Introduction

Oncology Reports

1021-335X 1791-2431

D.A. Spandidos

10.3892/or.2017.6139

or-39-02-0764

Articles

Ethanol extract of Patrinia scabiosaefolia induces the death of human renal cell carcinoma 786-O cells via SIRT-1 and mTOR signaling-mediated metabolic disruptions

Zeyan

1 * Tang

Yueqing

1 * Zhu

Shiqin

2 Li

Dawei

1 Han

Xiao

1 Gu

Gangli

1 Xing

Naidong

1 Ren

Juchao

1 Guo

Zhaoxin

1 Jiao

Wei

1 Yan

Lei

1 Xu

Zhonghua

1 Zhang

Wenhua

1Department of Urology, Qilu Hospital of Shandong University, Jinan, Shandong 250012, P.R. China 2Department of Biochemistry, Shandong University, Jinan, Shandong 250012, P.R. China

Correspondence to: Dr Zhonghua Xu or Dr Wenhua Zhang, Department of Urology, Qilu Hospital of Shandong University, Wenhuaxi Road 44, Jinan, Shandong 250012, P.R. China, E-mail: xuzhonghua1963@163.com, E-mail: summerdays123@126.com *

Contributed equally

022018

07122017

39 2 764 772 16042017 17102017

2018

Recently, natural plant extracts have shown tremendous potential as novel antitumor drugs. Patrinia scabiosaefolia, a traditional prescription for inflammatory diseases, has been reported to effectively suppress various types of cancers. However, the mechanisms underlying its antitumor properties remain elusive. In the present study, we investigated the antitumor effects of an ethanol extract of Patrinia scabiosaefolia (EPS) on human renal cell carcinoma 786-O cells. After 24 h of incubation with EPS, the cell viability and colony number of 786-O cells were significantly decreased in a concentration-dependent manner as compared to the control group as determined by MTT and colony formation assays, respectively. The necrotic rate and apoptotic rate in the EPS exposure group were significantly higher than these rates noted in the control group as revealed by LDH release assay and Hoechst 33342/PI double staining, respectively. At the concentration of 1.0 mg/ml, the necrotic and apoptotic rates reached 41.7±6.6 and 7.8±1.4%, respectively (P<0.01). However, the fluorescence intensity of intracellular calcium concentration ([Ca²⁺]_i) was markedly elevated from 0.029±0.0007 to 0.060±0.003 (P<0.001) after the intervention of EPS. Moreover, the fluorescence intensity of intracellular ROS in the EPS exposure group was significantly higher (0.074±0.005) compared to that observed in the control group (0.033±0.001, P<0.001), which was partly attenuated by the specific antioxidant N-acetylcysteine (NAC). Furthermore, our results demonstrated that EPS significantly downregulated the expression of SIRT-1 and obviously induced the dephosphorylation of mTOR. Moreover, combined treatment with the SIRT-1 inhibitor nicotinamide and EPS was able to significantly enhance the induction of necrosis and reduction in cell viability of 786-O cells noted following treatment with EPS alone. In summary, we conclude that EPS induced the death of 786-O cells via SIRT-1 and mTOR signaling-mediated metabolic disruptions, which provide novel insight into the application of natural plant extracts for the treatment of cancers.

Patrinia scabiosaefolia 786-O cells antitumor metabolic disruptions SIRT-1 mTOR

Introduction

Traditional Chinese medicine (TCM), including various natural plant extracts, has shown tremendous potential for the discovery of alternative drugs for the treatment of cancer (1,2). Fewer adverse effects and cost effectiveness endow TCM great advantages over Western chemotherapeutics (3). Patrinia scabiosaefolia, a perennial plant mainly distributed in East Asia, is extensively prescribed for various diseases, such as oxidative damage (4), inflammation (5), tumors (6) and edema (7). Of all the above-mentioned effects, antitumor activity is a prominent characteristic of Patrinia scabiosaefolia. Peng et al reported the Patrinia scabiosaefolia inhibited the growth of different cancer cell models including colorectal cancer mouse tumor tissues, HT-29 and U266 through G1/S cell cycle arrest, suppression of tumor angiogenesis and the STAT3 pathway, respectively (6,8,9). A previous study demonstrated that Patrinia scabiosaefolia induced the apoptosis of breast carcinoma MCF-7 cells without caspase-9 activation (10). However, the mechanisms underlying its antitumor properties emain elusive, and the antitumor effects of Patrinia scabiosaefolia deserve increased attention.

Recently, research which has focused on exploiting metabolic perturbations in the area of cancer therapy has been extensively investigated (11). ROS homeostasis serves as a critical factor for the survival of cancer cells. There is evidence indicating that cancer cells are more vulnerable to high levels of ROS (12), which further evokes a series of metabolic dysfunction, such as apoptosis, calcium overload and bio-macromolecule degradation (13–15). The calcium ion, as a ubiquitous secondary messenger, induces intracellular ROS generation. In turn, ROS can regulate the Ca²⁺ signaling pathway (14). Excessive accumulation of ROS and calcium overload can initiate the apoptotic pathway (16). The reciprocal interactions of the above three alterations intensively promote the death processes of cancer cells. Thus, manipulation of metabolic disruptions of cancer cells may be a promising target for cancer treatment.

