Biomolecules & Therapeutics 2024; 32(6): 759-766  https://doi.org/10.4062/biomolther.2024.137
Isoorientin Suppresses Invasion of Breast and Colon Cancer Cells by Inhibition of CXC Chemokine Receptor 4 Expression
Buyun Kim1 and Byoungduck Park2,*
1Korean Medicine Application Center, Korea Institute of Oriental Medicine (KIOM), Daegu 41062,
2College of Pharmacy, Sahmyook University, Seoul 01795, Republic of Korea
*E-mail: bdpark@syu.ac.kr
Tel: +82-2-3399-1623
Received: August 13, 2024; Revised: September 6, 2024; Accepted: September 7, 2024; Published online: October 7, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Cancer metastasis still accounts for up to 90% of cancer-related deaths, but the molecular mechanism for metastasis is unclear. Several chemokines and their receptors mediate tumor cell metastasis, particularly through long-term effects that regulate angiogenesis, tumor cell proliferation and apoptosis. Among them, CXC chemokine receptor 4 (CXCR4) has been shown to play a pivotal role in cancer metastasis through interaction with a ligand (CXCL12), also known as stromal cell-derived factor 1α (SDF-1α). The CXCR4 promoter region is well characterized, and its expression is controlled by various transcriptional factors, including NF-κB, HIF−1α, and so forth. Isoorientin (ISO) is a 3’, 4’, 5, 7-tetrahydroxy-6-C-glucopyranosyl flavone. ISO has been reported to exhibit anti-oxidant, anti-cancer, and anti-inflammatory properties. However, the anti-metastatic effect of ISO following downregulation of CXCR4 is unknown, and the mechanism underlying the antitumor activity has yet to be elucidated. In our present study, we showed that ISO inhibited the expression of CXCR4 through NF-κB regulation in breast and colon cancer cells. We have also demonstrated that ISO inhibits CXCR4 expression in a variety of tumor cells. Furthermore, we found that CXCR4 expression is regulated through inhibition of the transcription process. Inhibition of CXCR4 expression also reduced the invasion of cancer cells by CXCL12. In conclusion, our results suggest that ISO is a novel inhibitor to regulate CXCR4 expression and the key molecule contributing to antitumor activity.
Keywords: Isoorientin, CXCR4, CXCL12, Invasion, Metastasis
INTRODUCTION

Metastatic breast cancer is a leading cause of cancer-related deaths among women worldwide. Several chemokines and corresponding receptors mediate tumor cell metastasis via long-term effects specifically regulating angiogenesis, tumor cell proliferation, and apoptosis (Yue et al., 2024). Among them, CXC chemokine receptor 4 (CXCR4) has been shown to play a key role in breast cancer metastasis via interaction with its ligand (CXCL12), also known as stromal cell-derived factor 1α (SDF-1α). The CXCR4/CXCL12 axis has been shown to play an important role in the migration, invasion and attachment of breast cancer cells to promote tumor growth and metastasis (Zielińska and Katanaev, 2020). The expression of CXCR4 in malignant breast cancer cells induces metastasis of cancer cells to local lymph nodes, lung, liver or bone, which express high levels of CXCR12 (Garg et al., 2024). The CXCR4 promoter region is well characterized, and its expression is controlled by various transcriptional factors, including nuclear respiratory factor-1 (NRF-1), NF-κB, HIF−1α, and so forth (Chen et al., 2017; Bradley, 2018; Jiang et al., 2018). Additionally, CXCR4 expression has been known to be regulated by diverse cytokines and growth factors (Lee et al., 2018; Wang et al., 2018a). In particular, aberrant NF-κB signaling activation leads to the upregulation of CXCR4, contributing to autoimmune disorders, chronic inflammation, and diverse cancers (Ebrahimi et al., 2024). Therefore, the pursuit of novel therapeutics aimed at modulating the CXCR4/CXCL12 signaling axis holds considerable promise for combatting cancer metastasis.

