Technological watch

Objective. To investigate the possible development of radix hedysari polysaccharide as an antiglioma drug, we studied the effect of radix hedysari polysaccharide on glioma cells in vitro and the growth of glioma in nude mice and on the phagocytosis of macrophages in nude mice with glioma. Methods. The effect of radix hedysari polysaccharide on the growth of glioma was studied based on U251 cell line in vitro. The effect of radix hedysari polysaccharide on the growth of glioma was studied in vivo. The growth inhibition rate of radix hedysari polysaccharide on U251 cell line was determined by the MTT assay. The cell cycle of U251 was analyzed by flow cytometry. The expression of cytokines in U251 cells and tumor tissues was detected using PCR. The phagocytosis of macrophages in the serum of glioma nude mice was detected by Giemsa staining. TNF-? signaling pathway proteins in the serum of glioma nude mice were detected by ELISA. Results. Radix hedysari polysaccharide inhibited the growth of U251 cells, induced apoptosis in G1 phase by cell cycle arrest, and facilitated apoptosis in glioma mice by regulating cell cycle. Mice injected with radix hedysari polysaccharide showed delayed tumor growth and grew slowly. Radix hedysari polysaccharide enhanced the phagocytosis of macrophages in glioma nude mice. Radix hedysari polysaccharides could inhibit tumor development by regulating the immune function of tumor mice and affecting the TNF-? signaling pathway. Conclusion. Radix hedysari polysaccharide can effectively inhibit the growth of glioma and affect the TNF-? signaling pathway, thus playing an antiglioma role.

1. IntroductionGlioma is the most common and fatal primary central nervous system tumor in adults. Although the number of the therapeutic drugs for glioma is increasing, the therapeutic effect on glioma is still unsatisfactory. It was investigated that the survival time after a combined approach of surgical resection followed by chemoradiotherapy is short and the prognosis of patients with glioma is extremely poor [1]. Therefore, it is still urgent to seek for the excellent drug for the treatment of glioma. The primary task of developing new drugs is to explore the effect of drugs on proliferation and invasion of glioma and molecular mechanisms involved. Polysaccharides that widely exist in many plants are macromolecular substances with various biological activities, which have not only attracted extensive attention as excellent biological packaging materials but have also been widely used in enhancing immunity, anticancer, and anti-inflammation [2–5]. It was reported that polysaccharides induced tumor cell cycle arrest by regulating cyclin E and D [3] or facilitated apoptosis in glioma cells by altering the Bax/Bcl-2-p53 axis [6]. Polysaccharides extracted from radix hedysari have attracted much attention due to their antitumor activity, immunomodulatory effects, and antioxidant properties [7], but there are few reports on the effects of radix hedysari polysaccharide on glioma.

In the occurrence and development of glioma, macrophages have two different characters, which perform functions of promoting growth and invasion of tumor or killing tumor cells in different microenvironments [8]. And it was reported that radix hedysari polysaccharide can significantly improve the phagocytosis of macrophages [9]. Tumor necrosis factor (TNF-?) is a key cytokine involved in inflammation, immunity, cellular homeostasis, and tumor progression. TNF-? plays an essential role on the normal cell proliferation and function of macrophages, NK cells, T cells, B cells, and dendritic cells, and it is an important effector molecule for cell-mediated killing of some tumors [1, the growth inhibition rate of radix hedysari polysaccharide on glioma cell lines showed dose dependency. The inhibition rate in the low-dose group was 29.5%, and the rate in the middle-dose group was 45.3%. The inhibition rate in the high-dose group was 66.0%, revealing that the growth of cells in the group was significantly suppressed compared with that of other groups (Figure 1). These results indicated that radix hedysari polysaccharide obviously suppressed the growth of U251 cells and 500 ?g/ml of radix hedysari polysaccharide had a better effect of inhibiting cell growth.

Figure 1: The growth inhibition of different concentrations of radix hedysari polysaccharide on U251 cells detected by the MTT assay. , comparing the 50 ?g/ml RHPS experimental groups.3.2. Effect of Radix Hedysari Polysaccharide on Cell Cycle of Glioma Cell LinesIn order to further explore the inhibitory effect of radix hedysari polysaccharide on U251 cells, we studied the changes in the cell cycle of U251 cells and analyzed their cell cycle distribution by flow cytometry. Figure 2 shows the effects of radix hedysari polysaccharide on the cell cycle of U251 cells (G1, S, and G2). As compared with the control group (the cell ratio in the G1 phase was 61.4%), the ratio of U251 cells in the G1 phase was significantly increased in the experimental group treated with 500 ?g/ml of radix hedysari polysaccharide (the cell ratio in the G1 phase was 71.1%, ). The results indicated that radix hedysari polysaccharide can suppress the growth of U251 cells and induce apoptosis in the G1 phase by cell cycle arrest (Figure 2).