SIRT-1, as an NAD⁺-dependent deacetylase, has been demonstrated to regulate extensive cellular metabolic processes including cell stress response, apoptosis and lifespan extension (17–19). A previous study indicated that excessive expression of SIRT-1 in large B-cell lymphoma was closely related to poor patient prognosis (20). Recently, SIRT-1 has been demonstrated to function as an oncoprotein and tumor promoter in various types of cancer cells (21,22). Mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, has been widely accepted to regulate cell growth and proliferation (23). Various chronic diseases, such as cancer, ageing and diabetes, are closely correlated with mTOR (24). There is evidence indicating that mTOR-dependent mechanisms are responsible for the tumorigenesis of many types of cancers (25). Thus, we speculated that SIRT-1 and mTOR may be involved in the EPS-induced antitumor effects.

In the present study, we investigated the antitumor effects of an ethanol extract of Patrinia scabiosaefolia (EPS) on 786-O cells. MTT assay, colony formation assay and Hoechst/propidium iodide (PI) staining were performed to detect the inhibition of proliferation and the pro-apoptotic effects of EPS. Intracellular ROS and Ca²⁺ were examined to identify the metabolic disruptions induced by EPS. However, we conducted western blotting to explore the expression of SIRT-1 and mTOR after EPS stimulation. Furthermore, the above antitumor effects were found to be augmented by the SIRT-1 inhibitor nicotinamide. Thus, we conclude that EPS induced the death of 786-O cells via SIRT-1 and mTOR signaling-mediated metabolic disruptions.

Materials and methods <sec> <title>Cell culture and reagents

Human renal cell carcinoma 786-O cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), and human proximal tubular cells (HK-2) were cultured in Dulbeccos modified Eagles medium (DMEM)/F12 (1:1) supplemented with 10% FBS (all from HyClone, Logan, UT, USA). Both types of cells were cultured under the condition of 95% humidity and 5% CO₂ at 37°C. After passaging, the cells were cultured for 24 h and then treated with different concentrations of EPS for 24 h. MTT was provided by Solarbio (Beijing, China). Hoechst 33342 and PI, reactive oxygen species assay kit, Fluo-3 AM probe, N-acetylcysteine (NAC) and LDH cytotoxicity assay kit were purchased from Beyotime Biotechnology (Shanghai, China). Antibodies for SIRT-1, p-mTOR, mTOR and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). All other reagents used were commercially available.

Preparation of an ethanol extract of Patrinia scabiosaefolia

Patrinia scabiosaefolia Link (also named Patrinia hispida Bunge and Patrinia scabiosaefolia Fisch. ex Trev (Caprifoliaceae family) was provided by Shennong Bencao Pharmacy (Shandong, China) and authenticated by Professor Aidong Lang, the analyst of the School of Pharmacy at Shandong University. Dry herbs of Patrinia scabiosaefolia (the plant name has already been verified with http://www.theplantlist.org) of 1 kg were drenched in 95% ethanol (10 l) for 3 days, and then filtered. The filtrates were evaporated in a vacuum evaporator. The relative density was determined as 1.05. The EPS powder was obtained by a freeze-dryer. Subsequently, EPS was dissolved in dimethyl sulfoxide (DMSO) to produce a stock solution with the concentration of 300 mg/ml. The final concentrations of DMSO in all the culture medium were <0.5%. Patrinia scabiosaefolia specimen was deposited at the Urology Laboratory of Cardiovascular Research Center in Qilu Hospital of Shandong University.

MTT assay

MTT assay was performed to evaluate the cell viability of 786-O and HK-2 cells. 786-O and HK-2 cells were seeded into 96-well plates at a density of 5×10³/well, and incubated with different concentrations of EPS for 24 h. Subsequently, MTT (0.5 mg/ml) was added into each well and the cells were incubated for 4 h under conditions of 37°C and 5% CO₂. Then, the supernatant was removed and the formazan crystals were dissolved in 100 µl DMSO. The absorption value was measured at 570 nm by an ELISA reader (Thermo Multiskan MK3; Thermo Fisher Scientific, Waltham, MA, USA).

Colony formation assay

786-O cells were seeded in 6-well plates at the density of 1×10³/well, and were then cultured for 10–14 days after stimulations. When colonies were visible, the incubation was terminated. Subsequently, the cells were rinsed with phosphate-buffered saline (PBS) and stained with crystal violet after being fixed with methanol. Colonies >50 cells were counted under a microscope with low magnification.

Hoechst 33342 and PI double staining

Hoechst/PI double staining was conducted to examine the apoptotic rate of 786-O cells. In brief, 786-O cells were cultured with 10 µg/ml Hoechst 33342 and PI at 37°C for 15 min and then rinsed with PBS three times after stimulation. Morphological alterations of 786-O cells were observed by fluorescence microscope (Carl Zeiss SAS, Jena, Germany). Hoechst 33342 can penetrate the normal cell membrane, whereas PI merely penetrates the damaged cell membrane. Cell staining with Hoechst 33342 manifests blue fluorescence, whereas cells stained with PI manifest red fluorescence. The apoptotic rate and necrotic rate of 786-O cells were identified as the percentages of apoptotic cell nuclei and necrotic cell nuclei in five random fields.

