International Journal of Polymer Science

International Journal of Polymer Science / 2019 / Article
!A Erratum for this article has been published. To view the article details, please click the ‘Erratum’ tab above.
Special Issue

Polysaccharides for Biomedical Applications

View this Special Issue

Research Article | Open Access

Volume 2019 |Article ID 9543526 | https://doi.org/10.1155/2019/9543526

Cheng Peng, Bo Han, Baishan Wang, Gaoyang Zhao, Fang Yuan, Yeyu Zhao, Liang Liang, Dingge Liu, Zhaohui Zhai, Yanping Shen, "Therapy of Prostate Cancer by Nanoyam Polysaccharide", International Journal of Polymer Science, vol. 2019, Article ID 9543526, 5 pages, 2019. https://doi.org/10.1155/2019/9543526

Therapy of Prostate Cancer by Nanoyam Polysaccharide

Academic Editor: Jianxun Ding
Received20 Oct 2018
Revised22 Nov 2018
Accepted05 Dec 2018
Published31 Jan 2019

Abstract

We evaluated the effect and mechanism of yam polysaccharide on the proliferation of the prostatic cancer cell line and tumor-bearing mice. The effect of nanoyam polysaccharide on prostatic cancer cell line PC-3 was measured using the scratch adhesion test and flow cytometry. The growth effect induced by nanoyam polysaccharide was detected with the CCK-8 test. The levels of caspase-3 protein were determined with Western blot. In our data, nanoyam polysaccharide presented inhibitory effect on the proliferation of PC-3. The scratch adhesion test showed that the rate of wound healing in the intervention group was significantly lower than that in the control group (). Flow cytometry assay showed that, after treatment with nanoyam polysaccharide, the apoptosis rate in the intervention group was significantly lower than that in the control group (, ; ). Western blot assay showed upregulated levels of caspase-3 in the intervention group, compared to the control group (). Our results suggested that nanoyam polysaccharide strongly suppressed the growth of prostatic cancer by inducing the overexpression of caspase-3 and may be a potent anticancer strategy.

1. Introduction

Prostate cancer, defined as one of the most common malignant tumors in male, ranks the second in global morbidity and the sixth in mortality [1]. Previously, prostate cancer was usually treated with surgery and local radiotherapy. However, due to the invisibility of early prostate cancer, most patients are diagnosed in the middle and late stage and miss the best opportunity for treatment. Testicular resection combined with endocrine therapy was mainly used in patients with advanced cancer. Patients may develop androgen-independent prostate cancer during endocrine therapy [2, 3].

With the continuous in-depth study of Chinese traditional pharmacology, which is often used in the comprehensive treatment of cancer and has achieved good results, Chinese medicine has become an important part of tumor treatment in China. Studies have shown that traditional Chinese medicine played multiple roles in preventing tumorigenesis and antitumor therapy, such as increasing efficacy and reducing toxicity, suppressing tumor recurrence and metastasis [4, 5]. Some studies have found that nanoyam polysaccharides could be used as antitumor active substances [68], but little research has been performed on prostate cancer.

Previous studies indicated that the hot water extraction could be used to separate pure Chinese yam polysaccharide [9]. There exists bioactive ingredients such as β-1,3-glucose, α-1-galactose, and α-1,6-galactose in the purified yam polysaccharide, which could be encapsulated into nanostructure. Nanoparticles are prepared by natural polymers with the size of approximately 100 nm [10]. In this study, the antitumor activity of nanoencapsulated yam polysaccharide was investigated in prostate cancer in vitro and in vivo.

2. Methods and Materials

2.1. Cell Line and Animals

Human prostatic cancer cell line was purchased from Shanghai Cell Institute, Chinese Academy of Sciences. Cells were cultured in medium RPMI1640 (Gibco) containing 10% bovine fetal serum (Thermo Fisher Scientific). Forty healthy male 5-6-week-old BALB/c mice (body weight, 18-22 g) were purchased from the Centre of Experimental Animals at Fudan University. All mice were kept on a 12 h light/dark cycle and given free access to food and water. The mice were divided into four groups randomly: control group (), low-dose group (), medium group (), and high-dose group (). The animal experiment and all associated procedures were approved by the Animal Ethical and Welfare Committee at Fudan University.

