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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 60  |  Page : 12-17  

Cytotoxicity and mitochondrial-mediated apoptosis induced by Fenugreek seed oil in human hepatocellular carcinoma cells via reactive oxygen species generation


1 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
2 Department of Zoology, College of Science, King Saud University; Al-Jeraisy Chair for DNA Research, Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia
3 Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia

Date of Submission26-Apr-2018
Date of Decision08-Jun-2018
Date of Web Publication23-Jan-2019

Correspondence Address:
Nida N Farshori
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh-11451
Saudi Arabia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_163_18

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   Abstract 


Background: Cancer has been recognized as a major cause of death globally. Traditional medicines have been used to treat cancer for several periods, and herbal medicines are currently being used for the treatment of cancer worldwide. Fenugreek is an annual plant (family: Fabaceae) and is documented as a medicinal herb since olden days. Objective: The present study was aimed to investigate the anticancer activity of Fenugreek seed oil (FSO) against human hepatocellular carcinoma (HepG2) cell line. Materials and Methods: The HepG2 cells were exposed to 10–1000 μg/ml of FSO for 24 h to assess the cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test, neutral red uptake assay, and morphological changes. The cytotoxic concentrations of FSO were used to study the induction in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels. Further, the expression of pro-and antiapoptotic genes (p53, caspase-3, caspase-9, Bax and Bcl-2) were studied. Results: A concentration-dependent decrease in cell viability of HepG2 cells was observed after FSO exposure. Our results showed that FSO decreased cell viability of HepG2 cells in a concentration-dependent manner. FSO was also found to increase the ROS generation and decrease the MMP level in HepG2 cells. Our gene expression study indicates upregulation in apoptotic marker genes and downregulation in antiapoptotic gene. Conclusion: The results showed that FSO is capable to induce mitochondrial-mediated apoptosis through ROS generation in HepG2 cells and could be a useful resource in developing effective remedies for the treatment of hepatocellular carcinoma.
Abbreviations used: FSO: Fenugreek seed oil; HepG2: Human hepatocellular carcinoma; ROS: Reactive oxygen species; NRU: Neutral red uptake; DMSO: Dimethyl sulfoxide; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MMP: Mitochondrial membrane potential; DNA: Deoxyribonucleic acid; RNA: Ribonucleic acid; DCFH-DA: 2,7-dichlorofluorescein diacetate; FBS: Fetal bovine serum; PBS: Phosphate-buffered saline; PCRq: quantitative real-time PCR; NaHCO3: Sodium bicarbonate; CO2: Carbon dioxide; DMEM: Dulbecco's modified eagle medium.

Keywords: Apoptosis, cytotoxicity, Fenugreek seed oil, gene expression, mitochondrial membrane potential level, reactive oxygen species generation


How to cite this article:
Al-Sheddi ES, Farshori NN, Al-Oqail MM, Al-Massarani SM, Siddiqui MA, Ahmad J, Al-Khedhairy AA. Cytotoxicity and mitochondrial-mediated apoptosis induced by Fenugreek seed oil in human hepatocellular carcinoma cells via reactive oxygen species generation. Phcog Mag 2019;15:12-7

How to cite this URL:
Al-Sheddi ES, Farshori NN, Al-Oqail MM, Al-Massarani SM, Siddiqui MA, Ahmad J, Al-Khedhairy AA. Cytotoxicity and mitochondrial-mediated apoptosis induced by Fenugreek seed oil in human hepatocellular carcinoma cells via reactive oxygen species generation. Phcog Mag [serial online] 2019 [cited 2019 Feb 21];15:12-7. Available from: http://www.phcog.com/text.asp?2019/15/60/12/250595





SUMMARY

  • Fenugreek seed oil (FSO) decreased the cell viability of human hepatocellular carcinoma (HepG2) cells in a concentration-dependent manner. FSO was also found to increase the reactive oxygen species (ROS) generation and decrease the mitochondrial membrane potential level in HepG2 cells. The gene expression studies indicated upregulation in the apoptotic marker genes (p53, Bax, caspase-9, and caspase-3) and downregulation in Bcl-2 gene. The results from the present study showed that FSO is capable to induce mitochondrial-mediated apoptosis through ROS generation in HepG2.