LDH release assay

To further verify the necrosis promoting effect of EPS, LDH release assay was conducted. 786-O cells were plated in a 96-well at the density of 5×10³/well. After a 24-h treatment with EPS, 100 µl of the supernatant was transferred to a clear 96-well plate, and then 50 µl LDH reaction working solution was added to each well for 30 min at room temperature. Subsequently, the absorption value was measured at 490 nm by an ELISA reader (Thermo Multiskan MK3). The relative LDH activity was represented by the following equation: (OD_sample - OD_blank)/(OD_max - OD_blank).

ROS assay

ROS assay was performed to investigate the level of intracellular ROS. 786-O cells were seeded into a 24-well at the density of 3×10⁴/well. The cells were co-incubated with 250 µl DCFH-DA probe (10 µM, 1:1,000 diluted with RPMI-1640 medium) at 37°C for 20 min after stimulation. Then, the cells were rinsed with medium for three times and 200 µl of the medium was added into every well. All the images were captured by fluorescence microscopy and the fluorescent intensity was analyzed by ImageJ software [National Institutes of Health (NIH), Bethesda, MD, USA].

[Ca<sup>2+</sup>]<sub>i</sub> measurement

786-O cells were plated into a 24-well plate at the density of 3×10⁴/well. After EPS stimulation, the cells were washed by PBS, and then incubated with 5 µM Fluo-3 AM probe for 30 min at 37°C. Subsequently, the cells were washed with PBS and then observed by fluorescence microscopy. The fluorescence intensity analysis was performed by ImageJ software.

Western blotting

786-O cells were harvested by RIPA buffer plus phenylmethylsulfonyl fluoride (PMSF), and then total proteins were collected after stimulation. The concentrations of proteins were measured using the BCA method. Protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands of interest were subsequently electrotransferred to a nitrocellulose (NC) membrane and blocked with 5% skim milk for 1 h at room temperature. Incubations were conducted with primary antibodies (1:1,000) against GAPDH, p-mTOR, mTOR and SIRT-1 at 4°C overnight and subsequently secondary antibodies (1:5000) under room temperature for 1 h. After that, the gray scale values of the bands were detected using western immunoblotting detection (ECL system) reagents and then semi-quantitative analyzed by ImageJ software.

Statistical analysis

All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were conducted with one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis and unpaired t-test. The value of P<0.05 was considered as statistically significant. In addition, GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) was used in all processes of the statistical analysis.

Results <sec> <title>EPS significantly inhibits the proliferation and growth of 786-O and HK-2 cells

The ethanol extract of Patrinia scabiosaefolia (EPS) exhibited significant antiproliferation effects on the 786-O cells. Under the stimulation of EPS, cell viability of the cells was markedly decreased in both concentration-dependent and time-dependent manners compared to the control group. At the concentration of 0.6 mg/ml, 786-O cells exposed to EPS had a cell viability of 0.49±0.12 (P<0.05), whereas, there was only 0.14±0.01 (P<0.001) viability when the concentration reached 1.0 mg/ml (Fig. 1B). Morphological alterations were consistent with the above results. The cells shrunk into a bright-round shape and less number of cells were noted with shorter processes following treatment with EPS. These damages were enhanced with the increasing EPS concentration (Fig. 1A). The time-dependent manner of the antiproliferation effects was also determined by MTT assay (Fig. 1C). Furthermore, as shown in Fig. 1D and E, the colony count significantly decreased from 100.0±1.5 to 20.3±0.9 when the cells were co-incubated with EPS at 0.2 mg/ml for 24 h (P<0.001). Whereas, almost no colonies could be observed under the concentration of 0.6 mg/ml EPS (P<0.001).

Human proximal tubular cells (HK-2) were also used to investigate the effects of EPS on normal cells. As shown in Fig. 1D, the cell viability of the HK-2 cells was significantly decreased in a concentration-dependent manner. Cell viabilities of 0.793±0.062 (P<001) and 0.257±0.013 (P<0.001), respectively were noted at concentrations of 0.6 and 1.0 mg/ml.

EPS significantly promotes apoptosis and necrosis of 786-O cells

Apoptosis, which is characterized by chromatin condensation and nuclear shrinkage, is identified by bright blue fluorescence following staining with Hoechst 33342/PI. In contrast, red fluorescence indicates necrotic cells due to the penetration of PI and viable cells show dull blue fluorescence. As shown in Fig. 2, more necrotic and apoptotic cells were detected in the EPS stimulation group as compared to the control group. At the concentration of 0.6 mg/ml, the apoptotic rate was 8.3±0.7% and the necrotic rate was 21.2±4.6% compared to 0.4±0.2% for apoptosis and 0.4±0.4% for necrosis in the control group. When the concentration of EPS increased to 1.0 mg/ml, the necrotic rate of cells was markedly increased from 21.2±4.6 to 41.7±6.6% (P<0.05). However, there was no significant difference regarding the apoptotic rate between treatment with EPS concentrations of 0.6 and 1.0 mg/ml (P>0.05).

To further verify the promotion effect of EPS on necrosis, LDH release assay was performed. There is a positive linear relationship between the level of LDH release and the necrotic rate. As shown in Fig. 2L, as the EPS concentration increased, the level of LDH release was significantly elevated in a concentration-dependent manner.

