Cardamonin inhibits breast cancer growth by repressing HIF-1α-dependent metabolic reprogramming

Background Cardamonin, a chalcone isolated from Alpiniae katsumadai, has anti-inflammatory and anti-tumor activities. However, the molecular mechanism by which cardamonin inhibits breast cancer progression largely remains to be determined. Methods CCK-8 and Hoechst 33258 staining were used to detect cell growth and apoptosis, respectively. HIF-1α driven transcription was measured by luciferase reporter assay. Glucose uptake and lactate content were detected with 2-NBDG and L-Lactate Assay Kit. Cell metabolism assays were performed on Agilent’s Seahorse Bioscience XF96 Extracellular Flux Analyzer. Mitochondrial membrane potential was measured with JC-1 probe. DCFH-DA was used to measure ROS level. Protein expression was detected by western blotting assay. Immunohistochemistry was performed to measure the expression of HIF-1α, LDHA and CD31 in tumor tissues. Results Cardamonin inhibited growth of the triple negative breast cancer cell line MDA-MB-231 in vitro and in vivo by suppressing HIF-1α mediated cell metabolism. Cardamonin inhibited the expression of HIF-1α at mRNA and protein levels by repressing the mTOR/p70S6K pathway, and subsequently enhanced mitochondrial oxidative phosphorylation and induced reactive oxygen species (ROS) accumulation. We also found that cardamonin inhibited the Nrf2-dependent ROS scavenging system which further increased intracellular ROS levels. Eventually, accumulation of the intracellular ROS induced apoptosis in breast cancer cells. In addition, cardamonin treatment reduced glucose uptake as well as lactic acid production and efflux, suggesting its function in repressing the glycolysis process. Conclusions These results reveal novel function of cardamonin in modulating cancer cell metabolism and suppressing breast cancer progression, and suggest its potential for breast cancer treatment. Electronic supplementary material The online version of this article (10.1186/s13046-019-1351-4) contains supplementary material, which is available to authorized users.


Background
Breast cancer is the second leading cause of cancer death among women across the world [1]. Chemotherapy is a common therapeutic strategy for breast cancer patients. However, serious adverse effects such as digestive tract toxicity, cardiovascular system toxicity, skin toxicity, hepatotoxicity, and delayed wound healing compromise the clinical effects of current chemotherapy drugs [2]. Thus, development of new chemotherapy drugs with fewer side effects remains to be an urgent need for breast cancer treatment.
Electron leakage from mitochondrial electron transport is the main source of intracellular reactive oxygen species (ROS) [3,4]. Although appropriate levels of ROS benefit cancer cell proliferation and growth, over-accumulation of ROS results in cell damage and apoptosis [3,5]. Metabolic reprogramming is one of the hallmarks of cancer [6]. Different from normal cells to use mitochondrial oxidative phosphorylation (OXPHOS) for energy supply, most of breast cancer cells have increased aerobic glycolysis in both normal and hypoxia conditions, which produces energy and intermediate metabolites to support proliferation and growth [6][7][8][9]. Compared with mitochondrial OXPHOS, aerobic glycolysis generates less ROS, while promotes glutaminolysis and enhances pentose phosphate pathway (PPP) metabolism to facilitate the biosynthetic process [10,11]. Therefore, targeting metabolic reprogramming of cancer cells may be a promising strategy for cancer treatment.
Cardamonin is a chalcone isolated from Alpinia katsumadai with significant anti-tumor activities in lung cancer, colon cancer, nasopharyngeal carcinoma and glioblastoma [22][23][24][25][26][27][28][29][30][31]. Cardamonin also inhibits growth of chemotherapy-resistant breast cancer cells [26]. Although cardamonin has been identified as a Wnt and NF-κB inhibitor [22,29,32], the detailed molecular mechanism by which cardamonin inhibits breast tumor growth largely remains to be determined. In the present study, we showed that cardamonin significantly inhibited the growth of breast cancer in vivo and in vitro, which is most likely mediated by reprogramming cancer metabolism through inhibition of the HIF-1 pathway. These findings may facilitate the clinical application of cardamonin in breast cancer treatment.

