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ORIGINAL ARTICLE
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Induction of apoptosis and suppression of Ras gene expression in MCF human breast cancer cells


1 Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
2 Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran
3 Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

Date of Submission13-May-2020
Date of Decision16-Jul-2020
Date of Acceptance15-Sep-2020
Date of Web Publication30-Jul-2021

Correspondence Address:
Maryam Kolahi,
Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz
Iran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_624_20

 > Abstract 


Breast cancer is the leading invasive cancer in women globally. This study aimed at evaluating the anti-apoptotic activity of p-Coumaric acid (PCA) on MCF-7 breast cancer cell line. Experiments were conducted in which the MCF-7 cell line was treated with PCA. which showed decreased cell viability, increased lactate dehydrogenase activity, and caspase-3 activation. The results were evaluated with real-time polymerase chain reaction which revealed that PCA reduced the amount of H-Ras and K-Ras transcript in MCF-7 breast cancer cells. In the presence of PCA there was a significant increase in the levels of mRNA gene Bax and late apoptotic cells which was dose dependent. It also retarded the relative expression of antiapoptotic gene, Bcl2 in treated cells. The results suggest that PCA exhibits anti-cancer properties against MCF-7 cells. PCA inhibited the growth of MCF7 cell. The optimum concentration of PCA was 75–150 mM. PCA can inhibit the growth of MCF-7 cells by reducing Ras expression and inducing cell apoptosis. Our results suggest that PCA could prove valuable in the search for possible inhibitors of Ras oncogene functionality and gain further support for its potential utilization in the treatment of patients with breast cancer. PCA is safe and could complement current treatments employed for the disease.

Keywords: Apoptosis, Caspase, H-Ras oncogenes, N-Ras oncogenes, p-Coumaric acid



How to cite this URL:
Saremi S, Kolahi M, Tabandeh MR, Hashemitabar M. Induction of apoptosis and suppression of Ras gene expression in MCF human breast cancer cells. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322710




 > Introduction Top


Breast cancer is the leading invasive cancer in women globally. In relation to its occurrence, it represents 16% of all female cancers and is also reflected in the 22.9% of invasive cancers in women. Another important point to note is that the disease does not only affect women but also men, with a total of 18.2% cancer deaths being associated with breast cancer on a global scale. The percentage occurrence of breast cancer is interestingly much higher in developed countries in contrast to that of developing ones. This may be due to a number of factors, with experts in the field postulating that differences in the lifestyles and consumption patterns of women from different demographic regions play a major role.[1],[2] Different approaches are required to avoid and treat such a deadly disease. A number of treatment options are available for patients suffering from breast cancer. These may include the surgical removal of the infected breasts and application of various types of therapy inclusive of radiation, chemotherapy, hormone therapy, as well as targeted therapy. In addition to the high cost of treatment, the majority of the treatment options may in some instances lead to side effects during treatment.[3] Apoptosis is the programmed death of cells in tissue. This procedure allows for various morphological modifications in the cells, for example enhanced rates condensation and budding of the cell, with the formation of embrane-enclosed apoptotic bod-ies containing cell-preserved organelles. The most significant sign of cytotoxic antitumor agents is that of induction of apoptosis.[4] Phenylpropanoids such as plant secondary metabolites have been identified as powerful antioxidants being able to block cancer cells through various mechanisms including apoptosis.[5] p-Coumaric acid (PCA), a member of the large group of phenylpropanoid compounds, is present in various fruits and vegetables that are consumed such as legumes (peanuts, navy beans), fruits (apples, tomatoes), vegetables (carrots), tea, and garlic and is also found in vinegar and honey.[6]

Current research focuses on the potential of new anticancer drugs that have the ability to target apoptotic defects.[7]

Investigation of gene expression in breast cancer revealed that treatment with select phenylpropanoid compounds can lead to the death of cancer cells via apoptosis, which was altered as a result of the dysregulation of the nuclear factor-kappa B (NF-κB) pathway, as revealed by a reduction in the expression of a number of κB-regulated gene targets.[8]

