Downregulation of Bcl-2 Family Expression and Enhanced DNA Fragmentation in HCT116 by Thymoquinone

Culture of Cells

King Abdulaziz University in Jeddah, Saudi Arabia, donated the HCT116 cell lines that they had acquired from ATCC. The obtained cells were cultivated in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 1% streptomycin and penicillin antibiotics (Sigma-Aldrich, St. Louis, MO, USA) and 10% fetal bovine serum (Gibco, Paisley, UK). Every cell was maintained in a CO₂ incubator at pH 7.4 and 37°C (New Brunswick, Scotland). In all the experiments, cells were seeded at 2 × 10⁵ cells/mL and treated with TQ at 50%–60% confluence. All chemicals were dissolved in ethanol.

Cell Proliferation Assay

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test has been used to assess the effects of different doses of TQ to cause cell death. ²¹ In summary, 96-well culture plates were filled with 10,000 cells/mL of culture medium. The cells were cultivated for a full day in a CO₂ incubator, which allowed them to multiply and adhere. The plates were removed from the incubator following the experiment’s first day, and new media with different concentrations of TQ were applied to the cells; the dose concentration varied from 1.56 to 100 µg/mL. The treated plates were then incubated for an additional 24 h. After the treatment was completed, 20 µL of MTT dye (5 mg/mL) (Invitrogen Corporation, San Diego, CA, USA) was added to each well and incubated for an additional 4 h. After the incubation period was over, the medium was carefully removed from the plates so as not to disturb the formazan crystals that had developed at the bottom of the plates. To facilitate the dissolution of the crystals, 100 µL of dimethyl sulfoxide (DMSO) (sourced from Fisher Chemicals in the UK) was then added to each well. The violet-hued final result at 570 nm was further examined using a microplate reader (BMG LABTECH, SpectroStar Nano, Ortenberg, Germany).

In order to find the cells’ vitality percentage, we used the raw data to calculate it from the relevant control values according to the method shown below. The experiments were repeated three times.

Cell Morphology

To view the morphological changes in the cultured cells, all of the treated and untreated cells in the flask (T25) were examined under an inverted microscope at 20× magnification after 24 h of TQ treatment.

Measurement of Reactive Oxygen Species Generation

Intracellular ROS generation was predicted using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). This non-fluorescent probe can freely infiltrate the intracellular matrix of cells, where it is oxidized by intracellular ROS to form the fluorescent DCF. Thus, fluorescence intensity is directly proportional to the quantity of ROS generated in the cells. ²² HCT116 cells were grown in a six‐well plate (3 × 10⁴ per well) and treated with different concentrations of TQ (8, 12, and 16 µg) for 24 h.

Following a 24-h incubation period, 1 mL of cells was incubated for 10 min at 37°C with 100 mL of DCFH-DA. A Shimadzu RF-5301 PC spectrofluorimeter was used to measure the fluorescent intensity under a FLoid cell imaging station (Invitrogen) with excitation and emission filters set at 485 ± 10 and 530 ± 12.5 nm, respectively. The increasing percentage (%) of fluorescence intensity was used to express the results.

Biochemical Analysis

TQ (8, 12, and 16 µg) was added to the HCT116 cells. The cells were then collected and utilized for the biochemical analyses. The methods of Ohkawa et al., Kakkar et al., and Sinha^23–25 were used to analyze the malondialdehyde (MDA) markers of lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were estimated by Moron et al. ²⁶

DNA Fragmentation

Researchers had previously described cells from which genomic DNA was extracted. ²⁷ In summary, 1 × 10⁶ cells were cultivated and exposed to 8, 12, and 16 µg of TQ. Trypsin was used to extract the treated cells, which were subsequently cleaned using Dulbecco’s phosphate-buffered saline (DPBS). Following the manufacturer’s instructions, the Qiagen DNA isolation kit was used to isolate DNA from the isolated cells. The nanodrop was used to measure the DNA concentration at 260 nm. Following that, 0.2% agarose gels containing 0.1 µg/mL ethidium bromide were used for DNA electrophoresis.

Gene Expression

The effect of TQ on mRNA expressions of Bcl-2, Bcl-xl, Bax, caspase-3, and caspase-9 was carried out by extracting mRNA from cells. mRNA was isolated by using the RNA extraction kit (BIO-RAD), and then cDNA was synthesized from the cDNA Reverse Transcription Kit (Applied Biosystems Cat# A25918). The cDNA templates were used to run the quantitative reverse transcription polymerase chain reaction (qRT-PCR) machine (Bio-Rad) by applying SYBR Green dye (Applied Biosystems). The sequences of the primers mentioned in Table 1 were obtained from Macrogen Inc. (Korea), and the reaction temperature and cycle duration were as per the manufacturer’s procedure. The β-actin expression is considered a reference standard. ²⁸

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Table 1.

List of Primer Sequences with Their Gene Accession Number.

Oligo	Primer Sequences	Tm (c)	Gene Accession Number
Bcl-2	5′-CGA CTT TGC AGA GAT GTC CA-3′ Forward 5′-CAT CCA CAG AGC GAT GTT GT-3′ Reverse	59.4 57.4	NM_000633.3
Bcl-xl	5′-CCA CTT AGT ACA GAT GTC CAT-3′ Forward 5′-GGT CCA TAA AGT CTA CTT GTA-3′ Reverse	58.3 58.9	NM 021633
Bax	5′-GCCTCCTTTCCTACTTCGGG-3′ Forward 5′-CTTTCCCCGTTCCCCATTCA-3′ Reverse	62.5 60.5	S76511
Caspase-3	5′-GCTACGATCCACCAGCATTT-3′ Forward 5′-ATGCCACCTCTCCTTTCCTT-3′ Reverse	58.4 58.4	NM_012922
Caspase-9	5′-TGT CCT ACT CTA CTT TCC CAGGT-3′ Forward 5′-GTG AGC CCA CTG CTC AAA GAT-3′ Reverse	62.5 60.5	P55211
Beta actin	5′-CAACCTTCTTGCAGCTCCTC-3′ Forward 5′-TTCTGACCCATACCCACCAT-3′ Reverse	60.5 58.4	AA955227

Source: Alam et al. ^{21, 29}.

