Paris saponin VII suppressed the growth of human cervical cancer Hela cells
© Zhang et al.; licensee BioMed Central Ltd. 2014
Received: 16 May 2014
Accepted: 21 July 2014
Published: 15 August 2014
Saponins of several herbs are known to induce apoptosis in many cancer cells. The present study aimed to investigate the growth inhibitory effect of Paris saponin VII (PS VII), a kind of steroidal saponins from Chonglou (Rhizoma Paridis Chonglou), on the human cervical cancer cell line Hela and the relative molecular mechanisms.
Hela cells were exposed to different concentrations of PS VII (1 to 100 μM). Inhibition of cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-ethynyl-2′-deoxyuridine (EdU) assays. The amount of apoptotic cells was evaluated by flow cytometric analysis. And the protein level of cleaved caspase-3, cleaved caspase-9, Bax, and Bcl-2 was evaluated by Western blot.
The half maximal inhibitory concentration (IC50) value of PS VII for the growth inhibition of Hela cells was 2.62 ± 0.11 μM. PS VII increased the expression of caspase-3, caspase-9, and Bax while decreased that of Bcl-2, suggesting that PS VII may induce apoptosis through intrinsic apoptotic ways.
These data indicate that PS VII has the potential for the treatment of cervical cancer.
Cervical cancer is the third most common type of cancer in women worldwide, and a leading cause of cancer-related mortality, with an estimated 275,000 deaths in 2008 . Despite considerable advances in surgical techniques and neoadjuvant chemotherapy, the survival rate of cervical cancer is still not remarkably improved. Platinum-based anticancer agents, represented by cisplatin, have therapeutic properties for patients with cervical cancer; however, toxicities, including myelosuppression, leukopenia, ototoxicity, neurotoxicity, and nephrotoxicity [2, 3], may limit their long-term use. Therefore, it is required to develop new drugs with a more specific effect and low toxicity.
Natural products have been shown to be excellent and reliable sources for the pharmaceutical development of anticancer drugs . Chonglou (Rhizoma Paridis Chonglou) is the root of Paris polyphylla. Phytochemical and pharmacological studies suggest that Chonglou has a wide range of medicinal activities, including anticancer, immunoregulatory, and cardiovascular effects . It has been applied in the treatment of malignant lymphomas, lung cancer, nasopharyngeal carcinoma, brain tumors, and digestive system carcinomas. Polyphyllin and extracts from Chonglou show good antitumor effects in vitro, through inducing apoptosis, affecting cell cycle distribution, inhibiting angiogenesis, and regulating the immune function [6, 7].
In the current study, we investigated the mechanism underlying the cytotoxic effects of a kind of steroidal saponins from Chonglou (Rhizoma Paridis Chonglou), namely, Paris saponin VII (PS VII), and its antitumor properties on Hela cell lines. The results indicated that PS VII inhibited the growth of the cell effectively. It upregulated cleaved caspase-3, cleaved caspase-9, and Bax expression in Hela cells. These preclinical studies indicated that PS VII may have potentials in the treatment of cervical cancer.
Chemicals and reagents
Hela, a human cervical cancer cell line, was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. The cells were maintained in a humidified atmosphere at 37°C in 5% CO2.
Cell proliferation assays
Cell viability was determined by MTT and 5-ethynyl-2′-deoxyuridine (EdU) assays. The Hela cells were seeded at a density of 2 × 104 cells/well in 96-well plates full of medium containing PS VII at various concentrations (1, 3, 10, 30, and 100 μM). After 24-h treatment, 20 μl of MTT solution (0.5 mg/ml) was added into each well and the mixture was incubated at 37°C for 4 h. The cells were then washed thrice with phosphate-buffered saline (PBS), and the formazan was resuspended in 150 μl DMSO. Absorbance was measured at 490 nm by using a Bio-Rad ELISA reader (Hercules, CA, USA). Half maximal inhibitory concentrations (IC50) of PS VII were determined by curve fitting analyses using Prism software (GraphPad Software, San Diego, CA, USA). For EdU assay, Hela cells (1 × 105 cells/well) were seeded in 24-well plates. After culture in a serum-free medium for 24 h, various concentrations of PS VII (0.8, 1.6, and 2.4 μM) were added and the cells were kept for another 24 h. Cell viability was determined using an EdU assay kit. Stained sections were examined under a microscope (Nikon, Tokyo, Japan). All experiments were repeated independently thrice.
Flow cytometric assays for annexin V
Hela cells were plated at a density of 5 × 106 per 10-cm2 dish and cultured with different concentrations of PS VII for 24 h. A total of 1 × 106 to 3 × 106 cells were washed with ice-cold PBS and resuspended in 1× binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2] at a concentration of 1 × 106 cells/ml. Five microliters of annexin V-fluorescein isothiocyanate (FITC) solution (25 μg/ml) and 5 μl of dissolved PI (250 μg/ml) were added to 100 μl of the cell suspensions. The cells were then gently vortexed and incubated at room temperature in the dark for 15 min. Then, 400 μl of ice-cold binding buffer was added and mixed gently before the cell preparations were examined by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA).
