Alpinetin inhibits breast cancer growth by ROS/NF‐κB/HIF‐1α axis

Abstract Alpinetin, the main active ingredient in the Chinese medicinal herb Alpinia katsumadai Hayata, has been found to have anticancer activity. However, the therapeutic efficacy of signalling cascades modulated by alpinetin remains unknown. Here, we showed that alpinetin provoked mitochondria‐associated apoptosis in a dose‐dependent manner in breast cancer cells. Mechanistic investigations revealed that alpinetin dampens hypoxia‐inducible factor‐1α (HIF‐1α) signalling due to a lack of NF‐κB activation through reduced mitochondrial reactive oxygen species (ROS) production, decreasing HIF‐1α transcription. In vivo, we also found alpinetin led to significant tumour regression by inhibiting NF‐κB pathway. Overall, our work uncovers a ROS/NF‐κB/HIF‐1α axis‐dependent mechanism underlying the anticancer effects of alpinetin and suggests that alpinetin could act as a novel therapeutic agent against breast cancer.


| INTRODUC TI ON
As expected, cancer is the leading cause of death and the single most important obstacle to increase life expectancy in every country in the world in the 21st century. 1 Chemotherapy is one of the effective treatments for metastatic cancers. However, serious side effects of chemotherapy drugs that kill normal cells and cancer cells without discrimination and the ability of cancer cells to evade apoptosis have remained significant impediments to successful chemotherapy. 2 For example, vinorelbine, paclitaxel and anthracyclines were found to be associated with multidrug resistance. 3,4 Therefore, revealing the anticancer mechanisms and screening novel chemotherapy drugs to circumvent drug resistance are likely to improve chemotherapy.
Alpinetin, a natural flavonoid, is the major active constituent of the traditional medicinal plant Alpinia katsumadai Hayata, which has been used medicinally since ancient times. 5,6 Alpinetin has been reported to have anticancer activity, including apoptosis induction, cell cycle arrest and proliferative suppression in many types of cancer, such as breast cancer, lung cancer, colon cancer and liver cancer. [7][8][9][10] In addition to anticancer activity, alpinetin has shown considerable anti-inflammatory activities. Alpinetin has been reported to inhibit NF-κB activation and decrease oxidative stress to suppress the release of proinflammatory cytokines, such as tumour necrosis factor (TNF)-α and interleukin (IL)-1. 11,12 However, the imprecise mechanisms of action and its unknown direct target have greatly hindered its clinical application in the treatment of cancer.
Mitochondria are the vital of energy metabolism that produces ATP through oxidative phosphorylation and acts as centre cellular signalling hubs. 13,14 It is generally believed that tumour cells are in a relatively hypoxic cellular environment in solid tumours, and energy harvesting is dependent on the glycolytic pathway. 15 Under conditions of low oxygen tension, the cellular metabolism, function and fate that is governed by altered gene expression initiated by the transcription factor HIF-1. 16 Therefore, HIF-1 was found to be highly expressed in a variety of cancers, thereby adapting to the hypoxic environment in the cancer microenvironment and avoiding cancer cell death. 17 In addition, mitochondria involved in the production of reactive oxygen species (ROS) that cause severe cellular damage. Studies have shown that ROS is involved in various biological processes of cancer cell development. 18,19 Thus, inhibiting the expression of HIF-1 and blocking ROS generation may be a potential target for cancer treatment.
In this present study, we examined the effect of alpinetin in three breast cancer cell lines and investigated the mechanisms by which alpinetin regulates HIF-1α expression and induces cancer cell death.
Data demonstrate that alpinetin dose-dependently inhibit NF-κB pathway activity by decreasing cellular ROS production, thereby inhibiting HIF-1 expression.

