Research ArticleExperimental Studies

Combinational Anti-tumor Effects of Chemicals from Paeonia lutea Leaf Extract in Oral Squamous Cell Carcinoma Cells

SHINSUKE NAKAMURA, YOSHIKI MUKUDAI, JUNICHIRO CHIKUDA, MEILIN ZHANG, HIDEYUKI SHIGEMORI, KAZUNAGA YAZAWA, SEIJI KONDO, TOSHIKAZU SHIMANE and TATSUO SHIROTA
Anticancer Research December 2021, 41 (12) 6077-6086; DOI: https://doi.org/10.21873/anticanres.15427

Abstract

Aim: We identified chemical components that exhibited antitumor activity against oral squamous cell carcinoma (OSCC) cells and examined their effective concentrations and additive and/or synergistic effects in combinational usage on the proliferation, apoptosis and cell cycle of OSCC cells. Materials and Methods: Using high-performance liquid chromatography, nuclear magnetic resonance spectroscopy and electrospray ionization-mass spectrometry, we identified the main chemical components of the methanol extracts from Paeonia lutea. We investigated the pharmaceutical effects of those components on the proliferation, apoptosis, and cell cycle of an OSCC cell line, SAS, using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and caspase assays, as well as flow cytometry cell cycle analysis. We also examined the effects of those components on the mitogen-activated protein kinase signal transduction pathway by western blotting. Finally, the effects on normal human epidermal keratinocyte cells were also examined in similar experiments. Results: Three chemicals have been identified in P. lutea leaves using high performance liquid chromatography: gallic acid methyl ester (GAME), pentagalloyl glucose (PGG) and paeoniflorin (PF). Both GAME and PGG significantly suppressed cell proliferation, and their combined effects were synergistic, while the effect of PF was minimal. However, those chemicals did not induce apoptosis. Cell cycle and western blotting analysis showed that the suppressive effects on cell proliferation resulted from G2 arrest and the suppression of phosphorylation of Akt/PKB. No effect was identified on normal human epidermal keratinocyte cells. Conclusion: These results indicate that GAME and PGG are the main chemical components of P. lutea leaves that have potential anti-cancer therapeutic effects.

Key Words:

Head and neck cancers are the sixth most common malignancies in the world, accounting for more than 500,000 new cases every year (1). Among them, most oral cancer is squamous cell carcinoma (SCC). The occurrence, progression, and treatment of oral cancer occasionally cause losses of function such as eating, swallowing and talking, and reduce patients’ quality of life (QOL). SCC is treated with radiation therapy and chemotherapy in addition to surgery, and in recent years, patients wishing to preserve functioning and QOL have preferred radiation therapy and chemotherapy (2). However, anticancer drugs such as cis-diamminedichloro-platinum (II) (CDDP) cause serious side effects (3-8). Therefore, it is imperative to find chemotherapeutic agents with minimal side effects. In our recent study (9), we focused on Paeonia lutea leaves, which are used in traditional Chinese medicine (10, 11). We revealed that methanol and butanol extracts of P. lutea leaves decrease the proliferation of oral squamous cell carcinoma (OSCC) cells in an anchorage-independent manner, and that those extracts repress bcl-2 expression in an anchorage-independent culture, resulting in apoptosis via the mitochondrial apoptotic pathway. In addition, we also found that those extracts alter the expression of integrin subunits and reduce chemotaxis and the adhesion of extracellular matrix (ECM), consequently suppressing invasion and metastasis. However, both the chemical composition and pharmaceutical mechanism of the extracts remain unclear.

In the present study, we identified chemical components that exhibited antitumor activity against OSCC cells and examined their effective concentrations and additive and/or synergistic effects in combinational usage on the proliferation, apoptosis, and cell cycle of OSCC cells. Furthermore, we investigated the effects on the Akt/PKB signaling pathway. We found that chemical components extracted from P. lutea leaves are potentially potent novel anticancer agents.