Cell viability assay
Cells were seeded in 96-well culture plates (2.0 × 10 3 cells/well) and grown overnight. After treatment with cardamonin at different concentrations for 24-72 h, the cells were incubated with CCK-8 (Cell Counting Kit-8, DOJINDO Laboratories, Cat. No.: CK04) solution (20 μl/ well) and cultured at 37°C for another 1 h. Absorbance of the dissolved solutions was detected at 450 nm on a Thermo Scientific Varioskan Flash microplate reader (USA). The cell viability rate was calculated as follows: (absorbance of drug-treated sample/absorbance of control sample) × 100.

Hoechst 33258 staining
MDA-MB-231 cells were seeded at a density of 1.5 × 10 5 cells/ml on coverslips in a 24-well plate and allowed to adhere to the coverslips overnight. After being treated with cardamonin (10, 20 and 40 μM) for 24 h, the cells were fixed with 4% PFA for 10 min. Then being gently rinsed with 1 × PBS, the cells were stained with 10 μg/ml Hoechst 33258 solution for 15 min. Finally the cells were rinsed with 1 × PBS and the cell morphology was observed under a fluorescence microscope.

Glucose uptake assay
Glucose uptake ability of MDA-MB-231 cells was evaluated by using the fluorescent glucose 2-NBDG (Thermo Fisher Scientific, Cat. No.: N13195). MDA-MB-231 cells seeded in 96-well plates were cultured in DMEM medium without glucose or carbon sources. After being treated with cardamonin (20 μM) for 3 and 6 h, respectively, the cells were gently rinsed with HBSS and incubated with 100 μM 2-NBDG at 37°C for 30 min. Consequently, the cells were rewashed with HBSS. Fluorescent intensity of the cells was detected on a microplate reader (Ex (λ) 465 nm; Em (λ) 540 nm). Meanwhile, after being treated with cardamonin (10, 20, 40, 80 μM) for 6 h, the glucose uptake of MDA-MB-231 cells was also measured according to the above method.

Lactate content measurement
Lactate, one of the end-products of aerobic glycolysis, can change the tumor microenvironment and have an impact on cancer-associated cells [10]. Intracellular and extracellular lactate contents of MDA-MB-231 cells were determined by using the L-Lactate Assay Kit (Cayman, Bath: 0521418, USA). MDA-MB-231 cells were cultured in the DMEM basic medium supplemented with 10% FBS and treated with cardamonin (20 μM) for 24 h. Then, the cells were cultured in DMEM basic medium containing 0.5% FBS for another 4 h. The cell pellet and culture medium were collected for lactate content measurement according to the manufacturer's instructions.

ROS measurement
The ROS level was measured according to the method described previously [33] with some modifications. Intracellular ROS level in MDA-MB-231 cells was detected by 2′, 7′-dichlorodihydrofluorescein (DCFH), which is oxidized into fluorescent dichlorofluorescein (DCF) in the presence of ROS. MDA-MB-231 cells were cultured in the 96-well plates at a density of 1 × 10 5 cells/ml in DMEM medium containing 10% FBS. After being treated with cardamonin (20 μM) for 2, 4, and 6 h, respectively, the cells were gently washed with HBSS followed by the incubation with 20 μM DCFH at 37°C for 30 min. The dye was then removed and replaced with fresh HBSS. Fluorescence of the cells was measured immediately on a microplate reader (Ex (λ) 485 nm; Em (λ) 535 nm). Meanwhile, after being treated with cardamonin (10, 20, 40 and 80 μM) for 6 h, intracellular ROS level in MDA-MB-231 cells was also measured according to the above method.

