Potential Metabolite Nymphayol Isolated from Water Lily (Nymphaea stellata) Flower Inhibits MCF-7 Human Breast Cancer Cell Growth via Upregulation of Cdkn2a, pRb2, p53 and Downregulation of PCNA mRNA Expressions

Controlled production of cyclin dependent kinases (CDK) and stabilization of tumor suppressor genes are the most important factors involved in preventing carcinogenesis. The present study aimed to explore the cyclin dependent apoptotic effect of nymphayol on breast cancer MCF-7 cells. In our previous study, we isolated the crystal from a chloroform extract of Nymphaea stellata flower petals and it was confirmed as nymphayol (17-(hexan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol) using x-ray diffraction (XRD), Fourier transform infrared (FTIR), and mass spectroscopy (MS) methods. The cytotoxic effect of nymphayol on MCF-7 cells were analyzed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cellular and nuclear damage was determined using propidium iodide (PI) and acridine orange/ethidium bromide (AO/ErBr) staining. Tumor suppressor and apoptosis related mRNA transcript levels were determined using real-time polymerase chain reaction (RT-PCR). Nymphayol potentially inhibits MCF-7 cell viability up to 78%, and the IC50 value was observed as 2.8 µM in 24 h and 1.4 µM in 48 h. Treatment with nymphayol significantly increased reactive oxygen species (ROS) level and the tunnel assay confirmed DNA damage. We found characteristically 76% apoptotic cells and 9% necrotic cells in PI and AO/ErBr staining after 48 h treatment with 2.8 µM of nymphayol. Gene expression analysis confirmed significantly (p ≤ 0.001) increased mRNA levels of cyclin dependent kinase inhibitor 2A (Cdkn2a), retinoblastoma protein 2 (pRb2), p53, nuclear factor erythroid 2-factor 2 (Nrf2), caspase-3, and decreased B-cell lymphoma 2 (Bcl-2), murine double minute 2 (mdm2), and proliferating cell nuclear antigen (PCNA) expression after 48 h. Nymphayol effectively inhibited breast cancer cell viability, and is associated with early expression of Cdkn2a, pRb2, and activation of p53 and caspases.


Introduction
Incidence of cancer are recognized with altered apoptosis mechanism, genetic mutations, oxidative stress, hypoxia, and sustained intra cellular inflammation, while environmental factors are linked to ultraviolet ray exposure, radiation, and lifestyle [1]. Aberrant cellular mechanisms in the apoptotic signaling pathway results in uncontrolled cell progression, leading to carcinogenesis [2]. Apoptosis is  (c-e) shows that the crystal structure refinement for the asymmetric unit consists of two molecules of 17-(hexan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol and one water molecule; (f) shows the structure of the novel compound, 25,26-dinorcholest-5-en-3b-ol. The isolated novel crystal was named nymphayol.

