Tumor Biol. DOI 10.1007/s13277-016-4912-6

ORIGINAL ARTICLE

Kaempferol inhibits cell proliferation and glycolysis in esophagus squamous cell carcinoma via targeting EGFR signaling pathway Shihua Yao 1,2 & Xiaowei Wang 2 & Chunguang Li 2 & Tiejun Zhao 2 & Hai Jin 2 & Wentao Fang 1

Received: 7 December 2015 / Accepted: 22 January 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016

Abstract Antitumor activity of kaempferol has been studied in various tumor types, but its potency in esophagus squamous cell carcinoma is rarely known. Here, we reported the activity of kaempferol against esophagus squamous cell carcinoma as well as its antitumor mechanisms. Results of cell proliferation and colony formation assay showed that kaempferol substantially inhibited tumor cell proliferation and clone formation in vitro. Flow cytometric analysis demonstrated that tumor cells were induced G0/G1 phase arrest after kaempferol treatment, and the expression of protein involved in cell cycle regulation was dramatically changed. Except the potency on cell proliferation, we also discovered that kaempferol had a significant inhibitory effect against tumor glycolysis. With the downregulation of hexokinase-2, glucose uptake and lactate production in tumor cells were dramatically declined. Mechanism studies revealed kaempferol had a direct effect on epidermal growth factor receptor (EGFR) activity, and along with the inhibition of EGFR, its downstream signaling pathways were also markedly suppressed. Further investigations found that exogenous overexpression of EGFR in tumor cells substantially attenuated glycolysis suppression induced by kaempferol, which implied that EGFR also played an important role in kaempferol-mediated glycolysis inhibition. Finally, the antitumor activity of kaempferol was validated Shihua Yao and Xiaowei Wang contributed equally to this work. * Wentao Fang [email protected]

1

Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiaotong University, No. 241 West Huaihai Road, Shanghai 200030, China

2

Department of Thoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China

in xenograft model and kaempferol prominently restrained tumor growth in vivo. Meanwhile, dramatic decrease of EGFR activity and hexokinase-2 expression were observed in kaempferol-treated tumor tissue, which confirmed these findings in vitro. Briefly, these studies suggested that kaempferol, or its analogues, may serve as effective candidates for esophagus squamous cell carcinoma management. Keywords Kaempferol . Esophagus squamous cell carcinoma . EGFR . Hexokinase-2 . Glycolysis

Introduction Esophageal cancer is the sixth leading cause of death from cancer and the eighth most common cancer worldwide. Based on the difference of histology, esophageal carcinoma consists of squamous cell carcinoma (ESCC) and adenocarcinoma (EA). In Asian countries, especially in China, ESCC is the predominate type [1]. Despite the advances in the field of surgery and tumor chemotherapy, the 5-year survival rate is still less than 14 % [2]. Therefore, there is an urgent demand to identify novel chemical entity with activity against esophagus carcinoma. In comparison with synthetic compounds, natural molecules have a wide range of sources, diversiform structures, multiple targets, and diversified pharmacological potential, which provide a considerable source for candidates. Kaempferol, which is found in tea, propoils, and vegetables, is one of the most common dietary flavonoids [3]. Except antioxidant and anti-inflammatory effect as reported [4], recent studies have demonstrated that kaempferol had antiproliferation activities and/or induced apoptosis in various human cancer cell lines, such as breast cancer [5, 6], hepatic cancer [7, 8], colon cancer [9, 10], ovarian cancer [11, 12], and prostate cancer [13]. Mechanism investigation manifested

Tumor Biol.