At the concentration of 1.0 mg/ml, the LDH release rate was 0.455±0.021 (P<0.001), which was consistent with the necrotic rate determined by Hoechst 33342/PI staining.

Effects of EPS on the intracellular ROS level

To investigate the oxidative damage effects of EPS on 786-O cells, ROS assay was performed to measure the intracellular ROS level. H₂DCFDA can be hydrolyzed by intracellular esterases, whereas ROS oxidates non-fluorescent H₂DCFDA to convert to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). Thus the level of green fluorescence can indicate the intracellular ROS level. As shown in Fig. 3, the intracellular ROS level was significantly elevated compared to that noted in the control group after EPS exposure (P<0.01). However, in the presence of 10 mM NAC, the level of intracellular ROS was significantly decreased compared to that following treatment with 0.6 mg/ml EPS alone (P<0.05). However, there was no obvious concentration-dependent trend in the EPS-induced ROS increase (P>0.05).

Effects of EPS on the intracellular calcium concentration

To explore the metabolic perturbation effects of EPS on 786-O cells, we detected the intracellular calcium concentration by Fluo-3 AM probe. These probes specifically bind to [Ca²⁺]_i and emit green fluorescence. As shown in Fig. 4, the mean fluorescence intensity of the EPS exposure group was significantly higher than that of the control group (P<0.01). In addition, the above effects were significantly enhanced with the increase in EPS concentration (P<0.001).

EPS downregulates the expression of SIRT-1 and induces the dephosphorylation of mTOR

Western blotting was performed to investigate the involvement of SIRT-1 and mTOR signaling in the antitumor effects of EPS. As shown in Fig. 5, EPS significantly downregulated the expression of SIRT-1 (P<0.05) and decreased the ratio of p-mTOR/mTOR (P<0.001) in the 786-O cells. Nevertheless, there was no statistically significant difference regarding the ratio of mTOR/GAPDH between the EPS exposure group and the control group (P>0.05).

SIRT-1 inhibitor nicotinamide partly enhances the antitumor effects of EPS

To further corroborate the involvement of SIRT-1 signaling in the antitumor effects of EPS, nicotinamide, a specific SIRT-1 inhibitor, was adopted for co-incubation with EPS. Cell viability assay and Hoechst 33342/PI double staining were carried out to explore the synergetic effects of nicotinamide. As shown in Fig. 6, the cells co-cultured with EPS plus nicotinamide had a significantly higher necrotic rate of 40.9±5.5% than that of 15.8±1.3% in the EPS group (without nicotinamide, P<0.01). The results of the MTT assay verified the above phenomena. The cell viability of the nicotinamide and EPS exposure group was significantly lower (0.33±0.02)compared to the EPS exposure group (0.54±0.02) (P<0.001). However, there was no significant difference regarding the apoptotic rate between the nicotinamide and EPS exposure group and the EPS exposure only group (P>0.05).

Discussion

In the present study, we initially investigated the antitumor effects of EPS on 786-O cells in vitro. Our results demonstrated that EPS showed vigorous antiproliferation and growth inhibitory effects even at a low concentration of 0.6 mg/ml, which needed a ×500 dilution of the stock solution. Certain fluorescence staining assays were performed to identify the manner of cell death and the involvement of metabolic disruption. The results indicated that an increase in ROS and calcium overload may be responsible for the processes of metabolic perturbations. Furthermore, the underlying molecular mechanism was explored and we found that SIRT-1 and mTOR signaling were involved in the antitumor effects of EPS.

Previous studies have reported that Patrinia scabiosaefolia showed marked antiproliferation effects on various types of tumor cells, including HT-29, MCF-7 and HeLa cells (4,10,26). Our results demonstrated that EPS significantly decreased the cell viability of 786-O cells in concentration- and time-dependent manners. As shown in Fig. 1A, the effects of EPS on cells involved not only antiproliferation, but also death-promotion based on morphological observation. Moreover, EPS markedly impeded the formation of cell colonies. However, the concentration-dependent tendency in the colony formation assay was not in accordance with the MTT results. We speculated that the lower initial cell seeding number may be responsible for this inconsistency. The interactions of cells are vital to their survival. Thus, a lower cell number made the cells more vulnerable to EPS. As shown in Fig. 1D, EPS showed significant antiproliferation effects on HK-2 cells. However, at the concentration of 0.6 and 1.0 mg/ml, the cell viabilities of HK-2 cells were 0.793±0.062 and 0.257±0.013, while cell viabilities of 786-O were 0.493±0.124 and 0.136±0.008. This result indicated that EPS exhibited, at least partly, specificity in regards to its antitumor effects.

Metabolic reprogramming, which refers to a series of metabolic alterations that stem from the molecular level, is a distinguishing characteristic of cancer cells (11). Changes in metabolism are critical for malignant proliferation and metastasis. However, these alterations also make the cells more susceptible to disruptions due to its metabolic dependency (27). As an essential role of metabolic regulation, ROS can affect the metabolism of cancer cells (12), and further cause cascaded reactions of metabolic perturbations including Ca²⁺ overload and apoptosis (14,16). Our results showed that EPS significantly induced apoptosis and necrosis of 786-O cells. However, intracellular Ca²⁺ and ROS levels were markedly elevated after the exposure of EPS, while the antioxidant NAC partly reversed the increase in ROS induced by EPS. Therefore, metabolic disruptions may mediate the antitumor effects of EPS, which indicate a new target for cancer intervention.