Results
In column chromatography, the Nymphaea stellata chloroform extract yielded a total of 74 fractions (each 150 mL); each fraction was spotted on a precoated Silica gel 60 F 254 , 0.25 mm thick thin layer chromatography (TLC) plate (Merck) and eluted in a hexane:ethyl acetate (4:1) system and fractions with similar Rf values in TLC pattern were pooled together. Finally, 17 major fractions were obtained and fraction 12 formed as a white colored amorphous crystal with traces of impurities. into two positions ( Figure 1c). In addition, Figure 1d,e confirmed that two molecules were not conformationally identical, particularly at the terminal side chain. A search in the crystallographic database with the unit cell parameter revealed no hits related to this crystal's unit cell parameter. The crystal was confirmed as a new crystal named nymphayol (25,26-dinorcholest-5-en-3b-ol [or] 17-(hexan-2-yl)-10,13-dimethylhexadecahydro-1Hcyclopenta[a]phenanthren-3-ol) and patented in Indian patent, 2007 [11]. In addition, the crystal was subjected to spectral analysis such as Fourier transform infrared spectroscopy (FT-IR) and mass spectrometry (MS) spectra analysis to reconfirm the functional group and molecular mass of the  The level of intracellular reactive oxygen species (ROS) with the potential to initiate oxidative stress and DNA damage may contribute to cell cycle arrest or cellular apoptosis. In our study, ROS generation was found to be at the basal level in dimethyl sulfoxide (DMSO) (vehicle control) treated cancer (MCF-7) and noncancerous (Vero and V79) cells. Furthermore, 0.7, 1.4, and 2.8 µM concentrations of nymphayol treated to MCF-7, Vero, and V79 cells for 48 h significantly (p ≤ 0.001) increased ROS level only in MCF-7 cells. In MCF-7 cells, 23% of ROS production was observed even at the lower (0.7 µM) dose of nymphayol. However, Vero and V79 cells produced 3% and 16% of ROS in the tested higher concentration (2.8 µM) of nymphayol, respectively. A samples of 2.8 µM of nymphayol treated MCF-7 cells showed 84% increased ROS generation as reflected by increasing dichlorofluorescein (DCF) fluorescence after an incubation of 30 min compared to the vehicle control ( Figure 2d). The cells pretreated with N-acetyl cysteine (NAC), an antioxidant, suppressed nymphayol-induced ROS generation (Figure 2d), which confirmed the nymphayol stimulated ROS production in MCF-7 cells.
Morphological changes of nymphayol treated MCF-7 cells after propidium iodide (PI) and acridine orange/ethidium bromide (AO/ErBr) staining were also investigated. PI and AO staining of 2.8 µM of nymphayol treated cells after 48 h showed abnormal nuclei, nuclei fragmentation, and horseshoe-shaped nucleus, which indicate apoptotic stimuli when compared to lower doses (Figure 3a  The manual counting of green florescence intensity indicates the degree of DNA damage induced by nymphayol. The cells stained with PI showed that 76% were apoptotic and 9% were in the necrotic stage ( Figure 4a). In the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, nymphayol-treated MCF-7 cells clearly exhibited increased green florescence intensity (45%), which confirmed the presence of terminal DNA damage and apoptosis (Figure 4b).   The expression of Cdkn2A, pRb1, and p53 is shown in Figure 5b. There was a significant (p ≤ 0.001) increase in Cdkn2A (1.72 fold) and pRb1 (1.30), and a 1.19-fold increase in p53 after nymphayol treatment when compared to the lower dose. Most interestingly, the p53 expression was around 2-fold higher when compared to the vehicle control. In addition, the mRNA expression level of mdm2 was significantly decreased to 2.36-fold in the 2.8 µM dose when compared to the 1.4 µM dose of nymphayol. Figure 6 shows the changes in the mRNA levels of Bax, Bcl-2, caspases, CDKN1A, and PCNA expression in nymphayol treated and the vehicle control MCF-7 cells. In Figure 5