2.2. Reagents

Nanoyam polysaccharides were purchased from Shanxi Ciyuan Biotech Company. Caspase-3 primary antibody (ab13847, 1 : 1000, Abcam) and GAPDH (#4970, 1 : 1000, Abcam) were purchased. HRP-conjugated rabbit anti-mouse IgG were from Jackson ImmunoResearch Laboratories Inc. (West Grove, Pennsylvania, USA). FITC-Annexin V/PI kit was purchased from Sigma (APOAF-20TST, USA). Flow cytometry assay was performed in FACS-san440 (BD, USA). CCK-8 kit was purchased from Beyotime.

2.3. Tumor Xenograft Mouse Model

All animal experiment protocols were approved by the Institutional Review Board at the Immune Disease Institute. Breast cancer xenograft model was established in nude mice by the injection of PC-3 cells. Next, the mice received intravenous injection of PBS (as negative control), low-dose nanoyam polysaccharide, medium-dose nanoyam polysaccharide, and high-dose nanoyam polysaccharide every day for 4 weeks. The tumor growth and body weight of the mice were monitored until day 30 when the mice were sacrificed and the tumor tissues were excised for analysis.

2.4. Scratch Adhesion Test

PC-3 cell suspension was prepared at a density of cells/mL. 2 mL cell suspension was inoculated in a 6-well plate and cultured for 12 h. After the cells were adhered to the wall, a straight line was drawn along the diameter of the hole with the nozzle of a 200 μL micropipette and the floating cells were washed away by PBS. The cells were divided into two groups. Normal medium was added to the control group, and nanoyam polysaccharide (80 mg/L) was added to the intervention group for 48 h after culture. The scratch was recorded at 0 h, 12 h, 24 h, and 48 h after microscopy. The relative ratio of the cell scratch spacing between the control group and the intervention group was calculated using the IPP software. The experiment was repeated three times, and the average value was calculated. The .

2.5. CCK-8 Assay

A cell counting kit-8 (CCK-8) analysis kit (Thermo Fisher Scientific) was used to determine the PC-3 viability. The CCK-8 kit was put into each well 0, 12, 24, and 48 h, respectively, based on the manufacturer’s protocol, and we recorded the absorbance at 450 nm.

2.6. Flow Cytometry Assay

Cells were obtained from the cell line, and mice were stained with FITC-labeled Annexin V and PI. After incubation at room temperature for 15 min, samples were washed once with PBS buffer. At least 10,000 cells were assayed by two-color FCM using flow cytometry. Data were analyzed by the CellQuest software (Becton Dickinson, Mountain View, CA).

2.7. Western Blot

Cells were then harvested and homogenized in ice-cold sodium dodecylsulfate (SDS) lysis buffer. After collection of total cell lysates, equal amount of protein was separated by 10% SDS-PAGE and blotted onto a PVDF membrane. The PVDF membranes were blocked with Tris-buffered saline (TBS) containing 5% skimmed milk powder for 1 h and then incubated at 4°C overnight with caspase-3 mAb. After that, the membranes were washed with 16Tris-buffered saline/Tween-20 (TBS/T) buffer for three times (5 min each time) and incubated with HRP-conjugated polycolonal secondary antibody for 1 h at room temperature. The membranes were developed with the enhanced plus chemiluminescence assay (Pierce, USA) according to the manufacturer’s instructions. Images were analyzed by the Image Pro Plus 6.0 software. The caspase-3 expressions were normalized to the GAPDH loading control.

2.8. Statistical Analysis

The data were presented as . All statistical analysis was performed using SPSS11.0. Unpaired Student’s -test was used for the comparisons between two different groups. values less than 0.05 were considered significant.

