abcArchives of Breast CancerArch Breast Cancer2383-04252383-0433Farname Inc.10.32768/abc.2024114408-417999version-of-recordOriginal ArticleEffects of Lupeol on Estrogen and Androgen Receptor-Positive Breast and Prostate Cancer CellsMajdMahdieh NezamiaAlizadehArashb*HashemElham ZadehbDVM Graduate Student, Faculty of Veterinary Medicine, Urmia University, Urmia, IranDivision of Pharmacology and Toxicology, Department of Basic Science, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
Address for correspondence:
Arash Alizadeh,
Division of Pharmacology and Toxicology, Department of Basic Science, Faculty of Veterinary Medicine, Urmia University,
Urmia,
Urmia,
Iran.
E-mail: dr.arash.alizadeh@gmail.com
The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.
Different reports have shown that prostate and breast cancers are the most common cancers worldwide. Lupeol, a dietary triterpene, provides various beneficial effects, including anti-cancer properties. The current study aims to investigate the anti-proliferative and antioxidant effects of lupeol, in line with the effects of lupeol on the expression of estrogen and androgen receptors in breast (MCF-7) and prostate (LNCaP) cancer cell lines.
Methods
MCF-7 and LNCaP cells were incubated with increasing concentrations of the lupeol (1, 10, and 100 µM) for 24 hours. The cytotoxicity of the lupeol was assessed by MTT and neutral red assays. Moreover, TAC (total antioxidant capacity) and gene expression of androgen and estrogen receptors were measured by spectrophotometric and qPCR methods, respectively. Overall, 17 beta-estradiol (E2) (9 nM) and dehydroepiandrosterone (DHEA) (5 µM) were selected as positive controls.
Result
The highest concentration of the lupeol induced cytotoxic effects on MCF-7 and LNCaP cells. Various levels of lupeol at specified time intervals increased TAC levels in comparison with the control group. Moreover, the expression levels of estrogen receptors (α and β) and androgen receptors were negatively affected by lupeol.
Conclusion
The findings of our study indicate that lupeol could serve as a promising and accessible multi-functional anti-tumor agent against hormone-positive breast and prostate cancers.
KeywordslupeolMCF-7LNCaPestrogen receptorsandrogen receptorantioxidant capacityNo specific grant from funding agencies in the public, commercial, or not-for-profit sectors was received for this study.Introduction
Epidemiological data suggested that triterpene-enriched diets could show beneficial effects on sex hormone-dependent cancers, including breast, prostate, ovarian, and endometrial cancers.1 Lupeol (Lpl) is a multi-source natural triterpene gaining more and more attention nowadays, due to its beneficial effects on different conditions such as infectious diseases, renal, cardiovascular, and inflammatory disorders, diabetes, hepatic toxicity, microbial arthritis, and cancer.2 Cancer, as one of the most aggressive diseases and the second cause of mortality, is a hyperproliferative disorder that involves various circumstances, including transformation, dysregulation of apoptosis, proliferation, invasion, angiogenesis, and metastasis.3,4
It has been revealed that Lpl can act as a sensitizer and chemotherapeutic agent in combination with conventional anti-neoplastic medicine through its modulatory effects on pivotal signaling pathways in cancer etiology and progression, such as the PI3K/AKT/mTOR and nuclear factor kappa B (NF-κB), downregulation of Bcl-2 and Bcl-Xl, MMP-9, as well as upregulation of caspase-3 that leads to apoptosis of apoptosis.5–7 Lpl has the potency to induce G2/M cell cycle arrest by inhibiting the cyclin-regulated signaling pathway in cancer cells.8 The structural similarity of Lpl with androgenic hormones makes it a potential candidate for modulating the androgen receptor signaling cascade.9 Previous investigations indicated different characteristics for Lpl, including inhibition of cell migration, decrease of cell proliferation, and induction of apoptosis.10 In addition, other mechanisms such as chemosensitization of tumor cells and inhibition of androgen receptor have also been reported as possible mechanisms of action for Lpl.10
