6-Gingerol induces cell-cycle G1-phase arrest through AKT–GSK 3β–cyclin D1 pathway in renal-cell carcinoma

Purpose 6-Gingerol, a major biochemical and pharmacological active ingredient of ginger, has shown anti-inflammatory and antitumor activities against various cancers. Searching for natural products with fewer side effects for developing adjunctive therapeutic options is necessary. Methods The effects of 6-gingerol on proliferation, colony formation, and cell cycle in RCC cells were detected by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and propidium iodide (PI) staining, respectively. Western blotting, an immunofluorescence assay, and immunohistochemical staining were performed to assess the expression of relevant proteins. A subcutaneous tumor model was set up to investigate the 6-gingerol effects on tumor growth in vivo, and the pharmacokinetics of 6-gingerol in mice were detected by LC/MS assays. Results 6-Gingerol treatment exerted time- and dose-dependent inhibition of the growth and colony formation of ACHN, 786-O, and 769-P cells, leading to a concomitant induction of cell-cycle G1-phase arrest and decrease in Ki-67 expression in the cell nucleus. Western-blotting results showed that 6-gingerol reduces phosphorylation of protein kinase B (AKT) Ser 473, cyclin-dependent kinases (CDK4), and cyclin D1 and, meanwhile, increases glycogen synthase kinase (GSK 3β) protein amount. Furthermore, the efficacy of 6-gingerol was demonstrated in an in vivo murine model of 786-O. Conclusion The above results indicate that 6-gingerol can induce cell-cycle arrest and cell-growth inhibition through the AKT–GSK 3β–cyclin D1 signaling pathway in vitro and in vivo, suggesting that 6-gingerol should be useful for renal-cell carcinoma treatment. Electronic supplementary material The online version of this article (10.1007/s00280-019-03999-9) contains supplementary material, which is available to authorized users.


Introduction
The incidence of renal-cell carcinoma (RCC) has increased rapidly among both men and women according to the American Cancer Society. In general, patients with RCC benefit from conventional therapies (surgical resection with or without chemotherapy) [1]. Nonetheless, approximately 30% of RCC patients have metastases at diagnosis [2] and fail to respond to cytotoxic chemotherapy and targeted chemotherapy [1,3,4], resulting in the 5-year survival rate less than 10% [2]. Most of ccRCC is initiated by mutation or missing of VHL tumor suppression gene. Function loss of VHL leads to VHL-HIF-mTOR pathway activation [5]. Tyrosine kinase inhibitors targeting VEGF (such as sunitinib and pazopanib) and mTOR inhibitors (such as everolimus and temsiromus) are the standard-of-care therapy for ccRCC patients [6]. However, many patients have progression disease treated with tyrosine kinase inhibitors or mTOR inhibitors. Immune Shan Xu and Haibao Zhang contributed equally to this work.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s0028 0-019-03999 -9) contains supplementary material, which is available to authorized users. checkpoint inhibitors (such as, nivolumab and ipimumab) have been shown to have acceptable safety and durable antitumor activity in ccRCC clinical treatment [6,7]. However, only ~ 20% patients had clinical benefits from immune clinical therapy [6,7]. There is increasing interest investigating non-toxic natural products for various types cancer treatment, searching for natural products with fewer side effects for developing adjunctive therapeutic options is urgently necessary [8].
In this study, we focused on the mechanism of 6-gingerol action on RCC in vitro and its antitumor effect in vivo. We found that 6-gingerol can inhibit cell growth by stalling the cell cycle at the G1-S transition via the AKT-GSK 3β-cyclin D1 pathway in vitro. Moreover, 6-gingerol can serve as a single agent for killing RCC cells in vitro and in vivo. Thus, our study suggests that 6-gingerol may be a promising agent for the treatment of RCC.

Materials and methods
Cell culture 786-O, 769-P, and ACHN cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 (Gibco) containing 10% (v/v) of fetal bovine serum (Hyclone) in a humidified incubator at 37 °C and 5% CO 2 .

MTT assay
786-O, 769-P, and ACHN cells (at 4000/well) were seeded in 96-well plates. After 24 h, 6-gingerol was added to the medium to achieve the indicated concentrations (0, 10,20,30,40, and 50 μM) in triplicate for 24, 48, and 72 h incubation with the three cell lines. Subsequently, 20 μL of MTT (5 mg/mL in phosphate-buffered normal saline; PBS) was added into each well, and the cells were incubated for 4 h. Then, cell formazan and the growth inhibitory rate were quantified and calculated as described before [19].

