2023 Impact Factor
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
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
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
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.
We performed western blot by reference to previous study (Giri
We performed RT-PCR by reference to previous studies (Kim
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
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.
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
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
Many reports suggest that CXCR4 was overexpressed in gastric, ovarian, pancreatic, cervical, and colon cancers (Xu
In a recent study, the mechanism of CXCR4 downregulation was reported to occur via degradation of lysine residues through ubiquitination (Caballero
In addition, the ubiquitination of CXCR4 acts as a target signal for lysosomal degradation (Caballero
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.
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
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
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
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.
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
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
Transcription factors HIF-1α, PPAR-γ and NF-κB mediate the upregulation of CXCR4 in human cancer cells (Wang
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
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.
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).
The authors declare no conflicts of interest.