The estrogen effects are associated with binding to the ERs: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), while the stimulation or inhibition of the underlined signaling pathway in the target organ depends on the balance between ERα and ERβ activities.13 Based on previous investigations, ERα is a transcriptional factor expressed in more than 70 percent of breast cancer patients and induces the proliferation of breast cancer cells.14 but there is limited information about the role of ERβ in the treatment and biology of breast cancer.15
Concerning prostate cancer, epidemiological studies have shown that the incidence and mortality of prostate cancer are among the top 5 important malignancies on a worldwide scale.16 Due to the fact that the androgen receptor plays an important role in the progression of prostate cancer; therefore, successful treatment can be achieved by the modulation of the androgen receptor and associated pathways.17
Various limitations have been documented concerning the standard therapeutic protocols in cancer therapy, such as surgery, chemotherapy, radiation, immune therapy, targeted therapy, and hormone therapy.18
Herbal medicines and nature-based medicinal approaches have provided a significant opportunity for the improvement of the applied treatment protocols because of broader beneficial effects with limited side effects in the prevention and treatment of cancer.19,20
Therefore, the purpose of this study was to investigate the antiproliferative, antioxidant, and pro-apoptotic effects of Lpl on two distinct types of cancer cells (MCF-7 as the ER-positive human breast cancer cell line and LNCaP as the androgen-sensitive human prostate adenocarcinoma cell line). Moreover, the possible effect of Lpl on gene expression of estrogen and androgen receptors was evaluated to open new venues in cancer therapy and improve the quality of life in cancer patients.
MethodsCell culture
This is an in vitro study on MCF-7 cells, as the ER-positive human breast cancer cell line, and LNCaP cells, as the androgen-sensitive human prostate adenocarcinoma cell line, were seeded in DMEM (Dulbecco's Modified Eagle Medium) and RPMI 1640 (Roswell Park Memorial Institute Medium) (Sigma-Aldrich, St. Louis, MO, USA), respectively. Both of the cell culture media were supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (100 IU/mL and 100 μg/mL). The cells were kept in a 5% CO2 and 37 °C incubator as optimal conditions. The subculture of the cells was performed after reaching 70% to 80% confluency according to the standard protocols.21 Cytotoxicity evaluations were conducted in 96-well plates with optimal density based on previous studies, while the samples for qPCR analysis were prepared by seeding the cells in 6-well plates. Lpl (L 5632) was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) and the stock solution was prepared with dimethyl sulfoxide (DMSO, Merck, Germany) while the final concentrations of Lpl (1, 10 and 100 μM) were used as experimental concentrations, and untreated cells were utilized as control cells (0.5% DMSO). Dehydroepiandrosterone (DHEA, D 4000) 5 µM and 17 beta-estradiol (E2) 9 nM (Sigma-Aldrich, St. Louis, MO, USA) were utilized as associated controls, and the stock solutions were prepared in DMSO.
Cytotoxicity assays (MTT)
Cell viability was quantified by the colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. MTT assay is based on the assessment of the capacity of living cells to reduce the yellow water-soluble substrate tetrazolium salt into a purple water-insoluble formazan product, which is considered an indicator of cell viability (22). MCF-7 and LNCaP cells were treated in 96-well plates (5×103 cells/well) with DMEM and RPMI culture medium, respectively. To this end, the cells were exposed to different concentrations of Lpl (1, 10, and 100 µM), DHEA (5 µM), and 17β-estradiol (9 nM) for 24, 48, and 72 hours. All the procedures were conducted based on the protocol of previous studies.23 Cell viability percentage was calculated by the following formula:
Neutral red (NRU) assay
Neutral red is a vital dye, which is preferentially absorbed and endocytosed by viable cells and internalized inside the lysosome; therefore, it can be considered as an indicator of lysosome and cell integrity.22 MCF-7 and LNCaP cells were treated in 96-well plates (5×103 cells/well) containing 100μl of DMEM and RPMI medium, respectively, by a concentration range of 1 to 100 μM of Lpl, 5 µM of DHEA, and 9 nM of E2 for 24, 48, and 72 hours. Then, 5 μL of Neutral red solution (4mg/ml) was added to the cells for 3 hours based on the previous protocol. As the final step, the absorbance was recorded at 540 nm.
