Cucurbitacin B Controls M2 Macrophage Polarization to Suppresses Metastasis via Targeting JAK-2/STAT3 Signalling Pathway

Haoyue Zhang Nanjing University of Chinese Medicine A liated to Nanjing University of Traditional Chinese Medicin Bei Zhao Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine Yu Yan Shanghai University of Traditional Chinese Medicine Hairong Zeng Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine Dongya Sheng Shanghai University of Traditional Chinese Medicine Yueyang Hospital of Integrated Traditional Chinese Medicine and Western Medicine Yang Zhang (  fsyy00239@njucm.edu.cn ) Nanjing University of Chinese Medicine A liated to Nanjing University of Traditional Chinese Medicine https://orcid.org/0000-0002-5992-5310


Introduction
Colorectal cancer (CRC), one of the leading causes of cancer morbidity and mortality in China, with around 1.8 million newly diagnosed cases worldwide annually [1]. The high mortality rate of CRC mainly is due to metastasis in 40 percent of cases. In most cases, this is because metastatic tumor cells are resistant to therapy and evade immune responses. Accumulating evidence suggests that the alterations of immune microenvironment and epigenetic contribute to the progression and metastasis of CRC [2]. Despite signi cant advance in therapies for CRC, many patients show a poor response to conventional therapy, leading to subsequent recurrence and a poor prognosis, highlighting the importance in innovative therapeutic approaches.
Tumor-associated macrophages (TAMs) are a pivotal component of the tumor microenvironment, which are emerging as a factor in the development and metastasis of CRC [3,4]. It is can divided into classically activated macrophages (M1) and alternatively activated macrophages (M2), which both have been observed in tumors [5,6]. It is generally believed that these two types of macrophages are functionally antagonistic [7]. Alternatively activated M2 macrophages promote the occurrence and development of tumors by involving in moderating in ammatory responses, promoting angiogenesis and contributing to tissue remodeling [8]. In respond to T helper type 2(Th2) cytokines such IL-4 and IL-13 results in alternative activation(M2), characterized by increase of mannose receptor (CD206), increased arginase-1 (Arg-1), found in in ammatory zone (Fizz-1), chitinase-like 3(Ym-1), Chemokine (CCL2) [9]. More importantly, increasing the number of M2 macrophages is a vital marker of CRC and is directly associated with poor clinical prognosis [10,11]. Thus, it is paramount to extensively and intensively understand the relationship between macrophage in ltration and colorectal patients' tumor progression, for the development of effective therapeutic strategies.
JAK/STAT pathway serves as multiple regulatory roles to modulating immune function, cell growth, differentiation and death [12][13][14]. Structure activation of JAKs contributes to phosphorylation of the signal transducer and activator of transcription (STAT) family [15,16]. STAT3 signaling is closely correlated with in building a protumor genic in ammatory microenvironment [17]. STAT3 activation in macrophages has been shown to enhance proliferation and survival of macrophage, and potentiate protumor functions such as immune tolerance and extracellular matrix remodeling inhibition of colon cancer cells [18]. Therefore, STAT3 inhibition is considered to be effective for patients with advanced malignant tumors.
Cucurbitacin B is a main active monomer of Cucurbitaceae, and has been widely used in the treatment of malignant tumors [19][20][21]. In vivo and in vitro experiments have shown that cucurbitacin B inhibited the proliferation of multiple cancers via NF-κB, JAK/STAT, PI3K/AKT, and MAPK/ERK signaling pathways [22,23]. To date, cucurbitacin B is a highly promising anti-tumor drug. However, the speci c molecular mechanism under of cucurbitacin B in CRC remains unclear.
Traditional herb medicines serve as growing vital roles in inhabitation and treatment of multiple cancers [24]. Here, we researched the anti-tumor and anti-metastasis effects of cucurbitacin B on the CRC, and we revealed the potential molecular mechanisms. In vitro assays results suggested that cucurbitacin B might inhibit the proliferation of CRC by JAK/STAT3 signaling pathway. According to the gap, herein, IL-4/13 or LPS was used to stimulate macrophages polarizing to M2 or M1 phenotype. The M2 polarized macrophages were co-cultured with CT-26 to develop a M2-like TAMs macroenvironment. The effects of cucurbitacin B on the metastatic behavior of CT-26 and HCT 116 in the co-culture system were investigated. In addition, the in vivo anti-cancer effects of cucurbitacin B were investigated in a subcutaneous xenograft tumor mice model. This study provided a novel mechanism of disrupting the M2 polarization and promoted the M1 polarization-induced signaling to inhibit the tumor growth, which can be utilized to develop therapeutic applications of cucurbitacin B as an anti-metastatic therapy for colon cancer.

