1. Introduction Lung cancer is the leading cause of cancer mortality in both man and woman all over the world. The effectiveness of current treatment is severely limited, with an age-standardized mortality rate of 30.0 per 100,000 [1, 2]. Despite improvements in screening [3] and surgical techniques, survival remains poor and new identify potent therapeutic substances from natural resources for therapy are urgently needed. Recently, polysaccharides isolated from plants have been found to possess a variety of biological activities, including antitumor activities, and have attracted enormous attention in biochemical and medical areas [4]. Numerous studies have reported that natural polysaccharides can inhibit tumor cell proliferation either by directly inducing apoptosis or by triggering immunopotentiation activity in 1

combination with chemotherapy [5, 6]. More importantly, many of the natural polysaccharides are found to be effective and relatively nontoxic substances. Thus, natural polysaccharides possessing the splendid activities of inducing proliferation, migration and apoptosis might be ideal candidates for therapy. Glehnia littoralis (G. littoralis), a perennial herb belonging to the Apiaceae, is a typical species growing at temperate sandy coasts around the North Pacific Ocean, widely in China. The dried roots and rhizomes of G. littoralis are an important Chinese medicine for the treatment of lung disease [7]. It was widely used to cure tuberculosis, lung cancer and other serious lung disease in traditional oriental medicine, which can rapidly relieve the symptoms of severe cough, hemoptysis and dyspnea [8]. Furthermore, G. littoralis was also used as a source of edible plant-based anticancer for food usage, such as porridge, soup. More and more studies showed that G. littoralis are packed with the polysaccharides. Based on prominent property of polysaccharides on various diseases, we hypothesize that the pharmacological activities of G. littoralis on lung disease treatment thank to its rich source of polysaccharides [9-11]. Nevertheless, little attention has been devoted to investigating the relationships between anticancer activities of G. littoralis and its polysaccharides. Therefore, the inhibitory effects of polysaccharide (PGL) from G. littoralis on human lung cancer cell line A549 were investigated in this study.

2. Materials and methods

2.1. Materials and reagents The Glehnia littoralisa was purchased from Yantai Herbal Medicine Market (Yantai, China). PGL was prepared and carried on the quality detection according to the reported methods [12-14]. H-DMEM and Fetal Bovine Serum (FBS) were provided by Thermo Fisher Scientific Co. (Gibco, USA). Deionized water was manufactured by a Milli-Q water purification system (Classic UVF MK2, ELGA, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Cisplatin (DDP), Transwell chamber, Hoechst 3342, RNase A were purchased from Sigma Chemical Co. (St. Louis, MD, USA). All other reagents were analytical grade. Phosphate Buffer solution (1×PBS) was prepared with 8.0 g NaCl, 0.2 g KCl, 3.63 g Na2HPO4·12H2O and 0.24 g KH2PO4 dissolved in ultrapure water, ajusted pH to 7.2 (EL20, Mettler toledo, Shanghai), diluted to 1000 mL, and then storied at 4 ℃.

2.2. Cell culture A549 cells were obtained from the Shanghai Institute of Cell Biology (Shanghai, China) and were cultured using H-DMEM with 10% FBS. They were maintained in 37℃ incubator with 5%CO2 saturation. Cell growth and morphology were examined every day and medium was replaced every other day. Cell passage: Serial sub-cultivation was performed when cell density reached 80%, firstly culture medium was discarded, and then washed with 1×PBS. Then the cells were added in 0.5 mL (Eppendorf, Germany) 0.25% trypsin, digested at 37℃ for 1-2 min. When the form of cells was roundness, the digestion was terminated by adding 3 mL medium with serum. Subsequent experiments were conducted 12 hours after cell adherence. Cell suspension was attenuated to 6-7 mL every dish and shaked. When the cells were distributed at the bottom of the culture dish evenly, then incubated the cells at 37℃with 5% CO2。 Cell cryopreservation: Logarithmic growth phase cells were chosen for cell cryopreservation. Cell suspension which prepared by the conventional method, was moved to EP tubes and centrifugated (1500 rpm) for 5 min. In general, 10% DMSO and 20% FBS added in 1640 medium was used as a freezing medium for the preservation of cells. Supernatant was discarded carefully, freezing medium was added in centrifuge tubes, the cell suspension was blowed gently with pipette and added into cryopreserved tubes respectively. In addition, cryopreserved tubes were firstly left at 4 ℃ for 30 min, then -20 ℃ for 2 h, lastly stored at -80 ℃ in refrigerator (THERMO Heto ULtra Freeze, USA). Cell recovery: cryopreserved tubes were put in water bath at 37 ℃ and shaked (WD-9405B, Liuyi instrument, Beijing) rapidly inorder to melt quickly. Cell suspension was transfered to EP tubes, added in five folds complete medium, and centrifugated (1500 rpm) for 5 min. They were moved in a new medium, incubated at 37℃with 5% CO2, and medium was replaced every other day. 2