Ras proteins (H-, N-, and K-Ras) containing GTP-regulated molecular switches that are integrally involved in cell signaling pathways contributes to various aspects of cell response such as proliferation, differentiation, motility, and death. In normal cells, Ras can be activated by extracellular stimuli through diverse cell surface receptors, leading to the activation of the enzymes such as mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK), kinase (MEK) 1, and MEK2. In many tumors, oncogenic mutations have reported excessive levels of Ras-GTP to mediate downregulation due to GAP and to stay constitutively GTP bound and active.[9]

Previous research has shown the interference of Ras genes in several major pathways of apoptosis such as phosphatidylinositol 3-kinase and NF-κB, promotion via RASSF1/Nore1/Mst1 and the Raf/MEK/ERK pathways.[10] Research has revealed that Ras paradoxically mediates both pro- and anti-apoptotic signaling depending significantly on the type of cell and other contributing factors.[11] In normal cells, activated Ras leads to the protection of proapoptotic reactions, allowing inactive oncogenes to respond to hyper proliferative signals, whereas in cancer cells, elevated levels of Ras have stimulated survival rather than death. The vital role of Ras proteins in cell proliferation has attracted much attention to their recruitment in Ras transformation.[12] Intensive efforts have been aimed at using anti-Ras strategies to inhibit oncogenic Ras in different cancers. Interestingly, screens with anti-Ras effect can be identified as Ras inhibitors with clinical function in cancer treatment in future. Previous studies have provided significant support that Ras can induce growth and progression of breast cancer.[13] In the current study, the aim was to evaluate the cytotoxic and anti-apoptotic effects of PCA and to determine their possible cell death properties on breast carcinoma cancer cells. Furthermore, for the first time, we investigated whether PCA can change the Ras and signaling pathways stimulated by Ras in human breast cancer cell lines via its effect on the rate of Ras gene expression.


 > MaterialS Top


Assay of cell viability using the 3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method

The viability of the cells was assessed utilizing the mitochondria enzyme-dependent reaction of 3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In this assay, metabolic active cells deactivated the yellow tetrazolium salt MTT, producing purple-colored formazan crystals. The resulting formazan was dissolved in DMSO and the absorbance was determined spectroscopically at a wavelength of 570 nm. MTT was solubilized in phosphate-buffered saline (PBS, pH 7.2) to a final concentration of 5 mg/mL. This solution (20 µL) was added to each well and incubated for 4 h. Thereafter, the supernatant from each well was cautiously removed, and DMSO (200 µL) was added to each well. The formazan produced was quantified spectroscopically by measuring the absorbance of the solution at a wavelength of 570 nm utilizing a micro titer plate reader (Bio-Tek SX2, Winooski, VT, USA). For the spectroscopic assay test, a wavelength of 570 nm and a reference wavelength of 690 nm were utilized. Optical density (OD) was determined by subtracting the absorbance reading at the reference wavelength from the absorbance reading at the test wavelength. The resulting data were expressed as a percentage of the control. Percentage growth inhibition and cell viability were calculated using Eqs. 1 and 2.

Cell inhibition (%) = 100−([At − Ab)/(Ac − Ab]) × 100 (1)

Cell viability (%) = ([At − Ab]/[Ac − Ab]) ×100 (2)

where At is the absorbance of the test compound, Ab is the absorbance of the blank, and Ac is the absorbance of the control.

Lactate dehydrogenase enzyme assay

Lactate dehydrogenase (LDH), a soluble cytosolic enzyme, is released upon loss of membrane integrity due to apoptosis. LDH activity can therefore be utilized to determine cell membrane integrity and serves as a means of assessing cell viability by measuring plasma membrane permeability (Haslam et al., 2000). The LDH activity in the cell supernatant was analyzed after centrifugation at 3000 ×g for a duration of 5 min, and the LDH activity was measured utilizing a LDH assay kit (Zist Chem, Tehran, Iran) based on the manufacturer's protocol. In the initial step of the reaction, LDH catalyzes the reduction of NAD+ to NADH and H+ through the oxidation of lactate to pyruvate. In the following step, diaphorase utilizes the newly formed NADH and H+ for the catalytic reduction of a tetrazolium salt (INT) to an intensely colored formazan, which absorbs strongly at 490 nm to 520 nm. The resulting data were expressed as a percentage of the control samples.

