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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 6  |  Page : 1294-1301

Citrullus colocynthis regulates de novo lipid biosynthesis in human breast cancer cells


1 School of Biological Sciences, University of the Punjab, Lahore, Pakistan
2 School of Biological Sciences, University of the Punjab; Department of Biotechnology, Virtual University of Pakistan, Lahore, Pakistan
3 Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan

Date of Submission21-Feb-2020
Date of Decision15-Jul-2020
Date of Acceptance12-Aug-2020
Date of Web Publication18-Dec-2020

Correspondence Address:
Asima Tayyeb
School of Biological Sciences, University of the Punjab, Lahore
Pakistan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_206_20

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 > Abstract 


Background: Reprogrammed energy metabolism is considered a hallmark of cancer and is proposed as an important target for therapy. Uncontrolled and infinite cell proliferation needs efficient energy sources. To meet the demands of cancer cells lipid metabolism is activated. Citrullus colocynthis is a traditional medicinal plant known for its anticancer and hypolipidemic effects.
Aims: Aim of the current study was to assess the effect of C. colocynthis leaves on regulation of lipid metabolism in MCF-7, a human breast cancer cell line.
Methods: Methanolic extract of leaves and its fractions in increasing polarity-based solvents (n-hexane, chloroform, ethyl acetate and n-butanol) were prepared and analyzed for the presence of secondary metabolites in each fraction. Bioassays and apoptosis genes expression analysis was conducted to evaluate the anticancer and cytotoxic effect of breast cancer cells treated with extract and its fractions, separately. Lipid quantification and gene expression regulation of genes involve in lipid metabolism was performed to evaluate regulation of lipid metabolism.
Results: Results showed a significant anticancer activity of methanolic extract of C. colocynthis and two of its fractions prepared with chloroform and ethyl acetate. Quantification of lipids depicted significant increase in cholesterol and increase in triglycerides of treated cells compared to control untreated cells. Expression regulation of genes further confirmed the lipid regulation through significant down regulation of genes involve in lipid metabolism (FASN, HMGCLL1, ACSL5 and ELOVL2).
Conclusion: The present study concludes that C. colocynthis holds strong anticancer potential through regulation of lipid metabolism and with further studies can be proposed for novel therapeutic approaches.

Keywords: Anticancer, breast cancer, Citrullus colocynthis, lipid metabolism, lipid regulation, traditional therapy


How to cite this article:
Perveen S, Ashfaq H, Shahjahan M, Manzoor A, Tayyeb A. Citrullus colocynthis regulates de novo lipid biosynthesis in human breast cancer cells. J Can Res Ther 2020;16:1294-301

How to cite this URL:
Perveen S, Ashfaq H, Shahjahan M, Manzoor A, Tayyeb A. Citrullus colocynthis regulates de novo lipid biosynthesis in human breast cancer cells. J Can Res Ther [serial online] 2020 [cited 2021 Dec 4];16:1294-301. Available from: https://www.cancerjournal.net/text.asp?2020/16/6/1294/303892




 > Introduction Top


In cancer cells increased synthesis of metabolic intermediates takes place as a result of metabolic reprograming for production of building blocks and signalling molecules. This altered metabolic phenomena is a prominent hallmark of cancer.[1] To meet the demands of high growth and survival rate in highly proliferating cancer cells, very active metabolic processes are required. Tumor microenvironment and oncogenic events derive altered metabolic processes in cancer cells. Therefore, a prolong metabolic repertoire is needed to withstand the tumor environment and meet cancer cell demands.[2]