Isoorientin (ISO) is a C-glycosyl flavone, also known as 3’, 4’, 5, 7-tetrahydroxy-6-C-glucopyranosyl flavone, and its chemical structure is shown in Fig. 1A. It can be extracted from different plant species, such as Phyllostachys pubescens, Patrinia, Fagopyrum esculentum, Eremurus spectabilis, and Drosophyllum lusitanicum (Yuan et al., 2016). ISO has been reported to exhibit anti-oxidant (Cui et al., 2023), anti-cancer (Gundogdu et al., 2018), and anti-inflammatory activities (Ong et al., 2022). According to a previous study, ISO induces apoptosis, decreases invasiveness, and downregulates vascular endothelial growth factor (VEGF) secretion by activating 5’ adenosine monophosphate-activated protein kinase (AMPK) signaling in pancreatic cancer cells (Ye et al., 2016). ISO also promotes autophagy and apoptosis simultaneously by reactive oxygen species (ROS) mediated p53, PI3K/AKT, JNK, and p38 signaling pathway in HepG2 cancer cells (Yuan et al., 2014). However, the anti-metastatic effect of ISO following downregulation of CXCR4 is unknown, and the mechanism underlying the antitumor activity has yet to be elucidated. In this study, we investigated the antitumor activity of ISO and demonstrated that ISO inhibits CXCL12-induced metastasis by decreasing the expression of CXCR4 in breast and colon cancer cells.

Figure 1. ISO inhibits CXCR4 expression in MDA-MB-231 cells. (A) Chemical structure of isoorientin (ISO). (B, C) UV spectrum and HPLC chromatogram at 330nm of isoorientin. (D, E) MDA-MB-231 cells were incubated with the ISO (20, 40, and 80 μM) for 24 h or with the 80 μM of ISO for the indicated time, after which Western blotting was done as described above. (F) Liver cancer (Hep3B), breast cancer (MCF7), pancreatic cancer (HPAC, PANC1), colon cancer (SW480), lung cancer (A549) and multiple myeloma (U266) cells were incubated with 80 μM of ISO for 24 h. The cell extracts were analyzed by Western blot analysis using CXCR4 antibody. β-actin was used to confirm equal sample loading.
MATERIALS AND METHODS

Reagents

Dr. Ki Yong Lee (College of Pharmacy, Korea University, Sejong, Korea) provided the isoorientin (ISO) and its chemical structure was shown in Fig. 1A. The purity analysis of ISO was described in our previous report (Kim et al., 2021). ISO was dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock solution and further dilution was done in cell culture medium. Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, 0.25% trypsin-EDTA, fetal bovine serum (FBS) and antibiotic-antimycotic were purchased from Gibco BRL (Grand Island, NY, USA). Lactacystin and chloroquine were obtained from SantaCruz Biotechnology (Santa Cruz, CA, USA). CXCR4 antibody was obtained from Abcam (Cambridge, MA, USA). CXCL12 was purchased from R&D Systems (Minneapolis, MN, USA).

Cell culture

Breast cancer cell (MDA-MB-231), pancreatic cancer cells (PANC1, HPAC), colon cancer cell (SW480) and liver cancer cell (Hep3B) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM containing 10% FBS and 1% antibiotics. Breast cancer cell (MCF7), lung cancer cell (A549), multiple myeloma cell (U266) were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 containing 10% FBS and 1% antibiotics. Cells were maintained at 37°C in an atmosphere of 5% CO2–95% air.

Western blot analysis

We performed western blot by reference to previous study (Giri et al., 2023). Cell lysates containing 20 μg protein samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto polyvinyl difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). They were then probed with respective primary antibodies. HRP-conjugated secondary antibodies were detected using chemiluminescence with ECL reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Reverse transcriptase–polymerase chain reaction (RT–PCR)

We performed RT-PCR by reference to previous studies (Kim et al., 2020; Ryu et al., 2023). The cDNA for CXCR4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified by PCR with specific primers. The sequences of the sense and antisense primers for CXCR4 were 5′-CCG TGG CAA ACT GGT ACT TT-3′ and 5′-TTT CAG CCA ACA GCT TCC TT-3′, respectively. Conditions for PCR reaction were 1× (94°C, 5 min); 30× (94°C, 30 s; 55°C, 30 s and 72°C, 30 s); and 1× (72°C, 10 min). PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide.