Results
Cucurbitacin B suppressed the proliferation of colorectal cancer cell We performed a CCK8 assay to investigate the ability of cucurbitacin B in inhibiting tumor proliferation.
Cucurbitacin B effectively inhibited the viability of HCT116 and CT26 cells in dose-dependent manner, with IC50 values of 435.70 nM and 364.20 nM respectively. To further investigate whether cucurbitacin B induced apoptosis, the percentage of Annexin V apoptosis cell was analyzed by ow cytometry with Annexin V/PI staining. HCT116 and CT26 cells were treated with cucurbitacin B at different concentrations (400 nM and 800 nM) for 48 h. The apoptosis of HCT116 and CT26 cells was dramatically increased after treatment with cucurbitacin B. Next, colony formation assay revealed that cucurbitacin B prohibited colonies in concentration-dependent manner, the signi cant different between control and cucurbitacin B group. Here, we also found that increase in apoptosis concomitant with the changes in expressions level of cleaved caspase-3, Bax and Bcl-2.
As a result, the above date demonstrated that cucurbitacin B exerted anti-tumor effects on CRC.
Cucurbitacin B inhibited JAK2/STAT3 signaling We next sought to determine conducted the expression of JAK2 and STAT3 in CRC cells treated with cucurbitacin B. Figure 2a shows signi cantly decrease on phosphorylated JAK2 and STAT3 in CRC cells treated with cucurbitacin B.
We next determined to investigate whether IL-6-triggered STAT3 activation differed between control and cucurbitacin B treatment. We therefore examined the expression level of total and phosphorylated STAT3 in CRC cells treated with cucurbitacin B. As anticipated, exposure of CT-26 and HCT116 cells to 25 ng/mL lead to increased nuclear level of phosphorylated STAT3 compared with unstimulated CT-26 and HCT116, whilst cucurbitacin B can suppressed this translocation. Mechanistically, to further validate the interaction between cucurbitacin B and STAT3, uorescence measurement and microscale thermophoresis (MST) were performed. We found cucurbitacin B bound to stat3 (K d = 39.75 µmol/L) ( Figure 2b). Moreover, the double IF staining of p-STAT3 was conducted to further con rm the importance of JAK-2/STAT3 signaling pathway. According to Figure 2c, that the nuclear p-STAT3 level was increased treated with IL-6 and decreased when cells were treated with both IL-6 and cucurbitacin. Collectively, these results supported that the inhibitory effect of cucurbitacin B on the normal or IL-6-triggered p-STAT3 in CRC cells.

Cucurbitacin B inhibited Macrophages switching to M2 phenotype
Multiple mechanisms of immune suppression can arise in tumor to facilitate the metastasis of cancer [25]. It is generally accepted that M2 phenotype is crucial in growth of tumor. To future order to investigated whether cucurbitacin B is involved in macrophage polarization,we performed ow cytometry analysis and RT-PCR assays. First of all, RAW264.7 and THP-1-derived macrophage cells were exposed with or without cucurbitacin B investigate how cucurbitacin B affected the distribution of M2 polarization. It is well known, CD206 is a marker for activated M2 macrophage [26]. It is well known that M2 polarization of macrophages are activated by cytokines such as IL-4 and IL-13. As shown in Figure 3a and 3b, on the one hand, stimulation of IL-4/IL-13 (without cucurbitacin B) signi cantly increased the expression of CD206 marker (p < 0.05). Afterwards, cucurbitacin B has shown to be able to suppress the IL-4/IL-13-induced expression of chitinase 3-like 3 (Ym-1), in ammatory zone (Fizz-1), arginase 1 (Arg-1), and CCR-2 ( Figure 3c and 3d) (p < 0.05).
Collectively, these observations suggest that cucurbitacin B inhibited polarization of TAMs to M2 phenotype in RAW264.7 macrophages.
The JAK-2/STAT3 signaling pathway involved in cucurbitacin B-mediated inhibition of M2 polarization Previous studies indicated the JAK2/STAT3 pathway plays a vital and in ammatory mediator in macrophage polarization. The JAK-2/STAT3 signaling pathway has shown to participate in IL-4/IL-13induced TAMs polarization. Herein, the phosphorylation status of JAK-2/STAT3 in IL-4/IL-13-induced RAW264.7 and THP-1-derived macrophages with or without cucurbitacin B was tested using western blotting assay. As described in Figure 4a, in RAW264.7 macrophages, the phosphorylation status of JAK-2/STAT3 was enhanced after treating cells with IL-4/IL-13. This increased phosphorylated level of JAK-2 and STAT3 was signi cantly inhibited by co-treatment with cucurbitacin B, suggesting that JAK-2/STAT3 signaling pathway might participate in cucurbitacin B-mediated inhibition of M2-like TAMs polarization. Therefore, collectively, these ndings support the hypothesis that JAK-2/STAT3 signaling pathway participated in cucurbitacin B-mediated inhibition of M2-like TAMs polarization.