The most striking alteration in cancer cells is increased glucose uptake, which is utilized by phenomenon of aerobic glycolysis called “Warburg effect.”[3] Metabolism of fatty acid is strongly coupled with the glucose and glutamine metabolism as both of them are precursors for synthesis of fatty acids.[4] Deregulated metabolism of lipids is common feature of cancer cells along with glucose metabolism.[5],[6],[7] Most of the lipid requirement of adult tissues is performed by the uptake of lipoproteins and free fatty acids from the blood stream. The de novo fatty acid and cholesterol synthesis is performed only by a small category of tissues like lactating breast, liver and adipose tissues.[8] Elevation of de novo fatty acid synthesis is main aberration of the lipid metabolism in cancer cells. Hence, targeting the reprogramming of lipid metabolism might be a positive approach in treating cancer.[9] The expression pattern of genes involved in fatty acid metabolism is also different in different molecular subtypes of breast cancer.[10]

Blocking the release of fatty acids from stored condition or redirecting fatty acids to store, could reverse the pool of fatty acids present for growth and proliferation of cancer cells. Cancer cells mostly store fatty acids in lipid droplets in the form of triglycerides. It has been reported that quantity of lipid droplets is elevated in cancer cells but function of lipid droplets in stored form is still not known.[11] These stored lipids supply pool of fatty acids which is used for energy production in cancer cells using enzyme lipases, i.e., adipose triglyceride lipase, hormone sensitive lipase, and monoacyl glycerol lipase.[12]

In last couple of decades, a large number of medicinal plants have been reported for their anticancer activities. However, very less is known for the operative molecular mechanisms. Citrullus colocynthis is a traditional medicinal plant known for its various pharmacological effects that includes antiobesity and hypolipidemic effect, antidiabetic, antimicrobial, antiparasitic, antioxidant, and anticancer.[13],[14],[15],[16],[17],[18],[19] Enhanced lipid metabolism is a key in providing energy to fast proliferating breast cancer cells.[20] Therefore, agents which can modulate lipid synthesis in cancer cells are getting attention in cancer therapeutics. C. colocynthis is already known for its antiadipogenic and hypolipidemic effects. Here, we aimed to investigate the C. colocynthis effect on regulation of lipid metabolism in breast cancer cells.


 > Materials and Methods Top


Cell culture

Human breast cancer cell line MCF-7 was obtained from cell culture bank of School of Biological Sciences, University of the Punjab, Pakistan. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum and 1% Pen-Strep under humidified environment with 5% CO2 in an incubator at 37°C. For harvesting cells, Trypsin-Ethylenediaminetetraacetic acid (0.25%) was used under standard procedure. All the cell culture reagents were purchased from Gibco, Thermo Fisher Scientific.

Preparation of plant extract and fractions

In this study, C. colocynthis used was obtained from Dera Ghazi Khan, Pakistan. The plant was confirmed from Department of Botany, University of the Punjab, Pakistan. Leaves were separated from plant and washed with autoclave distilled water to remove dirt and debris. Liquid nitrogen was used to dry leaves and with pestle and mortar they were crushed followed by grinding in electrical grinder to obtain fine powder. To prepare methanolic extract of leaves, 100 g powder was dissolved in 400 ml absolute methanol. It was placed on orbital shaker for 72 h at 37°C. Then it was kept at room temperature for 1 h to allow settlement of plant material. The supernatant was separated, filtered and further resuspended in 300 ml methanol. After 72 h shaking on orbital shaker, supernatant was again separated, filtered and further resuspended in 200 ml methanol. This was followed by shaking on orbital shaker for 72 h at 37°C and then finally supernatant was separated and filtered. From this supernatant, crude methanolic extract (CC-Met) was obtained by rotary evaporation of methanol. This extract was fractioned with solvents to prepare increasing polarity-based fractions by solvent-solvent extraction method. The fractions prepared include n-Hexane fraction (CC-Hex), Chloroform fraction (CC-Chl), Ethyl Acetate fraction (CC-EA) and n-Butanol fraction (CC-But). All fractions were rotary evaporated to obtain crude extract. Stock solutions (100 mg/ml) of CC-Met and fractions were prepared in sterile DMSO and filtered by 0.22 μ syringe filter. In DMEM, 1 mg/ml working solutions were prepared. Further dilutions were prepared from these working solutions to use in experiments.

