Panax Notoginseng Saponins Exert Anti- osteosarcoma Effect by Inhibiting G0 / G1 Phase and Affecting p53-mediated Autophagy and Mitochondrial Apoptosis


 BackgroundOsteosarcoma is the most common primary bone malignancy. Chemotherapy for osteosarcoma often induces severe complications to the patients. Thus, the identification of new effective antineoplastic agents with fewer side effects remain a necessity. Panax notoginseng saponins (PNS) were therapeutic active components of panax notoginseng and were reported taking the capability to inhibit the growth of several tumors in vitro and in vivo. However, its effect on osteosarcoma has not been studied. This study first investigated the effect of PNS on osteosarcoma cells.MethodsCCK-8 essay used to determine the appropriate working concentration of PNS on osteosarcoma,annixV-FITC/PI experiment used to measure the apoptosis of PNS on osteosarcoma, wound healing assay was used to detect the migration of PNS on osteosarcoma, cell invasiveness was measured by transwell essay,cell cycle was measured by PI,the expression of relative protein was shown by western blot.ResultsOur result indicated that PNS inhibited osteosarcoma cells’ proliferation, invasion and migration, promoted their apoptosis. Besides, PNS also increased mitochondrial membrane potential and the level of reactive oxygen species. Cell cycle of osteosarcoma was arrested in G0 / G1 phase after treatment with PNS. The expression of p53, and mitochondrial related apoptosis proteins were promoted; however, decreased autophagy in osteosarcoma cells with PNS treatment were observed.ConclusionTaking the above effect of PNS on osteosarcoma, PNS were of the potential therapeutic value for treatment of osteosarcoma.


Introduction
Osteosarcoma is a rare malignancy that occurs in children and adolescents and is diagnosed about 10 patients in per million people [1]. It is highly malignant and tends to metastasize at the early stage [2]. For patients with osteosarcoma, most current treatments mainly depend on neoadjuvant chemotherapy followed by surgical resection [3]. However, chemotherapy often introduces severe complications to the patients [4]. Thus, the identi cation of new effective antineoplastic agents with fewer side effects remain a necessity.
Autophagy is a process in which aging or damaged organisms were surrounded by autophagosomes, which fused with lysosomes and form autosomes for degradation [5,6]. Autophagy can prevent accumulation of damaged cell debris and can help keeping the micro-environment homeostasis [7].
Apoptosis is a classical mechanism of inducing cell death [8]. Generally, autophagy is considered to be a protective response to stress, and take the opposite effect on apoptosis: that is, autophagy inhibits the generation of apoptosis [9]. A well-balanced autophagy and apoptosis will be bene cial for limitation the development of tumor.
Panax notoginseng saponins (PNS) are the extract from panax notoginseng, its pharmaceutical effects are widely studied in recent years [10][11][12][13][14][15]. Based on the published literatures, PNS can facilitate both autophagy and apoptosis in several tumors [16,17]. However, different cells may take different biological characteristics when facing the same chemical treatment. Taking the reported autophagy and apoptosis regulation function of PNS on some other cells of the previous study,we explored for the rst time to know whether PNS in uence the growth of osteosarcoma and investigated its possible regulation mechanism related to cell apoptosis and autophagy. We found that PNS can inhibit osteosarcoma cell proliferation and migration, increase mitochondrial membrane potential, inhibit osteosarcoma in G 0 / G 1 phase.Different from the results of other tumor treatment studies, PNS enhance mitochondrial apoptosis but inhibit autophagy in osteosarcoma cells.

Cell culture
Osteosarcoma 143B and HOS cells were obtained from China Centre for Type Culture Collection (Wuhan, China). Cells were cultured in DMEM/F12 medium containing 1% penicillin/streptomycin and 10% FBS in a conventional incubator at 37 ℃ with 5% CO 2 .

Cell viability assay
Cell viability was measured by CCK-8 kit according to the manufacturer's instructions. Brie y, in a nutshell, cells were seeded in a 96-well plate with 5×10 3 cells/well. After 24 hours, the medium was replaced by the new medium containing 0, 100, 200, 300μg /ml PNS. After incubation for 24 hours or 48 hours, 10μL CCK-8 solution was added to each well. Cell viability was measured at 450 nm using a micro tablet reader (Tecan Sunrise, Salzburg, Austria) 1.5 hours later. All the experiments were carried out in triplicate.