Electrophoretic mobility shift assay (EMSA)

As described in our previous studies, the DIG Gel Shift Kit (Roche, Mannheim, Germany) was used to detect NF-κB-p65 binding activity, with the manufacturer’s instructions (Kim et al., 2020). The DNA−protein complexes were separated by electrophoresis in 6% non-denatured polyacrylamide gels using 0.5× TBE as a running buffer. After electrophoresis, the gels were transferred to nylon membranes and detected chemiluminescence.

Immunofluorescence assay

MDA-MB-231 cells were treated with ISO for 24 h and fixed in 3.7% formaldehyde. Membranes were treated with 0.1% Triton X-100 in PBS for 5 min, after washing briefly with PBS, the slides were blocked with 5% bovine serum albumin (BSA) for 1 h. The cells were then incubated with the anti-NF-κB-p65 antibody at room temperature for 1 h. After washing, the slides were incubated with secondary antibody Alexa Flour 488 for 30 min and counterstained against nuclei with Hoechst 33342 for 10 min. After fixation using the ProLong® Gold Antifade Mountant reagent (Life Technologies™, Molecular Probes® from Thermo Fisher Scientific, MA, USA), the fluorescence microscope (Nikon ECLIPSE Ti-U; Nikon Corporation, Tokyo, Japan) was used to photographically measure NF-kB translocation to the nucleus.

Invasion assay

In vitro invasion assays were performed by reference to previous study (Kim et al., 2020; Choi et al., 2023) using a Bio-Coat Matrigel invasion assay system (BD, Biosciences, Lexington, KY, USA) according to the manufacturer’s instructions. Cells were seeded in a Matrigel-precoated transwell chamber with polycarbonate membrane and then the lower chambers were filled with DMEM supplemented with CXCL12 (100 ng/mL) in the presence or absence of ISO (80 μM). After 24 h of incubation, the upper surface of the transwell chamber was wiped with a cotton swab and the invading cells were fixed and stained with Diff-Quick staining. The number of invading cells was counted in five randomly selected microscopic fields (100×).

Statistical analysis

The findings from each experiment are presented as the mean value along with the corresponding standard deviation (SD), derived from a minimum of three independent trials. Statistical significance was determined at p<0.05 using Student’s t-test for each analysis.

RESULTS

ISO inhibits CXCR4 expression in MDA-MB-231 cells

Recently, many studies have revealed the pivotal involvement of the chemokine receptor CXCR4 in cancer metastasis, highlighting its clinical significance across diverse cancer types (Shi et al., 2020). Elevated levels of CXCR4 in breast cancer are indicative of poor prognosis, contributing to metastasis, resistance to treatment, and recurrence (Ebbing et al., 2017). Both MDA-MB-231 and MCF-7 cells are breast adenocarcinoma. However, it has been reported that the MDA-MB-231 cells are more aggressive and invasive than MCF-7 cells (San-Millan et al., 2023). So, we performed most experiments with MDA-MB-231 cells throughout whole study. To investigate whether ISO modulates the expression of CXCR4 in breast cancer cells, we treated MDA-MB-231 cells with different concentrations of ISO (20, 40 and 80 μM) for 24 h, and for different durations (6, 12 and 24 h) with 80 μM of ISO (Fig. 1). We found that ISO significantly inhibited the CXCR4 expression in MDA-MB-231 cells in a dose- (Fig. 1D) and time-dependent manner (Fig. 1E).

ISO reduces CXCR4 expression in various types of cancer cells

Many reports suggest that CXCR4 was overexpressed in gastric, ovarian, pancreatic, cervical, and colon cancers (Xu et al., 2018). To determine whether ISO regulated the expression of CXCR4 in other cancer cells, we analyzed the expression of CXCR4 in the absence or presence of ISO in the following cancer cell lines: liver cancer (Hep3B), breast cancer (MCF7), pancreatic cancer (HPAC, PANC1), colon cancer (SW480), lung cancer (A549) and multiple myeloma (U266) (Fig. 1F). Cells were exposed to 80 μM of ISO for 24 hours, and the expression of CXCR4 was assessed using Western blot analysis with a specific anti-CXCR4 antibody. As depicted in Fig. 1F, ISO treatment led to a reduction in CXCR4 protein levels across all tested cancer cell lines, indicating a non-specific inhibition of CXCR4 expression by ISO.