| Cell viability wound-healing and apoptosis assays
The cells (5 × 10 3 cells/mL) were seeded onto 96-well plates at 37°C A wound-healing assay was used to assess cell migration. As a previously described method in a study, 20

| Quantitative RT-Qpcr
Total RNA was isolated using the TRIzol ® reagent (Invitrogen), and cDNA was synthesized using the HiScript ® II Q Select RT SuperMix for qPCR kit (Vazyme Biotech Co., Ltd). Quantitative RT-qPCR was All results are expressed as the mean ± SEM of three independent experiments. The symbols * and ** denote significant differences of P < .05 and P < .01, respectively normalization control to obtained relative expression data. The expression levels of miRNA were assessed using a Hairpin-it™ micro-RNA qPCR Quantitation Kit (GenePharma) according to the standard protocol. The expression of U6 was used as an endogenous control.
All primer sequences are shown in Table 1.

| Western blot analysis
Cell or tissue were lysed in RIPA solution involving a phosphatase inhibitor (Vazyme), immobilized protein on beads in sample reducing buffer followed by denaturing at 95°C for 5 minutes. The protein concentrations were determined using the BCA protein assay kit (Vazyme). The total protein was separated by SDS-PAGE and transferred onto PVDF membranes. Blots were successive incubated with antibodies and secondary antibodies. Anti-β-actin was used as control. Protein expression was detected using an Enhanced Chemiluminescence Detection System (ImageQuant LAS 4000 mini).

| Measurement of ROS production
The intracellular ROS level was measured by flow cytometry. Briefly, cells were seeded at a density of 1 × 10 6 cells/mL into 6-well plates and, after treatment as indicated, the intracellular ROS level was

| Immunofluorescence staining
Cells with or without alpinetin treatment were collected and fixed with 4% paraformaldehyde for 10 minutes, loaded onto coverslips and dried, and permeabilized with 0.2% Triton X-100 for 10 minutes before they were blocked with 5% BSA for 1 hour. Cells were incubated with an antibody overnight at 4°C and then incubated with secondary antibody in the dark for 2 hours at 25°C. After incubating cells with DAPI (5 mg/mL) for 10 minutes (DAPI, Beyotime) and observed using fluorescence microscopy (Olympus). PBS was used for all washing steps.

| Histological analyses and immunohistochemistry
Tissues were fixed in 4% formaldehyde solution, embedded in paraffin, and sections were stained with haematoxylin and eosin (H&E).
Immunohistochemistry detection using anti-Ki67 was performed on paraffin sections. The staining processes were performed according to standard methods. The sections were observed using an optical microscope (Olympus).

| TUNEL assay
TdT-UTP nick-end labelling (TUNEL) assay was performed a TUNEL assay kit (Roche Diagnostics GmbH) according to the manufacturer's instructions as described previously. 21

| Statistical analysis
The values are expressed as mean ± SEM and P values were calculated as detailed in the corresponding legends. The unpaired t test (GraphPad Software) was used for statistical analysis and to generate graphs. Replicates are biological replicates. Student's t test was used to assess statistical significance ( * P < .05, * * P < .01).  Figure 1C,D). Similarly, the wound-scraping assay indicated that alpinetin inhibited cell migration compared with DMSO treatment ( Figure 1E,F). These results demonstrate that alpinetin is cytotoxic to breast cancer cells.

| Alpinetin induces mitochondria-associated apoptosis in breast cancer cells
To observe whether the apoptotic effect of alpinetin was activated by a cascade of caspases, the cleavage of cleaved caspase-9/caspase-3/ PARP was detected by Western blot. As shown in Figure 2A, alpinetin caused activation of caspase-9, caspase-3, and PARP in 4T1 and MDA-MB-231 cells. Apoptosis consists of two classic pathways: the death receptor 'extrinsic' and mitochondrial 'intrinsic' pathways. 22 Apoptosis induced by activation of the caspase-9/3 cascade is suggested to involve the mitochondrial apoptotic pathway. Therefore, we determined the mitochondrial membrane potential (Δψm) via the JC-1 fluorescent probe. As shown in Figure 2B,C, we found that al-  Figure 2D,E). Altogether, these data suggest that the cellular uptake of alpinetin induces mitochondriaassociated apoptosis.