Materials and Methods

Identification of chemical components from methanol extracts of P. lutea. P. lutea leaves were collected from the Tsukuba Peony Garden (Tsukuba, Ibaraki, Japan). After pulverization of the leaves, methanol was added, extraction was performed at room temperature for one week, then the extract was obtained by suction filtering. This process was repeated twice. The resultant extracts were solidified under reduced pressure, concentrated, and dried at 35°C using a rotary evaporator to obtain the methanol extract. The extract was stored at –30°C until subsequent experiments. The methanol extract was partitioned between ethyl acetate and water, and the water-soluble portion was then partitioned with butanol. Each soluble portion was depressurized with a rotary evaporator to dryness. The ethyl acetate soluble portion was separated into eight fractions (PLE-1-8) by silica gel column chromatography (12-14). Purification was performed using high performance liquid chromatography (HPLC) to obtain simpler compounds. Furthermore, the butanol soluble fraction (PLB) was also separated into 9 fractions (PLB-1 to 9) by silica gel column chromatography (15). Structural analyses of the substances were performed using nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization-mass spectrometry (ESI-MS) (ADVANCE 500, Bruker, Billerica, MA, USA and Waters Synapt G2, Waters, Milford, MA, USA).

Cell cultures. SAS cells (human oral squamous cell carcinoma-derived cell lines) (16) were grown in high-glucose Dulbecco’s modified Eagles medium (HDMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Carlsbad, CA, USA) at 37°C in an atmosphere containing 5% CO2 and 100% humidity. Normal human epidermal keratinocytes (NHEK) cells were purchased from PromoCell (PromoCell, Heidelberg, Germany) and used at early passages. Cells were maintained in Keratinocyte Growth Medium 2 (PromoCell) supplemented with 0.06 mM calcium chloride and cultured as described above. Gallic acid methyl ester (GAME), pentagalloyl glucose (PGG), and paeoniflorin (PF) (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 100% ethanol. For addition of chemical solutions and 100% ethanol (as a vehicle control) to a culture, the final ethanol concentration was less than 0.1% in order to minimalize the cytotoxicity of ethanol.

Cell growth and apoptosis assays. Five thousand cells were seeded in each well of a 96-well tissue culture plate. After 24 h, 10 μM of treatment (individual or combined) were added to each well and incubated for an additional 48 h. The cells were then subjected to tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously (17), and to caspase 3/7 assays using a Caspase-Glo (Promega, Madison, WI, USA) and GloMax-Multi+ Detection System (Promega) according to the manufacturer’s protocol.

Cell cycle analysis. Two million cells were seeded in a 6-cm cell culture dish. After 24 h, 10 μM of chemicals (individual or combined) were added to each dish and incubated for an additional 24 hs. The cells were then subjected to cell cycle analysis using the Tali cell cycle solution (Life Technologies), and the cell cycle was determined using a Tali cell cycle kit and Image Based Cytometer (Life Technologies) according to the manufacturer’s protocol.

Protein preparation and western blotting analysis. Rabbit polyclonal extracellular signal regulated kinase (ERK) 1/2, anti-phospho (p)-ERK1/2, anti-Akt, anti-p-Akt, anti-c-jun-N-terminal-kinase (JNK), anti-p-JNK and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), and horseradish peroxidase-conjugated donkey anti-rabbit-IgG 2nd antibody was purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). The cells were seeded in a 24-well tissue culture plate at a density of 1×106 cells/well and were incubated with GAME, PGG and PF, alone or in combination, for 24 hours in the presence of 10% FBS. Then, 20 ng/ml of recombinant human epidermal growth factor (EGF) (R&D Systems, Minneapolis, MN, USA) was added to the culture to stimulate phosphorylation of Akt and MAPKs. After 0, 1, and 2 hours, total cellular protein was prepared as described previously (18). Protein concentrations were measured using Quick Start Bradford Reagent (Bio-Rad, Hercules, CA, USA) and bovine serum albumin (BSA, Bio-Rad) as a standard, and aliquots were stored at –80°C until use. Western blotting analysis was carried out as described previously (19). Briefly, 20 μg of total cellular protein was subjected to sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) on a 4 to 20% gradient gel (Bio-Rad); the blot was then transferred onto a polyvinylidene difluoride (PVDF) membrane using an iBlot 2 Dry Blotting System (Life Technologies). The chemiluminescence signals were visualized using Amersham ECL Western Blotting Detection Reagents (GE Healthcare) and a ChemiDoc XRS Plus ImageLab System (Bio-Rad).

