The Roswell Park Memorial Institute medium (RPMI-1640) cell culture medium, fetal bovine serum (FBS), additional sources like sodium pyruvate, nonessential amino acids, L-glutamine, vitamin solution, penicillin and streptomycin were purchased from Life Technologies, Inc., Grand Island, United States. Reagents like as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT), propidium iodide, mercury orange, RNase and GA were obtained from Sigma-Aldrich (United States). Supplementary stains such as merocyanine - 540 and YO-PRO-1 were acquired from Invitrogen Inc, United States.

Human colorectal adenocarcinoma cell line HCT-15 (Organ: Colon, Disease: Colorectal adenocarcinoma; Organism: Human; procured from National Centre for Cell Science, Pune, India) was grown in RPMI medium, while 10% FBS, sodium pyruvate, penicillin, L-glutamine, nonessential amino acids and vitamin solution was given as supplements. Adherent monolayer cultures of HCT-15 were preserved in T-25 flasks and incubated at 37 °C in 5% carbon dioxide (CO₂). The cultures were free of mycoplasma and maintained no longer than 12 wk after recovery from frozen stocks.

Cell numbers or cell proliferation inhibition by GA was determined by thiazolyl blue tetrazolium bromide (MTT) assay. In brief, colon cancer cells were trypsinized, counted and 1000 cells were seeded per well in 96-well plate. The subsequent day, 100 μL of the medium containing the preferred concentration of GA was added to the appropriate wells. The cells were then maintained at 37 °C in 5% CO₂ for the desired length of time. The untreated cells kept for 72 h was used as control for this experiment. At this moment, 100 μL of (5 mg/mL) MTT reagent was added to each well, and the plate was sustained at 37 °C in the incubator for 2 h. After aspirating the supernatant, 200 μL of dimethyl sulfoxide was added to each well to solubilize the formazan crystals formed in viable cells. The optical density was spectrophotometrically measured at 570 nm using enzyme-linked immunosorbent assay plate reader[12].

In order to assess the colony forming ability of GA treated colon cancer cells, the colony formation assay was executed. The cultured HCT-15 cells were treated with GA at a concentration of 740 μmol/L for definite time periods of 12 h, 24 h, 48 h and collected by trypsinization. The cells were counted and seeded again in triplicate on a 6-well tissue culture plate with 3000 cells/well. Following 15-d incubation at 37 °C, colonies were stained with 0.5% crystal violet in methanol and the number of colonies was counted[13]. Control used in this experiment was untreated cells kept for 72 h. For all the experiments performed below, control cells remained untreated and kept for the same duration as the longest time point of the respective experiment.

Cell cycle analysis is an exceptional type of test that involves the flow cytometry and the fluorescent propidium dye and distinguishes the different phases of the cell cycle. The sub-G₁ fraction of the cell cycle was used as a measure of the apoptotic cells. After the appropriate treatment with GA, HCT-15 cells were washed with phosphate-buffered saline (PBS), then re-suspended in 50 μg/mL of propidium iodide stain containing 0.1% sodium citrate with 0.1% Triton X-100 for 20 min at 4 °C. Analysis was performed in linear amplification mode in case of cell cycle analysis using FACScan; Becton Dickinson Immunocytometry Systems. Remaining experiments of flow cytometry were performed in the logarithmic amplification mode unless otherwise stated[14].

MMP (ΔΨ_m) levels of GA treated HCT-15 cells were measured by the rhodamine-123 fluorescent dye. The HCT-15 colon cancer cells were treated with GA (740 μmol/L) for different time points. Then cells were harvested and re-suspended in 1 mL of rhodamine-123 (5 μg/mL) for 1 h and maintained at 37 °C. The intensity of fluorescence from rhodamine-123 was measured by flow cytometry[12]. Obtained fluorescence values were normalized with respect to control as 100%.

The lipid bilayer breaks when the drugs induce an antiproliferative effect on the cancerous cells. To estimate the lipid layer breakage the fluorescent dye merocyanine-540 is used. The cultured HCT-15 cells were treated with GA at concentration of 740 μmol/L for different time points. Cells were harvested and re-suspended in 1 mL of merocyanine-540 (10 μg/mL) for 15 min at 37 °C. The intensity of fluorescence was measured by flow cytometry[13].