Cell metabolism assays
The Mito Stress Test Kit (Agilent, Cat. No.: 103015-100) was used to measure the oxygen consumption rate (OCR). The Glycolytic Rate Assay Kit (Agilent, Cat. No: 103344-100) was used for measuring the glycolytic proton efflux rate (GlycoPER). The Agilent Seahorse XF Real-Time ATP Rate Assay Kit (Agilent, Cat. No.: 103592-100) was used to detect the ATP production rates of mitochondrial oxidative phosphorylation and glycolysis, respectively. Before metabolism measurement, the probe plate was hydrated with HPLC grade water in a CO 2 -free incubator. The assay phenol red-free solution containing 10 mM glucose, 2 mM glutamine, 1 mM pyruvate and 5 mM HEPES was kept in a 37°C CO 2 -free incubator to maintain the pH value. Then the HPLC grade water in the hydration plate was replaced with calibration solution and kept in a 37°C CO 2 -free incubator. MDA-MB-231 cells were seeded into XF96 cell culture microplates (Seahorse Bioscience) at the density of 5000 cells/well (for measurement of OCR and Glyco-PER) or 7500 cells/well (for measurement of ATP production rates of oxidative phosphorylation and glycolysis), and allowed to adhere to plate overnight. Then the cells were incubated with cardamonin (20 μM) for 3, 6 and 12 h, and the cell culture medium was replaced with phenol red-free assay solution and placed in a 37°C CO 2 -free incubator for 1 h. Finally, OCR, GlycoPER and ATP production rates of mitochondrial oxidative phosphorylation and glycolysis were determined and analyzed on the Agilent's Seahorse Bioscience XF96 Extracellular Flux Analyzer (Agilent Technologies) according to the manufacturer's instructions and protocols (Seahorse Bioscience, North Billerica, MA, USA).
For the detection of GlycoPER value, Rot/AA (inhibitors of mitochondrial electron transport chain) and 2-deoxy-D-glucose (2-DG, inhibitor of glycolysis) were added according to the manufacturer's instructions and protocols (Seahorse Bioscience, North Billerica, MA, USA).
For the measurement of OCR value, oligomucin, FCCP, and Rot/AA, respectively, were added according to the manufacturer's instructions and protocols (Seahorse Bioscience, North Billerica, MA, USA).
For the determination of ATP production rates of mitochondrial oxidative phosphorylation and glycolysis, oligomycin and a mix of rotenone and antimycin A were added according to the manufacturer's instructions and protocols (Seahorse Bioscience, North Billerica, MA, USA).

Luciferase reporter assay
Luciferase reporter construct containing three HRE was purchased from Addgene (26731). HRE-luciferase reporter plasmid and Renilla luciferace vector were transiently transfected into HEK293 cells. The cells were treated with cardamonin (20 and 40 μM) for 24 h. Then the cells were harvested and subjected to luciferase activity assay by using Dual-Luciferase R Reporter Assay system (Promega, Cat. No. E1910).

Animal treatment
Female nude mice, 4-week-old, were purchased from Shanghai Slake Experimental Animal Co., Ltd. and kept under SPF level conditions. All animal experiments were carried out in accordance with the protocol approved by the Animal Ethics Committee in Shanghai University of Traditional Chinese Medicine (SHUTCM), which complies with international rules and policies for laboratory animal use and care as founded in the European Community guidelines (EEC Directive of 1986; 86/609/EEC). All animal experiments were approved by the institutional Ethics Committee of SHUTCM (SZY201704012).
After habituation for a week, the mice were inoculated subcutaneously with MDA-MB-231 cells (1.0 × 10 7 cells in 200 μL PBS per mouse). When the tumor grew to 50 mm 3 , the nude mice were randomly divided into three groups, namely control group, cardamonin group and 5-Fu group. The mice in control group and cardamonin group were intraperitoneally injected with physiological saline and cardamonin (3 mg/kg), respectively, once a day. But the mice in 5-Fu group were intraperitoneally injected with 5-Fu (50 mg/kg) twice a week. Body weight and tumor size of the nude mice were recorded every 3 days. The size of the tumor was calculated according to the formula: [length × (width) 2 ]/2. Four weeks after the drug administration, the nude mice were sacrificed and the tumor tissues were isolated for further analysis.
Hematoxylin and eosin (H&E) staining H&E staining was conducted to assess the pathological change of tumor tissues according to the procedure described previously [36]. And the images were captured with microscope (Olympus, BX61VS).