Caspase-3 activity in Hela cells was detected using the Caspase-3 Activity Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China). The assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3, resulting in the release of a pNA moiety. Absorbance values were measured at 405 nm. Results were adjusted to the total protein content, and activity was expressed as nanomoles of pNA per milligram of total protein.
Western blot analysis
Hela cells were incubated with 0.8, 1.6, or 2.4 μM of PS VII for 24 h. The cells were then harvested and resuspended in lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China, plus the protease inhibitors leupeptin 10 μg/ml, aprotinin 10 μg/ml, and PMSF 0.1 mmol/l). Protein lysates (30 μg) were electrophoresed on 15% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA) and blocked with Tris-buffered saline (TBS) buffer containing 0.05% Tween-20 and 5% nonfat milk for 2 h at room temperature. The membranes were then incubated overnight at 4°C with various primary antibodies, followed by HRP-conjugated secondary antibodies. After washing the membranes thrice for 10 min in TBS buffer containing 0.05% Tween-20, the immunoreactive bands were detected using the Immobilon Western HRP Substrate (Millipore, Billerica, MA, USA). The experiment was repeated independently three times.
Data were expressed as mean ± standard deviation (SD). Statistical analyses were done by using the one-way ANOVA test and Fisher’s least significant difference (LSD) t test to compare the different groups. Probability (P value) of less than 0.05 was considered to be statistically significant.
PS VII inhibited the cell growth of Hela cells
PS VII induced cell apoptosis in Hela cells
PS VII acted on the intrinsic apoptotic pathway
Chonglou (Rhizoma Paridis Chonglou) has been traditionally used for the treatment of microbial infection, hemorrhage, menometrorrhagia, and venomous poison [8–11]. The main bioactive ingredients of Chonglou are the steroidal saponins. Phytochemical and pharmacological studies have further revealed a novel therapeutic role as an anticancer agent for these steroid saponins . However, evidence-based researches into the mechanism underlying the cytotoxic effects of steroidal saponins are still undefined.
In the current study, we found that PS VII, a kind of steroidal saponins from Chonglou, could inhibit the growth of Hela cells in a concentration-dependent manner. We then attempted to investigate whether the growth-inhibiting effect of PS VII was caused by apoptosis.
Apoptosis, which is characterized by membrane blebbing, shrinkage of the cytoplasm and nucleus, and DNA fragmentation , helps to keep tissue homeostasis by eliminating potentially deleterious cells. Deregulated apoptotic cell death would lead to diseases such as cancer. In cancer cells, the incidence of apoptosis and the rate of cell proliferation are uncontrolled, which would cause tumor invasion. Therefore, it will be a reasonable way to induce cancer cells to undergo apoptosis by various anticancer agents.
Results of flow cytometric analysis showed that PS VII increased the apoptosis of Hela cells in a concentration-dependent manner.
Mammalian cells undergo apoptosis mainly through two ways: the death receptor-mediated or extrinsic pathway and the mitochondrial-mediated or intrinsic pathway . Caspases, a family of cysteine proteases, are central regulators of apoptosis. Caspase-8 is involved in the extrinsic and caspase-9 in the intrinsic pathway. After activation, they would cleave and activate downstream effectors such as caspase-3, which subsequently cleave cytoskeletal and nuclear proteins [15, 16]. Results of caspase-3 activity assay showed that PS VII treatments led to increased caspase-3 activity in Hela cells. And these effects seemed to be concentration dependent. Western blot analysis demonstrated that PS VII treatments caused increased cleaved caspase-3 and caspase-9 expression, but had no significant effect on cleaved caspase-8 expression. The cleavage of caspases was prevented by pretreatment of Hela cells with a pan-caspase inhibitor, Z-VAD-FMK. These findings suggest that PS VII induced the apoptosis of Hela cells through an intrinsic way involving caspase activation.
This was proved by decreased Bcl-2 expression and increased Bax expression. The Bcl-2 protein family, which plays an important role in the intrinsic pathway, is divided into two functional subfamilies: pro-apoptotic proteins (Bax and Bid) and anti-apoptotic proteins (Bcl-2 and Bcl-xL). The ratio of Bax/Bcl-2 appears to be a critical determinant of a cell’s threshold for undergoing apoptosis. Results obtained above were from in vitro experiments. In the next step, we will assess the effect of PS VII in vivo and find the possible mechanisms by using xenograft models.
In brief, the results reported herein demonstrated that PS VII inhibited the growth and induced the apoptosis of Hela cells effectively. One of the possible mechanisms involved is that PS VII triggered cell apoptosis via an intrinsic pathway depending on caspase activation. The precise signaling pathway remains to be further investigated. However, these data may provide an approach to treat cervical cancer.
This investigation was supported by Grant No. 2014JQ4152 from the Technology Gallery of Shaanxi Province and Grant No. 2012D45 from the Department of Public Health of Shaanxi Province.
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