Honokiol suppresses metastasis of renal cell carcinoma by targeting KISS1/KISS1R signaling

Renal cell carcinoma (RCC) is a common urological cancer worldwide and is known to have a high risk of metastasis, which is considered responsible for more than 90% of cancer associated deaths. Honokiol is a small-molecule biphenol isolated from Magnolia spp. bark and has been shown to be a potential anticancer agent involved in multiple facets of signal transduction. In this study, we demonstrated that honokiol inhibited the invasion and colony formation of highly metastatic RCC cell line 786-0 in a dose-dependent manner. DNA-microarray data showed the significant upregulation of metastasis-suppressor gene KISS1 and its receptor, KISS1R. The upregulation was confirmed by qRT-PCR analysis. Overexpression of KISS1 and KISS1R was detected by western blotting at the translation level as well. Of note, the decreased invasive and colonized capacities were reversed by KISS1 knockdown. Taken together, the results first indicate that activation of KISS1/KISS1R signaling by honokiol suppresses multistep process of metastasis, including invasion and colony formation, in RCC cells 786-0. Honokiol may be considered as a natural agent against RCC metastasis.

In this study, we found that honokiol inhibits the invasion and colony formation of highly metastatic RCC cells 786-0 (34) in a dose-dependent manner. DNA-microarray data showed significant upregulation of metastasis-suppressor gene KISS1 and its receptor, KISS1R. Both of the upregulation were confirmed by qRT-PCR analysis. Overexpression levels of KISS1 and KISS1R were detected by western blotting at the translation level as well. Of note, inhibition of invasion and colony formation were reversed by KISS1 knockdown. Taken together, our results indicate that honokiol suppresses the multistep process of metastasis, including invasion and colony formation, in RCC cells 786-0 via stimulation of KISS1/ KISS1R signaling pathway.
Data points represent the mean ± SD of three individual filters within one representative experiment repeated at least twice.
Colony formation assay. Colony formation of the 786-0 cells incubated in the presence of honokiol (0-40 µM) was evaluated as previously described (36). Data points represent the mean ± SD in one representative experiment repeated at least twice.
Western blot analysis. The 786-0 cells were treated with honokiol (0-40 µM) for 24 h. Whole protein extracts isolated from cells were prepared and western blot analysis with KISS1 and KISS1R antibodies were performed as previously described (39). Western blots were quantified with HP-Scanjet 550c and analyzed by UN-SCAN-IT software (Silk Scientific, Orem, UT, USA).
siRNA transfection. The 786-0 cells were transfected with human KISS1 siRNA or control siRNA-A as previously described (37). After 48 h of transfection, the cells were harvested and KISS1 knockdown was verified by western blot analysis.
Statistical analysis. All the statistical analysis was performed using SigmaPlot 11.2.0 (Systat Software Inc., San Jose, CA, USA). Data are presented as mean ± SD. Statistical comparisons were carried out using ANOVA with the significance level adjusted using the repeated t-tests with Bonferroni correction. P-value <0.05 was considered to be significant.

Honokiol inhibits invasion and colony formation of highly metastatic RCC cells.
To evaluate whether honokiol ( Fig. 1) suppresses invasive behavior of highly metastatic RCC cells, the 786-0 cells were treated with honokiol (0-20 µM) for 24 h and cell invasion was determined as described in Materials and methods. As shown in Fig. 2A, honokiol inhibits cell invasion through Matrigel in a dose-dependent manner. Moreover, honokiol significantly decreases the number of anchorageindependent colonies formed, which is a key step in cancer metastasis ( Fig. 2B and C). In summary, honokiol significantly inhibits invasion as well as colony formation of highly metastatic RCC 786-0 cells in a dose-dependent manner.
Honokiol activates KISS1/KISS1R signaling in highly metastatic RCC cells. Since recent studies showed that activation of KISS1/KISS1R signaling by kisspeptin treatment decreases the motility and invasive capacity of conventional RCC, and overexpression of KISS1 inhibits invasion of RCC cells Caki-1 (40,41), we confirmed the significant upregulation of KISS1 and KISS1R in the 786-0 cells treated with honokiol by qRT-PCR (Fig. 3). In accordance with the change in mRNA, western blot analysis showed that honokiol stimulates expression of KISS1 and KISS1R in the 786-0 cells dose-dependently at the protein level (Fig. 4).

Silencing KISS1 reverses suppression of invasion and colony formation.
To determine whether the suppression of invasion and colony formation by honokiol are associated with the activation of KISS1/KISS1R signaling in the 786-0 cells, we silenced KISS1 with siRNA as described in Materials and methods. As shown in Fig. 5, knockdown of KISS1 partially rescues the effect of honokiol on cell invasion by more than 40%. Moreover, the effect of honokiol on colony formation of the 786-0 cells is markedly reversed by KISS1 silencing (Fig. 6). These results further indicate that KISS1/KISS1R signaling is a major target of honokiol in suppressing metastasis of RCC cells.

Discussion
In the present study, we investigated the role of honokiol in the metastasis of RCC cells. Our results showed that honokiol significantly inhibited the invasion and colony formation of highly metastatic RCC cells 786-0 in a dose-dependent manner. Moreover, honokiol markedly upregulated metastasis-suppressor gene KISS1 and its receptor, KISS1R, at both transcription and translation levels. Interestingly, knockdown of KISS1 partially rescued the effect of honokiol on cell invasion and its effect on colony formation of the 786-0 cells is reversed as well, indicating that KISS1/KISS1R signaling  is a major target of honokiol in suppressing metastasis of RCC cells. Metastasis suppressors are defined as molecules whose expression results in the suppression of metastasis processes and since 1986, more than 13 metastasis suppressors have been identified (42). The KISS1 gene, initially discovered as a novel human malignant melanoma metastasis-suppressor gene (43), has been validated as an anti-metastatic gene by preclinical and clinical evidence in various types of cancer (44). The encoded KISS1 protein can be processed to a C-terminally amidated peptide termed metastin binding and activating the G-protein coupled receptor GPR54 (KISS1R) (45). Shoji et al found that metastin inhibited migration and invasion of RCC with overexpression of KISS1R (46). In addition, a recent study demonstrated that an absence of KISS1R expression was associated with rapid progression of conventional RCC in patients (40), suggesting KISS1/KISS1R signaling as a promising target in RCC.   Honokiol targets multiple signaling pathways such as nuclear factor κB (NF-κB), signal transducers and activator of transcription 3 (STAT3), mammalian target of rapamycin (mTOR) and epidermal growth factor receptor (EGFR), which have great relevance during cancer initiation and progression (47). Moreover, pharmacokinetic studies revealed that honokiol crossed the blood-brain barrier (BBB), the bloodcerebrospinal fluid barrier (BCSFB) and had a desirable bioavailability after intravenous administration in animal models (48) thus making it a suitable agent for clinical trials.
In summary, our results indicate that activation of KISS1/ KISS1R signaling by honokiol decreases the invasiveness and colonized capacity of highly metastatic RCC cells. Furthermore, we confirmed that honokiol stimulated the expression of TIMP4 dose-dependently (data not shown). It is in accordance with the finding that metastin suppresses the motility and invasive ability of RCC cells which possess KISS1R through the downregulation of MMP-2 (49). As emerging studies show that KISS1R activates a series of signaling molecules such as protein kinase C (PKC), extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and phosphatidylinositol-3-kinase (PI3K) (50), further studies are in progress to investigate the specific mechanism of honokiol, which may have the potential for use as a natural agent against RCC metastasis.