SIRT-1, as an NAD⁺-dependent deacetylase, has been demonstrated to mediate extensive cellular metabolic processes including cell stress response, apoptosis and cell cycle arrest (17–19). SIRT-1 deacetylates a series of downstream molecules and induces extensive alterations, including an increase in ROS and apoptosis (28,29). Previous research has reported that MHY2256, an SIRT inhibitor, showed significant anticancer effects on MCF-7 and SKOV-3 cells through p53 acetylation (30). Accumulated evidence has demonstrated that overexpression of SIRT-1 is found in many malignant tumors including gastric cancer, hepatocellular carcinoma tissues, ovarian epithelial tumors and is closely linked with poor survival outcomes (21,31,32). Our results showed that the expression of SIRT-1 was significantly downregulated after EPS exposure, which was consistent with the above literature. Mammalian target of rapamycin (mTOR), formed by mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), has been implicated in metabolism, tumorigenesis and aging (24,33,34). The mTOR signaling pathway is involved in energy conservation, growth and division based on its protein kinase characteristics. PI3K/Akt/mTOR and mTOR/P53/P21 pathways have been demonstrated to be involved in antitumor effects by induction of autophagy and inhibition of proliferation (35,36). Wu et al reported that the Akt/GSK3β/mTOR pathway executes the effects of neuroprotection through an antioxidative mechanism (37). However, previous studies and clinical practice have also demonstrated that rapamycin, a specific inhibitor of mTOR, shows marked antitumor effects (24). Our results indicated that EPS obviously decreased the ratio of pmTOR/mTOR compared to the control group, which suggests that dephosphorylation of mTOR may induce the antitumor effects of EPS.

SIRT-1 and mTOR both mediate crucial molecular pathways in the signaling transduction network. However, there is no direct and explicit link between the two regulators to date (17). Previous study has reported that SIRT-1 negatively regulated mTOR signaling potentially mediated by sclerosis complex 2 (TSC2) (17). Guo et al reported that SIRT-1 mediated neuron regeneration by suppressing mTOR signaling (38). Nevertheless, there is evidence indicating that plumbagin induced the apoptosis and autophagy in prostate cancer cells by inhibition of both SIRT-1 and mTOR signaling (39). Therefore, to further substantiate the involvement of SIRT-1 signaling, we employed nicotinamide, an inhibitor of SIRT-1, to block expression of SIRT-1 signaling. The results demonstrated that nicotinamide significantly enhanced the antitumor effects of EPS, which matched our original inference.

However, the role of SIRT-1 and mTOR regarding tumorigenesis is still controversial and there must be several intermediate molecules between SIRT-1 and mTOR. Zhang et al reported that the behaviors of SIRT-1 in tumorigenesis depend on p53 mutations (18). Thus, the alterations of P53 in cancer tissue, along with SIRT-1 and mTOR signaling, deserve much attention in future experiments. Otherwise, given to the involvement of intracellular ROS, the role of MAPKs needs to be further investigated. The MAPK cascade can be activated by ROS via various mechanisms (40). Thus, elucidation of MAPK alterations may provide new insight into the mechanism underlying the antitumor effects of EPS.

Based on previous literature, 10 bioactive agents have been identified (41), including iridoids (42), saponins (41,43), triterpenes (44,45) and lactones (46). Detailed chemical fingerprint of NMR can be reviewed in the above literature. Forty-four essential oils of Patrinia scabiosaefolia have been identified by chemical fingerprinting of GC (Gas Chromatography) (4). Thus, identification of these bioactive components can provide valuable directions for further research.

In summary, the present study investigated the effects of EPS on 786-O cells and partly validated the involvement of ROS and Ca²⁺-mediated SIRT-1 and mTOR signaling. We highlighted the markedly antitumor effects of EPS and provide novel insight in drug discovery for cancer treatments.

Acknowledgements

The present study was supported by grants of the National Natural Science Foundation of China (nos. 81372335, 81400696 and 81502213), a grant of the Shandong Provincial Natural Science Foundation (ZR2014HQ026), a grant of the Bureau of Science and Technology of Jinan (no. 201303040), and the Science and Technology Development Project of Shandong (no. 2011GSF11807).

Abbreviations

EPS

ethanol extract of Patrinia scabiosaefolia

ROS

reactive oxygen species

SIRT-1

sirtuin-1

mTOR

mammalian target of rapamycin

References 1

Kan

Chen

Wang

Yang

Xiao

Wang

Chen

Weng

A novel cell cycle blocker extracted from Stellera chamaejasme L. inhibits the proliferation of hepatocarcinoma cells

Oncol Rep35348034882016

10.3892/or.2016.4742

27109908

Gordaliza

Natural products as leads to anticancer drugs

Clin Transl Oncol97677762007

10.1007/s12094-007-0138-9

18158980

Brglez Mojzer

Knez Hrnčič

Škerget

Knez

Bren

Polyphenols: Extraction methods, antioxidative action, bioavailability and anticarcinogenic effects

Molecules21212016

10.3390/molecules21070901

Lin

Cai

Lin

Peng

Chemical composition, anticancer, anti-neuroinflammatory, and antioxidant activities of the essential oil of Patrinia scabiosaefolia