ISO downregulates CXCR4 transcription, but not via its protein degradation

In a recent study, the mechanism of CXCR4 downregulation was reported to occur via degradation of lysine residues through ubiquitination (Caballero et al., 2019). Therefore, we investigated whether ISO downregulates CXCR4 by increasing its proteasomal degradation. Toward this end, we treated MDA-MB-231 cells with lactacystin, a proteasome inhibitor, 1 h prior to ISO treatment. The ISO-induced degradation of CXCR4 (Fig. 2A) was minimally affected by lactacystin, indicating that the downregulation of CXCR4 by ISO does not primarily occur through proteasomal degradation.

Figure 2. ISO downregulates CXCR4 transcription, but not via its protein degradation. (A, B) MDA-MB-231 cells were treated with indicated concentrations of lactacystin or chloroquine for 1 h at 37°C, followed by treatment with 80 μM of ISO for 24 h. Whole-cell extracts were analyzed by Western blot analysis using CXCR4 antibody. β-actin was used to confirm equal sample loading. (C, D) MDA-MB-231 cells were treated with ISO (20, 40, and 80 μM) for 24 h or with the 80 μM of ISO for the indicated time. Total RNA was isolated and analyzed by RT-PCR assays. GAPDH was used to confirm equal sample loading.

In addition, the ubiquitination of CXCR4 acts as a target signal for lysosomal degradation (Caballero et al., 2019). MDA-MB-231 cells were pretreated with chloroquine, a lysosomal inhibitor, for 1 h before ISO treatment. Our results showed that even 200 μM of chloroquine failed to prevent the degradation of CXCR4 (Fig. 2B), suggesting that it may not be a major pathway for CXCR4 suppression.

Since ISO did not suppress the expression of CXCR4 by enhancing its degradation, we investigated whether the inhibition of CXCR4 occurred at the transcriptional level instead. Cells underwent treatment with varying ISO concentrations for 24 h or were exposed to 80 μM of ISO for different durations, after which mRNA levels were assessed via RT-PCR. As illustrated in Fig. 2C and 2D, ISO reduced CXCR4 mRNA levels in a concentration- and time-dependent manner.

ISO inhibits constitutive activation of NF-κB in MDA-MB-231 cells

The NF-κB signaling pathway significantly contributes to the progression and metastasis of breast cancer cells by promoting the expression of the chemokine receptor CXCR4 (Wang et al., 2015). Furthermore, the promoter region of the CXCR4 gene harbors multiple NF-κB binding sites (Li et al., 2018). Therefore, we investigated whether ISO attenuated CXCR4 expression by suppressing NF-κB activation. We used a DNA-binding assay to investigate whether ISO affected the constitutive NF-κB activation in MDA-MB-231 cells. Our results showed that constitutive activation of NF-κB was decreased by ISO treatment in a concentration- and time dependent manner (Fig. 3A, 3B), which suggested that ISO may inhibit CXCR4 expression by suppressing NF-κB activation. Because it is known that phosphorylation and nuclear transport of NF-κB are prerequisite to its oncogenic functions (Christian et al., 2016), we used an immunofluorescent assay to investigate whether ISO interrupted nuclear translocation of NF-κB. As shown in Fig. 3C, ISO clearly inhibited the nuclear translocation of NF-κB in MDA-MB-231 cells.

Figure 3. ISO inhibits constitutive activation of NF-κB in breast cancer cells. (A, B) MDA-MB-231 cells were incubated with the indicated concentrations of ISO for 24 h or with 80 μM of ISO for indicated time. The nuclear extracts were analyzed for NF-κB-DNA binding activity; bars give the standard error (**p<0.01, ***p<0.001 vs control). (C) MDA-MB-231 cells were incubated with 80 μM of ISO for 24 h. The intracellular distribution of NF-κB was analyzed by immunofluorescence assay. After cells were fixed and permeabilized, NF-κB (green) was stained with rabbit anti-NF-κB followed by FITC-conjugated secondary antibodies and nuclei (blue) were stained with Hoechst 33342. The third panels display the merged images of the first and second panels.