that kaempferol treatment resulted in DNA damage [14], cell cycle arrest [9, 15], induction of apoptosis [10, 16], induction of autophage [7, 17], and inhibition of angiogenesis [11, 18]. Nonetheless, the activity of kaempferol in esophagus squamous cell carcinoma, its potential target, as well as its impact on tumor metabolism have not been investigated. Glycolysis is the metabolic process in which glucose is converted into pyruvate. In normal cells, when oxygen is abundant, pyruvate enters the mitochondrial tricarboxylic acid (TCA) cycle to be fully oxidized to CO2 (oxidative phosphorylation). Under hypoxic conditions, pyruvate is instead converted into lactate. Different from normal cells, cancer cells preferentially take glycolysis even in the presence of oxygen. Therefore, tumor glycolysis is often named Baerobic glycolysis^ or the Warburg effect to separate from the normal glycolysis. Tumor glycolysis supplies energy for rapid tumor growth; moreover, the products of glycolysis such as lactate also provide an appropriate microenvironment to promote tumor metastasis [19]. Critical to the process of tumor glycolysis is the first enzyme step of glucose phosphorylation. Hexokinases catalyze this essential irreversible step where glucose is phosphorylated to glucose-6-phosphate. So far, four isoforms of hexokinase, denoted as hexokinase (1–4) have been characterized in mammalian tissue [20]. Among these hexokinases, hexokinase-2 is predominantly expressed in malignant tumors that exhibit the highly glycolytic phenotype. Such as in hepatocellular carcinoma, an approximately 100-fold enhancement of the message was observed over the background mRNA signal, implicated hexokinase-2 upregulation during tumorigenesis [21]. Consistently, overexpression of hexokinase-2 was also detected in diverse cancers, such as ovarian [22], gastric [23], and breast cancers [24] and esophageal adenocarcinoma [25]. Clinical retrospective analysis revealed that higher expression of hexokinase-2 in tumor tissue had positive relationship with patients’ poor prognosis [26–28]. In the present study, efficacy of kaempferol against esophagus squamous cell carcinoma was evaluated in vitro and in vivo. Moreover, this is the first time we also investigated the impact of kaempferol on tumor metabolism in ESCC cells. Mechanism studies demonstrated that the inhibition of cell proliferation and glycolysis exerted by kaempferol was mainly attributed to its effect on epidermal growth factor receptor (EGFR)-mediated signaling pathway.

Materials and methods Cell line and reagents KYSE150 and Eca109 cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai) and cultured with RPMI 1640 medium

supplemented with 10 % fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin in a 37 °C incubator with 5 % CO2. Kaempferol, anti-β-actin (A5316), and antiFlag tag (F1804) antibodies were products of Sigma (St. Louis, MO, USA). Anti-rabbit IgG-horseradish peroxidase (HRP) and anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antihexokinase-1 (#2024), anti-hexokinase-2 (#2867), antiG6PD (#12263), anti-p-EGFR (Tyr1068) (#3777), antiEGFR (#4267), anti-p-ERK1/2 (Thr202/Tyr204) (#4370), anti-ERK1/2 (#4695), anti-p-Akt (S473) (#4060), anti-Akt (#4691), anti-p21 (#2947), anti-CDK4 (#12790), and anticyclin D1 (#2978) antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-Ki67 (ab66155) antibody was product of Abcam (Cambridge, UK). Cell proliferation assay Cells were seeded (2000 cells/well) in 96-well plate and cultured for 24 h, after treated with different concentration of kaempferol, the plate was cultured in a 5 % CO2 incubator. At different time points (0, 24, 48, or 72 h), 20 μl/well of CellTiter 96 Aqueous One Solution (Promega, WI, USA) was added and incubated at 37 °C for 1 h, and the absorbance was measured at 490 nm. For anchorage-independent growth assay, cells were suspended (8000 cells/ml) in 1 ml of 0.3 % agar with basal medium Eagle’s medium, 10 % FBS, 1 % antibiotics, and different concentrations of kaempferol (0, 15, 30, and 60 μM) overlaid into six-well plates containing a 0.6 % agar base. Cell culture was maintained in a 37 °C, 5 % CO2 incubator for 1 to 2 weeks, and then colonies were counted under a microscope using the Image-Pro Plus software program (Media Cybernetics, Silver Spring, MD, USA). Flow cytometry For cell cycle analysis, tumor cells (2 × 105) were seeded into six-well plate and cultured for 24 h, then exposed to different concentrations of kaempferol for 24 h. Cells were then harvested and washed with phosphatebuffered saline (PBS) twice and fixed with cold 70 % ethanol overnight at 4 °C. Cells were counterstained in the dark with 50 μg/ml propidium iodide (Biolegend, San Diego, CA, USA) and ribonuclease A (QIAGEN, Venlo, Netherland) in 300 μl PBS at 25 °C for 30 min. Stained cells were detected and quantified with FACSort Flow Cytometer (BD, San Jose, CA, USA). Western blotting Cells were harvested by trypsinization and pelleted by centrifugation. The pellets were lysed in NP40 lysis buffer (50 mmol/L Tris–HCl, pH 8.0; 150 mmol/L NaCl; 0.5 % NP40) supplemented with protease cocktail (Roche, Germany). Protein concentrations were determined using the Bradford assay (Bio-Rad, Philadelphia, PA, USA). Protein samples were subjected to SDS-PAGE and then electrically transferred to a polyvinylidene difluoride membrane

Tumor Biol.