| Alpinetin reduced ROS generation and then inhibited NF-κB in breast cancer cell
Mitochondria are important sites for intracellular ROS production.
Thus, the level of ROS in cancer cells was evaluated upon alpinetin treatment. Our results showed that alpinetin reduces intracellular ROS production ( Figure 3A,B). Several studies have suggested that ROS is the key stimuli regulator of NF-κB signalling, 23 so we assay the activation of NF-κB pathway. We found that alpinetin inhibits NF-κB pathway activation in a dose-dependent manner ( Figure 3C) and increases ROS level to abolish the effect of alpinetin in inhibiting NF-κB pathway ( Figure 3D). As expected, inhibition of ROS levels with NAC or alpinetin suppressed the nuclear translocation of p65/ NF-κB ( Figure 3E).

| Alpinetin dampens the expression of HIF-1α by ROS/ NF-κB axis
Next, we look for functional effectors downstream of NF-κB to understand the molecular mechanisms by which alpinetin exert their synergistic effects on breast cancer cell lines. We found that HIF-1α levels were significantly lower in alpinetin treatment cells   Further studies have shown that alpinetin can inhibit the phosphorylation of p-p65 and interfere with its entry into the nucleus ( Figure 3). The NF-κB pathway exerts survival activity by inducing the expression of several anti-apoptotic genes, such as c-FLIP, XIAP and member of the Bcl-2 family. 29,30 It is worth noting that activation of the NF-κB signalling pathway led to increased DNA damage in inflammatory disease, 31,32 but it promotes cancer cell survival in many types of cancer. 33 The reason is that the effects of NF-κB depend on the stimulus factors and cell type, and it can suppress or induce autophagy in a context-dependent manner. 34 In the present study, inhibition of HIF-1α-mediated apoptosis and migratory characteristics of breast cancer cells, through inactivated of NF-κB pathway, was also confirmed ( Figure 4). HIF-1α

| Alpinetin inhibits tumour growth in vivo
is the master transcriptional regulator mediating the adaptive responses to intra-tumoral hypoxia to drive cancer progression, particularly in breast cancer, which the efficiency of cellular oxygen utilization is lower than normal cells in its microenvironment. 15 HIF-1α transcriptionally regulates hundreds of oncogenic genes that are involved in cell fate, angiogenesis, invasion, metastasis and metabolic adaptation, such as VEGF, CXCR4 and LOX. 17 knockdown of HIF-1α leads to reduced migration and invasion of various breast cancer and HIF-1α also vital maintenance survival and self-renewal in tumour stem cells and metastasis cancer cell. 35,36 We showed noted that alpinetin decreases the expression of HIF-1α on transcription level by inactivating NF-κB pathway.
However, there are other ways to affect the stability or degradation of HIF-1α, such as ubiquitination and proteasomal degradation, which needs further research to prove. Importantly, overexpression HIF-1α dose not to completely abrogate alpinetin-induced apoptosis ( Figure 4F), these findings indicated that other pathways may also contribute to alpinetin-induced cancer phenotypic changes.
In conclusion, we report the discovery of a traditional Chinese medicine, alpinetin, that induces mitochondria-associated apoptosis and suppresses proliferation in a dose-dependent manner.
The pharmacological effects of alpinetin were also confirmed in an MDA-MB-231 mouse xenograft model. Mechanistically, our study provides proof that alpinetin exerts an antitumour effect through the ROS/NF-κB/HIF-1α axis. Collectively, these observations suggest that alpinetin could act as a potential breast cancer chemotherapy agent.

ACK N OWLED G EM ENTS
We thank all members of the Laboratory of Veterinary Clinical Diagnosis for helpful discussions and suggestions. This work was supported by the National Major Project for Breeding of Transgenic Pig (2016ZX08006-002) and the Cooperative Innovation Center for Sustainable Pig Production.

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.

AUTH O R S' CO NTR I B UTI O N S
TZ, SG, GD and CQ conceived and designed the experiments. TZ, XZ, GD and CQ performed the experiments. TZ, SG, XZ, JQ and CQ analysed the data. TZ and GD wrote the paper. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.