ISO inhibits CXCL12-induced invasion of MDA-MB-231 and HCT116 cells

The binding of CXCL12 to CXCR4 leads to metastasis of breast cancer cells (Zielińska and Katanaev, 2020). Recent studies have also shown that inhibition of CXCR4 expression is effective in inhibiting breast cancer metastasis (Wang et al., 2018b). So, we conducted an in vitro invasion assay to assess the correlation between ISO-mediated downregulation of CXCR4 and breast cancer cell migration. Our findings revealed that CXCL12 stimulated the invasion of MDA-MB-231 cells, whereas ISO effectively inhibited this invasion (Fig. 4A, 4B).

Figure 4. ISO inhibits CXCL12-induced invasion of MDA-MB-231 and HCT116 cells. (A) MDA-MB-231 cells were seeded on the top of matrigel chamber. After incubation with or without 80 μM of ISO for 24 h, transwell chambers were placed in 24-well plate, in which we added the basal medium only or basal medium containing 100 ng/mL CXCL12. After incubation, cell invasion ability was observed. (B) Columns give the mean numbers of invaded cells; bars give the standard error (*p<0.05, CON vs CXCL12; ###p<0.001 CXCL12 vs treated ISO group). (C) HCT116 cells were seeded on the top of matrigel chamber. After incubation with or without 80 μM of ISO for 24 h, transwell chambers were placed into the wells of a 24-well plate, in which we added either the basal medium only or basal medium containing 100 ng/mL CXCL12. After that, cell invasion ability was observed. (D) Columns give the mean numbers of invaded cells; bars give the standard error (***p<0.001 CON vs CXCL12; ###p<0.001 CXCL12 vs treated ISO group).

Next, we investigated whether the suppression of cell invasion by ISO was not specific to breast cancer cells. It has been reported that CXCR4 affects colon cancer cell metastasis and CXCL12 exposure induces the mobility of colon cancer cells (Khare et al., 2021). We elucidated the role of ISO in colon cancer cell metastasis, by analyzing the effect of ISO on CXCL12-induced cell invasion. Treatment of HCT116 cells with ISO interfered with CXCL12-induced invasion of these cells (Fig. 4C, 4D).

ISO suppresses CXCR4 expression in colon cancer cells, affecting both protein and mRNA levels

We further investigated whether this suppression was due to the inhibitory effect of ISO against CXCR4 expression. The results show that ISO inhibits the expression of CXCR4 in a dose- and time-dependent manner in HCT116 cells (Fig. 5A, 5B). As shown in Fig. 5C and 5D, ISO also downregulated the CXCR4 mRNA expression. Also, our results showed that constitutive activation of NF-κB was decreased by ISO treatment in a concentration- and time dependent manner (Fig. 5E, 5F), which suggested that ISO may attenuate the expression of CXCR4 by suppressing NF-κB activation.

Figure 5. ISO inhibits CXCL12-induced invasion of colon cancer cells via inhibition of CXCR4 expression. (A, B) HCT116 cells were incubated with the indicated concentrations of ISO for 24 h or with 80 μM of ISO for indicated time. β-actin was used to confirm equal sample loading. (C, D) HCT116 cells were incubated with the indicated concentrations of ISO for 24 h or with 80 μM of ISO for indicated time. Total RNA was isolated and analyzed by real-time PCR assays. GAPDH was used to confirm equal sample loading. (E, F) HCT116 cells were incubated with the indicated concentrations of ISO for 24 h or with 80 μM of ISO for indicated time. The nuclear extracts were analyzed for NF-κB-DNA binding activity; bars give the standard error (*p<0.05, ***p<0.001 vs control).
DISCUSSION

The objective of this investigation was to assess the impact of ISO on the expression of CXCR4, a chemokine receptor associated with various aspects of cancer progression such as cell growth, invasion, angiogenesis, and metastasis. Our study, for the first time, demonstrates that ISO effectively suppresses the expression of CXCR4 in breast and colon cancer cells. Additionally, we observed this inhibitory effect of ISO on CXCR4 expression across a diverse range of cancer cell types. While ISO is known to possess mild proteolytic activity, our findings indicate that the downregulation of CXCR4 is not mediated through proteolytic degradation of the receptor but rather via transcriptional suppression. Moreover, we found that reduced expression of the CXCR4 receptor led to decreased invasion of cancer cells in response to CXCL12.