Chin J Integr MedSep12016(Epub ahead of print)

10.1007/s11655-016-2459-4

Cho

Shin

Noh

Cho

Hong

Park

Lee

Anti-inflammatory effects of methanol extract of Patrinia scabiosaefolia in mice with ulcerative colitis

J Ethnopharmacol1364284352011

10.1016/j.jep.2010.04.047

20573566

Zhang

Sun

Shen

Liu

Ding

Peng

Patrinia scabiosaefolia inhibits the proliferation of colorectal cancer in vitro and in vivo via G1/S cell cycle arrest

Oncol Rep338568602015

10.3892/or.2014.3663

25500894

Baek

Bae

Son

Kim

Kang

Lee

Son

Chang

Inhibitory effects of nardostachin on nitric oxide, prostaglandin E2, and tumor necrosis factor-α production in lipopolysaccharide activated macrophages

Biol Pharm Bull26137513782003

10.1248/bpb.26.1375

14519938

Chen

Liu

Shen

Chen

Sferra

Peng

Patrinia scabiosaefolia inhibits colorectal cancer growth through suppression of tumor angiogenesis

Oncol Rep30143914432013

10.3892/or.2013.2582

23820929

Peng

Chen

Lin

Zhuang

Hong

Sferra

Patrinia scabiosaefolia extract suppresses proliferation and promotes apoptosis by inhibiting the STAT3 pathway in human multiple myeloma cells

Mol Med Rep43133182011

21468570

Chiu

Wong

Ooi

Ethyl acetate extract of Patrinia scabiosaefolia downregulates anti-apoptotic Bcl-2/Bcl-XL expression, and induces apoptosis in human breast carcinoma MCF-7 cells independent of caspase-9 activation

J Ethnopharmacol1052632682006

10.1016/j.jep.2005.11.007

16361073

Schulze

Harris

How cancer metabolism is tuned for proliferation and vulnerable to disruption

Nature4913643732012

10.1038/nature11706

23151579

Trachootham

Alexandre

Huang

Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?

Nat Rev Drug Discov85795912009

10.1038/nrd2803

19478820

Panieri

Santoro

ROS homeostasis and metabolism: A dangerous liason in cancer cells

Cell Death Dis7e22532016

10.1038/cddis.2016.105

27277675

5143371

Yan

Wei

Zhang

Cheng

Liu

Cross-talk between calcium and reactive oxygen species signaling

Acta Pharmacol Sin278218262006

10.1111/j.1745-7254.2006.00390.x

16787564

Ganie

Dar

Bhat

Dar

Anees

Zargar

Masood

Melatonin: A potential anti-oxidant therapeutic agent for mitochondrial dysfunctions and related disorders

Rejuvenation Res1921402016

10.1089/rej.2015.1704

26087000

Wang

Sun

Buyang Huanwu decoction vigorously rescues PC12 cells against 6-OHDA-induced neurotoxicity via Akt/GSK3β pathway based on serum pharmacology methodology

Rejuvenation Res194674772016

10.1089/rej.2015.1798

26935342

Ghosh

McBurney

Robbins

SIRT1 negatively regulates the mammalian target of rapamycin

PLoS One5e91992010

10.1371/journal.pone.0009199

20169165

2821410

Zhang

Hong

Nam

Kim

Paik

Joh

Kwon

Park

Choi

Jang

SIRT1 regulates oncogenesis via a mutant p53-dependent pathway in hepatocellular carcinoma

J Hepatol621211302015

10.1016/j.jhep.2014.08.007

25131770

Liu

Gan

Feng

Saeed

Sun

Reducing Smad3/ATF4 was essential for Sirt1 inhibiting ER stress-induced apoptosis in mice brown adipose tissue

Oncotarget8926792792017

28030827

Jang

Hwang

Kwon

Kim

Choi

Lee

Kwak

Park

Chung

SIRT1 expression is associated with poor prognosis of diffuse large B-cell lymphoma

Am J Surg Pathol32152315312008

10.1097/PAS.0b013e31816b6478

18724249

Chen

Jeng

Yuan

Hsu

Chen

SIRT1 promotes tumorigenesis and resistance to chemotherapy in hepatocellular carcinoma and its expression predicts poor prognosis

Ann Surg Oncol19201120192012

10.1245/s10434-011-2159-4

22146883

Lee

Kim

Noh

Park

Kwon

Park

Jung

Youn

Lee

Chung

Expression of DBC1 and SIRT1 is associated with poor prognosis for breast carcinoma

Hum Pathol422042132011

10.1016/j.humpath.2010.05.023

21056897

Dibble

Cantley

Regulation of mTORC1 by PI3K signaling

Trends Cell Biol255455552015

10.1016/j.tcb.2015.06.002

26159692

4734635

Zoncu

Efeyan

Sabatini

mTOR: From growth signal integration to cancer, diabetes and ageing

Nat Rev Mol Cell Biol1221352011

10.1038/nrm3025

21157483

Menendez

Lupu

Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis

Nat Rev Cancer77637772007

10.1038/nrc2222

17882277

Liu

Shen

Chen

Wei

Lin

Sferra

Hong

Peng

Patrinia scabiosaefolia induces mitochondrial-dependent apoptosis in a mouse model of colorectal cancer