Cell apoptosis determination
Cells were collected for apoptosis determination by using AnnixV-uorescent isothiocyanate (FITC)/sodium propanodide (PI) cell apoptosis detection kit. In short, cells were resuspended in 1×binding buffer and added with 5 μL FITC solutions and 5 μL PI solutions. After incubated at 37℃ for 15 minutes, cell apoptosis were analyzed by ow cytometry.
2.5 Wound-healing assay Cells (10 6 cells) were seeded into six-well plates and allowed to fusion. The osteosarcoma cells monolayer was mechanically scratched using a 200 μL sterile pipette tip. After washing twice with phosphate-buffered saline(PBS), cells in DMEM/F12 medium (without FBS) were treated with different concentrations of PNS, and then cultured for a period of time. A series of images of cell migration were captured with a microscope (Olympus IX51) 0 and 24 hours after scratching.

Colony-formation assay
Osteosarcoma cells were treated in different concentrations of PNS. Cells were placed in a six-well plate with the density of 5 × 10 2 cells / well, and incubated for another 2 weeks with no PNS in control group and various concentrations of PNS in the treatment groups. Methanol was used to x the colonies and was stained with Wright-Giemsa solution. Plates were taken and the number of colonies were counted. All experiments were carried out in triplicate.

Nuclear staining analysis by Hoechst 33258
After 24 hours treatment with PNS, osteosarcoma cells were washed twice with PBS and incubated with Hoechst 33258 at room temperature in the dark for 15 minutes. After washing with PBS twice, cell photographs were taken under an inverted uorescent microscope (Olympus BX51; Olympus, Tokyo, Japan) (×200), apoptotic cells showed a bright-blue uorescence. All experiments were repeated in triplicate.

Measurement of mitochondrial membrane potential (MMP)
JC-1 was performed to evaluate mitochondrial membrane potential. 5×10 5 Cells were rinsed with PBS and trypsinized at 37 ° C for 2 min, and then were incubated with 0.5 mL JC-1 staining working solution at 37° C for 20 min. After staining, MMP was measured using ow cytometry.
2.9 Mitochondrial permeability transition pore (MPTP) Osteosarcoma cells were seed in the density of 5x10 5 cells / mL, 3 μL working solution and 5 μL uorescence quencher were added to each sample, then be placed in a dark environment at 37 ° C for 15 minutes. Next, after washing with 3 mL MPTP buffer, osteosarcoma cells were resuspended with 400 mL Hanks' balanced salt solution (HBSS). The change of uorescence intensity of each sample was measured by ow cytometry. The decrease in relative uorescence units was used to evaluate the openness of MPTP.

Reactive oxygen species (ROS)
Following the manufacturer's instructions,5x10 5 cells/mL cells were detected by dichlorodihydro uorescein diacetate (DCHF DA). Brie y, 10 μm DCHF DA was stained in medium containing cells at room temperature for 2 hours. After staining, cells were collected and were washed twice with pre-chilled PBS,the amount of reactive oxygen was determined by ow cytometry.

Transwell essay
Transwell essay was used to evaluate the cell migration ability. Chambers were rst washed with serumfree medium, and then 600 uL of treatment medium containing 10% FBS (control, 100 μg / ml PNS, 200 μg / ml PNS) was added to the lower chamber. Cells (200 µL; 10 6 cells / mL) were planted in the upper chamber. After incubating the cells for 8 hours, the chamber was xed with 4% paraformaldehyde for 15-20 minutes and then was stained with crystal violet for 15 minutess. Finally, wiping the cells off the upper membrane, and counting the lower membrane cells through an inverted microscopy.

Cell cycle
Firstly, about 2-3×10 5 osteosarcoma cells per well were planted into 6-well plate, and were incubated at 37℃ for 24 hours after adding PNS of different concentrations (0, 100 μg / ml, 200 μg / mL). Then,osteosarcoma cells were washed with PBS and were collected. After that, cells were immobilized in the 75% cold ethanol and kept at -20°C for at least 2 hours. After centrifugation at 12000 bpm for 5 minutes, the cells were hydrated with PBS for 15 minutes, and then the supernatant was aspirated. Finally, the cells were centrifuged at a speed of 12000 bpm for 5 minutes and then stained with 50 μg / mL PI (MultiSciences, Zhejiang Province) for 30 minutes. The samples were analyzed by ow cytometry (Becton-Dickinson, New Jersey, USA).