Statistical analysis. Unless otherwise specified, all experiments were repeated at least three times, with similar results being obtained. A statistical analysis of the repeatability of the assay results was carried out using a paired Student’s t-test. Data are expressed as means±standard deviation of triplicate datasets.

Results

Isolation of main chemical components from P. lutea leaves using high performance liquid chromatography. A schema of the extraction process from P. lutea leaves is shown in Figure 1. P. lutea leaves (dry weight 350 g) were pulverized, methanol (4,000 ml) was added, and extraction was performed at room temperature for a week. Then, the extract was obtained by suction filtering. This process was repeated twice. Subsequently, the extract was solidified under reduced pressure, concentrated, dried at 35°C using a rotary evaporator to give a methanol extract (80.14 g), and stored at –30°C until later experiments. The methanol extract was partitioned 3 times between ethyl acetate (800 ml) and water (800 ml). Next, the water-soluble portion was partitioned with butanol (600 ml) 3 times. Each soluble portion was depressurized with a rotary evaporator to dryness to obtain the ethyl acetate soluble portion (PLE, 28.46 g), butanol soluble portion (PLB, 20.52 g) and water-soluble portion (PLW, 16.51 g). Of the PLE (28.46 g), 4.036 g was subjected to silica gel column chromatography to separate it into 8 fractions (PLE-1 to 8). PLE-5 and 8, containing the simpler compounds, were purified using HPLC and were suggested to contain similar compounds respectively using 1H and 13C NMR spectroscopy; they were then refined and structurally analyzed. The 1H NMR spectrum of PLE-5 was compared with that of GAME, and a characteristic signal of-OCH3 and H at positions 2, 6 was observed in each, at around 3.8 and 7.0 ppm, respectively. In the 1H and 13C NMR spectra of PLE-5, each signal was identical to that in the literature (12, 13). Therefore, PLE-5 was identified as GAME, and the data suggested that PLE-6 also contained GAME. For 1H and 13C NMR spectra of PLE-8-7, each signal was identical to that of PGG seen in the literature (13, 14), so consequently PGG was identified as the main component of PSE-8-7 (Figure 1A).

Figure 1.
Figure 1.

Schema of the isolation of chemical components from P. lutea leaves. (A) The extraction procedure for gallic acid methyl ester (GAME) and pentagalloyl glucose (PGG) from the ethanol layer extracted from P. lutea leaves. (B) Extraction procedure for paeoniflorin (PF) from the butanol layer extracted from P. lutea leaves. For details, see Results.

The butanol soluble fraction was also separated into 9 fractions (PLS-1 to 9) by silica gel column chromatography. PLB-4 was also structurally analyzed by 1H and 13C NMR spectroscopy. In the 1H and 13C NMR spectra of PLB-4, each signal was identified as matching PF according to previous data (15) (Figure 1B).

GAME and PGG decrease proliferation of SAS cells in individual and combined treatments. Several traditional Chinese ingredients, including GAME, PGG and PF have been reported to exert chemotherapeutic effects and suppress proliferation of various tumors (10, 20-28). Thus, we examined the effects of these chemicals on the viability and proliferation of SAS cells (Figure 2). First, the effective concentration of each chemical was assessed by MTT assay (Figure 2A and B). GAME showed the most significant inhibitory effect at a concentration of more than 10 μM, and PGG also had a significant effect. However, PF barely decreased cell proliferation. The calculated half maximal inhibitory concentration (IC50) values of GAME, PGG and PF for SAS cells were 152, 208 and >1,000 μM, respectively. Since previous studies (10, 20-32) have reported that the effective concentration of those chemicals is 10 μM, this concentration was used in the following experiments. Next, the combinational effects of GAME, PGG, and PF were assessed by MTT assay (Figure 2C). The proliferation-suppressive effect of GAME and PGG alone was significantly enhanced by the combined administration of both. However, PF did not enhance the effect of GAME or PGG. Notably, the combination of GAME, PGG and PF showed the most significant proliferation-suppressive effect. These results suggest that GAME and PGG could be the main components of P. lutea leaves, with the potential to enhance each other to suppress the proliferation of OSCC cells, while the effect of PF is less and at most adds to that of GAME and PGG.

