Calycosin(CA) Inhibits Proliferation, Migration and Invasion by Suppression of CXCL10 Signaling Pathway in Glioma

Background: Glioblastoma is a frequently encountered central nervous system malignancy. Calycosin, a bioactive compound extracted from dried Radix astragali root has exhibited anticancer activity across several different types of cancers, including glioma, but the therapeutic mode of action has yet to be clari�ed. One ubiquitous in�ammatory chemokine implicated in cancer biology is the C-X-C chemokine ligand 10 (CXCL10). The current study seeks to investigate CXCL10 as a likely calycosin target in aiding its inhibitory effects on glioblastoma progression. Method: The clinical signi�cance of CXCL10 including pathologic grade and survival rate in GBM patients were accessed from TCGA database and our clinical samples. CCK-8 experiments and clone formation assay were performed to detect the cell proliferation. Transwell assay was conducted to examined cell migration and invasion. Western blotting and immuno�uorescence staining was used to analyze protein expression levels. Xenograft mouse model was used to evaluate the effect of CA in vivo. Results: Firstly, we demonstrated that CXCL10 expression is positively associated with glioma and correlated negatively to patient prognosis, supporting our hypothesis that CXCL10 was a potential target for GBM treatment. Additionally, U251 and U87 glioma cell lines were found to demonstrate inhibited invasive, migratory and proliferative abilities upon exposure to calycosin. Calycosin was then found to downregulate CXCL10 expression in a manner that increased with increasing doses, resulting in reduced downstream in�ammatory cytokines including NLRP3, NF-κB, IL-1β. Moreover, we demonstrated that overexpressing CXCL10 abolished the inhibitory impact of calycosin on cellular invasion, migration and proliferation in glioma cell lines. Meanwhile, CXCL10 knockdown enhanced calycosin effects on GBM cells. We also used in vivo xenograft mice models to determine the impact of calycosin on glioma growth and obtained results similar to our in vitro experiments. Conclusions: The current investigation underscores the role of CXCL10 as a promising therapeutic target for treating GBM, with calycosin functioning as a future treatment modality.


Introduction
Glioblastoma (GBM) is an aggressive and unfortunately frequently encountered cerebral malignancy.
GBM treatment primarily involves surgery, radiotherapy and chemotherapy.[1,2].However, most GBM present as multifocal disease with high degrees of invasiveness.Complete surgical resection is challenging, resulting in abysmally high rates of recurrence and an average survival of a mere 14.6 months after diagnosis [3].Improved therapeutic methods are necessary to treat GBM.Calycosin(CA) is a signi cant bioactive chemical extracted from dried Radix astragali root [4].As an iso avonoid and a phytoestrogen, calycosin exerts anti-in ammatory, neuroprotective, and cardiovascular protective properties [5][6][7].Recently, it has been reported that calycosin exhibits antitumor activities.For instance, calycosin appears to inhibit breast cancer progression by downregulating Foxp3/VEGF/MMP-9 signaling pathway [8].In addition, calycosin was demonstrated to induce apoptosis via ERβmediated PI3K/Akt signaling pathways in osteosarcoma [9].Nie et al. veri ed that calycosin was also effective in inhibiting GBM growth through TGFβ suppression [10,11].However, the underlying mechanisms have yet to be fully outlined.
Chemokines are small secretory immunoregulatory proteins which interact with a subset of seventransmembrane G protein-coupled receptors (GPCRs) in order to regulate cell tra cking.GPCRs also participate in oncogenesis, tumor development and metastatic spread.The C-X-C motif chemokine (CXCL10), also named as the interferon-gamma-induced protein 10 (IP-10), is a 10 kDa polypeptide belonging to the CXC chemokine subfamily.Like most chemokines, CXCL10 exerts multiple biological functions ranging from mediating immune cell chemotaxis, angiogenesis and the in ammatory response [12][13][14].In recent years, some studies have reported that CXCL10 contributed to the progression in various cancers such as colorectal, ovarian, breast and pancreatic cancer [15][16][17][18].
Nevertheless, the role of CXCL10 as a tumor suppressor or oncogenic factor in glioma has yet to be con rmed.
In ammation is a signi cant instigator in the onset and progression of tumorigenesis [19].In ammation accelerates GBM cancer progression while simultaneously promotes treatment-resistance [20].The NODlike receptor protein-3 (NLRP3) in ammasome is documented to be excessively activated in various cancers including glioma.NLRP3 suppression prolonged the survival of glioma-bearing mice as well as tumor growth.NLRP3 downstream effectors IL-1β and NF-κB were also abundantly found in the GBM tumor microenvironment (TME), and likely contributes to tumor development [21][22][23].However, there is limited knowledge regarding the relationship between CXCL10 and NLRP3.Whether or not CA can downregulate CXCL10 to inhibit GBM also reminds unknown.This series of investigations demonstrates that raised CXCL10 expression was associated with more advanced tumor pathological grade and a poorer prognosis.We also identi ed CXCL10 to be a potential treatment target of CA in suppressing GBM, which likely works by downregulating downstream molecules such as NLRP3, NF-κB and IL-1β both in vivo and in vitro.These ndings suggest that CXCL10 was a novel biomarker in glioblastoma and CA may be a potential treatment agent in GBM.