Western blot
Protein expression levels of p27, cyclin D1, CDK2, Bcl-2, P53, Bax, Cytochrome c, Cleaved caspase 9, Cleaved caspase 3, Apaf-1, LC3-I, LC3-II in osteosarcoma 143B and HOS cells were analyzed by Western blot. After treating with different concentrations of PNS, osteosarcoma cells were centrifuged and were washed twice with phosphate buffer solution (PBS). Cells were lysed and centrifuged at 4 ° C for 12 min at 12000 r / min to collect total protein. A certain amount of protein loading buffer was added and boiled at 100 ° C for 15 min. After that, BCA protein assay kit was used to calculate the amount of protein. The equal protein was denatured for polyacrylamide gel electrophoresis (12%, SDS-PAGE). After electrophoresis, the protein was transferred to a 0.45 μm polyvinylidene uoride membrane (PVDF). The PVDF membrane was blocked with 5% skimmed milk powder for 1 hour at room temperature, and primary antibody was added to incubate overnight. After the incubation, the PVDF membrane was washed with TBST buffer (TRIS-HCL balanced salt buffer + Tween) 3 times, and then incubated with a secondary antibody at room temperature in the dark for 1 h. Then washing the band twice with TBST buffer, using the Odyssey infrared laser imaging system to scan the lm, and utilizing the gel image processing system to analyze the optical density value of the target band.
2.14 Statistical analyses SPSS 20.0 statistical software package (SPSS Inc, Chicago, Illinois) was used to process the results. The experimental results were expressed in the form of mean ± standard deviation (SD), and all experiments were repeated three times. One-way analysis of variance was used for statistical analysis, followed by least signi cant difference (LSD). P <0.05 indicates a signi cant difference.

Ethical statement
All the data of this paper was obtained from cell lines, we did not get these data from patients or animals directly, nor intervene these patients. So the ethical approval was not necessary.

PNS inhibit the proliferation of osteosarcoma
Firstly, in order to determine the appropriate working concentration of PNS (chemical structure in gure 1A) on osteosarcoma, CCK-8 test was carried out with PNS treatment at different concentrations. As shown in gure 1B, 1E, 6A and 6B, the optimal concentrations of PNS in osteosarcoma 143B cells at 24h and 48h were (182.6±2.3) μg/ml and (193.6±3.5) μg/ml respectively, and the optimal concentrations of PNS in osteosarcoma HOS cells at 24 hours and 48hours were (293.8±3.5) μg/ml and (299.1±5.2) μg/ml respectively. According to the above results, the 143B cells were sub-divided into three groups, namely control (0μg/ml), low concentration group (100μg/ml) and normal concentration group (200μg/ml) respectively; and the HOS cells were sub-divided into three groups, namely nc group (0μg/ml), low concentration group (150μg/ml) and normal concentration group (300μg/ml) respectively. And secondly, we found that the proliferation of osteosarcoma decreased with the increasing concentration of PNS, as shown in gure 1C, D.

PNS promote the apoptosis of osteosarcoma
AnnixV-FITC/PI experiment indicated that with the increased concentration of PNS, the effect of PNS on apoptosis of osteosarcoma is more obvious, The difference between 100μg/ml group and nc group was statistically signi cant (P <0.05),the difference between 200μg/ml group and nc group was also statistically signi cant (P <0.05) ( gure 2A, B). Hochest 32258 was used to measure the apoptosis of osteosarcoma. Bright blue nuclei indicated apoptotic cells. The results showed that PNS promoted the production apoptosis in a concentration-dependent way ( gure. 2C).The difference between 100μg/ml group and nc group was statistically signi cant (P <0.05),the difference between 200μg/ml group and nc group was also statistically signi cant (P <0.05).PNS increase the protein expression levels of p53, cytochrome c, cleaved caspase 9, cleaved caspase 3, Apaf-1 and Bax in dose-dependent manner, but decrease the expression levels of Bcl-2 in dose-dependent manner by WB ( Figure.2D, 2E, 6C, 6D). To further verify the above conclusions, PFT-α (p53 inhibitor) was used to detect the apoptosis level of osteosarcoma. The results showed that 30 μm PFT-α could effectively inhibit the apoptosis of osteosarcoma ( Figure.2F, 6G). These results indicated that PNS could promote the apoptosis of osteosarcoma by activating the P53 mitochondrial pathway.