Total antioxidant capacity assay
Total antioxidant capacity (TAC) was assessed in the supernatant of MCF-7 and LNCaP cells with different concentrations of Lpl (1, 10, and 100 μM) at time points of 24, 48, and 72 hours by the method described by Koracevic et al.24 In brief, 490 μL of PBS solution was added to 10 μL of the sample. Additionally, sodium benzoate, acetic acid, Fe-EDTA, and H2O2 were added to the tubes, respectively. The tubes were then immersed at 37 °C for 60 minutes, and finally, after adding the thiobarbituric acid solution and placing the tubes in boiling water (10 minutes), the absorption of the samples was read at 532nm. A suitable solution (FeSO4.7H2O) of Fe2+ and ascorbic acid was used as a blank and standard solution.
RNA isolation and cDNA synthesis
The RNA isolation was performed by the standard TRIzol method.25 The RNA amount was determined spectrophotometrically, and RNA purity was determined by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA) with expected values between 1.8 and 2. The samples were stored at −70°C for cDNA synthesis. The 20-µL reaction mixture containing l µL RNA, 10 μL 2 × reaction buffers, 2 μL enzyme mix, and 7 μL RNase-Free water was prepared according to the instructions of Pars Tools Company. The cycling protocol for the 20μL reaction mixture was 10 minutes at 25 °C, followed by 60 minutes at 47 °C, and 5 minutes at 85 °C to terminate the reaction.
Real-time polymerase chain reaction
The PCR reaction was carried out in a total volume of 20 μL, containing PCR master mix (10 μL, FWD and REV specific primers (each 1 μL), and cDNA as a template (1 μL) and nuclease-free water (7 μL). PCR conditions were run as follows: denaturation at 95 °C for 10 minutes, 1 cycle, followed by 45 cycles at 95 °C for 20 seconds. The annealing temperature (45 to 65 °C) was 20 to 40 seconds, while elongation was 72 °C for 30 seconds and 72 °C for 10 minutes. Data were analyzed using the ∆∆Ct method, and expression values were normalized to the expression levels of the β-actin gene. Primer pairs for real-time PCR are depicted in Table 1.
Pairs of Real-Time Polymerase Chain Reaction Primers.
For statistical analyses, the mean and standard deviation of the measured parameters were calculated. All data are reported as the mean SD of triplicate experiments. The results were analyzed using GraphPad Prism software (version 8.02, GraphPad Software Inc., San Diego, California, USA). The comparisons between groups were made by analysis of variance (one-way ANOVA) followed by the Bonferroni post hoc test. The significant difference between the control and the treatment group is marked with an asterisk symbol () in the results section. A P-value less than 0.05 was considered significant.
ResultsCell viability
The MTT method was used to evaluate the effects of Lpl with increasing concentrations on breast (MCF-7) and prostate (LNCaP) cancer cell viability. The results of the MTT test showed that 100 μM Lpl significantly reduced the cell viability starting from 24-hour incubation and lasting for 72 hours (P<0.05). It should be noted that the decrease in the survival of LNCaP cells with the highest concentration of Lpl was not statistically significant at a 24-hour incubation period, but longer exposure resulted in a significant decrease in cell viability. As depicted in Figure 1, the control treatments with DHEA and E2 have shown no effect on cell viability.