2.3. MTT assay Cell viability was determined by measuring the ability of cells to transform MTT to a purple formazan dye [15]. A549 cells were plated at the density of 1×105 cells/ml in 96-well plate and incubated for 24 h at 37℃ with 5% CO2 saturation. After incubation, the cells were treated with various concentrations (20, 40, 80, 160, 260 or 380 µg/mL) of PGL and 8 µg/mL of DDP and incubated at 37℃ with 5% CO2 saturation for 48 h. Under the same conditions, the isopyknic solvent without PGL and DDP was as a control. After the drug treatment, 20 μL of MTT solution at 0.5 mg/ml was added to each well and incubated for 4 h. Medium was then removed and replaced with dimethyl sulfoxide (DMSO) in volume of 200 μL to dissolve. The colorimetric assay is measured and recorded at absorbance of 490 nm and the results were expressed as percentage of cell viability compared with the controls. Viability was defined as the ratio (expressed as a percentage) of absorbance of treated cells to untreated cells [16].

2.4. Transwell migration assay Transwell Boyden chamber was used to determine the migration of cancer cell. A549 cells were grown to 70-80% confluence and then serum starved 12 h before the experiment was carried out. Briefly, cells were harvested and resuspended in starvation medium, centrifuged and washed with PBS once, adjusted at a density of 1-2×105/ml containing 2% FBS solution. Matrigel was layed and cultivated at 37℃. This 24-well plate was added to 600 µL 10% FBS, 100 µL cell suspension was inoculated in Transwell chambers and cultivated 4 h. While cells adherented, samples according to the group were added. After cultivated in the incubator for 24 h to allow cell migration through the filter membrane to the lower side of the insert, 800 µL 4% paraformaldehyde was added in every well and the Transwell chambers were fixed with methanol for 20 min. Another 24-well plate was added in 800 µL 0.1% crystal violet staining solution, stained with stain solution for 1-2 min, and dried at room temperature. Chambers were washed and soaked with water for three times in order to decolorization, and then upper-chambers was dried. Cell migration was examined by observing three vision cells, counting, analyzing statistically under microscope [17, 18].

2.5. Flow cytometry detection on cell apoptosis and cycle Detection of PGL-induced apoptosis was performed by flow cytometry using a commercially available Annexin V-FITC or PI apoptosis detection kit. To analyse PGL induced apoptosis of A549 cells, cell concentration was adjusted to 1×105 cells/well. A549 cells were cultured for 24 h, different concentrations of PGL were added after culturing for another 72 h. The cells were washed with PBS once and centrifuged (1500 rpm) for 5 min. Subsequently, the cells were diluted 5×binding buffer with double distilled water and 500 μL suspension cells with 1×binding buffer were prepared. 5 μL of Annexin V-FITC binding buffer and 10 μL Propidium Iodide were added in every well, mixxed, and incubated for 5 min at room temperature out of direct sunlight. Flow cytometry were applied to detect cell apoptosis immediately after incubation [19, 20]. Then culture medium was discarded, followed by centrifugation (1500 rpm) for 5 min to collect cells. The cells were washed with PBS once, fixed with 75% pre-cooled ethanol in volume of 2 mL at 4℃ for 30 min or -20℃ overnight, and then centrifuged and discarded the supernatant. The fixed cells were washed once with 1 mL PBS, centrifuged, and then resuspended in 500 μL PBS containing 20 μg/mL RNase A (Sigma, New York, NY, USA) at 37℃ for 30 min. The cells were washed with PBS once, centrifuged, and then stained with PI working solution (50 μg/mL PI in 500 μL PBS) at room temperature for 30 min darkness. After mixed，the mixture was through 300 mesh sieve, and stored at 4℃ for test [19, 21]. The stained cells were analyzed with flow cytometer, and cell cycle distribution was evaluated using Multi-Cycle software (Phoenix Flow Systems, San Diego, CA).