(Millipore, Billerica, MA, USA). After blocking in 5 % nonfat dry milk in Tris-buffered saline (TBS), the membranes were probed with specific primary antibodies overnight at 4 °C, washed three times with TBS-Tween 20, and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Then, the membranes were washed with TBS-Tween 20 and the protein bands were visualized using ECL chemiluminescence reagents (Pierce Chemical Co., Rockford, IL, USA) according to the manufacturer’s protocol. Measurement of glucose uptake and lactate production Tumor cells were exposed to different concentrations of kaempferol for 24 h and then trypsinized and seeded in sixwell plates (5 × 105/well). After incubation for 4 h, media were discarded and then cells were incubated in fresh culture medium for another 8 h. Glucose and lactate levels were measured using the automatic biochemical analyzer (AU680, Beckman Coulter International, Brea, CA, USA). The relative glucose consumption rate and lactate production rate were normalized by the protein concentration of samples. Tumor xenograft experiment Five-week-old female Balb/c athymic nude mice were housed and maintained under specific-pathogen free (SPF) conditions in accordance with Institutional Animal Care and Use Committee. KYSE150 cells (1.5 × 106/mice) were subcutaneously injected into the right flank of nude mice. When the tumor reached a volume 50 to 100 mm3, the mice were randomly assigned into control and treatment group, six mice per group. Control groups were given vehicle alone, and treatment group received 100 mg/kg kaempferol. Kaempferol was administrated every 3 days by intraperitoneal injection. The sizes of the tumors were measured twice per week using microcalipers. The tumor volume (V) was calculated as follows: V = (length × width2) / 2. At the end of experiment, mice were sacrificed and the tumors are weighed and photographed. Immunohistochemical (IHC) staining Tumor tissues obtained from euthanized xenograft model were fixed in 10 % (v/v) formaldehyde in PBS, embedded in paraffin, and then cut into 5-mm sections. Paraffin sections of tumor were deparaffinized and hydrated, and endogenous peroxidase activity was blocked with 3 % H2O2. Antigen retrieval was performed using a microwave oven. The slides were blocked with blocking serum from the host of the secondary antibody and incubated with primary antibodies anti-p-EGFR (Tyr1068) (1:100), antihexokinase-2 (1:200), or anti-Ki67 (1:250) respectively at 4 °C overnight. Biotinylated secondary antibodies were added at a 1:100 dilution, followed by Vectastin ABC solution 1:100. The binding of the antibodies was visualized with 3,3-diaminobenzidine (DAB) solution. Slides were counterstained with Harris’ hematoxylin

a nd th en d e hy dr at ed . Sl i de s w er e vi ew e d an d photographed under a light microscope and analyzed using Image-Pro Plus software (version 6.2) program (Media Cybernetics). Statistical analysis All statistical analysis was performed by SPSS software (version 13.0). The experiments were performed in triplicate. All the quantitative data were expressed as mean values ± standard deviation, and the significant differences between two groups were assessed by a two-tailed Student’s t test. A probability value of p < 0.05 was considered to represent a statistically significant difference.

