Nobiletin inhibits cell growth through restraining aerobic glycolysis via PKA‐CREB pathway in oral squamous cell carcinoma

Abstract Background/Aim Nobiletin is a polymethoxylated flavone enriched in Citrus and is used as an important drug in traditional Chinese medicine for various kinds of diseases. Among its multiple functions, it has shown that nobiletin inhibits proliferation of various cancer cells. However, it is unclear whether nobiletin inhibits the growth of oral squamous cell carcinoma (OSCC) cells. Materials and Methods We explored the antitumor effects of nobiletin in TCA‐8113 and CAL‐27 oral squamous cells. The Cell Counting Kit‐8 (CCK8) assay was used to measure cell vitality. Flow cytometry was performed to measure the number of cells in the various phases of the cell cycle. PCR and Western blot were applied to determine mRNA and protein expression, respectively. Results Nobiletin inhibited proliferation of TCA‐8113 and CAL‐27 cells via inducing cell cycle arrest at the G1 phase. In addition, the levels of phosphorylated‐PKA and phosphorylated‐CREB were reduced in nobiletin‐treated TCA‐8113 and CAL‐27 cells. Importantly, our results showed that nobiletin treatment resulted in impaired mitochondrial function and altered glucose consumption, and pyruvate and lactate production. Lastly, nobiletin was found to inhibit the generation of xenografts in vivo. Interestingly, administration of 50 μmol/L Sp‐cAMP, a potent PKA activator, rescued all phenotypes caused by nobiletin. Conclusions Nobiletin inhibits OSCC cell proliferation in a mitochondria‐dependent manner, indicating that it may have a promising role in cancer treatment and attenuation of drug resistance.


| INTRODUC TI ON
'-hexamethoxyflavone) is a polymethoxylated flavone that is present in dietary fruits such as oranges and has been an important drug in traditional Chinese medicine.
Oral squamous cell carcinoma (OSCC) is one of the head and neck squamous cell cancers that now ranks as the sixth most prevalent cancer worldwide (Argiris, Karamouzis, Raben, & Ferris, 2008;Leemans, Braakhuis, & Brakenhoff, 2011). The 5-year survival of OSCC patients is only about 50% although current treatments are effective against early diagnosed OSCC (Montal et al., 2016;Torre et al., 2015). Recent findings have revealed that mitochondrial function is critical for the effectiveness of chemotherapeutic agents for OSCC (Ansari et al., 2018;Liu, Xiong, Tan, & Liu, 2016). In addition, the cAMP/PKA/CREB signaling has been involved in OSCC drug resistance and migration (Chien et al., 2012;Suzuki et al., 2009).

| Cell culture
Cells were maintained in RPMI-1640 medium with 10% fetal bovine serum at 37°C under 95% air and 5% CO 2 .

| Protein extraction and Western blot
Protein extraction and Western blot experiments were performed as previously described (Chen et al., 2016). 60 μl RIPA lysis buffer (Solarbio) was used per well of a 6-well plate. At least three independent experiments were performed. Cell lysates were centrifuged at 18630 g for 15 min. The supernatants were collected and mixed with 2X SDS-loading buffer and subjected to Western blot analysis. Protein samples were analyzed by SDS-PAGE. Western blot was carried out using standard procedures, and immune-reactive proteins were visualized by SuperSignal™ chemiluminescence (Thermo Scientific).

| In vivo analysis of tumor growth
For tumor growth assays, TCA-8113 cells were injected into the left flank of 8-week-old nude mice. Twenty-four hours after implantation, 40 mg/kg nobiletin, 40 mg/kg nobiletin with 250 μg/kg Sp-cAMP, or an equal volume of PBS were injected every 2 days for 12 days. Once palpable, tumors were measured every 2 days and volumes were calculated using the formula: a*b 2 /2 (a is the largest dimension and b is the smallest). After the mice were sacrificed, the xenografts were pictured and weighed.

| Immunohistochemical analysis
Formalin-fixed, paraffin-embedded xenografts were sectioned and used for the immunohistochemical analysis. Paraffin was removed from the tissues, and the sections were hydrated through a graded series of ethanol. Antigen retrieval was performed, and sections were blocked with 5% sheep serum for 60 min. Sections were incubated with anti-PCNA antibody overnight at 4°C. Then, signals were visualized using 3, 3'-diaminobenzidine on the second day.

| Cytochrome c oxidase activity measurement
Cytochrome oxidase activities were measured with Cytochrome Oxidase Activity Colorimetric Assay Kit according to the manufacturer's instructions. The working compounds were mixed thoroughly and subjected to a measurement at 550 nm using a microplate reader (Infinite F200, Tecan). All results were normalized to the protein concentrations of the respective samples.