Figure 2: G1 arrest in glioma cells induced by radix hedysari polysaccharide. , comparing the blank experimental groups.3.3. Effect of Radix Hedysari Polysaccharide on Cytokines in Glioma Cell LinesThe cytokine expressions of the control group and the administration group treated with 500 ?g/ml radix hedysari polysaccharide were detected by PCR. The results showed that the expression levels of P53 and Bax mRNA were significantly increased (), while the expression level of Bcl-2 mRNA was significantly decreased (), which indicated that radix hedysari polysaccharide induced the apoptosis of U251 cells by changing the expression of cytokine genes (see Figure 3). As compared with the control group, the expression levels of cyclin E and cyclin D in the administration group were significantly decreased () (see Figure 3(a)) and the results of the western blot assay were consistent with those of the PCR assay (see Figure 3(b)), which further indicated that radix hedysari polysaccharide induced apoptosis in the G1 phase through cell cycle arrest.

Figure 3: Effect of radix hedysari polysaccharide on cytokines in glioma cell lines. , comparing the blank experimental groups.3.4. Effect of Radix Hedysari Polysaccharide on Mean Mass and Volume of Tumors in Nude Mice with GliomaWe have demonstrated that radix hedysari polysaccharide inhibited the proliferation of U251 cells in vitro by inducing G1 cell cycle arrest. The glioma model of nude mice was established to study the effect of radix hedysari polysaccharide on the growth of glioma in vivo. As shown in Figure 4, the mice injected with radix hedysari polysaccharide grew slowly and the progression of tumors in vivo was delayed. As compared with the high-dose group (400?mg/kg), the growth delay of the low-dose group (150?mg/kg) was significantly increased and the volume of tumor after 24 days of administration was significantly decreased (). After 24 days of administration, the masses of the blank control group, the low-dose group, and the high-dose group were , , and , respectively. The tumor mass of mice injected with radix hedysari polysaccharide was decreased significantly (), and the tumor mass of the low-dose group was decreased significantly () compared with that of the high-dose group after 24 days of administration. These data suggested that radix hedysari polysaccharide can obviously inhibit tumorigenesis in nude mice.

Figure 4: Effect of radix hedysari polysaccharide on the volume of tumor in nude mice with glioma.3.5. Effect of Radix Hedysari Polysaccharide on Cytokines in Glioma TissuesWe examined the effect of radix hedysari polysaccharide on the changes in cytokine expression of tumor tissue cells by PCR. The results showed that the expression levels of P53 and Bax mRNA were significantly increased (), which was similar with the results in vitro, while the expression of Bcl-2 mRNA was significantly decreased (). The results indicated that the radix hedysari polysaccharide induced the apoptosis of tumor cells by changing the expression of cytokine genes in vivo (see Figure 5). As compared with the control group, the levels of cyclin E and cyclin D in tumor tissues of the administration group were significantly reduced (), indicating that the radix hedysari polysaccharide induced the apoptosis in mice with glioma by regulating the cell cycle (see Figure 5). The effect of radix hedysari polysaccharide on cytokine expression of tumor tissue cells was not dose dependent.

Figure 5: Effect of radix hedysari polysaccharide on cytokines in glioma tissues.3.6. Effect of Radix Hedysari Polysaccharide on Phagocytosis of Macrophages in the Serum of Nude Mice with GliomaDifferentiation and development of glioma are closely related to the phagocytosis of macrophages. We detected the phagocytosis of macrophages in the serum of nude mice with glioma by Giemsa staining. The results showed that the phagocytic rate and index of the nude mice with glioma were significantly lower than those of the normal nude mice (), while the phagocytic rate and index of nude mice with glioma significantly increased after administration of radix hedysari polysaccharide (). There were also remarkable differences between the high-dose group and the low-dose group (see Table 1). The results showed that radix hedysari polysaccharide enhanced the phagocytosis of macrophages in the nude mice with glioma.