GA treated HCT-15 cells (740 μmol/L) were harvested using trypsin/EDTA and re-suspended in PBS. Working solution (20 μmol/L) of Dichlorofluorescein-diacetate (DCFH-DA) was directly added to the cells and then it was incubated at 37 °C for 15 min. DCFH-DA was cleaved by the intracellular nonspecific esterase to form DCFH. DCFH are oxidized by ROS to form the fluorescent compound DCF. Cells were washed before re-suspending in PBS and kept on ice immediately before analyzing by flow cytometry[12]. The fluorescence intensity of DCF was measured and correlated with the ROS generated in the GA treated colon cancer cells.

Yo-Pro-1 staining helps in the analysis of apoptotic cells without interfering cell viability. The stain targets the nucleic acids of the cells and emits a green fluorescence that is detected. The HCT-15 cells after treatment with GA (740 μmol/L), were seeded in the cell pellets and mixed with 1 μmol/L Yo-Pro-1 for 20 min at room temperature. After incubation, the fluorescent intensity was measured using flow cytometry[14].

Fixed amount of HCT-15 cells were seeded in a sterilized glass slide and incubated for 24 h. GA at a concentration of 740 μmol/L was added for 72 h time interval. After incubation, cells were harvested by using trypsin/EDTA and centrifuged for 5 min at room temperature. Then the supernatant was decanted and pellet was dried. Pellet was treated with 2.5% glutaraldehyde in distilled water for 45 min in hybrid oven shaker at 37 °C. Cells were washed thrice with PBS for 5 min and then dehydrated by ethyl alcohol of different concentration (30%, 50%, 70%, 95% and 100%) for 5-10 min. Cell fixation was done with hexamethyl disilazane and the sample was taken for scanning electron microscope analysis. As well as the photomicrograph images of HCT-15 cells were also acquired using light microscope.

The results are expressed as the mean ± SD and repeated at least three independently (biological triplicates). The data were analyzed using Instat software (GraphPad Prism, San Diego, CA). The statistical significance was found using one-way analysis of variance.

The antiproliferative effect of GA on the cancer cells was assessed by MTT assay. The cell viability assay was performed on the GA treated cells after 72 h of treatment. GA inhibited the growth of HCT-15 colon cancer cells in a dose-dependent manner. The HCT-15 cell growth were inhibited significantly with an IC₅₀ of around 740 μmol/L (Figure 2). However, the growth of GA treated HCT-15 cells were fairly affected even at higher concentrations. Statistical analysis showed that GA treatment results in significant inhibition (P < 0.05) compared with untreated control cells at 200 μmol/L for HCT-15 cells (Figure 2).

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Figure 2 Cell proliferation inhibition by gallic acid of colon cancer cells. Human colorectal carcinoma HCT-15 cells grown in 96-well plate were treated with various concentration of gallic acid (GA) (0-1500 μmol/L) for 72 h. Percentage of mean cell viability along-with the SD is indicated (n = 3). Mean differences are significant compared with untreated control cells (P < 0.05 vs untreated control cells). Statistical analysis showed that GA treatment results in significant inhibition (^aP < 0.05) compared with untreated control cells starting at 200 μmol/L for HCT-15 cells. Data represents mean ± SD.

The HCT-15 cells found to form 192 colonies after 12 h treatment with GA (740 μmol/L). The colony numbers reduced with increase in the period of exposure to GA. The GA treated HCT-15 cells showed a maximum of 155 and 110 colonies after 24 h and 48 h of treatment respectively. In contrast, the untreated HCT-15 cells were found to produce a maximum of 210 colonies after 48 h. This is graphically represented in Figure 3. The figure depicts the time-dependent inhibition of colony formation by GA on the colon cancer cells. There was a significant reduction (P < 0.05) in the number of colonies formed under the various time intervals examined when compared with corresponding untreated cells (Figure 3).

