Nobiletin Down-regulates SKP2-p21/p27-CDK2 Axis to Inhibit Tumor Progression and Shows Synergistic Effect with Palbociclib on Renal Cell Carcinoma


 Background: Natural extracts can enhance the efficacy and sensitivity of chemotherapeutic drugs, including nobiletin. However, the possible role and underlying mechanisms of nobiletin in cancer including Renal cell carcinoma (RCC) are still unclear.Methods：Cell proliferation, cell cycle and apoptosis analysis, colony-forming assays, immunoblotting analysis and qRT-PCR analysis were performed to investigate how nobiletin inhibits RCC cell proliferation in vitro. Nude mouse model was used to measure in vivo efficacy of nobiletin and its combination with palbociclib.Results：Nobiletin induced G1 phase cell cycle arrest, apoptosis, and proliferative inhibition in RCC Cells. Mechanistically, nobiletin inhibited activation of oncogene SKP2, leading to the accumulation of tumor suppressors and apoptosis-inducing substrates such as p21 and p27 to inhibit cell proliferation, eventually inhibit tumor formation. Intriguingly, we further found that sensitivity of palbociclib was associated with SKP2 protein level. Furthermore, in vitro and vivo results, dual inhibition of nobiletin and palbociclib showed synergistic lethality.Conclusion：Combination nobiletin and palbociclib may serve as a potential therapeutic strategy for renal cancer.