Discussion
Nymphayol effectively inhibited breast cancer MCF-7 cell growth with the IC 50 range of 2.8 µM in 24 h and 1.4 µM in 48 h compared to normal Vero and V79 cells. This finding confirmed the selective cytotoxicity of nymphayol against MCF-7 cells without causing toxicity to normal (V79 and Vero) cells. In this context, Min et al. [15] found that fucoidan induced cytotoxicity only in hepatocellular carcinoma, without causing senescence to normal Chang Liver cells. Moreover, the 2.8 µM (24 h, IC 50 ) dose of nymphayol inhibited 92% of MCF-7 cells, but the same dose of tamoxifen and doxorubicin inhibited 35% and 46% of MCF-7 cells even after 48 h. The present finding was in line with previous reports such as Nymphaea pubescens [16] and phytosterols (sitosterol, campesterol, and stigmasterol) are well known for their anti-proliferative and anticancer activities [17].
Under physiological conditions, excessive accumulation of ROS plays a significant role in mediating cellular responses in cytotoxicity due to oxidative damage in hyper proliferative cancer cells [18]. Similarly, our previous reports demonstrated that at the molecular level, epoxy clerodane diterpene showed selective cytotoxicity via ROS-induced DNA damage in MCF-7 breast cancer cells [19]. In the present study, nymphayol increased ROS level and oxidative stress resulted in the initiation of cell, nuclear membrane damage, and DNA damage in MCF-7 cells was confirmed by the TUNEL assay. Recently, researchers have confirmed that the plant sterols (β-sitosterol, campesterol and stigmasterol) induced cytotoxicity in specific cancer cells via ROS stimulated signaling cascade involved apoptotic processes [17]. Fluorescent propidium iodide (PI) stain is permeable to the cell and nuclear membrane of damaged MCF-7 cells. PI staining of nymphayol treated MCF-7 cells showed chromatin condensation and horseshoe-shaped nuclei, and nuclear fragmentation confirmed the morphological changes related to those typical of apoptosis was observed using a fluorescent inverted microscope. The observed results suggest that induction of apoptosis by nymphayol was likely to be mediated by increased ROS production in hyper proliferating MCF-7 cells ending with oxidative stress. In this context, Yadav et al. [20] reported that oxidative stress mediated DNA damage associated with the activation of tumor suppressors and the mitochondria mediated caspase dependent apoptosis signaling pathway in MCF-7 cells.
The tumor suppressor p53 is a principal transcription factor regulating cellular pathways involved in apoptosis. In response to diverse stresses such as DNA damage, hypoxia, telomere shortening, oncogene, p53 is activated, leading to apoptosis induction [21]. Under normal cell physiology and unstressed cells, p53 is tightly regulated by murine double minute 2 (MDM2) by maintaining p53 at low levels. Thus, MDM2, a potent cellular antagonist of p53, limits p53 growth-suppressive function [22,23]. Our data showed upregulation of CDKN2A, pRb1, and p53 and downregulation of MDM2 in nymphayol-treated MCF-7 cells when compared with the control. A recent study showed that HepG2 cells treated with fucoidan induced apoptosis, which might be mediated by upregulating p16 (INK4a) -Rb and p14 (Arf) -p53 pathways [15]. Our results showed that nymphayol inhibits Bcl-2 expression, which plays a significant role in regulating cell proliferation and apoptosis [24]. Bcl-2 overexpression is observed in the majority of human cancers [25]. Anti-apoptotic family members such as Bcl-2 play a pivotal role in inhibiting apoptosis [26].
Caspases serve as primary effectors during the process of the apoptosis signaling pathway [18,27]. In response to apoptotic signals, caspases are rapidly activated [28]. Activation of caspases, in particular caspase 3 and 9, cleaves poly (ADP-ribose) polymerase-1 (PARP-1), leading to cell apoptosis. PARP has received considerable attention for use as a main target for many chemotherapeutic drugs, suggesting the significant role of PARP in maintaining genomic stability and repairing DNA [29]. Cyclin-dependent kinase inhibitor 1A (CDKN1A or p21) carries two functional domains, allowing them to bind to PCNA and Cdk/cyclins [30]. CDKN1A inhibits PCNA-dependent DNA replication by preventing PCNA from contributing to DNA polymerase δ and ε function. In addition, it induces cell growth arrest after DNA damage [30]. Consistent with the above findings, in our study, nymphayol treatment significantly increased the mRNA expression levels of caspase-3, caspase-8, caspase-9, and PARP and downregulated PCNA.
Overall, the cytotoxic effect of nymphayol on breast cancer MCF-7 cells and its possible mechanism of action were explored. We found that nymphayol increased ROS generation and stimulated hyper proliferative signals in MCF-7 cells, which effectively increased the early expression of cyclin dependent kinase (CDKN2A), aiding in the accumulation of active tumor suppressor p53. Furthermore, active p53 stimulated caspase dependent mitochondria mediated apoptotic signaling pathway related genes. This resulted in upregulated levels of caspase 3, caspase 9, CDKN1A expressions, and downregulated PCNA expressions was associated with cell growth arrest. Further studies are needed to confirm the anticancer mechanistic effect of nymphayol using Annexin V-FITC based apoptotic and necrotic cell sorting and western blot based protein quantification using in vitro and in vivo models.

Cell Culture Materials
Propidium iodide (PI), acridine orange (AO), and ethidium bromide (ErBr) were procured from Sigma-Aldrich (St. Louis, MO, USA). The DeadEnd Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) Assay Kit was procured from Promega (Madison, WI, USA). The QuantiTect Primer Assay, Fast Lane Cell cDNA Kit, and QuantiFast SYBR Green PCR Kit were procured from Qiagen (Hilden, Germany). All other chemicals used in this study were cell culture grade.