Figure 2.
Figure 2.

Combinational effects of GAME, PGG, and PF on proliferation of SAS cells. (A and B) Individual IC50 values of GAME, PGG, and PF. SAS cells were cultured in the presence or absence of 1, 10, and 100 μM of GAME, PGG, and PF alone for 24 h. Then, the cells were subjected to MTT assay (A) and the IC50 calculated (B). (C) Combination effects of GAME, PGG, and PF. SAS cells were cultured in the presence or absence of 10 μM of GAME, PGG, and PF alone or in combination for 24 h, then subjected to MTT assay. Data in (A) and (C) are means±standard deviations of 3 cultures shown as relative values, with the control designated as 1. *p<0.05 vs. control.

Neither GAME, PGG, nor PF induce apoptosis, but GAME and PGG cause G2 arrest. Based on the proliferation-suppressive effects of GAME, PGG and PF described above, we examined whether the effects were caused by the induction of apoptosis and/or alterations of the cell cycle (Figure 3). Neither GAME, PGG, nor PF in individual treatments changed caspase 3/7 (a marker for early apoptosis) activity, and neither did combinational treatments (Figure 3A). We then examined the cell cycle distribution (Figure 3B). GAME and PGG administration alone for 24 hours induced G2 arrest, whereas PF only had a slight effect. The combined administration of GAME and PGG also induced G2 arrest, causing an additive or synergistic effect. Furthermore, PF hardly changed the induction of G2 arrest caused by GAME and PGG. Taken together, these results indicate that the proliferation-suppressive effects of GAME and PGG are mainly caused by the induction of G2 arrest of the cell cycle, and not by apoptosis.

Figure 3.
Figure 3.
Figure 3.

Combined effects of GAME, PGG, and PF on apoptosis and the cell cycle in OSCC cells. SAS cells were cultured in the presence or absence of 10 μM of GAME, PGG, and PF alone or in combination for 24 h, then subjected to caspase 3/7 assay (A) and cell cycle analysis (B). Data in (A) are means±standard deviations of 3 cultures shown as relative values, with the control designated as 1. *p<0.05 vs. control. In (B), data are the percentages of cells found in different cell populations, G0/G1, S, and G2/M.

PGG strongly suppresses the phosphorylation of Akt, and this effect is reinforced in the presence of GAME. The effects of GAME, PGG, and PF on major mitogenic signal pathways were investigated by western blotting; namely, we examined the activation of Akt and two major mitogen-activated protein kinases (ERK and JNK) by EGF stimulation (Figure 4). While the presence of PGG alone in culture reduced the phosphorylation of Akt, GAME and PF showed weaker reduction effects on the phosphorylation of Akt than PGG did. The combination of PGG and GAME had an additional effect, while the addition of PGG and PF had little effect. Furthermore, the combination of PGG, GAME and PF had little effect on the suppression of Akt phosphorylation. This suggests that PGG and GAME could mainly suppress the phosphorylation of Akt, and that the effect of PF might be weak and collateral. However, none of the chemicals modulated the phosphorylation of ERK or JNK, suggesting that PGG and GAME, but not PF, mainly act as down-regulators and inhibit the activation of the Akt signaling pathway.

Figure 4.
Figure 4.

Effects of GAME, PGG, and PF on the phosphorylation of Akt, ERK, and JNK in OSCC cells. SAS cells were incubated with 10 μM of GAME, PGG, and PF alone or in combination 24 h in the presence of 10% FBS, then 20 ng/ml of EGF was added. After 0, 1, and 2 h, total cellular protein was collected and subjected to western blotting for phosphorylated (p-) and total Akt, ERK and JNK. GAPDH was used as a loading control.