Background
Renal cell carcinoma (RCC) is the sixth most common cancer in men and the 10th in women, accounting for 5% and 3% of all new cancer cases, respectively [1]. Despite higher proportion of indolent and localized tumors identified partly due to widespread use of abdominal imaging techniques, there are still up to 17% of patients present with distant metastases at the time of diagnosis [2]. In addition, approximately one quarter patients with localized RCC develops distant metastasis after radical surgery. Once progression to metastatic RCC (mRCC), the treatment is still an intractable problem, despite the introduction of novel agents targeting different pathways including angiogenesis, mTOR inhibitors, immune checkpoint inhibitors. Comparing with cytokine treatment (interferon alpha or interleukin-2), targeted therapies have really significantly improved the clinical outcomes of mRCC [3]. Sunitinib as first-line therapy form RCC achieved significantly longer progression-free survival (PFS) (11 versus 5 months), overall survival (OS) (26.4 versus 21.8 months) and a better response rate (31% versus 6%) than interferon alpha group [4,5]. However, RCC is the highly heterogeneous tumor. Ascribe to the intratumor and intertumor heterogeneity, the objective response rates of current first-line agents form RCC were about 30% and even lower [3,6]. Even more disastrous is resistance to drugs eventually develop in nearly all patients, which have driven sustained efforts to identify novel targets and treatment strategies for mRCC, in order to achieve the better clinical outcomes.
Many natural extracts are the potential antitumor drugs because of its low toxicity and few side effects [7,8]. Some extracts have anti-tumor pharmacological effects and can be used as a regulator of multi-drug resistance to increase the sensitivity of cancer cells to chemotherapy [9]. Nobiletin is a typical example, which is an O-methylated flavonoid found mainly in citrus peel. Previous studies have shown that nobiletin can significantly inhibit growth and metastasis of several cancer cell both in vitro and in vivo through multiple pathways [10,11]. Of note, nobiletin can extremely inhibit cell proliferation through attenuating the expression of cyclin D1, CKD2, CKD4 and E2F [12]. So far, few studies have reported the effects of nobiletin in kidney cancer. Limited data showed that nobiletin could inhibit cells viability and hypoxia-induced migration of RCC cell lines, and the SRC/AKT, NF-κB and Wnt/β-Catenin signaling pathways might involve in the effects [13,14]. A study reported that nobiletin induced G0/G1 cell cycle arrest in RCC cells, but did not explain mechanism of action of nobiletin in cell cycle arrest [14]. Therefore, the possible role and underlying mechanisms of nobiletin in cancer including RCC are still unclear.
Cyclin/CDK complexes phosphorylates the G1 gatekeeper Rb, causing the release of S-phase-specific transcription factors, such as E2F. E2F causes the transcriptional induction of Cyclin E, which in turn partners with CDK2 to further phosphorylate RB and irreversibly cause the transition into S phase [16].
Therefore, the exploration of targeted anti-tumor drugs capable of blocking the progression of tumor cells from G0/G1 phase to S phase has become one of the hot spots in the development of anti-tumor drugs. Palbociclib is an orally active, potent, and selective inhibitor of CDK4 and CDK6, which blocks RB phosphorylation at low drug concentrations [17]. In addition, many solid tumors are often directly inherited, epigenetic or transcriptional modification leads to loss of control of the CCND-CDK4/6-INK4-Rb signaling pathway [18][19][20][21][22]. Because of the central role in tumor genesis and progression, CDK4/6 might represent a valid therapeutic target for cancer treatment in a broad spectrum of solid tumors.
Several phase II studies tested palbociclib monotherapy in a broad variety of solid tumors, namely well-differentiated or dedifferentiated liposarcoma (WD/DDLS) [23,24], HR + and triple negative breast cancer (TNBC) [25]. Moreover, a study has shown that palbociclib could inhibit of proliferation in RCC at nanomolar concentrations [26]. However, sustained G1-S phase cyclin expression and other early and late adaptations mediated by bypass signal transduction limit the effectiveness of CDK4/6 inhibitors [27]. For instance, some papers showed that palbociclib blocked cells in the G1 phase by inhibiting CDK4/6 activity, but it did not inhibit CDK2 activity, and the inhibition effect of RB phosphorylation could be reversible by CDK2 to produce drug resistance [28]. One candidate that could compensate for loss of CDK4/6 activity is CDK2, so a therapy that simultaneously inhibits both kinases at the outset might offer therapeutic advantages of palbociclib.
In this study, we observed that nobiletin did induce G1-phase cell cycle arrest, apoptosis, and proliferative inhibition in RCC Cells. Mechanistically, nobiletin inhibited activation of oncogene SKP2, leading to the accumulation of tumor suppressors and apoptosis-inducing substrates such as p21 and p27 to inhibit cell proliferation, eventually inhibit tumor formation. Intriguingly, we further found that sensitivity of palbociclib was associated with SKP2 protein level. Furthermore, in vitro and vivo results, dual inhibition of nobiletin and palbociclib showed synergistic lethality, suggesting that nobiletin could reverse drug resistance of palbociclib. Therefore, this combined regimen may serve as a potential therapeutic strategy for renal cancer.
Cells were cultured at 37 °C in a humidified atmosphere containing 5% carbon dioxide. All cell lines were tested to be free of mycoplasma contamination.

Immunoblotting (ib) And Antibodies
For direct IB analysis, cells were lysed in Radio-Immunoprecipitation Assay (RIPA) buffer with phosphatase inhibitors. The following primary antibodies were used: Cleaved Caspase-3 (Asp175) Plate Colony-forming Assay Cells were seeded into six-well plates (Corning, NY, USA) at a density of 500 cells/well. After 24 h, cells were treated with or without nobiletin for 48 h. The nobiletin-containing medium was then removed and replaced by complete medium, followed by incubation at 37 °C for 10-14d. The colonies were fixed with 4% paraformaldehyde, stained with crystal violet, and counted. The colonies composed of fifty or more cells were counted under microscopy. Each experiment was conducted in triplicate.

Cell Cycle Analysis
Cells were collected at the indicated time points after nobiletin and/or palbociclib treated and fixed with ice-cold 70% ethanol, and then stored at 4 °C overnight. After washing twice with phosphate buffered saline, then FxCycle PI/RNAse staining solution (Invitrogen, F10797) was used for detection of cell cycle according to manufacturer's protocol. These stained cells were subjected to cell cycle analysis using flow cytometer and analyzed for cell cycle phases with C6 Accuri system software.
Each experiment was conducted in triplicate.