Cell viability assays

MTT assay was done to determine effect of CC-Met and its fractions on cytotoxicity and viability of MCF-7 cells. Briefly, in 96 well plate 10,000 cells/well were seeded and grown to reach 70% confluency. Then the media in each well was replaced with test media at different concentrations in independent experiments for 72 h. Similarly, in control wells media was also replaced with culture media. After treatment, 100 μl MTT reagent (5 mg/ml) was added in each well and incubated for 4 h at 37°C. Purple color formazan crystals formed were dissolved in 150 μl DMSO followed by absorption at 570 nm. The percentage cell viability was determined as absorbance of treated well vs absorbance of control. Trypan Blue Assay was also performed with cells treated with CC-Met for 24 h, 48 h and 72 h to study time dependent effect of CC-Met on viability of cells. All the experiments were performed in triplicates. Statistical analysis of data was done using GraphPad Prism 5 and presented as mean ± standard deviation.

Phytochemical analysis

The fractions were subjected to phytochemical analysis to identify and screen pharmacologically active secondary metabolites in independent experiments. For this purpose, phytochemical tests were performed for flavonoids, steroids, cardiac glycosides, saponins, alkaloids, and phenols.

Test for flavonoids

1 ml of 0.1 M NaOH was added in 2 ml of each fraction. This changed the color to intense yellow which disappeared after addition of 1 ml of 0.1 M HCl. This confirmed the presence of flavonoids.[21]

Test for steroids

In a test tube, 5 ml chloroform was dissolved in 100 mg of fraction. This was followed by addition of 1 ml conc. H2 SO 4 by side walls. Formation of reddish brown participates indicated the presence of steroids.[22]

Test for cardiac glycosides

Kellar– Kiliani test: In a test tube, 4 ml glacial acetic acid and 2 ml of 5% FeCl3 were added in 100 mg of fraction. This was followed by addition of 1 ml conc. H2 SO 4 by side walls. Appearance of green blue color indicated the presence of cardiac glycosides.[23]

Test for saponins

10 ml distilled H2 O was added in 100 mg fraction in a test tube and shake it vigorously for 10 min. Froth formed was allowed to settle for 15 min. Persistence of froth layer indicated the presence of saponins.[21]

Test for alkaloids

3 ml of 0.1 M HCl was dissolved in 2 ml of each fraction followed by addition of 1 ml Wagner's reagent. Presence of reddish-brown precipitates indicated the presence of alkaloids.[21] Wagner's reagent was freshly prepared by dissolving 0.5 g iodine and 1.5 g potassium iodide in 25 ml distilled water.

Test for phenols

3 ml of 5% FeCl3 was dissolved in 3 ml of fraction. Formation of bluish-black precipitates indicated the presence of phenols.[24]

Cholesterol and triglycerides quantification

MCF-7 cells were treated with LC50 dose of CC-Met and its fractions in independent experiments for 72 h. After treatment, equal number of cells were collected from control and the samples by counting cells using Countess II FL Automated Cell Counter (Thermo Fisher Scientific, Catalog number: AMQAF1000). Modified Bligh Dyer Method was performed for cholesterol and triglycerides quantification.

For cholesterol quantification

Intracellular lipids were extracted from the cells using 100 μl MgCl2 (1 M) and 800 μl chloroform-methanol (2:1) in an Eppendorf. The mixture was vortexed. Upper aqueous layer containing aqueous polar lipids like acyl-CoAs was removed and to the lower layer, 500 μl MgCl2 and 700 μl chloroform-methanol (2:1) were added and vortexed followed by removal of upper layer. To the lower layer again 200 μl MgCl2 and 400 μl of chloroform-methanol (2:1) were added and vortexed. Upper layer and interphase were removed. The lower layer was evaporated using Eppendorf concentrator to obtain lipid extract. Concentration of the cholesterol in this extract was determined using kit (Analyticon biotechnologies, Catalogue No. 4046) according to manufacturer's instructions. Standard calibration curve was generated using known concentrations of standard cholesterol (SUPLECO, Catalogue No. 47127-U). Cholesterol in each sample was determined spectrophotometrically from this standards curve.