GAME, PGG and PF had no effect on cell proliferation and apoptosis in NHEK cells. GAME, PGG and PF have been reported to exert chemotherapeutic effects and suppress the proliferation of various tumors (10, 20-28). However, to the best of our knowledge, there are no reports using normal cells. Thus, we utilized NHEK cells as normal keratinocytes in order to investigate the effects of these chemicals on viability, proliferation and apoptosis. First, the effects on viability and proliferation were evaluated using an MTT assay (Figure 5A). Neither GAME, PGG, nor PF in individual treatments changed MTT activity, and nor did combinational treatments. Next, these chemicals were also evaluated using the caspase3/7 assay to determine whether they affected the induction of apoptosis. Subsequently, little effect on caspase3/7 activity was observed, similar to the MTT assay (Figure 5B). Taken together, these results indicated that GAME, PGG, and PF have almost no effect on growth inhibition and apoptosis in normal cells, suggesting few side effects as anti-cancer drugs.

Figure 5.
Figure 5.

Combinational effects of GAME, PGG, and PF on proliferation and apoptosis of NHEK cells. NHEK cells were cultured in the presence or absence of 10 μM of GAME, PGG, and PF alone or in combination for 24 h, then subjected to MTT (A) or caspase 3/7 assays (B). Data are means±standard deviations of 3 cultures shown as relative values, with the control designated as 1. *p<0.05 vs. control.

Discussion

Oral cancer therapy research is essentially aimed at identifying new anticancer drugs that selectively target tumor cells without cytotoxic effects on normal cells and tissues. Although many clinically approved anticancer drugs (i.e., CDDP and 5 fluorouracil) used to treat OSCC are available, they do not always induce a positive response, because tumor cells are not dependent on a single receptor or signal transduction pathway for cell survival, proliferation, and growth. Therefore, finding new chemicals with anticancer effects against OSCC cells is of utmost importance. Previous studies have reported that ethanol and butanol extracts of P. lutea leaves repress the metastasis of SCC cells. However, to the best of our knowledge, there have been no investigations of the chemical components of P. lutea leaf extracts and their effective concentrations against OSCC cells. Therefore, in the present study, we first identified 3 main components of P. lutea leaves using HPLC, NMR, and ESI-MS: GAME, PGG, and PF (Figure 1). These chemicals are present in several herbs in traditional Chinese medicine, such as Rhus chinensis and Paeonia suffruticosa. In addition, GAME has been reported to induce anti-tumor effects through the suppression of CD4+/CD25+ regulatory T cell function (20), to act as an anti-inflammatory against arthritis (21) and to suppress glioma cell survival and migration through the suppression of Akt and ERK 1/2 phosphorylation (22). PGG has been shown to exhibit in vivo anti-cancer effects against prostate cancer (23, 24), and lung cancer (25). Similarly, it has been reported to have in vitro inhibitory effects on the growth and/or invasion of breast cancer (26), and leukemia (27). PF can inhibit pancreatic cancer growth by up-regulating HTRA3 (high temperature requirement) (28). In the present study, we first conducted in vitro experiments to determine the anti-tumor effects in OSCC cells by individual and combined administrations of these three chemicals.