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Figure 3 Colony inhibitory activity of gallic acid against colon cancer cells. After various incubation periods of gallic acid (GA) treatment, colonies formed were stained with 0.5% crystal violet and counted, and percentage of survival was calculated by normalizing the values. Data reported is the mean ± SD from three different observations. Mean differences are significant at 12 h, 24 h and 48 h compared with untreated control cells (P < 0.05 vs untreated control cells).

The effect of GA on the different phases of cell cycle of HCT-15 cells was estimated for time intervals of 24 h, 48 h and 72 h. The mean percentage of cells at various phases like sub-G₁, G₀/G₁, S and G₂/M phases are tabulated in Table 1. The Table showed an increase in the sub-G₁ phase arrest from 0.98% (control) to 58.01% after 72 h. Statistical analysis of the sub-G₁ column indicated a significant increase (P < 0.05) of cells in the sub-G₁ phase. Besides this, the time dependency on the cell cycle arrest induced by GA treatment on HCT-15 cells was also inferred.

The HCT-15 cells were treated with GA of concentration at 740 μmol/L at various time intervals (12 h and 24 h) and then stained with rhodamine-123 dye. The Figure 4 shows the mean fluorescent intensity of the control and GA treated HCT-15 cells. The untreated HCT-15 cells are considered to have maximum fluorescent intensity. In contrast, the normalized percentage of mean fluorescent intensity decreased to 81 ± 0.577 and 61 ± 1.528 compared to the untreated cells (100% after 24 h) after 12 h and 24 h of GA treatment respectively. There was also a statistically significant reduction (P < 0.05) of potential at the estimated intervals compared to untreated cells (Figure 4).

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Figure 4 Effects of gallic acid on MMP (ΔΨm) in HCT-15 cells. Human colorectal carcinoma (HCT-15) cells were treated with GA for specified time-periods and then mitochondrial membrane potential were determined using rhodamine-123 by flow cytometry. Mean differences are significant at 12 h and 24 h compared with untreated control cells (P < 0.05 vs untreated control cells).

The HCT-15 cells were treated with GA (740 μmol/L) at intervals of 12 h and 24 h. The fluorescent intensities detected from the merocyanine-540 stained cells were measured using flow cytometry. The untreated HCT-15 cells displayed a maximum mean fluorescence intensity at 40 after 24 h. The GA treated HCT-15 cells showed maximum intensity at 62 and 68 after 12 h and 24 h respectively. The maximum mean fluorescence intensities of the GA treated cells are given in Figure 5. It is evident from the above results that the GA treated HCT-15 cells displayed an increase in the lipid layer breaks with an increase in exposure time.

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Figure 5 Lipid layer break provoked by gallic acid. HCT-15 cells were treated with gallic acid (GA) and evaluated using merocyanine-540 to quantify the lipid layer breaks (LLBs). The fluorescence intensity was estimated using flow cytometry. Data is representative of three independent experiments, mean differences are significant at 12 h and 24 h compared with untreated control cells (P < 0.05 vs untreated control cells).

The ROS generated by the HCT-15 cells after GA (740 μmol/L) treatment was estimated at various time intervals such as 24 h, 48 h and 72 h. The ROS generated by the cells oxidized the DCFH-DA to dichlorofluoresin and the fluorescence emitted was measured. The fluorescent intensity obtained by flow cytometry is shown in the Figure 6. The maximum mean fluorescent intensity was found to be 112, 140, and 182 during 24 h, 48 h and 72 h respectively. Whilst, the maximum intensity of control HCT-15 cells was about 98 after 72 h. Moreover, the differences in the ROS levels at various hours examined were significant (Figure 6). The ROS generation from the HCT-15 cells was increased by GA treatment with respect to the increase in time of exposure.

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Figure 6 Gallic acid induced reactive oxygen species generation. HCT-15 cells were cultured in the presence or absence of gallic acid (GA) for the specified time points. DCFH-DA fluorescence intensity was detected by using flow cytometry. Data represented is the maximum of three independent experiments. Mean differences are significant at 24 h, 48 h and 72 h compared with untreated control cells (P < 0.05 vs untreated control cells).