Reagents
Calycosin was purchased from Tianjin Wanxiang Hengyuan Science and Technology Ltd (Tianjin, China).Dulbecco's Modi ed Eagle Medium (DMEM) and Fetal Bovine Serum (FBS) were purchased from Gibco™.Antibody against CXCL10, NLRP3 and IL-1β were purchased from Cell Signaling Technology.Antibodies against NF-κB were purchased from Wuhan Servicebio Technology Co. Ltd. (Wuhan, China)

Cell Culture
The human GBM cell lines U87 and U251 were purchased from iCell Bioscience Inc (Shangha, China).These cell lines were cultured in Dulbecco's Modi ed Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37℃ in a humidi ed atmosphere in 5% CO 2 .
Cell Viability Assay 4000 cells/well were planted in a 96-well plate and treated with different doses of calycosin for 24 h.Then, 10 µl of the CCK-8 solution (Beyotime, Shanghai, China) was added to each well and incubated for 1 h at 37℃.Then, the reaction mixture was measured by Multimode Reader.
Colony formation assay 500 cells/well were seeded in 60mm dishes and treated with different doses of calycosin for 2 weeks.
Colonies were counted after xing by methanol and staining with Crystal Violet Staining Solution (Beyotime, Shanghai, China).

Cell Invasion Assay
The transwell system for assay of cell migration and invasion was purchased from Corning (Corning, USA).2×10 5 cell/well (200 µl DMEM supplemented with 1% FBS) were seeded in the upper chamber (8 µm Pore Polycarbonate Membrane) coated with o 100 µl matrigel (BD Biosciences, CA, USA).The lower chamber was lled without or with 600 µl DMEM supplemented with 20% FBS and indicated different concentrations of calycosin.After 24 h, the cells in the lower chamber were xed by methanol and stained with Crystal Violet Staining Solution (Beyotime, Shanghai, China).Five independent elds in each well were photographed at 100×magni cation, after which the cells in each eld were counted.

Western Blotting
Western blotting was performed using glioblastoma cell lysates, clinical normal or tumor tissues homogenates and xenograft glioblastoma tissue homogenates.Protein was extracted using PRO-PREP™ Protein Extraction Solution (Cell/Tissue) (iNtRON Biotechnology, Korea) according to manufacturer's instructions.Equal amounts of total protein were separated on 10-12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene di uoride membranes (Merck, KGaA, Darmstadt, Germany).The membranes were blocked with 5% BSA at room temperature for 1 h, and then incubated with speci c primary antibodies overnight at 4℃.The appropriate secondary antibodies conjugated with HRP were incubated for 1 h at room temperature, signal was obtained using Immobilon Western HRP Substrate (Merck, KGaA, Darmstadt, Germany).