Caspase-3 enzyme assay

The caspase-3 assay was performed based on the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of cysteine l aspartate-specific proteinases 3 (caspase-3'in treated MCF7 cells using human cysteinyl aspartate-specific proteinases-3 (caspase-3) ELISA kit (Hangzhou Eastbiopharm Co., Ltd, Hangzhou, China). Briefly, after treatment, cells were spun and cell pellets were lysed with lysis buffer, followed by sample addition (40 μL), and then both caspase-3-antibody (10 μL) and streptavidin-HRP (50 μL) were added. The plate was sealed with the provided membrane, with gentle agitation and incubation at 37°C for 60 min after washing with 30× wash buffer. HRP substrate solution A (50 μL) and HRP substrate solution B (50 μL) were added to all wells. The reaction mixture was incubated for 10 min at 37°C in the dark on a shaker. Stop solution (50 μL) was added to all wells to terminate the reaction and the absorbance was measured at 450 nm using a plate reader (Model 680 microplate reade, California, USA). A standard calibration curve was prepared, and the linear regression equation was generated to calculate sample concentration.

RNA preparation

Total RNA was extracted from MCF7 cells using Cinagen total RNA isolation kit based on the manufacturer's procedure (Cinagen, IRAN). Samples were suspended in dimethyl pyrocarbonate-treated water and quantified by nanodrop spectrophotometry at a wavelength of 260 nm (Eppendorf, Hamburg, Germany). RNA with an OD absorption ratio (OD260 nm/OD280 nm) of between 1.8 and 2.0 was used for reverse transcription (RT) reaction. Genomic DNA was eliminated by treatment of the isolated RNA (1 μg) with 2 units of DNase I (Fermentas Inc, Vilnius, Lithuania).

Reverse transcription − Polymerase chain reaction

RT was performed in a final volume of 20 μL by utilizing an AmpliSence cDNA synthesis kit (AmpliSence Enterovirus-Eph, Russia) based on the manufacturer's recommendations. Polymerase chain reaction (PCR) reactions were conducted in a 25 μL reaction utilizing Taq DNA polymerase (Cinagen Co, Iran) and a thermal cycler (Eppendorf Mastercycler, Hamburg, Germany). The actual primers (Macrogen, Seoul, South Korea) utilized for gene amplification are shown in [Table 1]. Thermal conditions for gene amplification were 35 cycles that consisted of denaturation at 95°C for 1 min, annealing at 58°C–60°C for 1 min, extension at 72°C for 1 min, with an initial denaturing step at 95°C for 10 min, and final extension at 72°C for 10 min. Confirmation regarding the expression of the genes studied in MCF7 cells was obtained by visualizing the PCR product by agarose gel electrophoresis (1%).
Table 1: Selected apoptotic related genes used in real-time polymerase chain reaction

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Real-time-polymerase chain reaction

To determine the levels of Ras and apoptotic gene expression in MCF7 cells treated with PCA, quantitative real-time PCR (qRT-PCR) was conducted using the ABI Step One plus real-time PCR detection system (ABI plus; Applied Biosystems, California, USA), and qPCR™ Green master kit for SYBR Green I® (Applied Biosystems, USA). The relative level of expression of gene transcripts was compared to the housekeeping gene, GAPDH. The actual primers (Macrogen, Seoul, South Korea) utilized for amplification of the genes were designed using Beacon Designer 7.1. (San Francisco), USA. The sequences of the primers utilized for gene amplification are listed in [Table 1]. Real-time PCR reactions were conducted utilizing the settings: preincubation at 95°C for 5 min followed by 40 cycles at 95°C for 15 s and 45 s at 58°C–60°C in 45 cycles. Reactions were performed in triplicate. As negative control, a reaction without cDNA was conducted in parallel. Relative quantification was assessed according to the comparative 2−ΔΔCt. For the analysis of qRT-PCR results that were based on the ΔΔCt method, StepOne™ (California, USA). software was utilized. The result of the gene expression was given by a unit less value based on the formula 2−ΔΔCt. Validation of the assay to check the primers of six genes showed similar amplification efficiencies as was previously described.[14]