For triglycerides quantification

In cells pallet, 350 μl MgCl2 (1 m) and 400 μl of chloroform-methanol (2:1) were added followed by vortexing and then centrifugation for 2 min at 10,000 RPM. Upper layer was removed. To the lower layer 250 μl of MgCl2 and 350 μl of chloroform-methanol (2:1) were added. It was vortexed and centrifuged at the same speed. Upper layer was again separated and to the lower layer 200 μl of MgCl2 and 200 μl of chloroform-methanol (2:1) were added followed by vortexing and centrifugation. Upper layer and interphase (cellular debris) were removed and lower layer containing lipids was evaporated to obtain lipid extract. Triglycerides were quantified using ABCAM Triglyceride Assay Kit-Quantification (ab65336). Standard calibration curve was generated using known concentrations of standard triglycerides (ab103967) and triglycerides in each sample were determined from this curve spectrophotometrically.

Gene expression analysis

Trizol reagent (Thermofisher Scientific - 15596018) method was used to isolate RNA from MCF-7 breast cancer cells treated with LC50 dose of CC-Met for 24 h, 48 h and 72 h in independent experiments. RNA was also isolated from cells treated with LC50 dose of fractions for 72 h using the same method. Its quality and quantity were assessed by DeNovix DS-11 FX + spectrophotometer. RevertAid First Strand cDNA synthesis kit (Thermofisher Scientific - K1622) was used to synthesize cDNA from purified RNA for Real Time PCR. Expression analysis of mRNA levels of pro and antiapoptotic genes (Caspase-3, Bcl-2, FAS and lipid metabolism genes (Fatty acid synthase [FASN], 3-Hydroxymethyl-3-methylglutaryl-CoA lyase [HMGCLL1], Acyl-CoA synthetase long chain family member 5 [ACSL5], Elongation of very long chain fatty acids 2 [ELOVL2]) was performed using Thermo Fisher Syber Green master mix in PikoReal qPCR (Thermo Scientific) in triplicates. Expression level of target mRNA level was normalize using mRNA level of housekeeping gene GAPDH. List of primers used in the study are provided in [Table 1]. Graph Pad Prism 5 was used to analyze the data statistically using Student's t-test and one-way analysis of variance (ANOVA) with Tukey's as post hoc test. Data was represented in mean ± SEM of triplicates.
Table 1: List of primers use in the study

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 > Results Top


CC-Met reduce cell viability and induce cell cytotoxicity in MCF-7 breast cancer cells

MTT Assay revealed significant anticancer effect of CC-Met by inducing cytotoxicity and reducing cell viability in dose dependent manner. LC50 calculated for CC-Met on MCF-7 cell was 30 μg. MCF-7 cells treated with LC50 concentration of CC-Met for 24 h, 48 h and 72 h separately, depicted a significant time dependent reduction in cell viability. The cell viability was reduced from 76% at 24 h treatment to 65% after 48 h and further 50% after 72 h treatment [Figure 1]a. Gene expression analysis of apoptosis and antiapoptosis genes in cells treated with CC-Met in time dependent manner further emphasized the anticancer potential of CC-Met. Results showed a significant increase of caspase-3 mRNA level in CC-Met treated cells relative to the control cells. In contrast, Bcl-2, an antiapoptotic gene showed significant downregulated expression in treated cells relative to control cells. A death ligand receptor, FAS was also significantly upregulated in treated cells relative to control in a time dependent manner [Figure 1]b.
Figure 1: (a) Effect of CC-Met on MCF-7 cells in dose dependent and time dependent manner after 72 h treatment. (b) Expression analysis of Apoptosis genes in CC-Met treated cells show significant increase in expression of apoptotic genes and decrease in expression of ant-apoptotic gene in time dependent manner (*P < 0.05, **P < 0.01, ***P < 0.001)

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Fractions reduce cell viability of breast cancer cells in dose dependent manner