N. Stellata Flower Chloroform Extract (NSFCExt) Preparation
The fresh petals of N. stellate flower were collected, shade dried, coarsely powered, and used for extraction. A total of 500 gm of dry petal powder was kept in an aspirator bottle; 1.5 L of chloroform was used, and the mixture was kept in a shaker (200 rpm) for 48 h. Then, the liquid portion containing the extract was filtered using Whatman filter paper (no. 2) on a Buchner funnel. This procedure was repeated three times and all extracts were decanted and combined. The solvent was removed by vacuum distillation in a rotary evaporator at 60 • C and the extracts were placed in pre-weighed flasks before drying.

Isolation of Crystal by Column Chromatography
Chloroform extract (10 gm) was adsorbed on silica gel (Acmae's 60-120 mesh) and chromatographed on a silica gel (Acmae's 100-200 mesh) column initially eluted with a continuous suitable system and gradually increasing the polarity of the mixture of solvents [hexane (non-polar) to methanol (polar)]. Eluted fractions were evaluated using TLC and a similar TLC patterns were pooled into major fractions.

Crystal Structure Refinement Using X-Ray Diffraction Method
The x-ray data of the crystal was recorded using an Enraf Nonious CAD4 X-ray and BRUKER AXS Kappa Apex 2 diffractometer. XRD analysis was carried out at the Indian Institute of Technology-Madras, Chennai. A crystal of suitable size (0.3 × 0.1 × 0.1 mm 3 ) was inspected for single crystallinity using a LEICA DMLSP polarizing microscope and mounted on a Kappa Apex2, CCD diffractometer equipped with graphite monocromated Mo (Kα) radiation, (λ = 0.71073 Å). The unit cell parameters were obtained using reflections scanned from three different zones of the reciprocal lattice. The intensity data were collected using ω and ϕ scan with a frame width of 0.5 • . The frame integration and data reduction were performed using Bruker SAINT-Plus (Version 7.06a) software. Multiscan absorption corrections were applied to the data using SADABS (Bruker axs) software (this data published in an Indian patent, 2007) [11,13].

Cell Culture
The MCF-7 human breast cancer cell line was obtained as a gift from Mahatma Gandhi-Doerenkamp Center (MGDC), National Center for Alternatives to Animal Experiments (NCAAE), Bharathidasan University, India. Vero and V79 hamster lung fibroblast cell lines were obtained from the National Center for Cell Sciences (NCCS), Pune, India. RPMI-1640 supplemented with 10% (v/v) heat inactivated Fetal Bovine Serum (FBS), 2 mM l-glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin was used to culture the cells at 37 • C in a humidified atmosphere of 5% CO 2 (Thermo Scientific, Waltham, MA, USA).

In Vitro Cytotoxicity Assay Using 3-(4,5-Dimethylthiazol-2yl)-2,5-Diphenyl Tetrazolium Bromide (MTT)
MCF-7 cells were seeded (1 × 10 4 cells/mL) in a 96-well culture plate and cultured for 24 h before treatment. Vero cells and V79 hamster lung fibroblast were also cultured using the same protocol. After 24 h, nymphayol was treated with increasing concentrations (0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 µM) and continued to incubate for the next 24 h and 48 h, respectively. Doxorubicin and tamoxifen were used as the positive control. After incubation, 20 µL of mitochondrial dehydrogenase enzyme specific dye MTT (1 mg/mL) was added to the treated cells and incubated in the dark for 4 h at 37 • C. The reaction of MTT with viable cell mitochondrial dehydrogenase and produced purple formazan crystals were dissolved using 100 µL DMSO [31]. Then, the plates were absorbed under 492 nm using a micro plate reader.