2.6. Immunofluorescence assay When cell confluence reached 70% after the treatment of cell vaccination drug, take out the cells for experiment. Then cells were washed twice with 0.1M PB for 30 second everytime and then fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature. After washing three times in 0.1M PB for 10min everytime, the cells were rupture of membranes under ether circulation (0.2%-0.5% Triton in PB, 3

RT 15min; 0.2%-0.5% Saponin PB, RT 15min; 100% Methonal, -20℃ 8min). Then cells were treated three times in 0.3% Triton in PBS for 10min everytime and 5% Donkey serum blocking or 10% BSA, RT 1hr. The primary antibody solution contains 0.3% Triton diluted with PBS, the cells were incubated for overnight at 4 ℃, and the washed with 0.3% Triton diluted with PBS for 10min, 15min, 20min, 30min. The secondary antibody diluted in PBS only was added to the cells and incubated for 1 h. After washing four times in PBS, the cells were treated with DAPI for 10-15min and then washed again. Prolong mounting, the cells were saved at 4℃ or -20℃ for further application [22].

2.7. Hochst 3342 staining assay To investigate the effect of PGL on the occurrence of apoptosis from A549 cells, we isolated cells and stained using Hoechst 3342, and further photographed by fluorescence microscope. A549 cells were seeded into 24-well plates, and then the cells were fixed with 0.5 mL stationary liquid for 10 min after the cells were apoptosis, discarded the stationary liquid and washed twice with PBS for 3 min. Subsequently, the cells were stained with 0.5 mL Hoechst 3342 solution for another 5 min, and washed twice with PBS for 3 min again. Finally, to determine morphological changes, the cells which have blue nucleus were photographed by a fluorescence microscope.

2.8. Statistical analysis All experiments were performed at least three times. The significance of the differences between the two groups was determined using Student’s t test. All analyses were carried out using the PGLS 16.0 software package (PGLS, Chicago, IL, USA). All quantitative data were reported as mean ± SD and differences were considered significant at P values less than 0.05.

3. Results and discussion

3.1. PGL inhibited the proliferation of A549 cells Tumor cell proliferation is one of the most important mechanisms of incidence and development of tumor [23]. MTT assay was used to evaluate the proliferation inhibition by PGL of A549 cells at time points of 0, 8, 16, 32, 64 and 96 h. As shown in Table 1, PGL markedly exhibited a broad spectrum of inhibition against A549 cells in a time- and dose-dependent manner compared with control group. After incubation for 96 h, the inhibition rate of PGL increased from about 0.2 to 49.8%, and the maximal effect on proliferation inhibition was observed with 380 μg/mL PGL which inhibited proliferation in 49.8% and higher than DDP group. These results suggested that PGL possesses great potential application in inhibiting lung cancer cell proliferation.

3.2. PGL inhibited the migration of A549 cells To further confirm the effect of PGL on the migration of A549 cells, transwell migration assay was performed. Cells were treated with PGL at concentrations of 40, 160, 380 μg/mL for 24 h. The representative photographs are shown in Fig. 1. The results showed that cell migration of A549 cells was significantly inhibited by PGL treatment with a dose-dependent manner (Fig. 1A). The number of cell migration treated with PGL at concentrations of 40, 160, 380 μg/mL was 137.8±8.2, 109.4±3.1, 76.2±4.2, respectively, which was less compared with control group of 157.0±2.8 (Fig. 1B). Significant differences were observed in A549 cells between PGL and the control group. Moreover, the number of cell migration was treated with PGL when its concentration increased 380 μg/mL were less than DDP group (96.3±4.6). Transwell assays results indicated that PGL can suppress the migration of A549 cells better than DDP.