Cucurbitacin B inhibited the migration induced by inhibiting M2 polarization of TAMs
It is well known that M2-like macrophage polarization is a major obstacle to effective anti-metastasis immune response. According to our above study, cucurbitacin B can suppressed M2-like macrophage polarization. Therefore, collecting condition medium from cucurbitacin B-stimulated M2 macrophages to investigate the effect on CT-26 cells' migration and invasion. Herein, CT-26 and HCT116 were co-cultured with IL-4/IL-13-stimulated RAW264.7 and PMA induced THP-1 cells, and then the capacity of cell migration and invasion to explore the effects of M2-polarized TAMs on capacity of migration and invasion of CT-26 and HCT116. The capacity of CT-26 and HCT116 migration and invasion was detected by transwell assay and wounding heal assay. The results suggest that M2 polarized TAMs promoted the cell invasion abilities (Figure 5a) of CT-26 and HCT116. The 24 h wounding healing assay was also performed to further con rm the migration ability of colon cancer cells. Cucurbitacin B gradually decreased the healing rate of CT-26 in ltrated with M2 polarized TAMs (p < 0.05) (Figure 5b). These ndings together revealed that cucurbitacin B could inhibit the migration and invasion capacity of colon cancer cells induced by M2-like TAMs polarization.

Cucurbitacin B inhibited tumor growth and metastasis in vivo
To determine the effects of cucurbitacin B on CRC growth and metastasis in vivo, we injected luciferin labeled CT-26 cell in C57BL/6 and BALB/c mice receptively. Figure 6a and 6b showed that treatment of cucurbitacin B at dose of 0.5 and 1 mg/kg signi cantly inhibited tumor growth, which was re ected by the decrease of the volume and the weight of tumor, and the nal volume and the weight of tumor from mice administrated with cucurbitacin B were signi cantly lower than those from control mice. In conjunction, no signi cant difference in body weight was observed between control and the cucurbitacin B-treated groups ( Figure. To verify the applicability of the cucurbitacin B therapy to CRC, the effects upon colon cancer metastasis were appraised using a colon cancer lung metastasis model. As results, the number of pulmonary tumor nodules dramatically upregulated after intravenous injection of CT-26 cells. However, cucurbitacin B at dose of 0.5 and 1 mg/kg for 2 weeks prominently decreased the bioluminescence accumulation ( Figure  6f). This data supported the notion cucurbitacin B treatment inhibited CT-26 cell metastasis in BALB/c mice.
Cucurbitacin B regulated M2-like polarization and promoted M1-like polarization in C57BL/6 murine colon cancer model We further verify whether cucurbitacin B suppressed the progression of colon cancer by regulating macrophages. The colon sections from different groups were stained with the M2-macrophage marker CD206 by IHC staining. The results are shown in Figure 6a, administration of cucurbitacin B signi cantly decreased the expression of CD206 in a dose-dependent manner when compared to control groups (p < 0.05). In agreement with this, Figure 6b indicated that the expression of CD206/CD11c were remarkedly suppressed after cucurbitacin B treatment in mouse spleen.
It is reported that macrophage can produce cytokines that participated in CD4+T and CD8+T cell activation. To further investigate the effect on macrophage by cucurbitacin B, we examined the in ltration of the T cells population in the CT-26 allograft after cucurbitacin B treatment by ow cytometry. As shown in Figure 6c, signi cant increase in the population of CD4+T and CD8+T cells were observed after cucurbitacin B treatment. (p<0.05.) These ndings suggested that cucurbitacin B could relief TAMmediated immunosuppression.