MTT cell viability assay of fractions revealed very low LC50 of fractions as compare to CC-Met. The lowest LC50 is of CC-Chl i.e., 3 μg followed by 12 μg of CC-EA and 20 μg of CC-Hex [Figure 2]. CC-But fraction was labelled non cytotoxic as it had no effect on cell viability and cytotoxicity within range of 5.0–30 μg and was excluded from further studies.
Figure 2: MCF-7 cells after 72 hours treatment with CC-Met fractions and percentage viability of cells at different concentrations. MTT cell viability assay revealed decrease in cell viability in dose dependent manner in all the groups. The table provides a comparison of LC50 dose of CC-Met and its fractions

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Fractions possess pharmacologically active secondary metabolites

In fractions, phytochemical analysis confirmed presence of secondary metabolites such as flavonoids, steroids, cardiac glycosides, saponins, alkaloids and phenols. Results displayed that CC-Chl fraction has high amount of saponins, alkaloids and phenols, CC-Hex fraction has high amount of cardiac glycosides, whereas, ethyl acetate fraction has significantly high quantity of flavonoids and steroids. [Table 2] summarizes the result of all phytochemical tests performed with the fractions.
Table 2: Phytochemical tests with the fractions of crude methanolic extract revealed the presence of secondary metabolites

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CC-Met and its fractions decrease cholesterol and increase triglycerides in breast cancer cells

The significant decrease in cholesterol concentration was observed in all the groups as compare to control. The lowest concentration was in CC-Chl (18.5 μg) as compare to control (35.5 μg). In CC-Met, CC-Hex and CC-EA cholesterol concentration was 30.8 μg, 25.3 μg and 22.1 μg respectively [Figure 3]a. In contrast significant increase in triglycerides was observed in all the groups with maximum increase in CC-EA (26.7 μg). Concentration of triglycerides in control, CC-Met, CC-Hex and CC-EA was 8.6 μg, 25.3 μg, 12.2 μg, and 26.7 μg respectively [Figure 3]b.
Figure 3: (a) Cholesterol concentration in MCF-7 cells treated with CC-Met and its fractions (b) Triglycerides concentration in MCF-7 cells treated with CC-Met and its fractions (*P < 0.05, **P < 0.01, ***P < 0.001)

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CC-Met and its fraction downregulate mRNA level of genes involve in lipid metabolism

Expression analysis of genes involve in lipid metabolism HMGCLL1, ACSL-5, ELOVL2 and FASN showed a significant downregulation in cells treated with CC-Met and its fractions as compare to control. 3-Hydroxymethyl-3-Methylglutaryl-CoA Lyase (HMGCLL1) show maximum downregulated mRNA transcript in CC-Met and CC-Chl [Figure 4]a. Long chain fatty acid-CoA ligase 5 (ACSL-5) maximum downregulation was in CC-Chl group [Figure 4]b. ELOVL2 was also downregulated maximum in CC-Chl group [Figure 4]c. FASN relative expression was also down regulated in all the groups. Its maximum downregulation was observed in CC-EA group [Figure 4]d.
Figure 4: Effect of CC-Met and its fractions on relative levels of mRNA. (a) 3-Hydroxymethyl-3-methylglutaryl-CoA lyase (b) Acyl-CoA synthetase long chain family member 5 (c) Elongation of very long chain fatty acids 2 (d) Fatty acid synthase (*P < 0.05, **P < 0.01, ***P < 0.001)

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 > Discussion Top


Breast cancer is the most frequently diagnosed cancer in woman worldwide.[25] The current available treatments such as surgery, radiotherapy and chemotherapy pose serious limitations such as tumor recurrence, metastasis systemic toxicity, off-targets effect and resistivity.[26] Therefore, to look for novel and effective anticancer agents is the need of the hour. Secondary metabolites are natural plant products which can effectively suppress tumor growth at low dose with minimum side effects.[27] Cancer cells show completely different metabolism as compared to normal cells because metabolic machinery in cancer cells is reprogrammed to meet the demands of cancer cells. High amount of fatty acids is produced in cancer cells mostly by the process of de novo biosynthesis. Activated lipid metabolism in cancer cells promotes invasion, migration, tumour growth and survival.[9] The aim of the study was to investigate effect of C. colocynthis in metabolism of breast cancer cells.