3.3. PGL induce cell apoptosis in A549 cells In order to investigate whether the growth-inhibitory effect is related to the induction of apoptosis, A549 cells were treated with 40, 160 and 380 μg/mL PGL which was performed by flow cytometry based on Annexin V-FITC/PI doubl staining. Both early and late apoptosis of A549 cells could be observed after the treatment of different concentrations (40, 160 and 380 μg/mL) of PGL. The results of flow cytometry analysis (Fig. 2) showed that the apoptosis of A549 cells were remarkably induced after treated with 4

PGL, and treatment resulted in a dose-dependent increase respectively. Compared with the control cells, the cells treated by PGL presented typical induction of apoptosis. These data suggested that the induction of apoptosis at least partly accounted for the growth inhibition of A549 cells.

3.4. Flow cytometry detection on cell cycle Inhibition of cell proliferation activity is associated with the block of cell cycle. The cell cycle distribution of A549 treated with PGL was detected to determine whether cell cycle arrest is related to the PGL-induced proliferation inhibitory effect. As shown in Table 2 and Fig. 3, compared with the control group, PGL reduced the percentage of G0/G1 and increased the population of S and G2/M phase in a dose-dependent manner, which indicated that PGL mainly arrests cell cycle in S and G2/M phase.

3.5. Hochst 3342 staining assay on cell apoptosis In order to confirm whether the growth-inhibitory effect is related to the nuclear morphological changes of A549 cells, nuclear morphological changes were confirmed by Hoechst 3342 staining (Fig. 4). Compared with the normal nuclear morphology of the control cells, the cells were treated by PGL presented typical morphological characteristics of apoptosis (Fig. 4A). As shown in Fig. 4B, the cell apoptosis rate of PGL at concentrations of 40, 160 and 380 μg/mL was increased in a concentration-dependent manner (27.0%, 41.4%, 51.5%), which was much higher compared with the control group (19.7%). Moreover, the cell apoptosis rate of the highest-dosen PGL was close to the DDP (54.8%).

3.6. PGL down-regulate the expression of PCNA To investigate the effect and mechanism of PGL on the occurrence of apoptosis from A549 cells, we stained using DAPI and conducted immunofluorescence analysis to examine the expression of PCNA. A549 cells were incubated with increasing concentrations of PGL (40, 160 and 380 μg/mL). As shown in Fig. 5, the effects of PGL which stained by DAPI was significantly induced apoptosis in a concentration-dependent manner. The data demonstrated that 160 and 380 μg/mL PGL can significantly decrease the expression of PCNA in a concentration-dependent manner. As a result, the high-dose PGL treatment caused better down-regulation in PCNA expression than DDP. The immunofluorescence analysis results indicated that the PGL-induced apoptosis in A549 cells was involved in a down-regulation trend of PCNA expression compared with the control group.

4. Conclusions Cancer cells were uncontrolled growth of abnormal cells, compared to normal cells, which have more proliferation and migration ability. The proliferation and migration of cancer cell is the important mechanisms of incidence and development of tumor. Apoptosis has been postulated as one of the most critical mechanism for the anticancer action [24]. In this study, we investigated the anticancer activities of PGL silencing on the proliferation, migration and apoptosis of A549 lung cells. The mechanism of PGL was detected by transwell assay, flow cytometry technology, Hochst 3342 staining assay and immunofluorescence assay. The results showed that PGL inhibited the proliferation and migration of A549 cells in a dose- and time-dependent manner in vitro. The cell cycle experiment illustrated that PGL could induce A549 cell cycle to arrest in S and G2/M phase, while PGL had influence on apoptosis of A549 cells by flow cytometry analysis. According to Hochst 3342 staining assay, PGL could induce cell apoptosis. Moreover, immunofluorescence detection elucidated PGL could down-regulate the expression of PCNA. In conclusion, PGL can not only inhibit cell proliferation and migration, but also remarkable induce cell apoptosis, and the underlying mechanism might be related to the decreased PCNA expression, leading to cell cycle arrested in S and G2/M phase. However, the molecular mechanisms and pharmacokinetics of PGL still need to be further studied to provide more strong evidence in resistance to breast cancer. Based on this study, PGL may be a candidate for further evaluation as an antitumor agent for human cancers, especially lung cancer. Acknowledgements