Discussion
As reported, cucurbitacin B exhibited anti-tumor and anti-metastasis effect in diverse cancer such as lung cancer, pancreatic cancer, neuroblastoma cancer and colon cancer in vitro and in vivo [27][28][29]. In continuation to characterize that cucurbitacin B exert anti-tumor effect via various pathway, this study aimed to explore the effect of cucurbitacin B on tumor growth and metastasis in vitro and vivo. Consistent with the previous ndings, we observed the anti-tumor effects of cucurbitacin B on CRC cells CT-26 and HCT116 cells in vitro. In our study, we validated that cucurbitacin B markedly inhibited cell viability in CRC cells. At the same time, our data also showed that cucurbitacin B had a similar impact on apoptosis and colony formation of CT26 and HCT116 cells. In addition, these results demonstrated cucurbitacin B suppressed the growth of CRC cells by apoptosis. We then nd the cucurbitacin B decreased the expression levels of phosphorylated STAT3 in a concentration-dependent manner. Meanwhile, our data also showed that cucurbitacin B could inhibit the IL-6-induced phosphorylation of STAT3. Particularly, a strong binding a nity was observed between cucurbitacin B and STAT3 by microscale thermophoresis analysis. Moreover, cucurbitacin B diminish nuclear accumulation of p-STAT3 by immunouorescence analysis. Altogether, these results demonstrated suggested that cucurbitacin B could target STAT3 in CRCs.
Macrophages, one of the main immune cells in TMA, have been validated to correlated with the growth and metastasis of various type of tumor [30]. Within the progression of tumor, M1 activation and M2 activation in tumor elicit anti-tumor and pro-tumor functions respectively. Clinically, M2-like TAM are one of the amplest cell types in diverse solid tumor and an important role to play in modulating tumor progression and metastasis [31]. It has been proved that large numbers of macrophages in ltrating into colon adenocarcinoma tissues were related to the poor patient prognosis [32]. Therefore, reprogramming of M2-like macrophage toward the antitumoral might be potential new therapeutic targets. More and more studies have indicated the mechanism of monomeric components of traditional Chinese medicine exert anti-tumor effect by modulating M1 or M2 macrophage polarization [33,34].
Despite it has been reported that cucurbitacin B gains great attention as a natural product which exert anti-tumor immunity, the underlying mechanism of its macrophage regulation is still poorly unexplored [35]. The remarkable inhibition of M2-like RAW264.7 and THP-1-derived macrophages polarization by the administration of cucurbitacin B was observed in this study.
The M1 and M2 phenotypes were aim to de ne macrophages, however the speci c markers were ultimately expression on monocytes. Our data showed lower expression of CD206 (M2) and the M2 markers (Ym-1, Fizz-1, Arg-1, IL-10) extremely diminish after cucurbitacin B treatment. To date, it has been proved that CD206 M2 TAMs increase cancer progression by STAT3 activation, inducing and maintaining a microenvironment [36]. Therefore, JAK2/STAT3 signaling in TAM is a crucial component of response to M2-like TAMs macrophages [37]. Herein, the role of JAK2/STAT3 signaling pathway in inhibiting M2-like TAMs macrophages induced by cucurbitacin B was investigated. The ndings in this study show that cucurbitacin B-induced inhibition of M2 decreased the phosphorylation status of JAK2 and STAT3. We therefore conclude that JAK2 /STAT3 participate in the progression of cucurbitacin B-induced inhibition of M2-like macrophages.
CRC is considered as an in ammation-related cancer, likewise one of the most common reasons of cancer-associated deaths worldwide [38,39]. Therefore, we inferred that cucurbitacin B was more likely to alter the tumor microenvironment, inhibiting M2 polarization, and thus inhibiting tumor metastasis. A coculture system was performed to explore the effect of cucurbitacin B-stimulated M2 phenotype macrophages on migration in vitro. Herein,we were surprised to nd that cucurbitacin B suppress M2 macrophage polarization, which result in inhibiting migration and invasion of colon cancer cell in our established in vitro colon TAM model. The ndings in this study revealed that cucurbitacin B suppressed M2-like macrophage and to further enhance the anti-metastasis in vivo.
In vivo study, cucurbitacin B has shown to inhibit the tumor growth and metastasis in C57BL/6 and BALB/c murine model respectively, and be functioned as an apoptosis inducer, as the evidence of increased Ki67 and caspase-3 expression levels in cucurbitacin B-administrated murine groups. However, the potential mechanisms of inducing the cell apoptosis and inhibiting the cell proliferation still need to be further investigated via in vivo study. C57BL/6 murine bowel cancer model was employed in this study. Interestingly, macrophages were identi ed in TME, including M2 macrophages aimed at driving tumor progression. Due to the effect of cucurbitacin B on macrophage polarization, the effects of cucurbitacin B on altered TAMs polarization in tumor tissues in vivo were investigated. We found that under treatment of cucurbitacin B, the expression of M2 marker (CD206) diminish in TAM. TMA can modulate the killing effect of T cells on tumor cells. Increasing studies show that the inhibition of M2 polarization will ultimately induce the activation of CD4+ and CD8+T cell and its immune response [40,41]. In this study, we demonstrated that cucurbitacin B induced more T cell in ltration into the tumors.
Cumulatively, these results suggest TAMs emphasized the anti-tumor effect of cucurbitacin B, especially via the JAK2/STAT3 signaling pathway, provided a potent strategy to regulate colon cancer cells growth and metastasis, and gave a more rational and long-term support for the clinical application of cucurbitacin B.