Apoptosis study-annexin V/propidium iodide

An annexin V apoptosis detection fluorescein isothiocyanate (FITC) kit (eBioscience, 88-8005-72) was utilized in the experiment. Briefly, after treatment, the cells were harvested and rinsed with 1× PBS, followed by digestion with Accutase (Gibco, A11105-01) at 37°C for 7 min. These were centrifuged at 300 ×g for 4 min and the resulting suspension was discarded. Cells were washed once in 1× PBS, and once in binding buffer. Cells were resuspended in 1× binding buffer (100 μL) at 5 × 106/mL. FITC-conjugated annexin V (5 μL) and propidium iodide (PI) (5 μL) were added to the cell suspension (100 μL) and incubated for 15 min at room temperature. The reaction was terminated by centrifugation and the suspension was removed. Cells were washed in 1× binding buffer, and the cells were resuspended in 200 μL of 1× binding buffer and analyzed by flow cytometry within 4 h.

Statistical analysis

Data analyses were performed using the SPSS 16.0 software package (SPSS Inc., Chicago, IL, USA). Two-way analysis of variance (ANOVA) and general linear model were fit to determine the effect of various concentrations of PCA and incubation times on each variable. One-way ANOVA was used to test differences between various means (post hoc analysis LSD test). All experimental data were presented as the mean ± standard deviation. The level of significance for all tests was set at P < 0.05.


 > Results Top


3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay

The cytotoxic effects of PCA on the growth of MCF7 cells as determined by MTT assays are shown in [Figure 1]. The treated cells with PCA when compared to the untreated control cells displayed a significant decrease in viability. Besides, treatment of MCF7 cells with PCA demonstrated cell growth inhibition in a dose-dependent manner. At greater concentrations, more cytotoxicity was detected. Evaluation of the cytotoxicity data revealed that the IC50 (dose required for 50% inhibition) of PCA on MCF7 cells was 40 mM for 24 h (P < 0.05). The IC50 values powerfully supported the higher cytotoxic activity of PCA on MCF7 [Figure 1]a and [Figure 1]b. The LDH enzyme leakage assay was also performed to test the given response after treatment with various concentrations of PCA. Similar to the MTT assay, the LDH assay results displayed a dose-dependent decrease in the viability of MCF7 cells at 24 h (P < 0.05) [Figure 2]. The highest leakage of LDH was displayed in 37–70 mM PCA treatment.
Figure 1: The percentage of growth inhibition and viability of MCF7 cells treated with p-Coumaric acid using the 3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (a) The mean percentage of cell viability and (b) inhibitory rate of p-Coumaric acid at various concentrations compared with control group after 24 h treatment. Letters denote significant differences among values at P < 0.05

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Figure 2: The enzyme activity of lactate dehydrogenase in MCF7 cells exposed to p-Coumaric acid. Cells were exposed to 10 and 300 mM p-Coumaric acid for 24 h at 37°C. Results are represented as mean ± standard deviation for three independent experiments, each with a minimum of three cultures. Letters denote significant differences among values at P < 0.05

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Of note is that both bioactivity assays revealed PCA toxicity against the MCF7 cell line, regardless of their differences in mechanism. In the MTT assay, only the live cells decreased or reduced the MTT salt to purple formazan by the mitochondrial succinate dehydrogenase enzyme, while, in the LDH assay, the LDH enzyme results from distortion of cell membrane integrity in the culture medium.

Caspase-3 assay

[Figure 3] shows the level of caspase-3 protein in the MCF7 cell line treated with PCA based on ng per cell. Caspase-3 rates were significantly increased in MCF7 cells treated with PCA at doses between 10 and 300 mM in comparison to untreated cells. Maximal caspase-3 activity was detected in cells treated with 150 mM PCA [Figure 3] (P < 0.05).
Figure 3: Inhibitory effect of p-Coumaric acid-induced caspase-3 in MCF7 cells were treated with 10 and 300 mM p-Coumaric acid for 24 h. Results are represented as mean ± standard deviation for three independent experiments, each with a minimum of three cultures. Letters denote significant differences among values at P < 0.05