5

This work was partly supported by the Chinese Postdoctoral Science Foundation (No. 2017M612642) and Natural Science Foundation of Shandong Province (No. ZR2016HB20). References [1] Bepler G. Cancer Control,;1; 4 (2003) 275-276.. [2] R. Kumar, S.K. Lu, A. Minchom, A. Sharp, M. Davidson, R. Gunapala, T.A. Yap, J. Bhosle, S. Popat, M.E. O’Brien,;1; Cancer Chemoth. Pharm. 77 (2016) 375-383. [3] P.B. Bach, J.N. Mirkin, T.K. Oliver, C.G. Azzoli, D. Berry, O.W. Brawley, T. Byers, G.A. Colditz, M.K. Gould, J.R. Jett,;1; JAMA-J. Am. Med. Assoc. 307 (2012) 2418-2429. [4] A. Zong, Y. Liu, Y. Zhang, X. Song, Y. Shi, H. Cao, C. Liu, Y. Cheng, W. Jiang, F. Du,;1; Carbohyd. Polym. 129 (2015) 50-54. [5] S.R. Chowdhury, S. Sengupta, S. Biswas, R. Sen, T.K. Sinha, R.K. Basak, B. Adhikari, A. Bhattacharyya,;1; Mol. Carcinogen. 54 (2014) 1636-1655. [6] A. Zong, H. Cao, F. Wang,;1; Carbohyd. Polym. 90 (2012) 1395-1410. [7] B.R. Cassileth, N. Rizvi, G. Deng, K.S. Yeung, A. Vickers, S. Guillen, D. Woo, M. Coleton, M.G. Kris,;1; Cancer Chemoth. Pharm. 65 (2009) 67-71. [8] T. Ng, F. Liu, H. Wang,;1; J. Ethnopharmacol. 93 (2004) 285-288. [9] N. Hiraoka, J. I. Chang, L.R. Bohm, B.A. Bohm,;1; Biochem. Syst. Ecol. 30 (2002) 321-325. [10] T. Masuda, M. Takasugi, M. Anetai,;1; Phytochem. 47 (1998) 13-16. [11] Z. Yuan, Y. Tezuka, W. Fan, S. Kadota, X. Li,;1; Chem. Pharm. Bull. 50 (2002) 73-77. [12] Y. Shi, Q. Xiong, X. Wang, X. Li, C. Yu, J. Wu, J. Yi, X. Zhao, Y. Xu, H. Cui,;1; Carbohyd. Polym. 136 (2016) 875-883. [13] Q. Xiong, Y. Jiao, X. Zhao, X. Chen, Q. Zhang, C. Jiang,;1; Carbohyd. Polym. 98 (2013) 217-223. [14] C. Jiang, Y. Jiao, X. Chen, X. Li, W. Yan, B. Yu, Q. Xiong,;1; Food Chem. Toxicol. 59 (2013) 18-25. [15] T. Chen, Y. S. Wong,;1; Biomed. Pharmacother. 63 (2009) 105-113. [16] C.G. Lee, H.W. Lee, B.O. Kim, D.K. Rhee, S. Pyo,;1; J. Funct. Food. 15 (2015) 172-185. [17] S. Liang, L. He, X. Zhao, Y. Miao, Y. Gu, C. Guo, Z. Xue, W. Dou, F. Hu, K. Wu, PLoS One,;1; 6 (2011) e18409. [18] G. Liu, S. Kuang, S. Wu, W. Jin, C. Sun,;1; Sci. Rep. 6 (2016) 26722. [19] M. Tao, L. Gao, J. Pan, X. Wang,;1; African Journal of Traditional, Compl. Altern. Med. 11 (2014) 176-179. [20] M. Bonomo, L. Milella, G. Martelli, G. Salzano,;1; J. Appl. Microbiol. 115 (2013) 786-795. [21] F. Chen, H. Li, Y. Wang, M. Gao, Y. Cheng, D. Liu, M. Jia, J. Zhang,;1; J. Funct. Food. 25 (2016) 523-536. [22] S. Syam, A. Bustamam, R. Abdullah, M.A. Sukari, N.M. Hashim, M. Ghaderian, M. Rahmani, S. Mohan, S.I. Abdelwahab, H.M. Ali,;1; J. Funct. Food. 6 (2014) 290-304. [23] B. Singh, T.K.B. And, B. Singh,;1; J. Agr. Food Chem. 51 (2003) 5579-5597. [24] R. Sinha, K. El-Bayoumy, Curr.;1; Cancer Drug Tar. 4 (2004) 13-28.