LNCaP and MCF-7 cells’ viability, exposed to different concentrations of Lupeol (1, 10, and 100 μM), based on the MTT test assay. *P≤0.05, ***P≤0.01
Figure 1Neutral red
The NR method was used to evaluate the effects of Lpl with increasing concentrations on survival and Lysozyme activity of breast (MCF-7) and prostate (LNCaP) cancer cells. For this purpose, the cells were exposed to different concentrations of Lpl as well as single concentrations of E2 and DHEA for 24, 48, and 72 hours. As shown in Figure 2, the highest concentration of Lpl in MCF-7 cells after 24 hours showed a significant effect (P<0.05).
LNCaP and MCF-7 Lysosomal Enzyme Activity Exposed to Different Concentrations of Lupeol (1, 10, and 100 μM) According to the Neutral Red Assay. *P≤0.05
Figure 2Cell morphology of LNCaP cells
As shown in Figure 3, the morphology of LNCaP cells following 24h incubation with E2, DHEA, and various concentrations of Lpl show significant changes in the highest Lpl concentration.
Morphology of LNCaP Cells Exposed to Different Concentrations of Lupeol. DHEA, dehydroepiandrosterone; E2, estradiol; Lpl, lupeol.
Figure 3Cell morphology of MCF-7 cells
As shown in Figure 4, the morphology of MCF-7 cells following 24 hours of incubation with E2, DHEA, and different concentrations of Lpl shows significant changes in the DHEA group and the highest Lpl concentration.
Morphology of MCF-7 Cells Exposed to Different Concentrations of Lupeol
Figure 4Total antioxidant capacity assay
Table 2 shows the results of the TAC assay in MCF-7 and LNCaP cells. Compared to the control group, 1 μM and 10 μM of Lpl after 24, 48, and 72 hours and 100 μM of Lpl after 48 hours, in MCF-7 cells, and 1 μM and 10 μM of Lpl after 48 and 72 hours, and 100 μM of Lpl after 72 hours, in LNCaP cells, increased TAC level. The mean OD was measured at 532 nm using a spectrophotometer. Both E2 and DHEA increased TAC compared to the control group in 48 and 72 hours of treatment (P<0.05).
Total Antioxidant Capacity in Different Study Groups at 24, 48, and 72 Hours
Time Groups
24h
48h
72h
MCF-7
LNCaP
MCF-7
LNCaP
MCF-7
LNCaP
Control
0.376 ± 0.019
0.557 ± 0.073
0.188 ± 0.010
0.319 ± 0.019
0.471 ± 0.016
0.345 ± 0.008
E2
0.336 ± 0.016
0.693 ± 0.004
0.860*** ± 0.077
0.546*** ± 0.017
0.822*** ± 0.051
0.900*** ± 0.038
DHEA
0.458*** ± 0.017
0.584 ± 0.109
0.533*** ± 0.013
0.439* ± 0.021
0.851*** ± 0.043
0.804*** ± 0.091
Lpl 1µM
0.508*** ± 0.004
0.614 ± 0.094
0.695*** ± 0.012
0.456** ± 0.015
0.875*** ± 0.022
0.867*** ± 0.055
Lpl 10µM
0.478*** ± 0.010
0.743* ± 0.014
0.611*** ± 0.025
0.513*** ± 0.037
0.744*** ± 0.077
0.898*** ± 0.008
Lpl 100µM
0.307 ± 0.003
0.544 ± 0.080
0.463*** ± 0.022
0.373 ± 0.077
0.418 ± 0.071
0.515** ± 0.060
LNCaP and MCF-7 cells exposed to different concentrations of Lupeol (1, 10, 100 μM), and the total antioxidant capacity was evaluated in the supernatant of the treated cells. *P≤0.05, **P≤0.01, ***P≤0.001. DHEA, dehydroepiandrosterone; E2, estradiol; Lpl, lupeol.
qPCR
qPCR analysis was used to evaluate the gene expression levels of the androgen receptor and alpha and beta estrogen receptors. As shown in Figure 5, the expression of alpha estrogen receptor genes at concentrations of 10 μM and 100 μM of Lpl was significantly reduced compared to the control group, but the decreasing effects of beta estrogen receptor gene expression were observed at 1 μM and 10 μM Lpl.