Table 1: Enhancement of phagocytosis of macrophages in nude mice with glioma by radix hedysari polysaccharide.3.7. Effect of Radix Hedysari Polysaccharide on TNF-? Signaling Pathway in Nude Mice with GliomaAs radix hedysari polysaccharide can enhance the phagocytosis of macrophages, we further detected such TNF-? signaling pathway proteins as TNF-?, NF-KB P56, and caspase-3 that are closely related to the macrophages in the serum of the nude mice with glioma by ELISA. The results showed that the expressions of TNF-? and caspase-3 in the nude mice with glioma were significantly lower than those in normal nude mice (), while the expression of NF-KB P56 was significantly overexpressed (). And this was obviously improved after the administration of radix hedysari polysaccharide. As compared with the nude mice with glioma, the expressions of TNF-? and caspase-3 in the serum were significantly elevated in the administration group (), and the highly expressed NF-KB P56 was significantly inhibited () (see Table 2).

Table 2: Effect of radix hedysari polysaccharide on the TNF-? signaling pathway in nude mice with glioma.4. DiscussionPolysaccharides play an antitumor role by affecting tumor differentiation and apoptosis, changing intracellular signal transmission and immune regulation [11, 12]. This study found that radix hedysari polysaccharide inhibited the growth of U251 in vitro and arrested cells in the G1 phase, which is consistent with the results reported in other studies [13]. At present, there are many apoptosis-induced drugs, but their toxic and side effects are also serious [14]. As a macromolecular drug extracted from plants, radix hedysari polysaccharide has better effects in enhancing immunity, anticancer, and anti-inflammation and has less toxic and side effects, which therefore is a good candidate drug.

Studies have found that macrophages play an active role in the growth and invasion of glioma. However, as the main cells involved in the immune system, they can secrete TNF to induce apoptosis of tumor cells or directly engulf tumor cells under variable microenvironment [15]. The study found that phagocytosis of macrophages was significantly enhanced by the stimulation of radix hedysari polysaccharide. The link between inflammation and cancer has been well realized in the treatment of many cancers, and inflammation is considered the seventh characteristic of cancer. TNF-? is the main mediator of cancer-associated inflammation and is a factor that promotes tumor growth. The proinflammatory effect of TNF-? was mainly due to its ability to activate NF-?B, while its antitumor effect was due to the activation of caspase-3 and the induction of apoptosis. When exposed to TNF-?, NF-?B is activated and results in the expression of a variety of inflammation-related genes in almost every type of cell. However, the sustained activation of NF-?B is associated with several aspects of tumorigenesis, such as promoting tumor cell proliferation, preventing apoptosis of drug-resistant cells, and increasing tumor angiogenesis and metastasis [16]. However, it was reported that local and repeated injections of large amounts of human recombinant TNF-? can induce hemorrhagic tumor necrosis in homologous and xenograft tumors. By contrast, low-dose chronic TNF-? had angiogenic activity and promoted tumor progression [17]. In this study, the low dose of radix hedysari polysaccharide could significantly increase the levels of TNF-? and caspase-3 in the serum of glioma nude mice and reduce the content of NF-?B. The results suggest that radix hedysari polysaccharide can regulate the immune function of tumor mice, affecting the TNF-? signaling pathway to inhibit tumor development.

Data AvailabilityAll the data is available with the handwritten notebook documented in our lab.

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

AcknowledgmentsThis work is supported by the Weifang Science and Technology Development Plan Project (Grant no. 2016YX023).

References

L. M. DeAngelis, “Brain tumors,” The New England Journal of Medicine, vol. 344, no. 2, pp. 114–123, 2001. View at Publisher · View at Google Scholar · View at Scopus

X.-R. Feng, J.-X. Ding, R. Gref, and X.-S. Chen, “Poly(?-cyclodextrin)-mediated polylactide-cholesterol stereocomplex micelles for controlled drug delivery,” Chinese Journal of Polymer Science, vol. 35, no. 6, pp. 693–699, 2017. View at Publisher · View at Google Scholar · View at Scopus

S. Li, T. Zhang, W. Xu et al., “Sarcoma-targeting peptide-decorated polypeptide nanogel intracellularly delivers shikonin for upregulated osteosarcoma necroptosis and diminished pulmonary metastasis,” Theranostics, vol. 8, no. 5, pp. 1361–1375, 2018. View at Publisher · View at Google Scholar · View at Scopus