   Introduction Top


Cancer has been recognized as a cause of death worldwide, and number of cases of occurrence of cancer is increasing regularly.[1] More than 80% of the hepatocellular carcinoma cases arise in the Asian Pacific regions and Saharan Africa.[2] and more than 50% cases account in China alone.[3] Hepatocellular carcinoma is utmost cause of death in men globally due to the poor diagnosis.[4] There are various types of the formulations available to treat the different kind of cancer diseases, however, none of them is found to be effective and safe.[5],[6] The drawback with chemotherapy as a cancer treatment is the toxicity of the established drugs. The traditional therapeutic drugs used are frequently linked with harmful effects.[7] However, herbal or plant-derived natural products are known to be benign and effective for the management of cancer diseases.[8] Nowadays, maximum research are targeted toward the use of plants or natural products derived anticancer agents from plants.[9] Therefore, searching for new natural products and their analogs for the management of hepatocellular carcinoma is the need of the day. Many studies demonstrated that natural products could be useful for the anticancer drug development.[10],[11] Fenugreek, an annual plant of Fabaceae family, is being cultivated worldwide as a semiarid crop and its leaves and seeds are common ingredients in dishes from South Asia. Fenugreek is being used as a culinary and medicinal herb since ancient.[12] The Fenugreek has a strong flavor and aroma. It is used as a spice in the preparation of a variety of dishes, and pickles.[13] The Fenugreek seeds are found to possess restorative and nutritive properties and are helpful in stimulating digestive processes.[14] The seeds of Fenugreek are used for the treatment of diabetes and hyper cholesterol anemia.[15] Many studies have shown the efficacy of Fenugreek seeds that can benefit health and treat diseases. Fenugreek is very well documented for anti-oxidative, anti-inflammatory, antilipidemic, anticancer, antiulcerogenic, antipyretic, antibacterial, and antifungal activities.[16] The experimental evidences prove the efficacy of Fenugreek in diabetic patients.[17] Numerous studies also demonstrated the hepatoprotective potential of Fenugreek seeds in various experiments.[18],[19] In previous research, Fenugreek seeds have been found to be protective against breast[20] and colon[21] cancers. Many active ingredients isolated from Fenugreek seeds and their beneficial effects have previously been reported.[22] These studies clearly indicate that Fenugreek seeds could be beneficial against cancer cells. However, details on molecular mechanism(s) involved in hepatic cancer cell death are not fully studied. Therefore, this in vitro study, we aimed to explore the possible anticancer efficacy of Fenugreek seeds against human hepatocarcinoma (HepG2) cell line. The HepG2 cells are known to be a good model system for studying the protective effect against liver diseases.[23]


   Materials and Methods Top


Cell culture

HepG2 cells were grown in Dulbecco's modified Eagle medium (DMEM) high glucose medium with 10% fetal bovine serum (FBS), 0.2% NaHCO3, and antibiotic solution in a carbon dioxide (CO2) incubator at atmosphere of 5% CO2 and 95% humidity.

Reagents

DMEM culture medium, solvents, dyes, and chemicals were purchased from Sigma and FBS, antibiotic–atimycotic and trypsin from Invitrogen.

Extraction of plant material

Fenugreek seeds used in the present investigation were purchased from local market of Riyadh, KSA and were identified by plant taxonomist Dr. Jakob Thomas, College of Science, King Saud University. The seeds were screened, grounded, and stored for extraction. The oil was extracted following the protocol of Horwitz.[24]

Preparation of stock solution

Due to the nonsolubility of Fenugreek seed oil (FSO) in aqueous medium, the stock solution was dissolved in dimethyl sulfoxide (DMSO). The maximum DMSO concentration was 0.1% in medium.

Experimental design

The cytotoxic potential was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, neutral red uptake assay (NRU), and morphological alterations in HepG2 cells after exposing with 10, 25, 50, 100, 250, 500, and 1000 μg/ml of FSO for 24 h. Then, cytotoxic concentrations, i.e. 50, 100, and 250 μg/ml of FSO were used for analysis of induction in ROS and mitochondrial membrane potential (MMP) levels. Further, HepG2 cells were exposed to 50 and 100 μg/ml for the ribonucleic acid (mRNA) expression of apoptotic marker genes.