Oncol Rep308979032013

10.3892/or.2013.2528

23754194

Cairns

Harris

Mak

Regulation of cancer cell metabolism

Nat Rev Cancer1185952011

10.1038/nrc2981

21258394

Liu

Yang

Sun

Shao

SIRT1mediated regulation of oxidative stress induced by Pseudomonas aeruginosa lipopolysaccharides in human alveolar epithelial cells

Mol Med Rep158138182017

10.3892/mmr.2016.6045

28000862

Zhuang

Niu

Tang

Cui

Liu

Zhao

Melatonin ameliorates busulfan-induced spermatogonial stem cell oxidative apoptosis in mouse testes

Antioxid Redox SignalJan.272017(Epub ahead of print)

10.1089/ars.2016.6792

5488354

Park

Woo

Kim

Lee

Kim

Lee

Jung

Anticancer effects of a new SIRT inhibitor, MHY2256, against human breast cancer MCF-7 cells via regulation of MDM2-p53 binding

Int J Biol Sci12155515672016

10.7150/ijbs.13833

27994519

5166496

Zhang

Huang

Deng

Cao

Yang

Chen

Zhang

Duan

Shi

Kong

Co-ordinated overexpression of SIRT1 and STAT3 is associated with poor survival outcome in gastric cancer patients

Oncotarget818848188602017

28061480

5386652

Jang

Kim

Hwang

Kwon

Kim

Park

Chung

Kang

Lee

Expression and prognostic significance of SIRT1 in ovarian epithelial tumours

Pathology413663712009

10.1080/00313020902884451

19404850

Kim

Rodriguez-Enriquez

Lemasters

Selective degradation of mitochondria by mitophagy

Arch Biochem Biophys4622452532007

10.1016/j.abb.2007.03.034

17475204

2756107

Brachmann

Hofmann

Schnell

Fritsch

Wee

Lane

Wang

Garcia-Echeverria

Maira

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells

Proc Natl Acad Sci USA1062229922304

2009

10.1073/pnas.0905152106

20007781

2799764

Chen

Yuan

Zhou

Zhu

Shi

Song

Yin

Luo

Regulation of different components from Ophiopogon japonicus on autophagy in human lung adenocarcinoma A549 cells through PI3K/Akt/mTOR signaling pathway

Biomed Pharmacother871181262017

10.1016/j.biopha.2016.12.093

28049093

Lin

Lei

[Inhibitory effect of jianpi-jiedu prescription-contained serum on colorectal cancer SW48 cell proliferation by mTOR-P53-P21 signalling pathway]

Zhong Nan Da Xue Xue Bao Yi Xue Ban41112811362016(abstract in English)

27932756

Zhu

Zhang

Liu

Sun

Lithium protects against methamphetamine-induced neurotoxicity in PC12 cells via Akt/GSK3β/mTOR pathway

Biochem Biophys Res Commun4653683732015

10.1016/j.bbrc.2015.08.005

26271595

Guo

Qian

Zhang

Morrison

Hayes

Wilson

Chen

Zhao

Sirt1 overexpression in neurons promotes neurite outgrowth and cell survival through inhibition of the mTOR signaling

J Neurosci Res89172317362011

10.1002/jnr.22725

21826702

Zhou

Pan

Yang

Zhang

Chow

Yang

Qiu

Zhou

Induction of apoptosis and autophagy via sirtuin1- and PI3K/Akt/mTOR-mediated pathways by plumbagin in human prostate cancer cells

Drug Des Devel Ther9151115542015

10.2147/DDDT.S75976

25834399

4366042

Son

Cheong

Kim

Chung

Kang

Pae

Mitogen-activated protein kinases and reactive oxygen species: How can ROS activate MAPK pathways?

J Signal Transduct 20117926392011

Gao

Zhang

Liu

Cai

Yang

New triterpenoid saponins from Patrinia scabiosaefolia

Carbohydr Res346288128852011

10.1016/j.carres.2011.10.008

22050996

Taguchi

Endo

Letter: Patrinoside, a new iridoid glycoside from Patrinia scabiosaefolia

Chem Pharm Bull22193519371974

10.1248/cpb.22.1935

4430045

Nakanishi

Tanaka

Murata

Somekawa

Inada

Phytochemical studies of seeds of medicinal plants. III. Ursolic acid and oleanolic acid glycosides from seeds of Patrinia scabiosaefolia Fischer

Chem Pharm Bull411831861993

10.1248/cpb.41.183

8448818

Choi

Woo

Triterpenoid glycosides from the roots of Patrinia scabiosaefolia

Planta Med5362651987

10.1055/s-2006-962622

17268965

Inada

Yamada

Murata

Kobayashi

Toya

Kato

Nakanishi

Phytochemical studies of seeds of medicinal plants. I. Two sulfated triterpenoid glycosides, sulfapatrinosides I and II, from seeds of Patrinia scabiosaefolia FISCHER

Chem Pharm Bull36426942741988

10.1248/cpb.36.4269

3246001

Yang

Choi

Yeo

Kim

A new triterpene lactone from the roots of Patrinia scabiosaefolia

Arch Pharm Res244164172001

10.1007/BF02975186

11693542

Figure 1.