J. Chen, J. Ding, W. Xu et al., “Receptor and microenvironment dual-recognizable nanogel for targeted chemotherapy of highly metastatic malignancy,” Nano Letters, vol. 17, no. 7, pp. 4526–4533, 2017. View at Publisher · View at Google Scholar · View at Scopus

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 · View at Google Scholar · View at Scopus

S. Zhong, D. F. Ji, Y. G. Li, T. B. Lin, Z. Q. Lv, and H. P. Chen, “Activation of P27kip1-cyclin D1/E-CDK2 pathway by polysaccharide from Phellinus linteus leads to S-phase arrest in HT-29 cells,” Chemico-Biological Interactions, vol. 206, no. 2, pp. 222–229, 2013. View at Publisher · View at Google Scholar · View at Scopus

P. Sitarek, E. Ska?a, M. Toma et al., “A preliminary study of apoptosis induction in glioma cells via alteration of the Bax/Bcl-2-p53 axis by transformed and non-transformed root extracts of Leonurus sibiricus L.,” Tumor Biology, vol. 37, no. 7, pp. 8753–8764, 2016. View at Publisher · View at Google Scholar · View at Scopus

D. Wei, Y. Wei, W. Cheng, and L. Zhang, “Sulfated modification, characterization and antitumor activities of Radix hedysari polysaccharide,” International Journal of Biological Macromolecules, vol. 51, no. 4, pp. 471–476, 2012. View at Publisher · View at Google Scholar · View at Scopus

J. Xie, D. T. Wu, W. Z. Li et al., “Effects of polysaccharides in Lycium barbarum berries from different regions of China on macrophages function and their correlation to the glycosidic linkages,” Journal of Food Science, vol. 82, no. 10, pp. 2411–2420, 2017. View at Publisher · View at Google Scholar · View at Scopus

L. Pinton, E. Masetto, M. Vettore, and S. Solito, “The immune suppressive microenvironment of human gliomas depends on the accumulation of bone marrow-derived macrophages in the center of the lesion,” Journal for Immunotherapy of Cancer, vol. 7, no. 1, p. 58, 2019. View at Publisher · View at Google Scholar · View at Scopus

D. Li, W. G. Xu, P. Q. Li et al., “Self-targeted polysaccharide prodrug suppresses orthotopic hepatoma,” Molecular Pharmaceutics, vol. 13, no. 12, pp. 4231–4235, 2016. View at Publisher · View at Google Scholar · View at Scopus

L. Di, J. X. Ding, X. L. Zhuang, L. Chen, and X. S. Chen, “Drug binding rate regulates the properties of polysaccharide prodrugs,” Journal of Materials Chemistry B, vol. 4, no. 30, pp. 5167–5177, 2016. View at Publisher · View at Google Scholar · View at Scopus

P. W. Szlosarek and F. R. Balkwill, “Tumour necrosis factor alpha:a potential target for the therapy of solid tumours,” The Lancet Oncology, vol. 4, no. 9, pp. 565–573, 2003. View at Publisher · View at Google Scholar · View at Scopus

Y. L. Cheng, C. L. He, J. X. Ding, C. S. Xiao, X. L. Zhuang, and X. S. Chen, “Thermosensitive hydrogels based on polypeptides for localized and sustained delivery of anticancer drugs,” Biomaterials, vol. 34, no. 38, pp. 10338–10347, 2013. View at Publisher · View at Google Scholar · View at Scopus

V. Haage, M. Semtner, R. O. Vidal, D. P. Hernandez, W. W. Pong, and Z. Chen, “Comprehensive gene expression meta-analysis identifies signature genes that distinguish microglia from peripheral monocytes/macrophages in health and glioma,” Acta Neuropathologica Communications, vol. 7, no. 1, p. 20, 2019. View at Publisher · View at Google Scholar · View at Scopus

A. Mantovani, P. Allavena, A. Sica, and F. Balkwill, “Cancer-related inflammation,” Nature, vol. 454, no. 7203, pp. 436–444, 2008. View at Publisher · View at Google Scholar · View at Scopus

A. Katsman, K. Umezawa, and B. Bonavida, “Chemosensitization and immunosensitization of resistant cancer cells to apoptosis and inhibition of metastasis by the specific NF-?B inhibitor DHMEQ,” Current Pharmaceutical Design, vol. 15, no. 7, pp. 792–808, 2009. View at Publisher · View at Google Scholar · View at Scopus