Immunohistochemistry (IHC) analysis
The IHC experiments were performed according to the specific operations described previously [37][38][39][40][41]. In brief, tumor tissue sections, 5 μm thick, were deparaffinized with xylen and rehydrated in gradient alcohol (anhydrous, 95, 75%). After being washed three times in PBS, the sections were incubated with 0.3% H 2 O 2 for 10 min to block the endogenous peroxidase. Then the sections were placed in sodium citrate antigen retrieval solution for 10 min in microwave oven. Thereafter, they were incubated with 10% goat serum blocking solution for 30 min followed by the incubation with primary antibodies against HIF-1α, LDHA and CD31 overnight at 4°C. Subsequently, the slices were incubated with secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. After DAB coloring reaction, the images were captured by Olympus VS120 Virtual Slide Scanner. Positive expressions of target proteins were displayed in brown color, and the cell nuclei were stained in blue color by hematoxylin. Five randomly selected fields were evaluated using a BH2 microscope (Olympus, Tokyo, Japan) equipped with a Nikon 4500 digital camera. The area and optical density (OD) of HIF-1α, LDHA and CD31 in each field were processed by the computer-aided automatic image analysis system (Qiu Wei, Shanghai, China). The IHC index was calculated as the average integral OD: [(positive area×OD)/total area].

Statistical analysis
All data were presented as mean ± standard deviation. Differences among groups were analyzed by one-way ANOVA analysis of variance with Dunnett's test or the Student's t-test using GraphPad 5.0 software (La Jolla, CA, USA). The value of P < 0.05 was considered statistically significant.  1b). We found that treatement with cardamonin (20 and 40 μM) induced chromatin condensation and nuclear shrinkage or fragmentation (Fig. 1c). Cardamonin (20 μM) also reduced protein levels of Bcl-2, while increased protein levels of Bax and cleaved caspase 3 in MDA-MB-231 cells (Fig. 1d). These results indicated that cardamonin treatment induced apoptosis in MDA-MB-231 cells.

Results
Cardamonin also significantly inhibited cell viability of BT549 cells, and the IC 50 Figure S1).
Cardamonin inhibits expression of HIF-1α and its target genes in MDA-MB-231 cells HIF-1α over-expression is associated with advanced cancer progression and poor clinical outcomes in breast cancer patients [43,44]. Activation of the HIF-1α pathway induces expression of many metabolism-related target genes such as PDHK1 and LDHA, which enhances glycolysis but reduces mitochondrial OXPHOS [42]. Using the HRE luciferease reporter construct that contains three hypoxia response elements (HRE), we showed that treatment with cardamonin significantly reduced HIF-1α driven transcription (Fig. 2a). Protein and mRNA levels of HIF-1α were downregulated in cardamonin treated cells under both normoxia and hypoxia conditions (Fig. 2b and c). Importantly, cardamonin (20 μM) treatment significantly inhibited transcription of HIF-1α target genes PDHK1 and LDHA (Fig. 2d). The protein levels of PDHK1 and LDHA were also reduced in cardamonin treated cells (Fig. 2e). Cardamonin treatment significantly inhibited the protein expressions of HIF-1α and PDHK1 in BT549 cells, but had no significant effect on protein levels of LDHA and LDHB (Additional file 1: Figure S3a-b). Interestingly, cardamonin treatment enhanced protein expression of HIF-1α, but reduced protein expression of PDHK1 in MCF7 cells (Additional file 1: Figure S3e-f), suggesting that other targets may mediate the inhibitory activity of cardamonin in MCF7 cells.