Each of the chemicals decreased the proliferation of SAS cells (Figure 2), but none of them induced apoptotic cell death (Figure 3A), which has been observed in other cancer cells (33). Interestingly, the proliferation-decreasing effects resulted from G2 arrest in the cell cycle (Figure 3B). Loss of checkpoint control that regulates the normal passage of the cell cycle is thought to be involved in the progression of cancer cells. In particular, the G2/M checkpoint is a prominent target of some anticancer drugs and their adjuncts (34-36). Cell cycle arrest participates in the anti-cancer process of many drugs, such as cantharidin (35), curcumin (37), wentilactone A (38) and celastrol (39). Thus, we suspect that cell cycle arrest plays a pivotal role in the inhibitory effect of GAME and PGG in SAS cells. Therefore, we hypothesized that the G2 arrest was caused by effects of the compounds on the phosphorylation profiles of Akt and MAPKs, such as ERK and JNK. We discovered that these chemicals were capable of suppressing the phosphorylation of Akt individually, but the phosphorylations of ERK and JNK, were not affected (Figure 4). Akt has been considered a key regulator for various signal transduction pathways, modulating multiple processes such as cell survival, proliferation and growth (40, 41). In addition, clinical studies have clarified that cancer patients whose tumors have increased Akt expression tend to have more invasive and metastatic disease (42, 43). Furthermore, we also examined the synergistic effects of the compounds. As a result, GAME and PGG had a synergistic effect on G2 arrest of the cell cycle and on suppression of Akt phosphorylation; however, PF barely had an effect. These results indicated that GAME and PGG, and not PF, are the main components of P. lutea leaves that exert a proliferation-decreasing effect in OSCC cells.

The effects of these chemicals on normal keratinocyte cells were examined for clinical use, since there are no reports using normal cells to the best of our knowledge. The results indicated that GAME, PGG and PF have no effect on growth inhibition and apoptosis in NHEK cells.

In summary, we report here for the first time that GAME and PGG induce G2 cell cycle arrest and that this results from suppressing Akt phosphorylation. These findings suggest an important role for GAME and PGG in the anti-cancer effects of P. lutea extract in OSCC cells. However, the effect of PF was minimal. As shown by the IC50 (Figure 2B), PF might not show an antitumor effect unless its concentration is 1000 μM or more. In fact, several studies (28, 32) have used PF at concentrations of 1000 μM or higher. Regardless, in the present study, we revealed that P. lutea leaf extract is a novel and promising therapeutic agent for the treatment of OSCC. However, it remains unclear if the in vivo anti-cancer activities of GAME, PGG and PF are mediated by direct actions or through metabolites or other indirect mechanisms. In previous studies, the anti-tumor effects of P. lutea leaves were due to apoptosis induction (9); yet no induction of apoptosis was observed in the present study. P. lutea, a natural product, contains many chemical substances in addition to the three identified here, and its inhibitory effects may be due to the interactions of additional compounds. We must therefore consider the influence of chemicals that could not be separated or identified by HPLC. Further and more detailed studies, such as in vivo experiments, are required, and these studies are ongoing.

Acknowledgements

We thank all the staff at the Department of Oral and Maxillofacial Surgery, School of Dentistry, Showa University; the Graduate School of Life and Environmental Sciences, University of Tsukuba; and the Division of Healthfood Science, Institute for Nanoscience and Nanotechnology, Waseda University for helpful suggestions. We also appreciate Ms. Miho Yoshihara for her secretarial assistance. This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (KAKENHI C) to YM (15K11301).

Footnotes

  • Authors’ Contributions

    SN significantly contributed to the present study and performed experiments, prepared the figures and wrote the manuscript. YM, together with KY and HS, conceived and designed this study, and YM also applied for the grant supporting the study. JC helped perform biochemical assays. KY prepared the materials and purified the P. lutea extracts. MZ and HS analyzed and identified the chemicals from P. lutea leaves. SK, ToS and TaS helped draft the figures and manuscript. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests.

  • Received July 31, 2021.
  • Revision received November 4, 2021.
  • Accepted November 5, 2021.

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Combinational Anti-tumor Effects of Chemicals from Paeonia lutea Leaf Extract in Oral Squamous Cell Carcinoma Cells
SHINSUKE NAKAMURA, YOSHIKI MUKUDAI, JUNICHIRO CHIKUDA, MEILIN ZHANG, HIDEYUKI SHIGEMORI, KAZUNAGA YAZAWA, SEIJI KONDO, TOSHIKAZU SHIMANE, TATSUO SHIROTA
Anticancer Research Dec 2021, 41 (12) 6077-6086; DOI: 10.21873/anticanres.15427
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