Cytotoxicity assessment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay

Following the protocol of Mosmann,[25] MTT test was performed to determine the percent cell viability. Briefly, the HepG2 cells (1 × 104) were seeded and left overnight for adherence. After the respective treatment, 10 μl/well (5 mg/ml of MTT) was added and subjected to incubation for 4 h. Then, supernatant was discarded and formazone crystals were solubilized in 200 μl of DMSO. The absorbance of plate was then measured at 550 nm.

Cytotoxicity assessment by neutral red uptake assay

NRU assay was done using method described previously.[26] Briefly, HepG2 cells (1 × 104) were plated and left overnight for adherence. After treatment, cells were rinsed with phosphate buffer saline and incubated in 50 μg/ml neutral red dye for 3 h. Then, the cells were washed and dye was extracted in acetic acid solution. The developed color was measured at 550 nm.

Morphological assessment

Morphological changes in HepG2 cells were observed under the microscope. HepG2 cells were treated with 10, 25, 50, 100, 250, 500, and 1000 μg/ml concentrations of FSO for 24 h.

Reactive oxygen species generation

Following the protocol of Wang and Joseph,[27] the ROS generation was measured using 2,7-dichlorofluorescein diacetate (DCFH-DA) dye with desired modifications. In brief, cells were treated for 24 h at 50–250 μg/ml FSO. After the treatment, cells were rinsed and exposed with DCFH-DA (20 μM) for 1 h in dark. The fluorescence was measured using spectrofluorometer. The cells were also examined to see the intracellular fluorescence intensity under the fluorescence microscope.

Mitochondrial membrane potential

The MMP level in HepG2 cells exposed to FSO was measured according to protocol of Zhang et al.[28] Briefly, after exposure to 50–250 μg/ml of FSO for 24 h, cells were exposed to Rh123 dye (10 μg/ml) in dark for 1 h. The fluorescence was measured using spectrofluorometer and images were taken at 20× magnification.

Quantitative real-time polymerase chain reaction study of apoptotic marker genes

The RNA was isolated using the RNeasy kit from the HepG2 cells treated with 50 and 100 μg/ml FSO for 24 h. After cDNA synthesis using M-MLV (Promega) and oligo (dT) primers (Promega), the expression level of pro- and anti-apoptotic marker genes were quantified by real-time polymerase chain reaction using Light Cycler® 480 instrument (Roche Diagnostics, Rotcreuz, Switzerland) following the protocol.[29] The primer sequences used are detailed in our previous publication.[11]

Statistical analysis

Post hoc Dunnett's test (ANOVA) was used for statistical analysis.


   Results Top


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay

[Figure 1] represents the results of percent cell viability by MTT assay in HepG2 cells. The results showed that FSO exhibited a concentration-dependent decrease in the viability of HepG2 cells after 24 h exposure. The percent viability was recorded as 79%, 56%, 18%, 17% and 14% at 50, 100, 250, 500, and 1000 μg/ml concentrations of FSO, respectively. The IC50 values of FSO obtained by MTT assay was 113.6 μg/ml [Figure 1].
Figure 1: Cytotoxicity assessment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay in human hepatocellular carcinoma cell line following the exposure of various concentrations (10–1000 μg/ml) of Fenugreek seed oil for 24 h. Values are mean ± standard error of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.05 versus control group

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Neutral red uptake assay

The percent cell viability obtained by NRU assays are presented in [Figure 2]. As shown in figure, a concentration-dependent decreased in the percent cell viability was recorded in HepG2 cells exposed to FSO for 24 h. The percent cell viability in HepG2 cells was observed as 69%, 45%, 20%, 18%, and 15% at 50, 100, 250, 500, and 1000 μg/ml concentrations, respectively. The IC50 values of FSO obtained by NRU assay was 90.9 μg/ml [Figure 2].
Figure 2: Cytotoxicity assessment by neutral red uptake assay in human hepatocellular carcinoma cell line following the exposure of various concentrations (10–1000 μg/ml) of Fenugreek seed oil for 24 h. Values are mean ± standard error of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.05 versus control group