Results Kaempferol inhibited ESCC cell proliferation and anchorage-independent growth in vitro First, we investigated the activity of kaempferol against ESCC cell proliferation in KYSE150 and Eca109. At low concentration (15 μM), exposure to kaempferol had little effect on the growth inhibition, but higher concentration (30–60 μM) and long-time (48–72 h) treatment with kaempferol substantially inhibited cell proliferation (Fig. 1a, b). Except proliferation assay, colony formation in soft agar was used to study kaempferol’s effect on anchorage-independent growth. Anchorage-independent growth is one of the hallmarks of cell transformation and is considered to be the most accurate and stringent in vitro assay for detecting malignant transformation of cells. As the result has shown in Fig. 1c, d, kaempferol potently inhibited the anchorage-independent growth at 30 μM and the number of colonies formed in soft agar was significantly decreased; at high concentrations, there were very few colonies observed. All these results displayed that kaempferol had a profound antitumor efficacy in human esophagus squamous cell carcinoma in vitro. Kaempferol induced G0/G1 cell cycle arrest in ESCC cells Next, flow cytometry analysis was applied to study kaempferol’s impact on cell cycle. As the results have shown in Fig. 2a, when cell was treated with kaempferol at low concentration (15 μM) for 24 h, no obvious cell cycle arrest was observed. At higher concentration (30–60 μM), the percentage of G0/G1 phase cell was significantly increased. Consistent with the result of FACS, western blotting demonstrated that the expression of p21, which is a key regulator of cell cycle progression at G1 phase, was substantially increased at higher concentration, suggesting that kaempferol-induced G0/G1 arrest was closely correlated with p21 elevation. Meanwhile, we also detected the impact of kaempferol on cyclin D1 and CDK4 expression. It is well known that cyclin

Tumor Biol.

Fig. 1 Kaempferol inhibited the proliferation of ESCC cancer cells and anchorage-independent growth. a, b Kaempferol inhibited the proliferation of ESCC cells. Human ESCC cancer cells KYSE150 (a) and Eca109 (b) were treated with indicated concentrations of kaempferol for 0, 24, 48, or 72 h. Cell proliferation was analyzed by absorbance at OD490. The asterisk (*p < 0.05) indicated a significant decrease of ESCC cell proliferation after treated with kaempferol. c, d Kaempferol suppressed the anchorage-independent growth. KYSE150 (c) and Eca109 (d) were

incubated with various concentrations of kaempferol as described and subjected to anchorage-independent growth assay. Representative photographs (c and d, left panels) were shown, and the graph (c and d, right panel) showed the data of at least three independent experiments expressed as means ± D; the asterisks (*p < 0.05, **p < 0.01, Student’s t test) indicated a significant decrease of colony formation after kaempferol treatment in contrast with the control

D1-CDK4 interaction is necessary to promote the transition from G0 phase to G1. As shown in Fig. 2b, kaempferol treatment resulted in the decrease of cyclin D1 but had no effect on the expression of CDK4, implied that kaempferol-induced cell cycle arrest was also correlated with the downregulation of cyclin D1.

of KYSE150 and Eca109 cells, at a concentration of 30 μM kaempferol, the expression of hexokinase-2 was markedly suppressed. However, after kaempferol treatment, the expression of hexokinase-1, Glut-1, and G6PD, which were key enzyme or transporter protein involved in glucose metabolism, had no obvious change (shown in Fig. 3a, b). All these data validated that kaempferol demonstrated an inhibitory effect against tumor glycolysis in ESCC cells via reducing hexokinase-2 expression.

Kaempferol inhibited glycolysis in ESCC cells via decreasing hexokinase-2 Aerobic glycolysis is one of the metabolic features characterized by tumor cells and is pivotal for the survival and growth of cancer cells. In this study, we examined the impact of kaempferol on tumor glycolysis. KYSE150 cells treated with kaempferol (30 μM) showed significantly lower glucose uptake than the untreated control. In Eca109 cells, kaempferol also inhibited glucose consumption in a dose-dependent manner. With the decrease of glucose consumption, the secretion of lactate, which is the product of tumor glycolysis, was also substantially decreased after kaempferol treatment. In KYSE150 and Eca109 cells, kaempferol (30 μM) treatment resulted in the reduction of lactate production in cell culture supernatant compared to that in control group. Consistent with the inhibition of glucose uptake and lactate production, in both

EGFR was the potential target of kaempferol to exert its activity in ESCC cells As the results have shown in Fig. 4a, exposure of ESCC cells to kaempferol resulted in the inhibition of phosphorylation of EGFR in a dose-dependent manner. With the inhibition of EGFR activity, the phosphorylation of ERK1/2 and Akt, which are the main downstream signaling pathways of EGFR, were both suppressed. Moreover, we also examined the effect of kaempferol on EGF-induced EGFR activation. Similarly, EGF-driven phosphorylation of EGFR as well as the activation of its downstream signaling were also inhibited by kaempferol in a time- and dose-dependent way (Fig. 4b). In order to further elucidate the role of EGFR played in