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Gene expression study

The degree of gene expression in MCF7 cells after they were treated with PCA was compared with the control samples (untreated MCF7 cells) and the results were expressed as fold change. The fold changes of H-Ras in MCF7 cells treated with PCA at doses between 75 and 150 mM in relation to untreated cells decreased from 1.0032 to 0.268 and 0.460 at 24 h, respectively (P < 0.05) [Figure 4]a. The level of K-Ras expression was lesser in a dose-dependent manner in MCF7 cells, 24 h after exposure to PCA. Minimum K-Ras expression was found in cells that were treated with 150 mM PCA. K-Ras expression showed significant difference at 300 mM PCA as compared to the control treatment [Figure 4]b (P < 0.05).
Figure 4: Relative expression of (a) H RAS, (b) K RAS, (c) Bax, (d) Bcl2, (e) Casp and (f) Bax/Bcl2 genes in MCF 7 cells treated with 10 and 300 mM p Coumaric acid for 24 h. Results are represented as mean ± standard deviation for three independent experiments, each with a minimum of three cultures. Expression values were normalized to those of GAPDH. Data were expressed as the mean fold difference (mean ± standard deviation). Letters denote signifcant differences among values at P < 0.05

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An increase in fold change of proapoptotic gene, Bax, from 1.015 at untreated cells to 2.367, 5.598, 8.565, 3.471, and 4.441 at 24 h, was observable in the treated MCF7 cells at doses of 10–300 mM, respectively, with the greatest Bax expression being found in cells that were treated with 75 mM PCA [Figure 4]c (P < 0.05). The fold change of antiapoptotic gene, Bcl-2, was however significantly reduced from 1.006 for untreated cells to 0.746 and 0.515 at doses of 75–150 mM, respectively. The most common reduction of Bcl-2 expression was shown in cells treated with 150 mM PCA [Figure 4]d (P < 0.05). The Bax/Bcl-2 ratios of the mRNA levels are recorded in [Figure 4]e. The ratios were increased toward the doses of 37–150 mM in comparison to untreated cells. As [Figure 4]e shows, the highest ratio was observed for 150 mM PCA [Figure 4e] (P < 0.05). The fold changes of caspase-3 in treated MCF7 cells also increased significantly at doses of 75–300 mM. The most common expression of caspase-3 was detected in cells treated with 150 mM PCA [Figure 4]f (P < 0.05).

Annexin V/propidium iodide study

A significant difference (P < 0.05) was observed in cell viability, early apoptosis, and late apoptosis of MCF7 cells after treating with PCA for 24 h [Figure 5]. The incubation of MCF7 with 10 mM PCA (24 h) decreased cell viability to 60.45% ± 1.31%, 30.92% ± 1.8%, and 10.98% ± 2.05% for early apoptosis, late apoptosis, and necrosis cells, respectively. Increase in PCA concentration to 35 mM, resulted in a further decrease in cell viability to 60.19% ± 0.13%, 25.85% ± 1.51, and 15.13% ± 0.21% of early apoptosis, late apoptosis, and necrosis in MCF7 cells, respectively. A similar decrease in cell viability and increase in apoptotic cells were found in MCF7 cells after treating with 35 mM PCA with an incubation period of 24 h. Cells treated with 35 mM PCA showed 60.52% ± 0.25%, 25.82% ± 1.19%, and 15.58% ± 0.52% of early apoptosis, late apoptosis, and necrosis, respectively. Incubation of MCF7 with PCA (75 mM) after 24 h however resulted in decreased cell viability to 5.51% ± 0.54% with 85.55% ± 1.17% and 10.03 ± 0.14% of early apoptosis, late apoptosis, and necrosis, respectively [Figure 5]. Higher doses of PCA (150 mM) showed 25.155 ± 0.12%, 65.85% ± 0.85%, and 10.15% ± 0.10% of early apoptosis, late apoptosis, and necrosis, respectively, in MCF7 cells after 24-h incubation. At the highest dose (300 mM), 70% ± 1.22%, 20% ± 1.32%, and 105 ± 1.15% of early apoptosis, late apoptosis, and necrosis, respectively, in MCF7 cells were observed after 24-h incubation.
Figure 5: Annexin V fluorescein isothiocyanate/propidium iodide staining used to assess the mode of cell death. p-Coumari V/FLUOS (−)/propidium iodide (+) (UL) may be bare nuclei cells in late necrosis, or cellular debris (upper left)c acid-treated MCF7 cells (a) Control (b) 10 mM, (c) 37 mM, (d) 150 mM, (e) 150 mM, and (f) 300 mM for 24 h showed an increased percentage of apoptotic population after 24 h incubation. The population of early, late apoptotic, and necrosis cells in the control group was found to be lower compared with experimental groups. Living cells or Annexin-V/FLUOS (−)/propidium iodide (−) [LL] are seen in the lower left quadrant. Cells that are Annexin V/FLUOS (+)/propidium iodide (−) [LR] are apoptotic (lower right). The cell population with Annexin V/FLUOS (+)/propidium iodide (+) [UR] has been described as necrotic or advanced apoptotic (upper right) and Annexin