Cardamonin induces metabolic reprogramming in breast cancer cell before apoptosis occurring
To determine function of cardamonin in cell metabolism, we first used JC-1 to examine the mitochondrial membrane potential in MDA-MB-231 cells. A reduction of the mitochondrial membrane potential was detected after cardamonin (20 μM) treatment for 9 h (Fig. 3a and  b). Consistent with the decreased mitochondrial membrane potential, the cell viability was significantly decreased as early as 9 h after treatment (Fig. 3c). Next, we used fluorescent glucose 2-NBDG to measure glucose uptake in MDA-MB-231 cells. After 6 h treatment with cardamonin, glucose uptake was significantly decreased in MDA-MB-231 cells (Fig. 3d and e). Intracellular and extracellular L-lactate concentrations were also significantly decreased (Fig. 3f), indicating that cardamonin reduced Llactate production and efflux. Results from the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) demonstrated that cardamonin (20 μM) greatly enhanced mitochondrial OXPHOS, as evidenced by increased basal respiration, maximal respiration and spare respiratory capacity, as early as 3 h after treatment (Fig. 3g  and h). However, the basal PER and glyco-PER were significantly reduced at 12 h after cardamonin treatment (Fig. 3i  and j), suggesting that the decrease of glycolytic rate was after the increase of mitochondrial OXPHOS. Interestingly, protein levels of HIF-1α and PDHK1 but not LDHA and LDHB, were significantly down-regulated at 6 h after cardamonin treatment (Fig. 3k-l). Same treatment also increased the oxygen consumption rate (OCR) in BT549 cells (Additional file 1: Figure S2a-b) and MCF7 cells (Additional file 1: Figure S2h-i). The basal PER and glyco-PER were Mitochondrial OXPHOS generates significant amount of ATP, and ROS is a by-products of this process. Cardamonin treatment (20 μM) for 4 h elevated ROS levels in a dose-dependent manner ( Fig. 4a and b). We also found that ATP production from mitochondria was enhanced by cardamonin (20 μM) (Fig. 4c); however, ATP production from glycolysis was decreased as late as 12 h after cardamonin treatment (Fig. 4d).
Nrf2, a stress-response transcription factor, regulates expression of antioxidant defense genes such as HO-1 and NQO1 by binding to anti-oxidant response elements (AREs) in promoter regions in the stress condition [5,[45][46][47]. Thus, activation of the Nrf2 defense system mitigates ROS accumulation [48]. Therefore, we asked whether the ROS scavenging system contributes to the cardamonin-induced ROS accumulation. The protein levels of Nrf2, HO-1 and NQO-1 were significantly reduced after cardamonin treatment for 6 h (Fig. 4e and f), suggesting that Nrf2 antioxidant pathway is a potential target of cardamonin that contributes to the ROS accumulation. Similar results were also obtained in BT549 (Additional file 1: Figure S2e-g) and MCF7 cells (Additional file 1: Figure S2l-n).

Activation of HIF-1α pathway abolishes effects of cardamonin on mitochondrial OXPHOS and cell viability in MDA-MB-231 cells
To determine whether the HIF-1α pathway mediated cardamonin's function in regulating cell metabolism and viability, we performed rescue experiment by treating MDA-MB-231 cells with CoCl 2 . Treatment with CoCl 2 (100 μM) increased protein levels of HIF-1α and induced PDHK1 expression in MDA-MB-231 cells. Importantly, CoCl 2 treatment reversed the inhibitory effect of cardamonin on the protein expression of HIF-1α and PDHK1 (Fig. 5a). CoCl 2 stimulation also counteracted the increased mitochondrial OXPHOS induced by cardamonin (Fig. 5b and  c). Consistent with the above results, ROS production and accumulation were suppressed by CoCl 2 in cardamonin-treated cells; the cell viability was also rescued ( Fig. 5d and e). NAC (N-acetyl-cysteine, ROS scavenger; 5 mM) abolished the inhibitory effect of cardamonin on cell viability of MDA-MB-231 cells (Fig. 5f). To further confirm whether the downregulation of HIF-1α target genes mediated the inhibitory effect of cardamonin, we introduced the PDHK1 plasmid into MDA-MB-231 cells. We found that PDHK1 overexpression increased the cell viability of cardamonintreated MDA-MB-231 cells (Fig. 5g and h). Similar results were also obtained in BT549 (Additional file 1: Figure S3c  Data are shown as mean ± SD; *, P < 0.05, **, P < 0.01; ***, P < 0.001, ns means no statistical difference, compared with control. n ≥ 3 mTOR/p70S6K pathway is the target of cardamonin to inhibit the HIF-1α pathway We found that cardamonin treatment (20 μM) reduced the protein expression of p-PI3K, p-Akt, p-mTOR and p-P70S6K in MDA-MB-231 cells in a time-dependent manner (Fig. 6a). Cardamonin treatment also decreased protein levels of p-PI3K, p-Akt, p-mTOR and p-P70S6K in a dose-dependent manner (Fig. 6b). However, at the early time points such as 0.5 h and 1 h, protein levels of p-mTOR and p-P70S6K but not p-PI3K, were significantly reduced in cardamonin-treated cells (Fig. 6c). These results suggested that the mTOR/P70S6K pathway may be upstream of the HIF-1α pathway in response to cardamonin treatment. Importantly, enhancing mTOR activity with MHY1485 rescued protein expression of HIF-1α and PDHK1 in cardamonin-treated MDA-MB-231 cells (Fig. 6d-e). Pretreatment with mTOR activator also attenuated the inhibitory effect of cardamonin on cell viability (Fig. 6f). These results indicate that cardamonin inhibited the HIF-1α pathway and cell viability through the mTOR/P70S6K pathway in MDA-MB-231 cells.