A number of studies reported that CXCR4 was overexpressed in various types of cancers such as gastric, ovarian, pancreatic, cervical and colon cancers, and in melanoma and hematological malignancies (Xu et al., 2018). Although it is still unclear why CXCR4 overexpression occurred in a variety of tumor cells, previous studies suggested the role of various genetic and post-transcriptional factors (Shi et al., 2020). The CXCR4 overexpression in different tumor cells has been found to be regulated by PAX3- and PAX7-FKHR gene fusion (Boudjadi et al., 2018), genetic mutations in the von Hippel Lindau gene tumor suppressor gene (Kruizinga et al., 2016), angiogenic growth factors (Ge et al., 2023), hypoxic conditions in the tumor microenvironment (Chen et al., 2023), transcription factor NF-κB (Yang et al., 2023), and proinflammatory cytokines (Ullah et al., 2024). Because CXCR4 is associated with cancer metastases, poor prognosis, and low overall survival rates of cancer patients, it has been considered as an ideal molecular target for new therapeutic and preventive interventions against metastatic cancer.

Our results clearly indicate that ISO suppressed CXCR4 expression in various tumor cell lines, which suggested that the effect of ISO on CXCR4 was not limited to a single cell type. We then sought to explore the mechanisms responsible for the ISO-induced reduction in CXCR4 expression in breast cancer cells. Previous research proposed that CXCR4 undergoes degradation through ligand-dependent lysosomal pathways, as well as through polyubiquitination mediated by atrophin-interacting protein 4, followed by degradation (Giorgiutti et al., 2024). On the other hand, the ubiquitin E3 ligase WWP1 negatively regulated cell migration in response to CXCL12 by limiting CXCR4 degradation to promote cancer metastasis (Vitale et al., 2023). However, our results demonstrated that the downregulation of CXCR4 by ISO was not mediated via proteasomal or lysosomal degradation. Instead, we found that ISO downregulated the expression of CXCR4 mRNA in both MDA-MB-231 and HCT116 cells, based on RT-PCR analysis, which suggests that the CXCR4 downregulation occurred at the transcriptional level.

Transcription factors HIF-1α, PPAR-γ and NF-κB mediate the upregulation of CXCR4 in human cancer cells (Wang et al., 2015; Bradley, 2018; Yang et al., 2023). The NF-κB binding site has been identified in the adjacent region of the CXCR4 promoter and has been reported to play a key role in CXCR4 expression in human breast cancer cells (Wang et al., 2015). Thus, we examined whether downregulation of CXCR4 by ISO was related to NF-κB modulation. Our electrophoretic mobility shift assay (EMSA) data showed that constitutive NF-κB activation was inhibited by ISO treatment. We next confirmed that the ISO inhibited the nuclear translocation of NF-κB by immunofluorescence analysis. Activation of NF-κB also triggers the expression of various adhesion molecules that are associated with cancer cell metastasis in other organs. Because ISO inhibits constitutive activation of NF-κB, it is possible that ISO may block the expression of these adhesion molecules as well, which should be further investigated. Another paper reported that ISO inhibits epithelial-to-mesenchymal properties in oral squamous cell carcinoma by blocking Wnt/β-catenin/STAT3 axis (Liu et al., 2021). So, we need to explore more anti-cancer mechanism of ISO to understand its properties thoroughly.

Our findings indicate that ISO effectively attenuated the ligand-induced invasion of breast and colon cancer cells, underscoring the pivotal role of the CXCR4 receptor in cancer invasion. These results highlight ISO’s potential to inhibit tumor metastasis through CXCR4 downregulation. However, when compared with our previous studies (Kim and Park, 2014; Kim et al., 2016), the effective concentration of ISO is little higher than that of baohuoside and pomolic acid. This would be a hurdle for further approaches like in vivo study. So, we try to design the optimal experimental conditions for further study.

Overall, our data suggest that ISO modulates CXCR4 expression, a crucial mediator in the interaction between cancer cells and the microenvironment, thereby exerting its anti-metastatic effects (Fig. 6). These insights position ISO as a promising candidate for anti-cancer therapy.

Figure 6. Schematic diagram shows how ISO inhibits metastasis of cancer cells.
ACKNOWLEDGMENTS

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (RS-2020-KH087790).

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

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