Colony formation assay
786-O, 769-P, and ACHN cells were harvested, counted, and seeded in 6-well plates at 1000/well, and then, the cells were treated with 6-gingerol and maintained in the humidified incubator for 7 days until visible colonies appeared. After that, a 0.1% v/w crystal violet solution (Sigma) was used to stain the cells, and the colony formation capacity of the cells was tested in two-dimensional (2D) culture.

Cell-cycle assay
Human RCC cell lines 786-O, 769-P, and ACHN were grown in 6 cm dishes. The next day, the cells were treated with 6-gingerol at the concentrations indicated in the figure legends for 48 h. As described above, the cells were harvested, washed, and fixed overnight in 70% ethanol at 20 °C. Then, the cells were washed with PBS, incubated with propidium iodide (30 mg/mL) for 30 min, and were analyzed by flow cytometry on a FACS Calibur flow cytometer (BD Bioscience). The data were analyzed in the Cell Fit software.

Immunofluorescence assay
786-O, 769-P, and ACHN cells were harvested, counted, and seeded in an 8-well chamber slide. After 24 h, the cells were treated with 6-gingerol at the concentrations indicated in Fig. 3 legends for 48 h, and a Ki-67 protein indirect immunofluorescence assay was performed as described before [20]. Briefly, the cells were incubated with an anti-Ki-67 antibody (Proteintech, #27309-1-AP) overnight at 4 °C (primary antibody), washed with PBS, and then incubated with a secondary antibody (a fluorescein isothiocyanate-conjugated affinity-purified goat anti-rabbit IgG antibody, cat. #ZF-0315, Beijing Zhongshan Golden Bridge Biotechnology) for 60 min at room temperature, followed by F-actin and DAPI staining (1:5000) for 15 min in the dark. The cells were examined by laser scanning confocal microscopy (Nikon A1R/A1).

Animal experiments
These experiments were approved by the institutional review board of the First Affiliated Hospital of Xi'an Jiaotong University. Eighteen BALB/c nude mice (4 weeks, male) were randomly separated into three groups, and then, these nude mice were subcutaneously injected with 2 × 10 6 786-O cells into the right shoulder. After 3 days, the nude mice were treated with corn oil, 6-gingerol (diluted in corn oil, 2.5 mg/ [kg body weight]), or 6-gingerol (diluted in corn oil, 5 mg/ [kg body weight]) every 3 days by gavage. Kinetics of tumor formation were estimated by measuring tumor size and volume every 3 days for 38 days along with the body weight of the mice. Tumor volume was calculated using the following equation: tumor volume = length × width × height × 0.523. At the end of the experiment, the animals were euthanized, and tumor tissues were surgically excised from the nude mice. The tumors were weighed and divided into two parts, one part was fixed in 4% paraformaldehyde and embedded in paraffin, whereas from the other part, total protein was extracted as described above by means of a tissue grinder machine (Servicebio, China).

Pharmacokinetics of 6-gingerol in vivo
These experiments were approved by the institutional review board of the First Affiliated Hospital of Xi'an Jiaotong University. 54 BALB/c nude mice (4 weeks, male) were randomly separated into two groups, and treated with 6-gingerol (2.5 mg/[kg body weight], 5 mg/[kg body weight]) by gavage, or corn oil for control. Pharmacokinetics of 6-gingerol in vivo were measured at 9 timepoints (0, 0.5, 1, 1.5, 3, 4, 6, 12, and 24 h), and each timepoint had 3 mice for measure. Mice were euthanized at every interval of the timepoints, and then blood was collected by cardiac puncture, centrifuged at 3000 rpm for 15 min to obtain plasma, and then stored at − 80 °C before use.

Statistical analysis
The results were analyzed in the GraphPad Prism software, and the differences between two groups were compared by two-tailed, unpaired Student's t test. p < 0.05 was regarded as the threshold value of statistical significance.

6-Gingerol inhibits the growth of RCC cells
The chemical structure of 6-gingerol is shown in Fig. 1a.