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Morphological changes in human hepatocellular carcinoma cells

Morphological alterations of treated HepG2 are presented in [Figure 3]. Morphological changes in HepG2 cells were recorded under the microscope. The results showed that cells exposed to 50 μg/ml FSO and beyond become rounded, shrinked, less adherent than control, and lost their original morphology and shape. However, HepG2 cells exposed to 10 and 25 μg/ml of FSO did not show any change in the original morphology.
Figure 3: Alterations in the morphological of human hepatocellular carcinoma cells after the exposure of various concentrations (10–1000 μg/ml) of seed oil of Fenugreek for 24 h. Images were taken using an inverted phase contrast microscope (OLYMPUS CKX 41) at 20× magnification

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Fenugreek seed oil induced reactive oxygen species generation

The results of FSO-induced ROS generation are shown in [Figure 4]. The induction in ROS generation in HepG2 cells exposed for 24 h was observed at 50, 100 and 250 μg/ml FSO concentrations. The increase in ROS was examined as 134% ± 11%, 201% ± 12.40%, and 238% ± 17.8% at 50, 100 and 250 μg/ml concentrations of FSO, respectively [Figure 4].
Figure 4: (a) Seed oil of Fenugreek induced reactive oxygen species generation in human hepatocellular carcinoma cells. Reactive oxygen species generation was studied using dichlorofluorescein diacetate dye after the exposure of Fenugreek for 24 h. (b) Percentage increase in reactive oxygen species generation in human hepatocellular carcinoma cells following the exposure of various concentrations of Fenugreek seed oil for 24 h. *P < 0.01, **P < 0.001 versus control

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Fenugreek seed oil induced mitochondrial membrane potential

FSO induced changes in the level of MMP are summarized in [Figure 5]. For the measurement of MMP, cells were exposed for 24 h at 50–250 μg/ml of FSO. The results showed a dose-dependent decrease in the level of MMP after FSO exposure. The decrease observed in the level of MMP was as 82% ± 5.4%, 58% ± 4.2% and 41% ± 3.1% at 50, 100 and 250 μg/ml FSO concentrations, respectively [Figure 5].
Figure 5: (a) Seed oil of Fenugreek induced reduction in the intensity of mitochondrial membrane potential level in human hepatocellular carcinoma cells exposed for 24 h. mitochondrial membrane potential was studied using Rh123 fluorescent dye. (b) Percentage reduction in mitochondrial membrane potential level in human hepatocellular carcinoma cells following the exposure of various concentrations of Fenugreek seed oil for 24 h. *P < 0.01, **P < 0.001 versus control

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Fenugreek seed oil-induced changes in the expression of apoptosis marker genes

Result highlights of FSO induced mRNA expression of apoptosis marker genes are presented in [Figure 6]. A significant (P < 0.05) upregulation of 1.96 and 2.09 fold in the expression of pro-apoptotic gene p53, 2.1 and 2.5 fold in caspase-3, 1.8 and 1.87 in caspase-9, and 1.85 and 1.93 in Bax was found at 50 and 100 μg/ml concentration of FSO, respectively. The downregulation of 0.7 and 0.65 fold in anti-apoptotic gene Bcl-2 was found at 50 and 100 μg/ml of FSO concentrations, respectively [Figure 6].
Figure 6: Fold change in the level of mRNA expression of various apoptosis genes by real time-polymerase chain reaction in human hepatocellular carcinoma cells exposed to Fenugreek seed oil for 24 h. The data provided are mean ± standard error from three separate experiments. *P < 0.05 versus control