Conclusion
Taken together, our results revealed that cucurbitacin B suppressed CRC cell growth respectively by inhibiting JAK2/STAT3 signaling. Furthermore, this study presented an important role of the positive feedback loop between CRC cells and TAMs in tumor associated environment. The anti-metastasis effect of cucurbitacin B was targeting inhibiting M2-like TAMs polarization via JAK2/STAT3 signaling pathway.
This study provided a novel insight regarding the potential therapeutic application of cucurbitacin B as an anti-metastatic therapy for colon cancer, and possibly for other cancers as well.

Materials And Methods
Cell lines and cell culture Mouse macrophage cell line RAW 264.7, the human monocyte cell line THP-1, human colon cell line HCT116 and mouse colon cell line CT26 were kindly provided by the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). RAW264.7 Cell were maintained in DMEM, while THP-1, HCT116 and CT26 were maintained in RPMI-1640. All cultures were supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin. The macrophages were polarized into M2 by 20 ng /mL IL-4/IL-13 for 24 h. Phorbol-12-Myristate-13-acretate (PMA) was taken to utilized to differentiate THP-1 cells.
CCK-8 assays CT-26 and HCT116 were seed in a 96-well plate with a density of 5 × 10 3 . Cell Viability Assay was determined using CCK8 in accordance with the manufacture' s instruction. The absorbance was measured at 450 nm with a microplate reader.
Colony formation CT-26 and HCT116 were seed in a 12-well plate with a density of 1×10 3 . The fresh medium was changed every day to keep the cell growing. After 10 days, the colonies were xed with 4% paraformaldehyde and dyed with crystal violet (Beyotime, China).
RT-PCR analysis RNA was rstly isolated from cell using MolPure ® cell RNA Kit (Yesen, China). mRNA expression was performed using SYBR Green PC Master Mix (Yesen, China). Reaction was carried out using RT-PCR kits (Applied Biosystems, Canada). Expression of genes were analyzed as RQ = 2 − ΔΔCt . Primer sequences used for RT-PCR are listed in Table 1.
Microscale Thermophoresis The interaction between Cucurbitacin B and STAT3 was detected by Microscale Thermophoresis using the Nano Temper Monolith NT.115 instrument. Each measurement consists of 16 traction mixtures where uorescent-labeled stat3, and two-fold diluted cucurbitacin B ranging from 100µM to 3.1nM was used. The MO. a nity Analysis v2.3 software was used to measure the K d .
Wound healing assay CT-26 and HCT116 cells were planted in a 12-well plate (5 × 10 6 ) and grown until 80% con uent,and a wound was made by dragging a plastic pipette tip across the cell surface. Then wound healing image was photographed again the next day using microscope. The area of the wound was measured with image J software.
Migration assay The migration assay was conducted in a 24-well cell culture chamber employing inserts Immuno uorescence staining Cells that treat with above way were xed with 4% paraformaldehyde (Beyotime, China) for 30 min and were incubated with STAT3 antibody. The image was pictured under the confocal microscope.
Cell apoptosis assay Cell apoptosis was evaluated using an Annexin V-FITC Kit (Biolegend, USA). Cells were analyzed using a CytExpert ow cytometer (Beckman Coulter, USA).
C57BL/6 and BALB/c murine colon cancer modelIn vivo experiment was conducted in compliance with the relevant laws and institutional guidelines. In C57BL/6 model: CT-26 (8 × 10 5 ) in 0.2 mL were injected subcutaneously into the ank of each mouse. After 1 d, mice were intraperitoneally injected administrated with 0.2 mL of cucurbitacin B (0.5 mg/kg) and 0.2 mL of Cucurbitacin B 1 mg/kg) for 25 d according to previous study [20]. Control group received equal volumes of normal saline (NS). On day 14 the animals were euthanized. Table 1 Mouse Ym-1 Forward 5'-AGAAGGGAGTTTCAAACCTGGT-3'

Statistical analysis
Values are expressed as the mean ±SD. Data were analyzed with GraphPad Prism software 8.0. Statistical analysis was performed by Unpaired Student's t-test (two-tailed) and one-way ANOVA test.

Consent for publication
We would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. No con ict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.