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Only the percentages of early apoptosis, late apoptosis, and necrosis in MCF7 cells between the two different concentrations (35–70 mM) were significantly different, indicating that by increasing PCA concentration, viable cells were moderated, and an increased number of cells experienced late apoptosis at 24 h of incubation. As shown in [Figure 6], MCF-7 cells were exposed to various concentrations of PCA (10–300 mM) for 24 h after preparation with the annexin kit and staining with PI, with Annexin V being studied utilizing a florescent microscope. With the increase in the concentration of PCA in a dose-dependent manner, the viability of cells can be reduced. The greatest impact of PCA was observed at the concentrations of 150–300 mM that results in significant induced indications of apoptosis. PI staining showed increased accumulation of the cells in the last stages of apoptosis or annexin V+ and PI+ [Figure 6].
Figure 6: The results of fluorescent microscopic study MCF7 cells treated with (a) control (b) 10 mM, (c) 37 mM, (d) 150 mM, (e) 150 mM, and (f) 300 mM for 24 h after staining with propidium iodide and Annexin V. Living cells or Annexin-V/FLUOS (−)/propidium iodide (−) (LL) are without color. Cells that are necrosis are red, Annexin V/FLUOS (−)/propidium iodide (+) (LR). Early apoptosis cells are green, Annexin V/FLUOS (+)/propidium iodide (−) (UR) and late apoptotic cell are green and red Annexin V/FLUOS (−)/propidium iodide (−) (UL)

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 > Discussion Top


The current study evaluated the effectiveness of PCA on the apoptosis process in MCF7 cells and changes in gene expression involved in apoptosis. The effects of antioxidants from herbal phenylpropanoids on cancer cell are of significant interest to researchers. The role of these compounds in apoptosis and their impact on oncogenes was evaluated in the present study. It is not yet known whether phenolic compounds of herbal origin can have an effect on oncogenes as Ras genes. To the best of our knowledge, this is the first report on the ability of PCA to inhibit the gene expression of Ras genes. This research has revealed that phenolic compounds in conjugation with their role as antioxidants in cancer cells could also change the expression of genes that are an integral part of the molecular pathway of cancerous cells. Studying the expression of Bax and Bcl2 genes as the index genes in cancer cells and assessment of caspase-3 activity as a biochemical indicator of apoptosis were deemed necessary to determine the mode of action and mechanism of PCA in our study. In addition, by studying caspase activity, one can assess the apoptosis path (internal and external). The viability assay revealed that the IC50 of PCA in MCF-7 cells was approximately 40 mM. Real-time PCR analysis showed that PCA decreased the levels of H-Ras and K-RAS transcript in MCF-7 breast cancer cells. Maximal inhibitory effect of PCA was observed on K-Ras expression at a concentration of 150 mM. PCA at a dose of 75–150 mM significantly induced apoptosis after 24 h treatment, resulting in increased Bax and caspase-3 mRNA levels and late apoptotic cells with annexin V + and PI + characteristic features based on the incidence of protein bands associated with annexin. It also inhibited the relative expression of antiapoptotic gene, Bcl2 in treated cells. An evaluation of the ratio of gene expression of Bax/bcl2 as an indicator of the apoptosis status revealed that PCA at a dose of 37–150 mM leads to an increase in this ratio that represents the progress of apoptosis in treated cancer cells. Overall, a survey of the data showed that PCA is one of the most abundant herbal ingredients used in human nutrition. PCA reduced the gene expression of H-Ras and K-Ras which is one of the most critical growth factors in cancer cells and mutations in the occurrence of many tumors. Our findings are similar to Jaganathan et al. who investigated the influence of PCA on human colorectal carcinoma cells. HCT 15 cells treated with PCA gave rise to an accumulation at the G1 phase of the cell cycle. In this study, it was observed that there was growth inhibition by PCA on treated HCT 15 cells, which is due to ROS generation and a plunge in mitochondrial membrane potential. These data were supported by PI and YO-PRO-1 staining and photomicrograph and scanning electron microscope observations.[15] PCA also has an antiproliferative effect on Caco-2 and MCF7 cells.[16],[17] Mitochondrial malfunction is one of the earlier events of apoptosis and is induced in PCA-treated cancer cells mediated by ROS generation.[18] Some other phenolic phytochemicals such as EGCG and resveratrol could improve p53 activation by the Ras/MAPK kinase/MAPK pathway. Data from this research show that PCA resulted in the upregulation of Bax and downregulation of Bcl2 in treated cells and that both of them are affected by p53 activation in the apoptosis process. [19, 20] Nakayama et al. showed that phenolic components such as cinnamic acid, have inhibitory effects on DNA fragmentation initiated by hydrogen peroxide stress in V79 cells.[21] Enhanced expression of Bax, caspase-8, raspase-9, and Fas genes and reduction in Bcl-2 and Bcl-xL gene expression in myocardial cells treated with PCA have been reported.[22] Caspase 9 is an initiator caspase that induces the stimulation of effector caspases, which includes caspase-3 and caspase-7.[23],[24] The activation of caspase-9 supports the data that were obtained from HT-144 cells that were treated with cinnamic acid. Micronuclei resulted from chromosomal breakage and/or whole chromosomal loss in NGM and HT-144 cells treated with phenolic components.[22] Researchers following the discovery of anticancer drugs postulate that inhibition of Ras proteins may be due to compounds having the ability to remove K-Ras from the plasma membrane as Ras proteins are thought to be fixed to the plasma membrane.[25]