PNS affect the expression of P53 and autophagy related gene proteins of osteosarcoma
To investigate the effect of PNS on p53 on autophagy, expression of p53 and autophagy related proteins LC3-I and LC3-II were studied. The results showed that PNS increased the expression of P53 and reduced the proportion of LC3-II/LC3-I in a concentration-dependent manner ( Figure.2D, 2E, 6E, 6F). To con rm the above conclusion, PFT-α (p53 inhibitor) was used to detect the autophagy level of osteosarcoma. The results showed that 30μm PFT-α could effectively promote autophagy of osteosarcoma ( Figure. 2G, 2H, 6H, 6I). These results con rmed that PNS could affect the expression of autophagy related gene proteins by P53 pathway.

PNS inhibit the migration and invasion of osteosarcoma
Wound healing assay was used to detect the migration of tumor cells. The experiment indicated that with the increasing concentration of PNS, the migration rate of osteosarcoma was lower ( gure 3A, B). The difference between 100μg/ml group and nc group was statistically signi cant (P <0.05),the difference between 200μg/ml group and nc group was also statistically signi cant (P <0.05).Cell invasiveness was measured by transwell essay. PNS can signi cantly inhibit the invasiveness of osteosarcoma ( gure 3C, D).The difference between 100μg/ml group and nc group was statistically signi cant (P <0.05),the difference between 200μg/ml group and nc group was also statistically signi cant (P <0.05).

PNS affected the G 0 /G 1 phase of osteosarcoma
Cell cycle assay results showed that PNS take signi cant effect on the G0/G1 phase of osteosarcoma cells ( gure 4A, B).The difference between 100μg/ml group and nc group was statistically signi cant (P <0.05),the difference between 200μg/ml group and nc group was also statistically signi cant (P <0.05). Proteins involved in cell cycle were studied, and the results showed that PNS reduced the expression of cyclinD1 and CDK2, but increased the expression of P27 ( gure 4C, 4D, 6E, 6F).
3.6 PNS increase the ROS level, decrease mitochondrial membrane potential and promote the opening of MPTP on osteosarcoma ROS levels were a sign of early apoptosis. The increasing level of ROS will result in loss of mitochondrial membrane potential and change of MPTP. After 24hours treatment with PNS, ow cytometry was used to measure the level of ROS. PNS increased the ROS level of osteosarcoma ( gure 5C, D).The difference between 100μg/ml group and nc group was statistically signi cant (P <0.05),the difference between 200μg/ml group and nc group was also statistically signi cant (P <0.05). MMP and MPTP were used to verify the effect of PNS on osteosarcoma. PNS promoted the opening of MPTP and the decrease of mitochondrial membrane potential in a dose-dependent manner ( gure 5A,5B,5E).The difference between 100μg/ml group and nc group was statistically signi cant in MPTP(P <0.05),the difference between 200μg/ml group and nc group was also statistically signi cant in MPTP(P <0.05).The difference between 100μg/ml group and nc group was statistically signi cant in MMP(P <0.05),the difference between 200μg/ml group and nc group was also statistically signi cant in MMP(P <0.05).