Measurement of Intracellular Reactive Oxygen Species (ROS)
Cellular reactive oxygen species (ROS) was quantified using 2 ,7 -dichlorofluorescin diacetate (DCFH-DA) [32]. MCF-7 cells were cultured in a 24-well plate and treated with 0.7, 1.4, and 2.8 µM nymphayol. In addition, MCF-7 cells were treated with the positive control of 20 mM N-acetyl cysteine. After 48 h incubation, the cells were washed twice with HBSS and then incubated in 2 mL of the DCFH-DA working solution at 37 • C for 30 min. The stable compound DCFH-DA diffuses into the cell, then hydrolyzes to form DCHF by the action of intracellular esterase. The presence of hydrogen peroxide or low molecular-weight peroxides present in cell oxidizes DCHF to the highly fluorescent green colored 2V,7V-dichlorofluorescein (DCF) compound. The fluorescent green color was measured using a SpectraMax Gemini XS fluorometer (Molecular Devices, Cambridge Scientific, Watertown, MA, USA) with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

Apoptosis Related Cellular and Nuclear Morphology Analysis
Cellular morphology for characteristic apoptotic and necrotic morphological changes after nymphayol treatment were determined using propidium iodide (PI) and acridine Orange/ethidium bromide (AO/ErBr) described by Leite et al. [33]. Briefly, MCF-7 cells were treated with nymphayol at 0.7, 1.4, and 2.8 µM for 48 h in 24 well plates. After incubation, cells were rinsed with PBS and stained with 500 µL of PI (1 mg/mL) or AO:ErBr (1:1.4 mg/mL) solutions. Within a few seconds of staining, cells were gently rinsed with PBS and images were captured using an inverted fluorescent microscope (Carl Zeiss, Jena, Germany) fitted with a 530/620 nm filter and observed at 200× magnification. The percentage of apoptotic and necrotic cells were determined by using a random sample of 300 stained cells, examined under inverted fluorescence microscope, and the pathological changes was counted manually.

Terminal Deoxynucleotidyl Transferase (TdT)-Mediated dUTP Nick End Labeling (TUNEL) Assay
Cells were allowed to grow on cover slips and treated with nymphayol at 0.7, 1.4 and 2.8 µM for 48 h along with a control. After 48 h, the cells were fixed with 4% paraformaldehyde, and DNA fragmentation was observed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling technique as per the manufacturer's protocol. The results were presented as a representation from a series of three separate experiments. The cDNA was directly prepared from nymphayol treated cells using a Fastlane®Cell cDNA kit (QIAGEN, Hilden, Germany) after 48 h. The mRNA expression levels of oxidative stress-related genes [cytochrome P450 1A (CYP1A)], glutathione peroxidase (GPx), glutathione synthase kinase 3 beta (GSK3β), tissue necrotic factor-alpha (TNF-α), and nuclear factor kappa B (NF-κB), tumor suppressor genes [cyclin dependent kinase inhibitor 2A (Cdkn2A), retinoblastoma protein 2 (pRb1), p53, and murine double minute 2 (mdm2)], apoptotic genes (B-cell associated X (Bax), B-cell lymphoma-2 (Bcl-2), cyclin dependent kinase inhibitor 1A (Cdkn1A), caspases, and proliferating cell nuclear antigen (PCNA) as well as the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were assayed using gene-specific SYBR Green-based QuantiTect®primer assays (QIAGEN, Germany). Primer sequences for the antioxidant and apoptosis related genes are provided in Table 2. The gene expression level was then calculated as previously described by Yuan et al. [34]. To determine the relative expression levels, the following formula was used: ∆∆Ct (comparative threshold) = ∆Ct (Treated) − ∆Ct (Control).

Statistical Analysis
SPSS/11.5 software was used for the statistical significance evaluation. The values were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test [35]. All results were four replicates in each group (mean ± SD) and the differences were presented statistically significant at p ≤ 0.01 and p ≤ 0.001.

Conclusions
Nymphayol possesses potent anti-proliferative effects in breast cancer MCF-7 cells. Nymphayol, a sterol triterpenoid, has been found to induce apoptosis in human breast cancer cells by the modulation of mitochondria mediated pathways involved and the activation of caspases linked with the apoptotic mechanism. The mechanistic anticancer effects were associated with the early expression of CDKN2A and pRb2 and activation of p53 and caspases. High expression of CDKN2A arrest p53-mdm2 amalgamation led to a higher availability of active p53. The sterol triterpenoid, nymphayol may play a complementary role against breast cancer therapy or synergistic role with currently used anticancer drugs for chemoprevention.

Conflicts of Interest:
The authors declare that they have no conflicts of interest.