Effect of lupeol on gene expression of alpha and beta estrogen receptors relative to the ß-actin reference gene in MCF-7 cells. *P≤0.05, **P≤0.01, ***P≤0.001.
Figure 5
It should be noted that DHEA (5µM) induced a decrease in gene expression levels of both receptors compared to the control group (P < 0.05).
As shown in Figure 6, the gene expression of androgen receptor at concentrations of 1 μM and 10 μM of Lpl was significantly reduced compared to the control group. However, DHEA showed decreasing effects and E2 showed increasing effects on the gene expression of the androgen receptor compared to the control group.
Effect of Lupeol on Gene Expression of Androgen Receptors Relative to the ß-Actin Reference Gene in LNCaP cells. *P≤0.05, **P≤0.01, ***P≤0.001.
Figure 6Discussion
Breast and prostate cancers, as hormone-dependent tumors, are the most common malignancies in women and men, respectively.26,27 In men and women, increased levels of androgens/estrogens and mutations in their receptors have been shown to increase the risk of prostate and breast hormone-positive (HR+) cancers, respectively.28,29
The current study was set up to investigate the cytotoxic effects of Lpl on MCF-7 cells as a human breast cancer cellular model with estrogen receptors, and LNCaP cells as an androgen-sensitive human prostate adenocarcinoma cellular model. Then, the effects of Lpl on estrogen and androgen receptor expressions and the antioxidant capacity of Lpl-exposed cells were evaluated to shed more light on the potential beneficial effects of Lpl.
The obtained results from the MTT assay showed that Lpl can reduce cell viability at the highest concentration (100 μM) in MCF-7 and LNCaP cell lines after 24, 48, and 72 hours. These results are in line with findings from a study by Pitchai et al.7 which showed that Lpl isolated from Elephantopus scaber plant has the ability to reduce the viability of MCF-7 cells with an IC50 value of 80μM.
The results from the NR assay in the current study were not identical in association with time and concentrations. Results from a previous study that investigated the effect of betulinic acid (BA) and oleanolic acid (OA) as triterpenoids on the function and morphology of mitochondria and lysosomes in human skin keratinocyte (HaCaT) cells showed that despite the structural similarity of these triterpenoid compounds, betulinic acid was capable of damaging lysosomal and mitochondrial membranes but oleanolic acid was not capable of this function. This different function of these triterpenoids that was observed in this study can be related to the specific structure–activity relationships of these two compounds.30 It seems that the chemical structure of Lpl as a triterpenoid compound can be an important factor concerning the NR results.
The current investigation demonstrated that Lpl has the ability to considerably alter the total antioxidant capacity (TAC) in a time- and dose-dependent manner in MCF-7 and LNCaP cell lines, so that at 1 and 10 µM concentrations it increases the TAC, but decreases the TAC at 100 µM concentration. Analysis of the ethanolic extraction of the Ficus pseudopalma plant showed that the plant contains significant amounts of Lpl with antioxidant properties.31 It has been reported that the antioxidant capacity of Lpl can be related to the free radicals/ROS species-scavenging activity and lipid peroxidation inhibitory effects of Lpl addressed by studies utilizing DPPH and ABTS assays.32,33 Moreover, DHEA and E2 in the present study, which were used to affect androgen and estrogen receptors, also showed the ability to increase total antioxidant capacity (TAC) levels in sample supernatants at time points of 48 and 72 hours. The results are in line with previous studies confirming the ability of DHEA and E2 to inhibit oxidative-stress responses and modulate the redox balance.34,35
A study by Ding et al.36 showed that pretreatment of Leydig cells isolated from rats with DHEA could reduce oxidative stress and DNA injury induced by hydrogen peroxide (H2O2). In another study using mouse decidual endometrial stromal cells (ESCs), DHEA showed the potency to reduce intracellular reactive oxygen species (ROS) levels in a dose-dependent manner.34 Considering the ameliorative effects of E2 on TAC levels in sample supernatants, the results of the current study are in agreement with the results from investigations using E2 in colon epithelial cells (CCD841CoN cells), where E2 at a dose of 8 nM could induce antioxidant enzymes including heme oxygenase-1 (HO-1) and NAD(P)H-quinone oxidoreductase-1 (NQO-1).37 It should be noted that E2 has the key role in the augmentation of antioxidant capacity via activation of the Nrf2 signaling pathway.38