Ethanol extract of Patrinia scabiosaefolia (EPS) significantly inhibits the proliferation and growth of 786-O and HK-2 cells. (A and B) 786-O cells were cultured with different concentrations of EPS (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml) for 24 h. (B) Then, cell viability was measured by MTT assay and (A) images of morphological alterations were captured by microscopy; *P<0.05, ***p<0.001, vs. 0 mg/ml. (C) 786-O cells were incubated with EPS at 0.6 mg/ml, and then cell viability was measured at 0, 4, 8, 16 and 24 h, respectively; *P<0.05, vs. 0 h. (D) HK-2 cells were cultured with different concentrations of EPS (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/ml) for 24 h and then cell viability was measured by MTT assay; **P<0.01, ***p<0.001, vs. 0 mg/ml. (E and F) Colony formation assay was performed. After incubation with different concentrations of EPS (0, 0.2, 0.4 and 0.6 mg/ml) for 24 h, 786-O cells were cultured for 10–14 days, and then the colony formation was determined. (E) The difference in the colony count was analyzed; **P<0.01, ***p<0.001. All data were analyzed by one-way ANOVA with post hoc test (n=3). Scale bars in A, 100 µm.

Figure 2.

Ethanol extract of Patrinia scabiosaefolia (EPS) markedly promotes the apoptosis and necrosis of 786-O cells. 786-O cells were cultured with different concentrations of EPS (0, 0.6 and 1.0 mg/ml) for 24 h before Hoechst/PI staining and LDH release measurement. (A-I) Morphological changes were observed by fluorescence microcopy. Red fluorescence indicates necrotic cells whereas bright blue fluorescence indicates apoptotic cells. White arrows indicate necrotic cells whereas yellow arrows indicate apoptotic cells. (J) Apoptotic rate; **P<0.01, vs. 0 mg/ml. (K) Necrosis rate; *P<0.05, 0.6 mg/ml vs. 0 mg/ml and 1.0 vs. 0.6 mg/ml; **P<0.01, 0 mg/ml vs. 1.0 mg/ml. (L) LDH release; *P<0.05, vs. 0 mg/ml; ***P<0.01, vs. 0 mg/ml. All data were analyzed by one-way ANOVA with post hoc test (n=3). Scale bars: A-I, 100 µm.

Figure 3.

Ethanol extract of Patrinia scabiosaefolia (EPS) significantly induces intracellular ROS increase in 786-O cells. 786-O cells were incubated with different concentrations of EPS (0, 0.6 and 1.0 mg/ml) for 24 h (A-C) and with the presence of 10 m NAC (D) before staining. The intensity of green fluorescence indicates the intracellular ROS level. (A-D) The morphological alterations were captured by fluorescence microcopy. (E) The mean fluorescent intensity was analyzed by ImageJ; *P<0.05, vs. 0.6 mg/ml EPS only; ***P<0.001, vs. 0 mg/ml). All data were analyzed by one-way ANOVA with post hoc test (n=3). Scale bars: A-D, 100 µm.

Figure 4.

Effects of an ethanol extract of Patrinia scabiosaefolia (EPS) on intracellular calcium concentration. After incubation with different concentrations of EPS (0, 0.6 and 1.0 mg/ml), 786-O cells were stained for [Ca²⁺]_i. The intensity of green fluorescence represents the intracellular calcium concentration. (A-C) The morphological alterations were captured by fluorescence microcopy. Scale bars: A-C, 100 µm. (D) The mean fluorescent intensity was analyzed by ImageJ. All data were analyzed by one-way ANOVA with post hoc test (n=3); *P<0.05, ***p<0.001.

Figure 5.

Ethanol extract of Patrinia scabiosaefolia (EPS) significantly suppresses the expression of SIRT-1 and induces the dephosphorylation of mTOR. 786-O cells were cultured with different concentrations of EPS (0 and 0.6 mg/ml) for 24 h and then the proteins were collected for western blotting. GAPDH was used as a loading control. (A) The representative bands of interest and (B-D) the statistical analyses of the gray scale are represented. SIRT-1 was normalized to GAPDH. Phosphorylated mTOR was normalized to total mTOR. Total mTOR was normalized to GAPDH; *P<0.05, ***P<0.001, vs. 0 mg/ml. All data were analyzed by unpaired t-test (n=3).

Figure 6.

Nicotinamide partly enhances the antitumor effects of EPS. 786-O cells were incubated at different concentrations (0 and 0.6 mg/ml) of EPS with or without the presence of 5 mM nicotinamide for 24 h. (A-I) The morphological changes were observed by fluorescent microcopy. White arrows indicate necrotic cells whereas yellow arrows indicate apoptotic cells. Scale bars: A-I, 100 µm. (J) Apoptotic rate as detected by Hoechst 33342/PI staining; **P<0.01 vs. 0 mg/ml. (K) Necrotic rate as detected by Hoechst 33342/PI staining; *P<0.05, vs. 0 mg/ml; **P<0.01, vs. 0.6 mg/ml EPS; ***P<0.001 vs. 0.6 mg/ml EPS + 5 mM nicotinamide. (L) MTT assay was performed to measure the cell viability; ***P<0.001 vs. 0.6 mg/ml EPS. All data were analyzed by one-way ANOVA with post hoc test (n=3).