Cell Viability Assay And Cell Proliferation Assay
The Cell Counting Kit-8 (CCK-8) assay (MCE, HY-K0301) was used to assess cell viability. The cell concentration was adjusted to 2 × 10 3 cells/well, and the cells were seeded into 96-well plates, followed by 24 h of culture at 37 °C in an atmosphere with 5% carbon dioxide. And then were treated with various concentrations of palbociclib or nobiletin and maintained in culture for 48 h. After removing the culture medium, the CCK-8 reaction solution was added according to the manufacturer's instructions, and then the absorbance was measured at 450 nm. Half-maximal inhibitory concentration (IC50) value is a critical index of the dose-response curve. Prism statistical software was used to calculate the IC50 values and to plot dose-response curves. According to IC50 values, cells was treated with the given concentration of nobiletin, and cultivation continued for a further 0, 1, 2, 3, and 4d. At different time after cell plating, cells were subjected to the proliferation assay by CCK-8 assay, according to the manufacturer's instructions. Each experiment was conducted in triplicate.

Cycloheximide Chase Analysis
Cycloheximide chase analysis was performed as described previously to define the effect of nobiletin on the stability of SKP2 protein [29]. Briefly, 786-O and 769-P cell lines (5 × 10 5 ) were exposed to 100 µM and 25 µM nobiletin for 24 h, respectively, and followed by 24 h-treatment with cycloheximide (50 µg/mL) (MCE, #HY-12320) to stop de novo protein synthesis. SKP2 levels at 0, 4, 8, 12 and 24 h following cycloheximide co-treatment were then determined by IB analysis. Each experiment was conducted in triplicate.

Quantitative Real-time Pcr
Total RNA was isolated from control and nobiletin-treated cells using RNAisoPlus (Takara, #9108), according to the manufacturer's instructions. Reverse transcription of the extracted RNA to corresponding complementary DNA was performed using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan). RT-qPCR was performed with QuantiNova™ SYBR® Green PCR Kit (Qiagen GmbH, Hilden, Germany) on an Applied Biosystems 7900HT Real-Time PCR System. The housekeeping gene, GAPDH, was used as loading controls. The following forward (F) primers, and reverse (R) primers were used: SKP2 forward-CAGGCCTAAGCTAAA TCGAGAG, SKP2 reverse-CTGGCAATGGTGGTGAAATG; GAPDH forward-AG CCTTCTCCATGGTTGGTGAAGAC, GAPDH reverse-CGGAGTCAACGGATTTG GTCGTAT. Each experiment was conducted in triplicate.

Immunohistochemical Staining
Immunohistochemical staining of mice tumors was performed as described previously [30]. Briefly, after deparaffinization, rehydration, antigen retrieval and blocking, the tissue slides were incubated overnight at 4 °C with indicated antibodies. The following primary antibodies were used: Ki-67 (Cell Signaling Technology, #9449), p27 and Cleaved-capase-3.

In Vivo Xenograft Model
Four-to six-week-old BALB/c athymic nude mice (nu/nu, female) were used with each experimental group consisting of five mice. All animal experiments were carried out according to a protocol approved by the University Committee for Use and Care of Animals. 2 × 10 6 786-O cells were mixed 1:1 with matrigel (BD Biocoat #354230) in a total volume of 200 ul and were injected subcutaneously into right flank side of nude mice. Nude mice were treated with vehicle, nobiletin (40 mg/kg/days, every day, per gavage), palbociclib (120 mg/kg/days, every day, per gavage) or nobiletin + palbociclib when the tumor size reached about 100 mm 3 . Nude mice were killed after 21 days of treatment. The growth of tumor was measured twice a week and average tumor volume (TV) was calculated according to the equation: TV= (L × W 2 )/2.