Tumor Biol. Fig. 2 Kaempferol induced G0/ G1 cell cycle arrest in ESCC cancer cells. a KYSE150 cells were treated with various concentrations of kaempferol for 24 h. Cellular phase distribution was analyzed by staining with propidium iodide. Representative FACS results (left panels) were shown, and the graph (right panel) showed the data of at least three independent experiments expressed as means ± SD; the asterisks (*p < 0.05, Student’s t test) indicated a significant difference. b KYSE150 were treated with indicated concentration of kaempferol for 24 h, and the cell lysates were subjected to western blotting to detect the change of G0/G1 phase-related protein

kaempferol-mediated activity in ESCC cells, ESCC cells were transfected with pcDNA3.0-Flag-EGFR to overexpress EGFR and then tested kaempferol’s activity in EGFR overexpression cells. As shown in Fig. 4c, after transfected with pcDNA3.0-Flag-EGFR, the expression of EGFR was substantially increased. With the increase of EGFR activity, kaempferol-mediated decrease of hexokinase-2 was significantly attenuated in contrast with the mock group, which confirmed that EGFR signaling pathway was involved in the regulation of hexokinase-2. Along with the increase of hexokinase-2 in EGFR-flag-transfected cells, kaempferolinduced suppression of glucose consumption and lactate secretion in EGFR overexpression cells were significantly recovered (Fig. 4c), which verified that kaempferol-mediated inhibition of glycolysis in ESCC cells was attributed to the reduction of hexokinase-2. Kaempferol suppressed xenograft tumor growth in vivo In order to further confirm the antitumor activity of kaempferol, the potency of kaempferol against ESCC cell growth was investigated in vivo. As shown in Fig. 5a–d, in contrast with vehicle group, tumor growth in kaempferoltreated group was dramatically delayed. Twenty-five days after tumor cell injection, the tumor volume of vehicle group had reached about 600 mm3, but in kaempferol group, the

average tumor volume was around 200 mm3. Meanwhile, no obvious toxicity was observed as evaluating the change of body weight of tumor-bearing mice between vehicle and kaempferol-treated group. Immunohistochemistry analysis in kaempferol-treated tumor tissue revealed that the phosphorylation of EGFR in tumor tissue was substantially decreased after kaempferol treatment, which confirmed the activity of kaempferol on EGFR in vivo. In consistent with the decrease of EGFR activity, hexokinase-2 expression in kaempferol group was also significantly declined. Ki-67 is an important marker to detect the capability of tumor cell proliferation; as the result has shown in Fig. 5e, the expression of Ki67 in kaempferol group was decreased over 80 % compared to control group. Therefore, we can speculate that, with the inhibition of glycolysis via EGFR-mediated reduction of hexokinase-2 in tumor tissue, energy supply to support tumor growth was hindered, and the proliferation ability of tumor cells was also weakened.

Discussion In present study, our results demonstrated that kaempferol exerted a substantial antitumor activity against esophagus squamous cell carcinoma in vitro and in vivo. For the first time, we also uncovered that, with the downregulation of

Tumor Biol. Fig. 3 Kaempferol suppressed glycolysis in ESCC cell via reducing hexokinase-2. KYSE150 (a) and Eca109 (b) cells were treated with various concentrations of kaempferol, and the cell lysates were subjected to SDS-PAGE to examine the change of indicated protein. βactin was used as loading control (left panels). Glucose consumption and lactate production (right panels) in cell culture medium were analyzed using the automatic biochemical analyzer (AU680, Beckman Coulter International, Brea, CA, USA). The graph showed the data of at least three independent experiments expressed as means ± SD; the asterisks (*p < 0.05, **p < 0.01, Student’s t test) indicated significant inhibition of glucose consumption and lactate secretion after kaempferol treatment

hexokinase-2 after kaempferol treatment, the glycolysis in esophagus squamous cell carcinoma was markedly suppressed. Further studies revealed the inhibition of proliferation and glycolysis caused by kaempferol in esophagus squamous cell carcinoma was closely correlated with kaempferol’s effect on EGFR activity. So far, it is widely accepted that EGFR plays an important role in tumor development and progression. In esophagus carcinoma, meta-analysis demonstrated that EGFR overexpression was identified in patients (62.8 %) and was a valuable predictor for the T stage, vascular invasion, and overall survival [29]. Moreover, Aichler et al. reported that EGFR also was an independent adverse prognostic factor in esophagus carcinoma patients treated with cisplatin-based neoadjuvant chemotherapy [30]. These studies demonstrated that