3. Results

3.1. Effect of Nanoyam Polysaccharide on Tumor Growth

The effect of nanoyam polysaccharide intervention on tumor growth in tumor-bearing mice was observed by detecting the volume and weight of the tumor body. Compared with the control group, different doses of nanoyam polysaccharide intervention could significantly inhibit the tumor weight of PC-3 tumor-bearing mice (), as shown in Figure 1(a). In the HD group and MD group, the tumor weight inhibition rate in the LD group was , , and , respectively. Compared with the negative control group, the nanoyam polysaccharide could significantly inhibit the volume of prostate cancer in tumor-bearing mice (), as shown in Figure 1(b).

3.2. Effect of Nanoyam Polysaccharide on Cell Viability

To determine the effect of nanoyam polysaccharide on apoptosis of prostatic cancer cells, Annexin V/PI staining was performed. As shown in Figures 2(a) and 2(b), apoptosis of prostatic cancer cells in the intervention group was significantly higher than that in the control group (, ; ). CCK-8 assay indicated that in the HD group and MD group, tumor cell activity in the LD group decreased to , , and of the control group, respectively, and the difference was significant (), as shown in Figure 2(d).

3.3. Effect of Nanoyam Polysaccharide on Cell Migration

Scratch adhesion test showed that the rate of wound healing in the intervention group at 12 h, 24 h, and 48 h were significantly lower than that in the control group (Figure 3, ), indicating that nanoyam polysaccharide had a inhibitory effect on prostatic cancer cells.

3.4. Effect of Nanoyam Polysaccharide on Protein Levels

The expression of caspase-3 protein of prostate cancer in mice was detected by Western blotting. Compared with the control group, the expression of caspase-3 in prostate cancer tissues was downregulated in the HD group, MD group, and LD group, with statistical difference (Figure 4, ).

4. Discussion

This study investigated the anticancer effect of polysaccharides of nanoyam. PC-3 cell line is one of the representative cell lines of human prostate cancer. In vitro study showed that the nanoyam polysaccharide presented inhibitory on the human prostate cancer cell line PC-3. Furthermore, the scratch test showed that nanoyam polysaccharide can significantly inhibit the migration of pc-3 cells. By using flow cytometry analysis, we found that the intervention of nanoyam polysaccharide could induce apoptosis of pc-3 cells. Subsequently, in vivo study of PC-3 tumor-bearing mice, we explored the inhibitory effect of nanoyam polysaccharide on human prostate cancer cells and its potential mechanism. Four weeks after the intervention of nanoyam polysaccharide, the weight and volume of the tumor were significantly reduced. The results of CCK-8 assay showed that the nanoyam polysaccharides had an inhibitory effect on the proliferation of prostate tumors, which was consistent with the results of in vitro flow cytometry analysis.

Tumor development and prognosis are closely related to apoptosis. Abnormal apoptosis is considered as one of the causes of tumor formation, and inducing apoptosis of tumor cells has become a new trend of tumor treatment over the years [11, 12]. Classic apoptotic pathways include exogenous death receptor pathways and endogenous mitochondrial cytochrome release pathways. Existing studies have shown that these two pathways are interrelated, rather than independent, and both of these pathways will eventually activate the apoptotic end-effector caspase 3 [13, 14]. In this study, the protein level of caspase-3 in tumor cells of PC-3 tumor-bearing mice was detected by Western blot. We found that the intervention of nanoyam polysaccharide could inhibit tumor development and metastasis by inducing the upregulated expression of caspase-3 protein. The cell apoptosis signal pathway of cancer cells is very complex, and the nanoyam polysaccharide contains many active components. Therefore, the signal mechanism of nanoyam polysaccharide inducing cell apoptosis needs further exploration.

To sum up, nanoyam polysaccharides are potential therapeutic agents for prostate tumors by inducing high levels of caspase-3 protein expression and inhibiting the proliferation of prostate tumors.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors’ Contributions

Cheng Peng, Bo Han, Zhaohui Zhai, and Yanping Shen contributed equally to this work.

Acknowledgments

This work was supported by the Science and Technology Plan Project of Liaoning Province (Grant no. 20170540607).