Use of herbal phenolic compounds on cancer cells showed that some of them including quercetin, can induce specific functions of K-Ras-p53 such as suppressing K-Ras-induced cancer.[26] Huang in 2013 proved that quercetin prevents mTOR signaling by stopping PI3K and Ras activity, activating AMPK, and upregulating TSC1.[27] Currently, scientists have studied some molecular pathways, as well as the PI3K/AKT/mTOR pathway for the treatment of cancer and anticancer drug design. The PI3K/AKT/mTOR pathways play a critical role in the malignancy of human tumors and their resulting growth, proliferation, and metastasis.[28],[29] Bharate et al. in 2012 reviewed 157 natural products in order to study their capability to regulate K-Ras posttranslational targets. They showed that the polycarboxylic class of natural products has more potential as anticancer agents through change of K-Ras signaling.[30],[31],[32] Researchers recommend that compounds possessing polyphenol hydroxyl, a new source in the mevalonate pathway, can inhibit the function of Ras proteins in human solid tumor cell lines as a novel source of FPTase inhibitors. Chen et al. proposed that the anticancer effect of the phenolic compound, Salvia miltiorrhiza derivative, may be connected to the prevention of P21 Ras membrane association and augmentation of gap junction intercellular communication.[33],[34]


 > Conclusion Top


Our results suggest that although PCA may be applicable for in vitro toxicity, research in human breast cancer MCF-7 cells through engaging in the apoptosis pathway could also change the outcome of gene and protein expression. PCA inhibited the growth of MCF7 cells, with the optimum concentration of PCA being 75–150 mM. This finding suggests that PCA can affect the activity of some critical enzymes utilized in cancer therapy. Our results propose that PCA may be a new source which may prove valuable in the search for potential inhibitors of Ras oncogene function as anti-Ras drugs. However, due to limited bioavailability and pharmacokinetics, innovative formulations or chemical alterations of PCA are required for clinical cancer treatment.

Acknowledgements

This work was funded by a grant from the Ahvaz Jundishapour University of Medical Sciences (Project No: CMRC-115), Shahid Chamran University of Ahvaz Research Council (Grant NO: 94/3/02/31580). We are grateful to Dr. Andrea Goldson for editing the manuscript. The authors declare that there is no conflict of interest. Last and not least an emotional, massive thank you to my late uncle Dr. Reza Oboodi for his never ending support and guidance to the author (Dr. Maryam Kolahi). I hope he is somehow watching this from somewhere with a satisfactory smile.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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