Cardamonin inhibits tumor growth and protein expression of HIF-1α in the MDA-MB-231 xenograft model
To verify the inhibitory effect of cardamonin on breast cancer growth in vivo, we performed drug treatment experiment with the MDA-MB-231 xenograft model. Cardamonin or 5-Fu administered by intraperitoneal injection significantly inhibited tumor growth in vivo compared with the control group (Fig. 7a, b and c). 5-Fu and cardamonin had little effect on the body weight of nude mice (Fig. 7d). Furthermore, the increased protein levels of cleaved caspase3 and Bax were upregulated in the cardamonin-treated group, while protein expression of Bcl2 and the ratio of Bcl2/ Bax were reduced ( Fig. 7e and f).
H&E staining results showed that tumor cells appeared necrotic and the cytoplasmic ratio of tumor cells were decreased after cardamonin treatment (Fig.  7g). To determine whether cardamonin inhibited the HIF-1α pathway in vivo, we assessed HIF-1α protein levels and its downstream targets in tumor tissues. IHC and western blot results showed that cardamonin treatment reduced protein levels of HIF-1α and LDHA in vivo (Fig. 7g-j). A reduction in protein levels of p-PI3K, p-AKT, p-mTOR, and p-P70S6K was detected in cardamonin-treated tumor tissues (Fig.  7k-l). We also found that CD31 staining reduced by 53.10% in cardamonin-treated breast cancer tissues ( Fig. 7g and h), suggesting that cardamonin inhibits tumor angiogenesis.