Discussion
Compounds from medicinal plants may take pharmacological function. PNS play an important role in the clinical application in myocardial ischemia, cerebral infarction, atherosclerosis and several metabolic diseases [18][19][20][21]. Recently, PNS have been reported to have anti-tumor activity. P Wang et al. pointed out that the PNS can inhibit the metastasis of breast cancer [22]. ZG Yang et al. believed that PNS treated cervical cancer by inducing Hela cell apoptosis [23]. CZ Wang demonstrated that PNS could inhibit the proliferation of colon cancer [24]. Q Yang et al. pointed out that PNS could slow down the proliferation of lung cancer [25]. Some early tumor researches results indicated PNS can inhibit tumor cell proliferation with both increased cell apoptosis and autophagy [16,17]. However, chemicals may take different function in different cells; the therapeutic effect of PNS on osteosarcoma has not been studied yet.
In the present study, we rst demonstrate that PNS inhibited the proliferation of osteosarcoma and determined the appropriate treatment concentration by CCK-8 essay, and such inhibitory effect was in a time-dependent and concentration-dependent way. After PNS treatment, cell apoptosis was promoted. These results suggested that PNS may be of potential medical value for osteosarcoma therapy. P53 is the guardian human genes and plays as an important tumor suppressor gene [26]. It is involved in several cellular processes, including cell cycle control, apoptosis and autophagy regulation [27][28][29]. P53 can induce cell cycle arrest and can promote apoptosis related gene expression; it can induce mitochondrial pathway by affecting Bcl-2 and Bax, which result in the loss of MMP and openness of MPTP, and lead to mitochondrial dysfunction. As a result, cytochrome C is leaked into the cytoplasm, which activates the expression of Apaf-1, cleaved caspase 9 and cleaved caspase 3, and eventually lead to the activation of apoptotic pathways. But p53 can also inhibit autophagy by inhibiting the expression of LC3-II. In the present study, different from the early research, osteosarcoma cells showed an increased apoptosis and decreased autophagy after PNS treatment.
Cell metastasis is a sign of malignancy in tumor cells [30]. Malignant metastasis of tumor cells will lead to the failure of treatment in osteosarcoma, and will cause death of about 90% patients. In the present experiment, PNS could effectively reduce the invasion and migration of osteosarcoma cells in a concentration-dependent manner.
Cell cycle is mainly composed of G 1 , S, G 2 and M phase, which plays a role in inhibiting the growth and development of cells [31]. We observed a signi cant increase in the ratio of the G 0 /G 1 phase in osteosarcoma with the treatment of PNS, which indicating that PNS inhibited the growth and development of the osteosarcoma by inducing G 0 /G 1 arrest of the osteosarcoma. Different cell cycles are in uenced by different cell cycle regulatory proteins. The combination of CDK and Cyclin D promotes the formation of G1 phase. P27 inhibits this process by binding to the D-CDK4 and E-CDK2 complexes. In our experiment, we found that PNS could promote the expression of p27 and reduce the expression of cyclin D1 and CDK2 to arrest osteosarcoma in the G 0 /G 1 phase and inhibit the growth and development of osteosarcoma.
Increasing apoptosis is often accompanied with proliferation inhibition in tumors [32], and osteosarcoma is not an exception. Apoptosis can be divided into death receptor-mediated and mitochondrial-mediated pathways. This study just focused on the effect of PNS on mitochondria-mediated apoptosis. Loss of MMP will increase the mitochondrial membrane permeability and increase the production of intracellular ROS. This study indicated that PNS can cause MMP loss by promoting intracellular ROS production and increaseing mitochondrial membrane permeability, together with promoted MPTP opening and upregulated expression of apoptosis-related proteins, which eventually increase mitochondrial apoptosis in osteosarcoma cells.
Mitochondrial function, autophagy and apoptosis are critical in maintaining the normal physiological function [33,34]. On the one hand, excessive autophagy can induce apoptosis. However, it should not be ignored that inhibiting autophagy can also increase apoptosis [35]. In the present study, PNS treatment reducted the autophagy in osteosarcoma cells which lead to a signi cant increase in the adaptability of tumor cells to the neighbor micro-environment, leading to damage in normal cells, such as abnormal metabolism of normal cells, and cause more death of normal cells. In this context, our results are in consistent with some early researches on osteosarcoma [36][37][38].
For the rst time, our study demonstrated that PNS can cause G0/G1 arrest, apoptosis increment, but can inhibit autophagy via the p53 pathway in the osteosarcoma 143B and HOS cells. Although the current research hasn't proved the PNS effect in some other osteosarcoma cell lines and hasn't be veri ed by in vivo experiment, and the death receptor-mediated pathway was not fully investigated; these encouraging ndings still open a new way of taking PNS as a potential anti-tumor drug for further investigation. PNS promote the apoptosis of osteosarcoma 143B cells in vitro. A-B. Annix V / PI double staining method was used to detect and analyze the apoptosis level of osteosarcoma 143B cells. As the concentration of PNS increased, the apoptosis rate of osteosarcoma gradually increased. C. Hochest 32258 analyzes and quantitatively analyzes the morphology of osteosarcoma. The bright blue uorescent nuclei represent apoptotic osteosarcoma. D-F. Western Blot detected and analyzed p53, apoptosis-related proteins and autophagy-related proteins. Before treating osteosarcoma 143B cells with PNS, PTF-α was used for treatment, and the cell proliferation rate was analyzed by CCK-8. G-H. Western Blot detects and analyzes autophagy-related proteins. Before treating osteosarcoma with PNS, PTF-α was used for treatment. The expression of autophagy-related genes was analyzed by Western Blot. Data were expressed as mean ± standard deviation. * P means P <0.05 vs nc, ** P means P <0.01 vs nc. All experiments were performed three times.  were expressed as mean ± standard deviation. * P means P <0.05 vs nc, ** P means P <0.01 vs nc. All experiments were performed three times.  Blot. Before treating osteosarcoma HOS cells with PNS, PTF-α was used for treatment, and the cell proliferation rate was analyzed by CCK8.* P means P <0.05 vs nc, ** P means P <0.01 vs nc. All experiments were performed three times.