capacity to cause apoptosis in HT-29 cancer cells. Its potential use in anticancer treatments is highlighted by the requirement for additional research into its mechanisms of action through thorough studies and preclinical trials. The future of improving cancer treatment potentially lies in further investigating tricyclohexyltin p-methoxycinnamate’s mechanism in detail, including signaling pathways triggered in various cell types and toxicity of a compound, safety profile keeps in check to consider it with having therapeutic potential.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.