Analysis of Statistics

The findings are presented as mean ± SD and were subjected to one-way analysis of variance (ANOVA) and post hoc analysis using Tukey’s techniques. A Student’s t-test without pairing was used to compare the two groups. A value of p < .05 is regarded as statistically significant.

MTT Test

The MTT assay was used to determine the cytotoxic effect of TQ in HCT116 cells. The cells were exposed to varying concentrations of TQ (1.56, 3.125, 6.25, 12, 25, 50, and 100 µg/mL) for 24 h. The outcome demonstrated that TQ significantly and concentration-dependently inhibited cell proliferation. Thus, the growth inhibition curve (Figure 1) clearly shows that the inhibitory concentration (IC₅₀) of TQ for HCT116 cells was 16 µg. Based on this outcome, we selected TQ concentrations of 8, 12, and 16 µg/mL for additional research.

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Figure 1.

Source: Alam et al. ^{21, 29}.

Morphological Studies

There were significant differences between the treated and control cells. Treated cells showed several morphological changes that were visible at 20× magnification (Figures 2A–2D), in contrast to the untreated cells depicted in Figure 2A, which were observed 24 h after treatment. This provides a graphical depiction of these changes. More cells with traits like blebbing (purple arrow) of the cell membrane, greater growth inhibition, and cell lysis/shrinking (black arrow) were seen in cells treated with higher doses. Conversely, untreated cells persisted in adhering to one another throughout the whole incubation period.

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Figure 2.

Source: Alam et al. ^{21, 29}.

Intracellular Reactive Oxygen Species

The measurement of intracellular ROS offers strong evidence that DCFH-DA dye caused oxidative stress in cancer cells (Figure 3). TQ concentrations of 8, 12, and 16 µg/mL were applied to HCT116 cells. As TQ dosage increases, ROS production is proportionately increased. In comparison to the control, at the 12 µg significance level (p < .01) and at the 16 µg level (p < .001).

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Figure 3.

Source: Alam et al. ^{21, 29}.

Lipid Peroxidation and Antioxidant Status

One well-known biomarker for oxidative stress is elevated LPO and reduced antioxidant status. In comparison to the control, we found that HCT116 cells treated with TQ (8, 12, and 16 µg/mL) had higher levels of the LPO marker (MDA) dose dependently and lower levels of antioxidant enzymes (GSH, SOD, and CAT) with an increase in dose (Table 2). The results indicate significant changes in every parameter at 12 and 16 µg/mL (p < .01, p < .001).

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Table 2.

Displaying Chemoprotective Effects of Thymoquinone (TQ) in HCT116.

	Control	8 µg	12 µg	16 µg
MDA (nmol/mg protein)	2.19 ± 0.08	12.09 ± 1.23##	33.94 ± 3.34###	48.67 ± 3.84###
GSH (units/mg protein)	19.63 ± 2.28	14.03 ± 3.08#	9.31 ± 1.87##	5.76 ± 0.89###
SOD (units/mg protein)	22.66 ± 4.51	16.45 ± 3.57#	7.08 ± 1.24###	3.38 ± 0.83###
CAT (units/mg protein)	17.11 ± 2.79	12.89 ± 1.84#	6.46 ± 0.48##	4.0 ± 0.74###

Source: Alam et al. ^{21, 29}.

Note: CAT: Catalase; GSH: Glutathione; MDA: Malondialdehyde; SOD: Superoxide dismutase. Significant #p <0.05; ##p < 0.01; ###p < 0.001 vs control.

DNA Fragmentation

Figure 4A displays the agarose gel electrophoretic pattern of the DNA degradation caused by TQ doses (lanes 16-8). The compact DNA (lane-C) of the control cell shows that the DNA of the cancerous cell is not fragmented, and is similar to low dosage (8 µg/mL). It was discovered that the ladder-like (smear-like) pattern of the DNA increased with the doses of TQ (lanes 16 and 12), corresponding to the 16 and 12 µg/mL, respectively, a common marker of apoptosis. Apoptosis can also be expressed as a percentage (%) of DNA degradation, with 100% as a control (Figure 4B). The study demonstrates that the DNA degradation of cancer cells increases from 124% to 288% with an increase in dose from 4 to 16 µg/mL, respectively (p < .05, p < .01, p < .001).

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Figure 4.

Source: Alam et al. ^{21, 29}.

Real-time Quantitative Reverse Transcription Polymerase Chain Reaction

A change in the amplification of gene expression varies with an increase in the dose of TQ (Figure 5). Expression of anti-apoptotic markers like Bcl-2 and Bcl-xl is reduced with an increase in TQ doses (p < .01, p < .001). On the other hand, the pro-apoptotic markers such as Bax, caspase-3, and caspase-9 are increased with TQ. Our results are more significant at 12 and 16 µg/mL concentration (p < .01, p < .001), as compared to the control cell.

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Figure 5.

Source: Alam et al. ^{21, 29}.