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   Discussion Top


Traditional therapeutic drugs used are frequently linked with harmful effects and triggered damage to healthy cells. Although many researches have been carried out for progressions in the management and regulation of cancer, significant effort and possibility for its enhancement remains. Natural therapies, such as plants or plant-derived natural products, have been used for health benefits and are beneficial to combat cancer.[6] A number of studies have shown that the efficacy of Fenugreek seeds that can benefit human health.[16] There have been numerous research studies conducted on qualitative and quantitative analysis of Fenugreek seeds and oils.[30],[31],[32] Therefore, in this study, we targeted to study the anticancer effect of FSO against HepG2 cells. The cytotoxicity of the seed oil was determined by MTT and NRU assays. These colorimetric assays are frequently used parameters for cytotoxicity measurements. Since, MTT assay and NRU assess different cellular functions;[25],[26] therefore, we implied both assays to observe the possible cytotoxicity of FSO. From both assays, MTT and NRU showed a concentration-dependent decrease in the percent cell viability of HepG2 cells exposed to FSO for 24 h. Our cytotoxicity data are in agreement with the published reports that showed cytotoxic potential of Fenugreek seed extract against cancer cell lines, T-cell lymphoma in vitro.[33] In another study, alcoholic extracts of Fenugreek exhibited cytotoxic response in vitro against various human cancer cell lines such as, neuroblastoma (IMR-32) and colon adenocarcinoma cell line (HT29).[34] The anticancer potential of seed oil of Fenugreek against human breast adenocarcinoma cells and human epidermoid cancer cells have also shown that Fenugreek seeds oil significantly decreased cell viability and altered the morphology in a concentration-dependent manner.[35] We also evaluated that the effect of cytotoxic concentrations of FSO on ROS generation, MMP level and the expression of apoptosis genes in human liver cancer (HepG2) cells. Studies showed that the exposure of plants or plant-derived products promotes oxidative stress.[36] In this study, we have shown a concentration-dependent induction in ROS. It is well known that an induction in ROS is one of the common causes of apoptotic cell death.[37],[38] Our results are coherent with the earlier reports where an induction in ROS has been shown after the treatment with natural products.[39] It is also observed that cell death can occur by mitochondrial damage with increase in ROS generation.[40] In this study, a dose-dependent decrease in the MMP level in HepG2 cells showed that the exposure of FSO could damage the mitochondrial membrane. This decrease in MMP level clearly indicates the role of ROS generation in cell death of HepG2 cells.

The mitochondrial-mediated pathways associated with anticancer drugs,[41] utilize caspase-9, cytochrome c and protease activating factor 1.[42] Bcl-2 proteins; (pro-apoptotic- Bax, Bad and Bak) and anti-apoptotic proteins (Bcl-2, BclxL, and Bcl-w) are known to regulate the mitochondrial apoptotic pathways.[43] The p53 gene plays an important role in apoptosis and is the most frequent genetic change in human cancer.[44] Reports suggest p53 induces apoptosis through transcription-dependent and independent mechanisms.[45] Many of the pro-apoptotic Bcl-2 family genes, for example, Bax, are known targets of p53.[46] The mitochondrial disruption through Bax activation and Bcl-2 inhibition and the release of cytochrome-c into the cytosol, activate caspase 9 that triggers caspase-3 activation.[47] In this investigation, we have evaluated the expression of several mitochondria-related apoptotic genes at transcriptional level. Our results showed upregulation in the expression of pro-apoptotic genes (p53, Bax, caspase-3 and caspase-9) and downregulation in anti-apoptosis gene, Bcl-2. These results demonstrated that FSO-induced apoptosis in HepG2 cells involving mitochondria-mediated pathway. Therefore, results from this study suggested that the role of mitochondrial pathway in FSO induced apoptosis in HepG2 cells. Other natural products have also observed these kinds of cancer cell growth inhibitory and apoptotic activities in HepG2 cells through mitochondrial-mediated pathways.[11]


   Conclusion Top


Our results demonstrated that FSO exerts apoptosis in HepG2 cells. The FSO was found to exhibit cytotoxicity in a concentration-dependent manner in HepG2 cells and induced apoptosis via mitochondrial pathway in hepatocellular carcinoma cells. The apoptosis induction was found to be associated with ROS generation and MMP. The gene expression study indicates upregulation in apoptotic marker genes (p53, caspase-3, caspase-9, and Bax) and downregulation in Bcl-2 gene. These results suggested that FSO is capable of inducing mitochondrial-mediated apoptosis in HepG2 cells through ROS generation cells and could be a useful resource to develop an effective remedy for hepatocellular carcinoma.

Acknowledgements

The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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