Fig. 1. Effects of PGL on abilities of migration of A549 cells. (A) Representative photographs of control and different concentrations PGL-treated A549 cells on transwell chambers at 200 magnifications. (B) Quantitative analysis of numbers for the migration cell from control and different concentrations PGL-treated A549 cells. Experiments were carried out as described in Section 2.4. Data represent mean ± SD for three independent experiments. Superscript letters a and b designate a 6

significant differences. a P < 0.05 compared with the control group. b P < 0.01 compared with the normal the control group.

Fig. 2. Effects of PGL on early apoptosis in A549 cells by flow cytometry.

Fig. 3. Effects of PGL on cell circle distribution of A549 cells by flow cytometry.

Fig. 4. Effects of PGL on cell apoptosis in A549 cells. (A) Representative photographs of apoptosis from control and different concentrations PGL-treated A549 cells at 200 magnifications. (B) Quantitative analysis for rate of apoptosis (%) from control and different concentrations PGL-treated A549 cells. Experiments were carried out as described in Section 2.7. Data represent mean ± SD for three independent experiments. Superscript letters a and b designate a significant differences. a P < 0.05 compared with the control group. b P < 0.01 compared with the normal the control group.

Fig. 5. Effects of PGL on the expression of PCNA of the A549 cells

Table 1 Effect of PGL on the proliferation of A549 cells. 8h

Concen Gro

tration

up

(μg/mL

16 h Inhib

A490

)

itory rate

Inhib A490

(%) Cro ntro

-

l DD P

8

20

PG L

40

80

160

0.324± 0.0027 0.300± 0.0054 0.336± 0.0025 0.324± 0.0015 0.312± 0.0035 0.300± 0.0014

-

7.4

-3.7

0.07

3.8

7.4

32 h Inhib

itory

A490

rate (%)

0.434± 0.012 0.394± 0.0057 0.448± 0.0026 0.408± 0.0047 0.403± 0.012 0.397± 0.0059

64 h

itory rate

Inhib A490

(%) 0.588±

-

0.011 0.468±

9.1

0.014 0.591±

-3.3

0.021 0.555±

5.9

0.014 0.552±

7.0

0.017 0.543±

8.5

0.0037

7

-

20.5

-0.5

5.6

6.1

7.6

96 h

itory rate

Inhib A490

(%) 1.306± 0.021 0.835± 0.014 1.297± 0.031 1.220± 0.025 1.169± 0.015 1.116± 0.0075

-

36.1

0.7

6.6

10.5

14.6

itory rate (%)

1.211± 0.0057 0.632± 0.0078 1.209± 0.0094 1.109± 0.0050 1.053± 0.029 0.951± 0.0035

-

47.8

0.2

8.4

13.1

21.4

260

380

0.298± 0.0016 0.289± 0.0037

7.8

10.8

0.392± 0.0043 0.388± 0.0047

0.501±

9.5

0.012 0.473±

10.5

0.016

14.7

19.6

0.972± 0.0091 0.760± 0.016

25.6

41.8

0.809± 0.0087 0.608± 0.0034

Table 2 Effect of PGL on apoptis and cell circle of A549 cells. Percentage of cell (%)

Concentration (μg/mL)

G0/G1

S

G2/M

Crontrol

-

59.54

34.94

1.28

DDP

8

4.16

94.51

3.31

40

62.86

42.44

0.88

160

54.07

51.8

1.19

380

4.83

95.22

3.85

Group

PGL

TDENDOFDOCTD

8

33.2

49.8

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