Statistical analysis
All data were shown as the average ± standard deviation (Mean ± SD) and each experiment was repeated at least three times independently. All Statistical analyses were performed using the Of note, in both tested RCC cell lines, MG-132 co-treatment didn't rescue SKP2 expression to the levels comparable to those of drug-free controls (Fig. 4C). To further substantiated nobiletin-induced SKP2 degradation, we conducted cycloheximide chase analyses, which cycloheximide almost unchanged in the rate of degradation of SKP2 protein in nobiletin-treated RCC cell lines (Fig. 4D). Next, with respect to transcriptional control, we found that SKP2 mRNA levels were noticeably reduced by nobiletin in 786-O and 769-P cell lines (Fig. 4E).
Therefore, these results supported that nobiletin-induced SKP2 down-regulation was mainly achieved by targeting SKP2 for transcriptional degradation. Intriguingly, FOXO3 levels were dose-dependently up-regulated in 786-O and 769-P cell lines by nobiletin treated (Fig. 4F). Some papers demonstrated that FOXO3 transcription factor is a negative regulator of SKP2 and SKP2 SCF complex [36]. Based on this, we speculated that the down-regulation of SKP2 might be associated with increasing of FOXO3 levels. Altogether, our results showed that nobiletin regulated SKP2-p21/p27-CDK2 axis to inhibit tumor progression in RCC.
Insensitivity of CDK4/6 inhibitor palbociclib is associated with higher SKP2 levels in RCC cells and palbociclib is targeted degradation of CDK4/6. Therefore, we speculated that RCC cells with higher SKP2 levels might require a higher concentration of palbociclib to achieve an IC50 response.
We first exogenously overexpressed SKP2 in Caki-1 and OSRC-2 cell lines, followed by treating palbociclib for 48 h. It was showed that SKP2 overexpression of Caki-1and OSRC-2 required higher concentration of palbociclib to achieve an IC50 response than control group (Figs. 6B&C).