U87 Xenograft Mouse Model Paeoni orin Treatment
Female BALB/c nude mice were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China).Mice were aged 6-8 weeks and kept under a standard protocol approved by the Laboratory Animal Center of the Second A liated Hospital of Guangzhou Medical University.All procedures performed in studies involving animals were in accord with the ethical standards of the Ethics Committee.Each mouse was injected subcutaneously with cultured U87 cells (5×10 6 cells/mouse) into the dorsum.The tumor size was measured in two orthogonal directions using calipers, and the tumor volume (mm 3 ) was calculated using the equation: 1/2×length×width 2 .When the tumors grew to about 150 mm 3 , the tumor-bearing mice were distributed into two groups (n = 5 each) and orally fed with calycosin (10mg/kg/day) or vehicle (equivalent amount of PBS) Tumor sizes and body weights were measured once every 5 days.At the end of these experiments, the mice were sacri ced and the tumors were resected and homogenized for western blotting.

Statistical analysis
All data are composites of three repeated experiments and were analyzed using the SPSS 20.0 software.An independent T-test was used to perform simple comparisons between 2 groups and multiple comparisons between the groups were evaluated using one-way analysis of variance, followed by post hoc analyses, which were carried out using Dunnett's T3 test or Turkey test.P < 0.05 was interpreted as achieving statistical signi cance.

Results
CXCL10 is upregulated in glioma tissues and contributes to poor prognosis CXCL10 mRNA expressions were rst quanti ed in glioma and normal brain tissues based on publicly accessible data from The Cancer Genome Atlas (TCGA) and GTEx database as well as in 24 pairs of glioma tissue samples and matched adjacent normal brain tissue samples.CXCL10 expression levels were signi cantly raised in glioma tissues in contrast to their adjacent healthy tissues (Fig. 1A, D-E).CXCL10 mRNA expression were signi cantly increased based on more advanced WHO GBM grade (Fig. 1B, F-G).Furthermore, as shown in Fig. 1C and H, patients with elevated CXCL10 expression had markedly decreased overall survival (OS) in contrast to patients with lower CXCL10 expressions.These results suggest that high CXCL10 expression correlates positively to poorer prognosis in GBM patients.

CA inhibited cells growth and downregulated CXCL10 signaling in GBM cells
We evaluated the impact of CA on GBM, CCK-8 experiments was performed.CA treatment markedly decreased the degree of cell proliferation in U87 and U251 GBM cells in a time-and dose-dependent manner.An increase in CA concentration from 100 to 400 µM resulted in reduced cell viability from 90-40% in both cell lines (Fig. 2A-B).CA effects on GBM cell growth were evaluated using colony formation assays.Likewise, the number of viable cell colonies were reduced from 50 to 5 in a dose-dependent manner.In conclusion, CA appears to have a suppressive effect on in vitro GBM cell growth (Fig. 2C-D).
CA effects on the migratory and invasive abilities of cells were also studied.In contrast to the untreated groups, the CA treated groups exhibited reduced cell invasiveness and cell migration in a dose-dependent manner (Fig. 2E-F).These results demonstrate that CA signi cantly inhibits GBM cell migration and invasion.
As mentioned above, CXCL10 is upregulated in GBM tissues and contributes to tumor progression and a poorer patient prognosis.Suppressing CXCL10 appears to be a promising therapeutic modality for GBM.GBM cell exposed to CA demonstrated downregulated expressions of CXCL10 and its downstream molecules such as NLRP3, NF-κB and IL-1β.As shown in Fig. 2I, CA reduced CXCL10, NLRP3, NF-κB and IL-1β expressions in a dose-dependent manner.

Overexpression of CXCL10 reduced the effects of calycosin in GBM cells
To further characterize whether CA inhibited GBM cell growth by downregulating CXCL10, U87 and U251 cells with a lentiviral vector carrying CXCL10 cDNA.The results showed that overexpressed CXCL10 enhanced U87 and U251 cells invasive, migratory and proliferative abilities.Furthermore, overexpressing CXCL10 reduced the suppressive properties of CA on U87 and U251 cells (Fig. 3A-H).We also found that overexpressing CXCL10 promoted NLRP3, NF-κB, IL-1β expression (Fig. I).