References

  1. A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward, and D. Forman, “Global cancer statistics,” CA: A Cancer Journal for Clinicians, vol. 61, no. 2, pp. 69–90, 2011. View at: Publisher Site | Google Scholar
  2. H. H. Cheng, D. W. Lin, and E. Y. Yu, “Advanced clinical states in prostate cancer,” The Urologic Clinics of North America, vol. 39, no. 4, pp. 561–571, 2012. View at: Publisher Site | Google Scholar
  3. H. A. Adeola, J. M. Blackburn, T. R. Rebbeck, and L. F. Zerbini, “Emerging proteomics biomarkers and prostate cancer burden in Africa,” Oncotarget, vol. 8, no. 23, pp. 37991–38007, 2017. View at: Publisher Site | Google Scholar
  4. C.-q. Ling, X.-q. Yue, and C. Ling, “Three advantages of using traditional Chinese medicine to prevent and treat tumor,” Journal of Integrative Medicine, vol. 12, no. 4, pp. 331–335, 2014. View at: Publisher Site | Google Scholar
  5. X. Feng, D. Li, J. Han, X. Zhuang, and J. Ding, “Schiff base bond-linked polysaccharide–doxorubicin conjugate for upregulated cancer therapy,” Materials Science and Engineering: C, vol. 76, pp. 1121–1128, 2017. View at: Publisher Site | Google Scholar
  6. L. Liu, M. Li, M. Yu et al., “Natural polysaccharides exhibit anti-tumor activity by targeting gut microbiota,” International Journal of Biological Macromolecules, vol. 121, pp. 743–751, 2019. View at: Publisher Site | Google Scholar
  7. J. S. Chang, H. P. Kuo, K. L. B. Chang, and Z. L. Kong, “Apoptosis of hepatocellular carcinoma cells induced by nanoencapsulated polysaccharides extracted from Antrodia Camphorata,” PLoS One, vol. 10, no. 9, article e0136782, 2015. View at: Publisher Site | Google Scholar
  8. J. Su, L. Jiang, J. Wu, Z. Liu, and Y. Wu, “Anti-tumor and anti-virus activity of polysaccharides extracted from Sipunculus nudus (SNP) on Hepg2.2.15,” International Journal of Biological Macromolecules, vol. 87, pp. 597–602, 2016. View at: Publisher Site | Google Scholar
  9. W. Yang, Y. Wang, X. Li, and P. Yu, “Purification and structural characterization of Chinese yam polysaccharide and its activities,” Carbohydrate Polymers, vol. 117, pp. 1021–1027, 2015. View at: Publisher Site | Google Scholar
  10. Y. Huang, Y. Zhao, F. Liu, and S. Liu, “Nano traditional Chinese medicine: current progresses and future challenges,” Current Drug Targets, vol. 16, no. 13, pp. 1548–1562, 2015. View at: Publisher Site | Google Scholar
  11. K. Zhao, D. Li, W. Xu et al., “Targeted hydroxyethyl starch prodrug for inhibiting the growth and metastasis of prostate cancer,” Biomaterials, vol. 116, pp. 82–94, 2017. View at: Publisher Site | Google Scholar
  12. M. Qiu, L. Chen, G. Tan et al., “JS-K promotes apoptosis by inducing ROS production in human prostate cancer cells,” Oncology Letters, vol. 13, no. 3, pp. 1137–1142, 2017. View at: Publisher Site | Google Scholar
  13. Y. S. Kim, S. K. Kim, and S. J. Park, “Apoptotic effect of demethoxyfumitremorgin C from marine fungus Aspergillus fumigatus on PC3 human prostate cancer cells,” Chemico-Biological Interactions, vol. 269, no. 269, pp. 18–24, 2017. View at: Publisher Site | Google Scholar
  14. J. C. Lee, E. A. Shin, B. Kim et al., “Auraptene induces apoptosis via myeloid cell leukemia 1-mediated activation of caspases in PC3 and DU145 prostate cancer cells,” Phytotherapy Research, vol. 31, no. 6, pp. 891–898, 2017. View at: Publisher Site | Google Scholar

Copyright © 2019 Cheng Peng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


More related articles

672 Views | 296 Downloads | 2 Citations
 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly as possible. We will be providing unlimited waivers of publication charges for accepted research articles as well as case reports and case series related to COVID-19. Review articles are excluded from this waiver policy. Sign up here as a reviewer to help fast-track new submissions.