6-Gingerol blocks cell cycle by arresting it at the G1 transition
Next, to determine the mechanism of 6-gingerol action on renal cancer cell growth, DNA-based cell-cycle analysis was performed on ACHN, 786-O, and 769-P cells after treatment with 10, 30, or 50 μM 6-gingerol for 48 h. As shown in Fig. 2a, b, the indicated concentrations, 0, 10

6-Gingerol reduces Ki-67 nuclear staining in RCC cells
Ki-67 is known to be a cell proliferation marker for research and cancer histopathology and a marker for a cell response to drugs that target cell proliferation [21,22]. In our study, Ki-67 immunofluorescent staining was evaluated to reveal the RCC cell response to 6-gingerol. As expected, Ki-67 immunofluorescent staining was weakened in a dosedependent manner in ACHN and 786-O cells after treatment with 30 μM or 50 μM 6-gingerol for 48 h (Fig. 3).

6-Gingerol reduces AKT phosphorylation and cyclin D1 and CDK4 expression in RCC cells
The expression of cyclin D1 and CDK4, which forming a complex with CDK6 for the G1-S phase transition [23], was measured by western blotting. As illustrated in Fig. 4, cyclin D1 and CDK4 protein levels were markedly downregulated in ACHN, 786-O, and 769-P cells after treatment with 30 or 50 μM 6-gingerol for 48 h. Western blotting also revealed that the signaling proteins upstream of cyclin D1, and p-AKT Ser 473 was inhibited and GSK 3β was upregulated by 30 or 50 μM 6-gingerol treatment. The above results suggested that 6-gingerol could suppressed AKT-GSK 3β signaling and, furthermore, reduced the expression of cyclin D1 and caused G1 arrest in RCC cells.

6-Gingerol inhibits tumor xenograft growth through AKT-GSK 3β signaling
The antitumor effect of 6-gingerol in vivo was validated in the 786-O xenograft model. The nude mice were subcutaneously injected with 2 × 10 6 cultured 786-O cells, and the tumor-bearing mice were treated with corn oil, a low dose of 6-gingerol (2.5 mg/kg), or a high dose of 6-gingerol (5 mg/ kg) every 3 days; meanwhile, the tumor growth was monitored by means of tumor volume and tumor weight (Fig. 5a). We observed that the mice treated with 6-gingerol (2.5 or 5 mg/kg) experienced no significant body weight loss relative to the corn oil group (Fig. 5b). On the contrary, the mice from the 6-gingerol 2.5 and 5 mg/kg groups gained some weight (Fig. 5b). Moreover, tumor growth was inhibited significantly between days 18 and 38 (Fig. 5c). The experiment and treatment were stopped on day 38 when one control mouse had an almost 2 cm diameter tumor on its shoulder, even though no mice showed clear signs of ill health due to 6-gingerol treatment or tumors. As expected, tumor weight and tumor volume were significantly changed at the final timepoint (day 38; Fig. 5d, e). The concentration of 6-gingerol in plasma was also measured in mice, and the main pharmacokinetic parameters of 6-gingerol were shown in Fig. 5f, and Table S1. In high dose of 6-gingerol group, the half-time and Tmax of 6-gingerol were about 3.6 h and 1.5 h, respectively, and C max was (181.37 ± 76.31) ng/mL. In low Fig. 3 Ki-67 is under expressed after treatment with 6-gingerol. a Cells were seeded in an 8-well chamber slide and treated with 0, 30, and 50 μM of 6-gingerol for 48 h. Then, Ki-67 immunofluorescent staining (green), F-actin staining (red) for the cytoskeleton, and DAPI (blue) staining for cell nuclei were all photographed by a laser confocal microscope. b ACHN and 786-O cells were seeded in 10 cm dishes and treated with 0, 30, and 50 μM of 6-gingerol for 48 h. Then, the cells were lysed, and the protein levels of Ki-67 were analyzed by a western blot assay ◂ Fig. 4 AKT-GSK 3β-cyclin D1-signaling pathway is involved in 6-gingerol-induced G1 arrest of ACHN, 786-O, and 769-P cells. Cells were seeded in 10 cm dishes and treated with 30 or 50 μM 6-gingerol for 48 h. Then, the cells were lysed, and the protein levels of AKT, p-AKT Ser 473, GSK 3β, p-GSK 3β Ser 9, cyclin D1, and CDK4 were analyzed by a western blot assay 1 3 dose of 6-gingerol group, the concentrations of 6-gingerol at 12 h and 24 h were not detectable; thus, the pharmacokinetic parameters could not be calculated.
The expression levels of AKT, p-AKT Ser 473, GSK 3β, p-GSK 3β Ser 9, CDK4, and cyclin D1 in tumor tissue removed from the mouse groups treated or not treated with 6-gingerol were measured next. As presented in Fig. 6a, both doses of 6-gingerol attenuated immunohistochemical staining of p-AKT Ser 473 and cyclin D1 in the tumor tissues, whereas strong staining of GSK 3β was observed in high dose of 6-gingerol treatment group. We also measured AKT, p-AKT Ser 473, GSK 3β, p-GSK 3β Ser 9, CDK4, and cyclin D1 in tumor tissue by western blotting (Fig. 6b). The above results suggested that 6-gingerol can inhibit tumor cell growth by downregulating cyclin D1 through inhibition of the Akt-GSK 3β-signaling pathway in vivo.