Downregulation of CXCL10 enhances the effects of calycosin in GBM cells
To clarify the tumorigenic role of CXCL10, CXCL10 expression was knocked down in CA-treated GBM tumors using speci c siRNA.CCK-8 and colony formation experiments showed that knockdown of CXCL10 enhanced CA-induced suppression of cells proliferation (Fig. 4A-D).Both CXCL10 siRNA or CA strongly inhibited cell migratory and invasive capabilities (Fig. 4E-H).These results indicated that combination treatments demonstrated a signi cantly greater effect compared to any agent given in isolation.Our results also demonstrated that CA treatment decreased NLRP3, NF-κB and IL-1β expressions while additional CXCL10 suppression resulted in even lower in ammatory marker expressions (Fig. 4I).Taken together, these experiments highlight the that the anticancer CA property was at least partly mediated through downregulating CXCL10.
CA inhibits GBM Growth in a U87 Xenograft Mouse Model A U87 xenograft mouse model was used to evaluate the inhibitory effects of CA on the progression of GBM.As shown in Fig. 5A-C, mice treated with CA had much smaller tumor volumes in contrast to the control group.Body weights of mice from both groups were similar after 20 days of treatment with either CA or vehicle (Fig. 5D).Furthermore, the U87 xenograft mouse models treated with CA had lower CXCL10, NLRP3, NF-κB and IL-1β tumor expressions.CA appeared to remarkably suppress the CXCL10 pathway (Fig. 5E).Our results from the in vivo studies are similar to those gained in our in vitro experiments, further highlighting the central role of CA in inhibiting GBM progression.

Discussion
In present study, we proved that overexpressing CXCL10 promoted GBM progression.Moreover, we demonstrated that CA was able to inhibit growth, migration and invasion in GBM via downregulation of CXCL10 pathway.We also veri ed that CA exerts anti-tumor effect in a GBM xenograft mouse model.Similar ndings were seen in both our in vitro and in vivo experiments.
Current treatment modalities available to treat GBM comprises primarily of surgical resection along with supporting chemoradiotherapy.Curative resection is challenging given the multifocal and aggressive nature of disease [1].This study identi es calycosin, a natural bioactive chemical extracted from dried Radix astragali root to exert anti-tumor properties through suppressing CXCL10 expression in GBM.
Calycosin has shown anti-tumor effects across several different types of malignancies, including glioma [8][9][10].A previous study by our group found that CA exerted anti-GBM effects by downregulating TGFβ and c-Met [10,11].Furthermore, Ni Q et al reported that the combination of CA and temozolomide enhanced anti-tumor effects in GBM [24].However, the underlying molecular target of CA in GBM remains to be completely investigated.In our study, we veri ed that CA inhibited GBM growth both in vitro and in vivo through inhibition of the CXCL10 signaling pathway, indicating that the CXCL10 pathway may be critical in treating GBM.CXCL10 is involved in the regulation of multiple in ammatory signaling pathways, which are closely linked with tumor development and occurrence.However, the role of CXCL10 in GBM progression remains controversial.Maru SV et al reported that overexpression CXCL10 signi cantly impacts glioma cell proliferation [25].A recent immunohistochemical study found that CXCL10 was overexpressed in GBM [26].Kenji Shono et al revealed that CXCL10 exerts anti-tumor impact in a mouse model of malignant glioma [27].CXCL10 appears to be critical regulator of GBM growth and progression.Suppressing CXCL10 may potentially impart positive effects in treating GBM.However, there are contradictory studies which have found that CXCL10 upregulation inhibits GBM progression [28,29].In current study, we demonstrated that CXCL10 was obviously elevated in GBM and is negatively associated with patient prognosis, based on data gained from publicly accessible databases and actual patient tissue samples.
We therefore hypothesize that CXCL10 functions as an oncogene in GBM.Furthermore, we con rmed that downregulating CXCL10 suppressed the in ammatory signaling pathway which further inhibited GBM progression.
Many agents exert in vitro anti-GBM effects but fail to yield similar results in GBM orthotopic xenograft mouse models due to the existence of blood-brain barrier (BBB).Previous studies on cerebral ischemic mouse models and brain reperfusion injury models found that CA exerted remarkable therapeutic effects in brain disease and showed a good safety pro le [5].This study nds that CA inhibited GBM growth by downregulating the CXCL10 signaling pathway in a GBM mouse model.CA also decreased tumor volume without signi cant changes in body weight, which further veri es the in vivo safety and e cacy pro les of CA.
Overall, we demonstrated that CXCL10 functions as an oncogene and holds to potential as a potential therapeutic target of CA in glioma.

Figure 5 Effects
Figure 5