In general, similar to drugs such as tamoxifen, phytoestrogenic bioactive compounds may act as selective estrogen receptor modulators (SERMs), exhibiting estrogen agonist or antagonist effects in a dose and target organ tissue-dependent manner through changes in the expression of particular co-activators or co-repressors of ERα and ERβ activity.39,40 In this regard, a study by Thongon et al.41 indicated that phytoestrogenic extract obtained from Curcuma comosa plant had estrogenic activity at low doses (0.1–1 μM) and anti-estrogenic activity at high doses (10–50 μM) on HEK-293T cells while showing antagonistic effects on estrogen receptors in MCF-7 cells. In an in vitro study, results of gene expression analysis indicated that Lpl could induce the expression of endogenous estrogen receptor at a concentration of 1μM in a way that the effects on ERβ were more pronounced than those on ERα. However, Lpl alone (10−9 and 10−8μM) has been shown to serve as an antagonist in HEK293T‐Erα.42 Interestingly, co-administration of Lpl with an estrogen receptor antagonist (fulvestrant) resulted in the Lpl-effect as an estrogen agonist in the tissue of vagina.38
The results of the present study showed that Lpl at high concentrations (10 and 100μM) has a decreasing effect on ERα expression levels, which is in line with previous reports.42 It is known that DHEA and metabolites could act as ligands for estrogen and androgen receptors.43 On the other hand, based on the function of aromatase enzymes, androgens can be converted to estrogens, which can also affect estrogen receptors. Therefore, under certain circumstances, they can produce more estrogens (E2) as well as estrogenic effects.44
In the present study, the gene expression analysis showed that Lpl acts as an anti-androgen on the androgen receptor. Based on the structural similarity of Lpl with androgens and its interactions with androgen receptor signaling function through competitive antagonism and reducing the expression level of prostate-specific antigen (PSA) as an androgen receptor target, it can be concluded that a decrease in androgen receptor transcription is inevitable.9,45
The microscopic images of MCF-7 and LNCaP cells demonstrated that the highest concentration of Lpl (100 μM) after 24 hours damages the cells. These results are in line with the findings of previous studies that have examined the cytotoxic effects of Lpl derived from different natural sources on different cell lines where the cytotoxic effects of Lpl on cancer cells were documented in changes in the expression of proteins involved in cell cycle (cyclin proteins and cyclin-dependent kinases), apoptosis, autophagy and epithelial–mesenchymal transition (EMT) processes.46–48
Conclusion
Based on the results obtained from this study, it can be concluded that Lpl has significant effects on the cell viability of ER-positive breast (MCF-7) and AR-positive prostate (LNCaP) cancer cells. Furthermore, Lpl exerted inhibitory effects on the expression levels of androgen and estrogen receptors, which might be an important factor in the development of cancer cells. In addition to these effects, evaluating the TAC induced by Lpl can account for the complementary beneficial effects of Lpl in cancer patients. The findings of our study indicate that Lpl could serve as a promising and accessible multi-functional anti-tumor agent against hormone-positive breast and prostate cancers. Further studies are warranted to unravel the detailed mechanism of action behind the beneficial effects of Lpl.
Ethical considerations
Not applicable.
The Research Deputy of Urmia University funded the present experiment. We are grateful to Dr Hassan Malekinezhad for his kind assistance during the experiment.
Data availability
The data used in the current study are available from the corresponding author upon reasonable request.
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