Discussion
In the present study, we identified novel function of cardamonin in regulating the HIF-1 pathway and . i, j Western blotting analysis showed that cardamonin (3 mg/kg) inhibited the protein expression of HIF-1α, PDHK1 and LDHA in tumor tissues. k, l Cardamonin (3 mg/kg) inhibited the protein expression of p-PI3K, p-AKT, p-mTOR and p-p70S6K in tumor tissues. Data are shown as mean ± SD; *, P < 0.05, **, P < 0.01; ***, P < 0.001, ns means no statistical difference, compared with control. n ≥ 4 cancer cell metabolism. We showed that cardamonin treatment reduced HIF-1α expression in TNBC cell lines, and subsequently inhibited glycolysis and increased mitochondrial OXPHOS and ROS accumulation, finally induced cell apoptosis in breast cancer cells in vitro and in vivo. These results suggest that cardamonin inhibits breast cancer progression by targeting the HIF-1 pathway and its mediated cancer cell metabolism.
ROS is mainly generated during mitochondrial OXPHOS [3,4], and aerobic glycolysis may reduce ROS accumulation in tumor cells and enhance cancer cell survival during chemotherapy. Integrity of mitochondrial membrane is very important for mitochondrial OXPHOS; disruption of the membrane integrity not only impairs mitochondrial OXPHOS, but also induces cell apoptosis [49][50][51][52][53]. Treatment with cardamonin as late as for 9 h in breast cancer cells significantly reduced MMP, which was accompanied by a decrease in cell viability. And, enhanced mitochondrial OXPHOS and a reduction in glucose uptake, lactate production and lactate efflux were detected in cardamonin-treated MDA-MB-231 cells as early as for 6 h. These results suggest that cardamonin reduced cell viability by enhancing ROS generation in mitochondria.
Increased levels of ROS have been detected in many cancers, in which they promote tumor development and progression by inducing genome instability and multiple signaling pathways. However, accumulation of ROS to the high level also induces cell apoptosis, suggesting that a delicate balance of intracellular ROS levels is required for cancer cell function [53][54][55]. Most of the chemotherapeutic drugs and radiotherapy drugs induce cytotoxicity and cell cycle arrest by increasing oxidative stress [56][57][58][59][60]. We identified cardamonin as a ROS inducer in breast cancer cells. Cardamonin increased ROS at least partially through reducing the ROS scavenging system because Nrf2 and NQO1 expression is reduced upon treatment. Importantly, NAC, the active oxygen scavenger, reversed the inhibitory effect of cardamonin on cell viability in breast cancer cells. These results suggest that ROS is the critical mediator of inhibitory function of cardamonin in breast cancer cells.
Tumor recurrence and metastasis are major challenges in the treatment of tumors. Accumulation of lactate acid induced by glycolysis, which acidifies the tumor microenvironment and subsequently promotes tumor metastasis [60]. It has been shown that reduction of lactate acid level slowed down or even block cancer metastasis [60,61]. We found that the lactate acid efflux was inhibited by cardamonin, implicating a potential function of cardamonin in suppressing cancer metastasis. Indeed, our unpublished data showed that cardamonin treatment inhibit breast cancer metastasis in vitro.
HIF-1α is a crucial regulator of cell metabolism by modulating glycolytic enzyme gene expression [12,62,63]. At the low level of HIF-1α, pyruvate, one of the end products of glycolysis, is mainly converted into acetyl-CoA and enters the citric acid cycle. However, the majority of pyruvate enters aerobic glycolysis when the HIF-1 pathway is activated. This regulation is mainly through PDHK1, a gene inhibiting PDH activity to reduce the entrance of acetyl-CoA into citric acid cycle [12,62]. We found that cardamonin treatment decreased the mRNA and protein expressions of HIF-1α and inhibited its target genes in MDA-MB-231 and BT549 cells. Rescue experiments showed that introduction of HIF-1α and PDHK1 prevented the increase of mitochondrial OXPHOS and ROS accumulation induced by cardamonin. Together these results indicate that cardamonin inhibits cancer growth at least partially through targeting HIF-1α-mediated metabolic reprogramming in TNBC cells. In ER positive MCF7 cells, cardamonin failed to reduce protein levels of HIF-1α, but the protein expression of PDHK1 was still inhibited by cardamonin. However, PDHK1 overexpression or NAC both abolished the inhibitory effect of cardamonin on cell viability in MCF7 cells, suggesting that other molecular mechanisms are involved in PDHK1 regulation and metabolic reprogramming in MCF7 cells.
It has been shown that the mTOR/p70S6K pathway [19], NF-κB pathway [64,65] and MAPK pathway [66,67] regulate the HIF-1 pathway during cancer progression [68][69][70]. Our study showed that cardamonin inhibited activation of the mTOR/p70S6K pathway in MDA-MB-231 cells. Rescue experiments demonstrated that mTOR activation abolished the inhibitory activity of cardamonin on the cell viability and protein expression of HIF-1α and PDHK1 in MDA-MB-231 cells. Given roles of the NF-κB pathway in enhancing cell survival and HIF-1 activation, we also determined whether NF-κB mediates function of cardamonin in breast cancer cells. We found that cardamonin treatment reduced p-NF-κB levels at very early time point (Additional file 1: Figure S4). Interestingly, TNF-α (agonist of NF-κB) pretreatment reversed the inhibitory effect of cardamonin on cell viability and protein levels of p-NF-κB, HIF-1α and PDHK1 in MDA-MB-231 cells. This results suggest that the NF-κB pathway is another potential target of cardamonin that regulates HIF-1α/PDHK1 axis in breast cancer cells.

Conclusions
Our results reveal novel function of cardamonin in inhibiting the HIF-1α pathway and its dependent metabolic reprogramming in breast cancer cells (Fig. 8). We also identified the mTOR/p70S6K pathway as one of the cardamonin targets to repress HIF-1α/PDHK1 axis. These findings provide the mechanistic insight into tumor inhibitory activity of cardamonin and suggest the therapeutic potential of cardamonin in breast cancer treatment.