| Immunofluorescent staining of mitochondria
Mitochondria were stained with MitoTracker® Green FM KIT according to the manufacturer's instructions. Images were captured with a laser-scanning confocal microscope (True Confocal Scanner SP5; Leica; HCX Plan Apochromat confocal scanning 20×/0.7 NA objective lens) by LAS AF software (Leica).

| Metabolism related tests
Glucose consumption experiments were performed with Glucose Uptake Assay Kit as previously described (Hai, Shin, Bi, Ye, & Jin, 2018;Sun & Zhang, 2017). Briefly, TCA-8113 cells cultured in serum-free medium containing 100 μmol/L nobiletin with or without 50 μmol/L Sp-cAMP were incubated overnight in 96-well plate with a density of 5,000 cells/well. On the next day, cells were washed and incubated in KRPH with 2% BSA for 40 min and stimulated with insulin for 20 min, followed by 2-DG treatment for 20 min. Cells were then lysed and heat at 85°C to degrade endogenous NAD(P), followed by NADPH generation at 37°C for 1 hr. After recycling amplification reaction, the glucose uptake was quantified by optical density (OD) at 412 nm in a kinetic mode.
Lactate production was examined with L-Lactate Assay Kit according to the manufacturer's instructions. Briefly, 1 × 10 6 TCA-8113 cells treated with 100 μmol/L nobiletin with or without 50 μmol/L Sp-cAMP were harvested and washed with PBS. Then, the cells were homogenized in lactate assay buffer; after deproteinization, the samples were further incubated with reaction mix for 30 min at room temperature. Lactate production was quantified by measuring OD 450 nm.
Pyruvate production was detected with Pyruvate Assay Kit according to the manufacturer's instructions. Briefly, 1 × 10 6 TCA-8113 cells treated with 100 μmol/L nobiletin with or without 50 μmol/L Sp-cAMP were harvested and homogenized in pyruvate assay buffer. After deproteinization, the samples were further incubated with the reaction mix for 30 min at room temperature. Pyruvate production was quantified by measuring OD 570 nm.
Pyruvate dehydrogenase activity was assayed using the PDH Enzyme Activity Microplate Assay Kit according to the manufacturer's instructions. Briefly, TCA-8113 cells treated with 100 μmol/L nobiletin with or without 50 μmol/L Sp-cAMP were harvested and total proteins were extracted. Then, 200 µl of 13.5 mg/ml proteins was loaded onto measuring plates and incubated for 3 hr at room temperature. After rinsing with the stabilizer solution, samples were incubated with the assay solution and the kinetics of OD at 450 nm was read for 30 min.

| Statistical analysis
All experiments were repeated at least three times, and all data are presented as means ± SD. Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc.). The statistical comparisons were performed by one-way ANOVA followed by Tukey's post hoc test. p < .05 was considered to indicate statistically significant differences. *p < .05, **p < .01.

| Nobiletin treatment inhibits OSCC cell proliferation
To study the effect of nobiletin on OSCCs, we treated the OSCCs cell lines TCA-8113 and CAL-27 with 50, 100, or 150 μmol/L of nobiletin.
We first analyzed the viability of TCA-8113 and CAL-27 cells treated

| Nobiletin treatment inhibits PKA/ CREB pathway
To further dissect the molecular mechanism underlying nobiletin-

| Nobiletin treatment inhibits mitochondrial activity
Since PKA/CREB activity is directly associated with mitochondrial activity (Xie et al., 2018), we next evaluated the mitochondrial activ-

| Nobiletin treatment inhibits aerobic glycolysis in OSCC cells
Apart from the mitochondrial activity, glucose consumption is another index used to evaluate the respiration process. Thus, we next analyzed the metabolic alterations in TCA-8113 and CAL-27 cells treated with 100 μmol/L nobiletin with or without 50 μmol/L Sp-cAMP. Interestingly, our results showed that when OSCC cells were treated with nobiletin, glucose consumption and pyruvate dehydrogenase activity were significantly reduced, while lactate and pyruvate production were significantly increased (Figure 4a-d).
Importantly, treatment with Sp-cAMP attenuated the metabolic effects of nobiletin, which returned to the basal levels (Figure 4a-d), suggesting that nobiletin-mediated metabolic changes mainly relied on the cAMP pathway. Taken together, our results suggested a model where nobiletin inhibited the PKA/CREB pathway and in turn impeded mitochondrial activity. As a result, impaired pyruvate dehydrogenase activity increased the accumulation of pyruvate, which inhibited the tricarboxylic acid cycle (TCA) cycle and finally inhibited proliferation (Figure 4e).