kaempferol had a direct inhibitory effect on EGFR activity. EGF-induced phosphorylation of EGFR and its downstream signaling pathway was dramatically inhibited by kaempferol in a time- and dose-dependent way (Fig. 4a, b). Meanwhile, the result of immunohistochemical analysis also confirmed the activity of EGFR in tumor tissue was significantly suppressed after kaempferol treatment (Fig. 5e). With the inhibition of EGFR, kaempferol-induced cell cycle arrest further proved that EGFR maybe the potential target for kaempferol to exert its biological effects in esophagus squamous cell carcinoma (Fig. 2a). Given kaempferol is a kind of flavonoids which is abundantly distributed in fruits, vegetables, and tea, our findings suggested that a high daily dietary intake of kaempferol would be beneficial for esophagus squamous cell carcinoma patients, especially for those who are EGFR

Tumor Biol.

Fig. 4 Kaempferol exerted its activity in ESCC cell via mediating EGFR signaling pathway. a KYSE150 cells were incubated with various concentrations of kaempferol for 24 h, and the cell lysates were subjected to western blotting to detect the change of indicated protein. β-Actin was used as loading control. b KYSE150 cells were starved for 24 h and then exposed to 30 μM kaempferol for 2 h. After stimulating with 100 ng/ml EGF for indicated times, cell lysates were collected and western blotting was used to examine kaempferol’s impact on the activity on EGFR, ERK1/2, and Akt. β-Actin was used as loading control. c

Exogenous overexpression of EGFR attenuated kaempferol’s activity in ESCC cells. KYSE150 cells were transfected with pcDNA3.0-FlagEGFR or pcDNA3.0 vector (vehicle) for 24 h and then treated with 60 μM kaempferol for 24 h; glucose consumption (middle panel) and lactate production (right panel) in cell culture medium were analyzed as previous described. Cell lysates were analyzed by western blot as indicated antibodies (left panel). The graph showed the data of at least three independent experiments expressed as means ± SD; the asterisks (*p < 0.05, Student’s t test) indicated significant difference

positive. Actually, epidemiological evidence indicated that, compared to other daily dietary flavonoids, kaempferol was reported to be associated with a decrease risk of various cancers [31–34]. Different from other study reported by Zhang et al. that kaempferol induced G2/M phase arrest in esophagus carcinoma cell KYSE-510 [35], in our studies, kaempferol induced G0/G1 arrest in KYSE-150. Meanwhile, our results showed that cyclin D1 was substantially decreased after kaempferol treatment in KYSE-150 cells (Fig. 2b). However, as reported by Zhang et al., cyclin B which promotes G2/M phase transition was significantly inhibited by kaempferol in KYSE-510, the discrepancy of kaempferol’s effect on cell cycle maybe due to the difference of EGFR expression in these two cell lines, and EGFR overexpression was detected in KYSE-150, whereas the EGFR expression in KYSE-510 was very low (data not shown). Our studies also uncovered kaempferol’s tumor-fighting effect was correlated with its effect on glycolysis. It is well known that cancer cells preferentially take glycolysis as the main way to provide energy for its rapid growth, even in the abundance of oxygen. Hexokinase-2 plays a pivotal role in the

process of tumor glycolysis. Our results demonstrated that glucose consumption and lactate production were significantly decreased after kaempferol treatment (Fig. 3a, b), which was attributed to the suppression of hexokinase-2. In Hela cells, Filomeni et al. [17] reported that kaempferol had been demonstrated to block cellular intake of glucose, and even in the presence of insulin, the increase of glucose transporter was still unable to maintain glucose influx, suggesting the inhibition of glucose intake caused by kaempferol was not due to the prevention of glucose entry but by the blockade of glucose metabolism. Generally, hexokinase-2 is bound to the outer mitochondrial membrane and interacts with the pore-like protein voltage-dependent anion channel (VDAC) to form a complex. The HK-VDAC binding interaction is crucial for the supply of ATP to the phosphorylation reaction. Previous studies confirmed that in human osteosarcoma cells, kaempferol treatments lowered mitochondrial membrane potential, which destroyed the proton gradient ATP synthase required [16]. Consistent with our results, with the decrease of HK-2, interaction between HK-VDAC was weakened, the permeability of mitochondrial membrane was changed, and the process of