Dual inhibition of nobiletin and palbociclib shows synergistic lethality in vivo
We finally validate the above in vitro findings by using an in vivo xenograft model. In the present study, we found that nobiletin could inhibit the proliferation of RCC lines in a dose-and time-dependent manners (Fig. 1). Further experiments confirmed that the inhibitory effect was due to the fact that nobiletin could induce G1-S phase arrest in cells ( Fig. 2A and Supplementary Fig. 1A).
The mechanism was that nobiletin could increase the protein levels of p27 and p21 and then inhibit the activity of CDK2 phosphatase, ultimately leading to the decrease of p-RB (Fig. 3A). Collectively, nobiletin hold great potential for treatment or to serve as a natural and anti-cancer compound by targeting SKP2.
Denovo or acquired resistance to CDK4/6 inhibitors is an almost ubiquitous inevitability. A study has found that palbociclib also inhibited proliferative activity in RCC cell lines, but not all RCC cells were sensitive to palbociclib [26]. We found that 786-O and 769-P cell lines needed high concentration of palbociclib to achieve an IC50 response (Fig. 5A). We observed that CDK4, CDK6, CDK2 and p-CDK2 basal levels were higher (Fig. 5B), as well as lower of p27 levels with higher of SKP2 levels in 786-O and 769-P cell lines (Fig. 5B). As we known, G1/S phase arrest is mainly regulated by two classical cyclin/CDK complex, including cyclinD-CDK4/6 and cyclinE-CDK2 [16]. p27 could influence CDK2, and be targeted degradation by SKP2 [28,34,35]. A study confirmed that while ER positive breast cancer cell lines were inhibited by palbociclib in culture, they quickly adapted because they permitted the degradation of p27 and a subsequent increase in CDK2 activity, allowing compensatory phosphorylation of RB and passage into S phase [28]. Meanwhile, another study also confirmed that CDK4/6 blockade was mediated by the noncanonical CDK2/cyclinD1 complex promoting p-RB phosphorylation recovery. The Cyclin E rebound is likely a consequence of CDK2/cyclinD1 activity and eventually triggering S-phase entry [27]. According to those, we hold that SKP2 was a key target that might provide a therapeutic benefit based on potential resistance of palbociclib. Next, our results showed that palbociclib was more sensitive to silencing of SKP2 group (Figs. 6E&F), and vice versa (Figs. 6B&C). Therefore, sensitivity of palbociclib was associated with SKP2 levels in RCC. High levels of SKP2 are a poor prognostic factor in multiple human cancers [29]. With these notions in mind, searching for agents that reverse resistance of palbociclib represents a promising strategy to discover novel cancer therapeutics [28,50]. Intriguingly, we investigated that nobiletin could target to downregulate SKP2 levels to suppressed activation of CDK2 phosphatase ( Fig. 3 and Fig. 4). Based on this, 786-O and 769-P cell lines as models to investigate whether nobiletin-palbociclib combination induced apoptosis in RCC cells by dual inhibition. The increase of cleavage of caspase-8 and caspase-3 appeared in combination nobiletin with palbociclib treated RCC cells (Fig. 7E and Supplementary   Fig. 3C). Similarly, a significant increase of p27 was observed with the combination treatment compared with palbociclib treatment alone ( Fig. 7E and Supplementary Fig. 3C). Similarly, dual combination of nobiletin and palbociclib significantly inhibited tumor growth in xenograft model ( Fig. 8A-E). It is noteworthy that natural compounds could improve the sensitivity and efficacy of chemotherapy drugs[51-53]. To this end, our finding that nobiletin as active component holds great possibilities for reversion of resistance of palbociclib.
To the best of our knowledge, the notion that nobiletin elicits G1 cell-cycle arrest for induction of anti-proliferation (Fig. 8F) through promoting SKP2 degradation has never been reported previously. In conclusion, for the first time, our results demonstrated that nobiletin regulated SKP2-p21/p27-CDK2 axis to inhibit tumor progression and showed synergistic chemopreventive effects with palbociclib in RCC cells. The enhanced anti-cancer effects by the combination treatment were associated with inhibition of cell cycle progression, induction of cellular apoptosis, as well as inhibition of proinflammatory cytokines. The fact that combination of nobiletin and palbociclib at their half doses produced stronger anti-cancer effects than nobiletin or palbociclib alone at their full doses provided a strong basis to utilize nobiletin/palbociclib combination for renal cell carcinoma chemoprevention.

Conclusions
In this study, we reported that nobiletin down-regulated SKP2-p21/p27-CDK2 axis to inhibit tumor progression; and nobiletin down-regulated the protein level of SKP2 from the transcriptional level by up-regulating the expression of FOXO3. Meanwhile, Sensitivity of CDK4/6 inhibitor palbociclib was associated with SKP2 levels. Furthermore, dual inhibition of nobiletin and palbociclib showed synergistic lethality in vitro renal cell carcinoma cell model and in vivo nude mice model. To our best knowledge, this is the first study reporting that the mechanism by which nobiletin regulates G1/S block of cell cycle and shows synergistic chemopreventive effects with palbociclib in RCC.         Nobiletin induced G1-phase cell cycle arrest, apoptosis, and colony-forming inhibition in         RCC cell lines were cultured, and followed by determination of protein expression using IB analysis. Data were presented as means ± SD. *p< 0.05, **p< 0.01, ***p< 0.001, compared with the control group; n=3. GAPDH levels served as the control for equal loading. RCC cell lines were cultured, and followed by determination of protein expression using IB analysis. Data were presented as means ± SD. *p< 0.05, **p< 0.01, ***p< 0.001, compared   palbociclib (120 mg/kg/days for every day for 4 weeks), n = 5; nobiletin + palbociclib, n = 5.

List Of Abbreviations
The tumor growth was monitored and growth curve was plotted (B) and tumors were harvested and photographed (C) Body weight was measured during the treatment and plotted (D). E. Immunohistochemical staining of xenograft tumor tissues. Tumor tissues from four groups of mice were fixed, sectioned, and stained with indicated antibodies. Scale bars: 100μm. Data were presented as means ± SD.