| D ISCUSS I ON
Incidence of oral cancer accounts for approximately 2.1% of all cancers (Nishiyama et al., 2018). The discovery of chemicals with better clinical efficacy is an ultimate goal for drug development (Wu, Song, et al., 2018). Nobiletin is a common flavone enriched in daily consumed fruits such as oranges. Here, our data showed that nobiletin alone was able to restrain the growth of OSCC cells through inducing F I G U R E 3 Nobiletin treatment inhibits mitochondrial activity. (a) TCA-8113 and CAL-27 cells were incubated with 100 μmol/L nobiletin for 24 hr in the absence or the presence of 50 μmol/L Sp-cAMP, and then cytochrome oxidase activity was measured. Data from three independent experiments are shown as mean ± SD. *p < .05 compared to control group. (b) Representative immunofluorescent images of TCA-8113 and Cal-27 cells labeled with MitoTracker Green. Images were captured at 200× magnification cell cycle arrest. When OSCC cells were treated with 150 μmol/L nobiletin, the G2 cell population decreased from 33.38% to 4.90% and the expression of PCNA and cyclin D1 was almost hardly detectable. Our data agree with previous data that nobiletin inhibits cell growth via inducing G1 cell cycle arrest in a dose-dependent manner Lien et al., 2016;Morley et al., 2007;Uesato et al., 2014). Our data further validated the anti-proliferative role of nobiletin and suggested that nobiletin alone can be utilized as an anti-cancer drug. However, both previous reports and our data did not explore the cytotoxicity of nobiletin on normal cells at the concentration of 150 μmol/L, although the in vivo assay did not reveal any abnormalities in the mice except that nobiletin restrained the growth of xenografts ( Figure 5). Future studies may examine nobiletin toxicity in normal cells.
At the molecular level, our data illustrated that nobiletin reduced the protein levels of both PKA and phosphorylated-PKA. However, treatment of PC12 cells with 50 µmol/L nobiletin (Lai et al., 2011) and hippocampal neurons with 100 µmol/L nobiletin for less than 15 min augmented PKA signaling (Matsuzaki et al., 2008). In this study, nobiletin concentrations less than 50 μmol/L did not have a significant effect (data not shown). Further, concentrations of nobiletin higher than 50 μmol/L resulted in significant inhibition of OSCC cell proliferation in vitro. Besides, we demonstrated, in 2 OSCC cell lines, that nobiletin decreased the protein levels of PKA. The differences may also be due to different cell types. Here, we found that oral administration of 40 mg/kg nobiletin inhibited in vivo OSCC tumor formation, which is consistent with previous studies that low-dose of nobiletin (20 mg and 40 mg/kg oral administration) is effective in controlling tumor progression in different disease models (Da et al., 2016;Xie et al., 2019). Nonetheless, our data demonstrated through various experiments that Sp-cAMP, a PKA activator, functions against nobiletin, which validated our results that the PKA/ CREB pathway is impaired when OSCC cells were treated with nobiletin. More importantly, our data revealed that mitochondrial function was dramatically affected by nobiletin. It has been reported that the PKA/CREB pathway tightly regulates mitochondrial functions (J. Lee et al., 2005;Rim & Kozak, 2002). Thus, our data link nobiletin, CREB, and mitochondrial function in OSCC cells, which may illuminate a novel aspect of OSCC treatment. However, since nobiletin severely impaired mitochondrial function, it is worth exploring how much influence nobiletin has on mitochondria in normal cells. Hence, our results showed that the respiration process of OSCC cells was F I G U R E 4 Nobiletin treatment inhibits aerobic glycolysis in TCA-8113 and CAL-27 cells. TCA-8113 and CAL-27 cells were incubated with 100 μmol/L nobiletin for 24 hr in the absence or the presence of 50 μmol/L Sp-cAMP. Glucose consumption (a), lactate production (b), and pyruvate production (c) in the cell culture medium were analyzed. Data from three independent experiments are shown as mean ± SD. *p < .05 compared to control group. (d) Total pyruvate dehydrogenase activity was assessed in TCA-8113 and CAL-27 cells. Data from three independent experiments were normalized to control group and are shown as mean ± SD. *p < .05 compared to control group.

| Statement of human and animal rights
The experimental procedures in this study were conducted in accordance with the Ethics Committee of the Wenzhou Medical University's approved protocols (wydw2017-0126).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.