Photomicrograph images of untreated and GA treated colon cancer cells (72 h) were acquired. The obtained images are shown in Figure 7A. In comparison to the untreated cells, the characteristic changes such as membrane blebbing and cell shrinkage are visible in the GA treated cells. These changes embrace the induction of apoptosis after GA treatment. Along with the digital microscopic image obtained, the HCT-15 cells were also examined with a scanning electron microscope to provide topographical or morphological images with high resolution. The images of untreated HCT-15 cells and GA treated HCT-15 cells (72 h) are given in the Figure 7B. It is evident from the images that the GA treated cells showed typical signs of apoptosis like membrane blebbing and shrinkage. In contrast, normal cells did not show any marked shrinkage. Hence, the microscopic images further corroborated the signs of apoptosis induced by GA. The images given the Figure 7 is representative of three independent experiments.

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Figure 7 Representative photomicrograph and scanning electron microscopic images of three independent experiments. A: Photomicrograph images of untreated and gallic acid (GA) treated HCT-15 cells. The arrow mark indicates cell death after GA treatment; B: Scanning electron microscopic images of untreated and GA treated HCT-15 cells. The arrow mark represents the rounding up of HCT-15 cells after GA treatment.

The HCT-15 cells were treated with GA (740 μmol/L) at various intervals of time such as 48 h and 72 h. The green fluorescence emitted by the Yo-Pro-1 stain from the GA treated HCT-15 cells is measured using flow cytometry. The result of fluorescence detected from the colon cancer cell line is given in the Figure 8. The maximum mean fluorescence intensity of GA treated HCT-15 cells at M2 phase was found to be 25.32 and 54.61 after 48 h and 72 h. In contrast, the maximum mean fluorescence intensity of control cells at M2 phase was found to be 7.75 after 72 h.

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Figure 8 Apoptosis assessment using Yo-Pro-1 dye by flow cytometry. HCT-15 cells was treated with gallic acid (GA) for specified time points. The distribution of the cell population changed according to the exposure time as indicated by M1 and M2. Data represented is the maximum of three independent experiments and the differences in the values of M2 were significant at 48 h and 72 h compared to untreated control cells (P < 0.05 vs untreated control cells).

Many surveys have shown that all types of cancers have a link with the diets the authors consume. Especially, in the case of colon cancer, diet plays a crucial role as the colonic epithelial cells are exposed to diets directly. Because of this reason, scientists explore various natural compounds present in food substances to treat colon cancer. Some studies have already shown that these natural compounds are absorbed by the body and have the potential to reduce the risk of colon cancer. The current study deals with examining the growth inhibitory effect of the dietary phenolic phytochemical gallic acid (GA) against HCT-15 colon cancer cells.

Despite the several anticancer drugs that are available for colon cancer, scientists continuously search for a novel anticancer drug with enhanced efficacy. Previous experiments have investigated the anticancer effect of GA against colon cancer, while the various reactions induced by GA on colon cancer cells have never been examined.

The study emphasizes the mechanism related to the anticancer effect of GA against colon cancer cell. GA was found to inhibit the growth of colon cancer cells through reactive oxygen species (ROS)-mediated apoptosis with notable morphological changes.

A series of events associated with the anticancer activity of GA are clearly depicted. Moreover, in-depth experimentation in preclinical and clinical settings is needed to promote GA as a plausible candidate for chemotherapy of colon cancer. Further, an in-depth proteomics study in relation to the GA induced apoptosis would be of great interest.

Apoptosis or programmed cell death, the death of cells that occurs as a normal and controlled part of an organism’s growth or development which can be Apoptosis can be induced either by a stimulus, such as irradiation or toxic drugs, or by removal of a repressor agent.

Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. In the past, a large number of substances derived from plants have been studied in antitumor research fields and many have proven to exhibit chemopreventive properties which could be used as adjuvant chemotherapy. In this manuscript, the authors found that diet-derived phenolic compound GA inhibited the proliferation and induced the apoptosis of HCT-15 cancer cells through increased generation of ROS. The study is important and may advance the field of chemoprevention.