In the present study, methanolic extract of C. colocynthis leaves and its fractions showed significant anticancer potential against MCF-7 breast cancer cells in a dose dependent and time dependent manner. Expression of apoptosis genes showed the upregulation of apoptotic genes caspase-3 and FAS and downregulation of antiapoptotic gene Bcl-2, indicating that apoptosis was induced in the cells. In 2017, a study on fruit extract of this plant also reported upregulation of caspase-3 and down regulation of Bcl-2 protein on breast cancer cell lines.[19] Phytochemical analysis of fractions revealed the presence of high amount of cardiac glycosides in CC-Hex fraction, saponins, alkaloids and phenols in CC-Chl fraction and flavonoids and steroids in CC-EA fraction. A study in 2019 on C. colocynthis leaves extracts elucidated presence of alkaloids, glycosides, steroids and flavonoids.[28] Glycosides, flavonoids, steroids and alkaloids has shown potential anticancer activity in various studies.[29] In 2006, cucurbitacin glycosides and flavone C-glycosides were isolated from butanol fraction of methanolic extract of C. colocynthis fruit.[30] In 2007 anticancer activity of cucurbitacin glucosides isolated from C. colocynthis leaves chloroform/methanol extract was evaluated in breast cancer cells.[31] The present study is the first one to report the anticancer potential and phytochemicals constituents of fractions of C. colocynthis leaves.

In our study, altered cholesterol and triglycerides levels were observed in breast cancer cells as compare to control cells. C. colocynthis has shown antilipidemic activity in various patients. In hyperlipidemic patients its seed powder has shown decrease concentration of cholesterol.[15] In 2013 a study identified β-sitosterol in seed oil of C. colocynthis and reported its antihyperlipidemic effects in rabbits.[32] In 2018, hypoglycemic and antilipidemic effect of glycosides and alkaloids present in seeds of C. colocynthis were reported in rat liver cells.[14] Increase cholesterol level promotes breast cancer progression.[33] A study on pancreatic cancer cells presented that triglyceride accumulation suppress the growth in pancreatic cancer cells.[34] Altering cholesterol and triglyceride level in cancer cells can be a therapeutic intervention in breast cancer cells.

In present study, downregulation of relative mRNA level of HMGCLL1, ACSL-5, ELOVL2 and FASN indicate downregulation of lipid metabolism in breast cancer cells. FASN is involve in synthesis of fatty acids and its expression has relation with aggressiveness, progression and metastasis of cancer.[35] ACSL-5 plays role in lipid biosynthesis by converting free long chain fatty acid into fatty acyl CoA ester. Expression of this gene is upregulated in breast cancer.[36] ELOVL2 in involve in elongating fatty acid chains. Its expression is increased through ERα by estrogen in breast cancer.[37] HMGCLL1 converts 3-hydroxy-3-methylglutaryl-CoA into acetyl-CoA and acetoacetate. This is a key step in ketogenesis to provide energy from fatty acids to the cells. A study reported inhibition of HMGCLL1 as effective therapeutic approach in chronic myeloid leukemia.[38] It's reduced expression in our study propose that it can be an effective target for breast cancer therapy.


 > Conclusion Top


Current research highlights the anticancer potential of C. colocynthis leaves and its role in regulation of lipid metabolism in breast cancer cells. Cell viability assays together with cholesterol and triglycerides quantification and expression of lipid metabolism genes revealed that C. colocynthis leaves extract and its fractions reduce cell viability, induce apoptosis and downregulate de novo fatty acid synthesis pathway in breast cancer cells. Therefore, with further comprehensive studies most active compounds in C. colocynthis leaves can be identified and isolated and can be proposed for therapeutic interventions of breast cancer. cells.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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