Discussion
Our study addressed the anticancer capacity of 6-gingerol against RCC in vitro and in vivo. The results proved that 6-gingerol inhibits RCC growth (Fig. 1). Furthermore, we found that 6-gingerol suppresses cell growth primarily by inhibiting the AKT-GSK 3β-cyclin D1 pathway, as evidenced by a significant decrease in p-AKT Ser 473, CDK4, and cyclin D1 protein amounts, meanwhile, a significant increase GSK 3β protein amounts in vitro and in vivo (Figs. 4, 5, 6), with concentration-dependent accumulation of cells at the G1 phase in vitro (Fig. 2). The above results are consistent with those of other studies about 6-gingerol effects on colon cancer cells, in which 6-gingerol induced the G2-M arrest of the cell cycle at concentrations (34-51 μM) similar to those in our study [13].
Some studies have proved that 6-gingerol inhibits the growth of other cancer cells at concentrations as high as 500 μM in the culture medium [17]. Note that 125 μg/mL (424 μM) 6-gingerol is IC 50 reported for HeLa cells [16]; those authors reported that 6-gingerol induces apoptosis and autophagy by binding to DNA [16]. Moreover, other investigators have studied a high concentration (up to 200 μM) of 6-gingerol as a therapeutic agent killing HT-29, HCT-116, SW480, and Caco-2 cells [24]. Nevertheless, 5-15 μg/ mL (up to 50 μM) 6-gingerol has been used to treat LoVo cells [25], and the results showed that a low concentration of 6-gingerol (34 μM) can cause significant growth inhibition in agreement with our results (Fig. 2). The possible reasons for the discrepancies between these studies and our study may be the differences in the purity of 6-gingerol provided by different companies, differences in sensitivity of different cell lines, differences in 6-gingerol duration of treatment of different cell lines, and dissimilarity of the mechanisms of action in different cell lines.
6-Gingerol has been previously shown to inhibit tumor cell growth through downregulation of cell-cycle regulators CDK1, cyclin A, and cyclin B1 and by repressing cyclin D1 in colorectal cancer cells [24], by activating caspases 3 and 9 and modulating mitochondrial functions in human gastric cancer cells [11], by inhibiting protein expression of the cyclin family and mTOR pathway in human cervical adenocarcinoma cells [15], by decreasing cyclin A and CDK expression, and by altering MAPK and PI3K-AKT pathways in pancreatic cancer cells [12]. The previous research results have indicated that the AKT-GSK 3β pathway and cyclins may be the common mechanism of action of 6-gingerol in its antitumor activities. Nevertheless, there is no study about the antitumor action of 6-gingerol in RCC.
In our experiments, we also found that the AKT-GSK 3β-cyclin D1-signaling pathway is involved in the cellcycle arrest induced by 6-gingerol. Because cyclin D1 is the key cell-cycle regulatory protein controlling the G1 cell-cycle phase to the S phase [26,27], we measured its expression after a G1 cell-cycle arrest was observed in ACHN, 786-O, and 769-P cells (Fig. 2a). As expected, cyclin D1 protein expression was decreased in a dosedependent manner by 6-gingerol treatment in our study (Fig. 4), and these results were consistent with the reports by Seong-Ho Lee et al. about human colorectal cancer cells [24]. Furthermore, evidence indicated that GSK 3β controls the cell cycle by regulating cyclin D1 degradation [28,29], whereas GSK 3β is one of the major effectors of AKT and is inactivated by phosphorylation at Ser9 by AKT [30]. In our study, proteins p-AKT Ser 473 and cyclin D1 were significantly downregulated after 50 μM  18 BALB/c nude mice were randomly separated into three groups and treated with vehicle (corn oil; control, n = 6), 2.5 mg/kg 6-gingerol (n = 6), or 5 mg/kg 6-gingerol (n = 6) every 3 days after subcutaneous injection of tumor