Tumor Biol. Fig. 5 Kaempferol suppressed ESCC cells growth in vivo. Activity of kaempferol was determined in KYSE150 xenograft model. Nude mice were randomly divided to groups when tumor volume reached 50 to 100 mm3. The dosage of kaempferol was 100 mg/kg and was administrated every 3 days by intraperitoneal injection. a Photograph of tumors in vehicle and kaempferol-treated group. b The weight of tumors in vehicle and kaempferol group. c The change of body weight of tumorbearing mice during the experiment. d Tumor growth curve in vehicle and treated group. e Tumor tissues underwent immunohistochemistry analysis by staining with anti-Ki67, anti-pEGFR, and anti-hexokinase-2 antibody to detect the change of related protein after kaempferol treatment. Left panel: representative photograph of tumor tissue per group (×200); right panel: the expression of indicated protein in per group was quantified. The asterisks (*p < 0.05, Student’s t test) indicated significant difference

glycolysis in mitochondrial was influenced. In addition to the impact on glycolysis, multiple studies also identified HKVDAC interaction was critical for preventing induction of apoptosis in tumor [36–38]. Kaempferol-induced apoptosis was reported in various cancer cells, such as colon cancer, kidney cancer, and bladder cancer [10, 15, 39]. Given kaempferol’s effect on hexokinase-2, we think it cannot exclude the role of hexokinase-2 decrease played in kaempferolinduced apoptosis. In ovarian and colon cancer, it had shown that the activity of hexokinase-2 was associated with

chemoresistance [22, 40]. Through inhibition of glycolysis by targeting hexokinase-2, the sensitivity of tumor cells to chemotherapy was substantially enhanced [41, 42]. Given the effect of kaempferol on hexokinase and glycolysis, we speculate that kaempferol maybe have a role to strengthen the efficacy of other chemotherapies. Exogenous overexpression of EGFR in esophagus squamous cell carcinoma cells substantially rescued the decrease of hexokinase-2 and the inhibition of glycolysis caused by kaempferol (Fig. 4c), implied the blockade of glycolysis by

Tumor Biol.

kaempferol was closely associated its effective inhibition of EGFR signaling pathway. In EGFR-mutated lung adenocarcinoma, it was reported that EGFR signaling regulated global metabolic pathway [43]. Similarly, De Rosa et al. have shown that differential inhibition of EGFR signaling pathway has reversed the Warburg effect and reactivated oxidative phosphorylation in non-small cell lung cancer cells, selective inhibition of AKT and ERK1/2, and caused mitochondrial complex subunits (OXPHOS) upregulation and glycolysis inhibition, respectively [44]. Conversely, in another study, it revealed that suppression of AKT/mTORC1 signaling pathway was involved in the inhibition of glucose metabolism in colorectal cancer [45]. In pediatric osteosarcoma, it also demonstrated PI3K/AKT-mediated hexokinase-2 expression played a part in the control of glucose uptake [46]. In the present study, the results showed that, with the inhibition of EGFR activation, the activities of AKT and ERK1/2 were both notably decreased after treatment (Fig. 4a, b). Therefore, more detailed investigation will be needed to elaborate which downstream signaling pathway plays a dominant role in the regulation of glycolysis, or both of them are involved. Whatever, all these dates proved that kaempferol’s biological effect in esophagus carcinoma was mainly attributed to its effect on EGFR. Briefly, for the first time, the antitumor activity of kaempferol against ESCC was investigated in vitro and in vivo. Different from the mechanism reported by previous studies, we demonstrated that the EGFR-hexokinase-2 signaling pathway was the underlying mechanism for kaempferol to exert its tumor-fighting effect. Our study provided a preclinical rational for kaempferol, or its analogue, to be applied to prevention and clinical treatment of human esophagus squamous cell carcinoma. Acknowledgments This work was funded by the National Natural Science Foundation of China (81301829) and the Key Project of Science and Technology Commission of Shanghai Municipality (15411951700).

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Compliance with Ethical Standards Conflicts of interest None

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