cells until euthanasia. b Body weight changes of the xenografted mice treated or not treated with 6-gingerol; Statistical analysis of the data was calculated using student's t test between two groups, error bars indicate ± SD, n = 6. ns no significance. c Tumor volume changes in the xenografted mice during the treatment. Statistical analysis of the data was calculated using student's t test between two groups, error bars indicate ± SD, n = 6. **p < 0.01 as compared with the control group. d Tumor weight of xenografted mice treated or not treated with 6-gingerol. Statistical analysis of the data was calculated using student's t test between two groups, error bars indicate ± SD, n = 6. **p < 0.01 as compared with the control group. e Tumor images from groups treated or not treated with 6-gingerol. Ctr represents control group, n = 6; LD represents 2.5 mg/kg 6-gingerol treatment group, n = 6; and HD represents 5 mg/kg 6-gingerol treatment group, n = 6. f Plasma concentration-time curves of 6-gingerol. 6-gingerol was given by gavage, and blood was obtained and detected as described in the "Methods". LD represents 2.5 mg/kg 6-gingerol treatment group (n = 3), and HD represents 5 mg/kg 6-gingerol treatment group (n = 3). Statistical analysis of the data was calculated using student's t test between two groups, error bars indicate ± SD ◂ Fig. 6 6-Gingerol suppresses the AKT-GSK 3β-cyclin D1-signaling pathway in vivo. a Immunohistochemical staining of p-AKT Ser 473, GSK 3β, and cyclin D1 in subcutaneous tumors with or without 6-gingerol treatment. b AKT, p-AKT Ser 473, GSK 3β, p-GSK 3β Ser 9, cyclin D1, and CDK4 expression levels were detected by a western blot assay in subcutaneous tumors from the mice with or without 6-gingerol treatment. Ctr represents control group (n = 6), LD represents 2.5 mg/kg 6-gingerol treatment group (n = 6), and HD represents 5 mg/kg 6-gingerol treatment group (n = 6). CM-1 means control group mouse and the mouse numbered 1, LM-1 means 2.5 mg/kg 6-gingerol treatment group mouse and the mouse numbered 1, HM-1 means 5 mg/kg 6-gingerol treatment group mouse and the mouse numbered 1 6-gingerol treatment of RCC cells (Fig. 4). Meanwhile, protein of GSK 3β was increased after 50 μM 6-gingerol treatment (Fig. 4). The decreased protein amounts of p-AKT Ser 473 and cyclin D1 and increased protein amount of GSK 3β were detected by western blotting and immunohistochemical staining in the tumor tissue removed from the mice treated with 6-gingerol as compared to that removed from control untreated mice (Fig. 6). Therefore, we concluded that 6-gingerol induces the G1 cell cycle arrest of RCC cells though interaction with the AKT-GSK 3β-cyclin D1-signaling pathway in vivo and in vitro.
As far as we know, ginger is a food product and has been used in medicine [31]. In our study, a significant decrease in tumor weight and tumor volume was observed in tumorbearing mice treated with 6-gingerol at 2.5 and 5 mg/kg doses (Fig. 5). Nonetheless, our results suggest that the higher dose has no effect on mouse body weight and lifespan when we compared the control group and the 6-gingerol treatment group (Fig. 5b). These findings were verified in other cancers. Because there was no detectable cytotoxicity of 6-gingerol in vivo, the anticancer capacity of 6-gingerol should be evaluated in future animal studies and clinical trials.

Conclusion
Our results indicate that 6-gingerol, as a natural compound, exerts an antitumor action by inhibiting the cell cycle through the AKT-GSK 3β-cyclin D1-signaling pathway in RCC. Various data combined with our study suggest that 6-gingerol may be a safe and useful complementary therapy for ccRCC and warrant further investigation of its antitumor efficacy in vivo and in vitro, either alone or in combination with TK inhibitors and mTOR inhibitors.