Curcumin derivative WZ35 inhibits tumor cell growth via ROS-YAP-JNK signaling pathway in breast cancer
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Breast cancer is the most prevalent cancer among women worldwide. WZ35, an analog of curcumin, has been demonstrated to remarkably improve the pharmacokinetic profiles in vivo compared with curcumin. WZ35 exhibits promising antitumor activity in gastric cancer, HCC, colon cancer. However, antitumor effects of WZ35 in breast cancer and its underlying molecular mechanisms remain unclear.
CCK8, Flow cytometry and transwell assays were used to measure cell proliferation, cell cycle arrest, apoptosis, cell migration and invasion. We constructed xenograft mouse model and lung metastasis model to assess the antitumor activities of WZ35 in vivo. To explore the underlying molecular mechanisms of WZ35, we performed a series of overexpression and knockdown experiments. The cellular oxygen consumption rates (OCRs) was measured to assess mitochondrial dysfunction.
We found that treatment of breast cancer cells with WZ35 exerts stronger anti-tumor activities than curcumin both in vitro and in vivo. Mechanistically, our research showed that WZ35 induced reactive oxygen species (ROS) generation and subsequent YAP mediated JNK activation in breast cancer cells. Abrogation of ROS production markedly attenuated WZ35 induced anti-tumor activities as well as YAP and JNK activation. In addition, ROS mediated YAP and JNK activation induced mitochondrial dysfunction in breast cancer cells.
Our study showed that novel anti-cancer mechanisms of WZ35 in breast cancer cells and ROS-YAP-JNK pathway might be a potential therapeutic target for the treatment of breast cancer patients.
KeywordsBreast Cancer WZ35 YAP ROS JNK Mitochondrial dysfunction
B cell lymphoma 2
Dulbecco’s modified eagle medium
mitochondrial translation factor
Extracellular regulated protein kinases
Fetal bovine serum
c-Jun N-terminal kinase
Kirsten rat sarcoma viral onco
Large tumour suppressor 1
Mammalian STE20-like protein kinase 1
Mammalian STE20-like protein kinase 2
Normal adjacent tissues
Nuclear respiratory factor 1
Nuclear respiratory factor 2
Oxygen consumption rate
Phosphate buffer solution
DNA polymerase subunit gamma
Reactive oxygen species
Salvador family WW domain-containing protein 1
The Cancer Genome Atlas
Breast cancer is a heterogeneous disease that is considered as the most frequently diagnosed cancer among women with high mortality and morbidity worldwide. The incidence of breast cancer is increasing year by year in developed countries and developing countries [1, 2]. Hormone-responsive breast cancer could benefit from currently available endocrine therapy, however, breast cancer cells lacking hormone receptors generally using chemotherapeutic drugs, such as taxol and doxorubicin . These chemotherapeutic drugs exhibit high dose-limiting toxicity to tumor cells as well as normal cells, which limit their clinical usage . In addition, endocrine therapy resistance has become the biggest limitation for treatment of breast cancer . Thus, searching for less toxic and effective therapeutics is urgently needed.
Curcumin is a natural polyphenolic compound, obtained and purified from the powdered rhizome of the Curcuma longa L . Substantial studies have reported that curcumin plays an essential role in anti-bacterial, anti-proliferative, anti-inflammatory, antioxidant, anti-carcinogenic and anti-amyloidogenic effects in vitro and in vivo through targeting various molecules [6, 7]. Meanwhile, it has been reported that anti-cancer activity of curcumin is mainly through the stimulation of the innate and adaptive immune systems [8, 9, 10]. However, poor bioavailability in vivo of curcumin per se has impeded its use in cancer therapy [11, 12]. To solve this problem, a new compound of curcumin analog WZ35, 1-(4-hydroxy-3-methoxyphenyl)-5-(2-nitrophenyl) penta-1,4-dien − 3-one, has been designed and synthesized by our lab. WZ35 has been proved possessing anti-cancer activities in gastric cancer by activating ROS-dependent ER stress and JNK mitochondrial pathways . Similar anti-cancer effects have been found in colon cancer and hepatocellular carcinoma (HCC) [14, 15]. However, the function of WZ35 in breast cancer remains unclear. There is considerable evidence showing that loss of Hippo pathway or overexpression of YAP/TAZ was associated with human cancers including lung, liver and intestine cancers through promoting cancer cell growth and suppressing cell apoptosis [16, 17, 18, 19]. On the contrary, hyperactivation of YAP is associated with a better prognosis in breast cancer patients, which suggests that YAP might act as a tumor suppressor in breast cancer .
Here, we demonstrated that WZ35 inhibits breast cancer cell growth, migration and invasion through activating ROS-YAP-JNK pathway. We further found that ROS-YAP-JNK pathway was involved in mitochondrial dysfunction in breast cancer cells. Our results suggest that WZ35 might be an effective therapeutic agent and targeting ROS-YAP-JNK pathway could be a potential therapeutic method for the treatment of breast cancer patients.
Materials and methods
Reagents and antibodies
Curcumin was purchased from Sigma (St. Louis, MO). WZ35, an analogue of curcumin, was synthesized by our lab and its structure has been described previously . Oligomycin, carbonylcyanide-p-trifluorometh oxyphenylhydrazone (FCCP), antimycin A and rotenone were purchased from sigma (St. Louis, MO). CCK-8 (CK04) were obtained from DOJINDO. Horseradish peroxidase (HRP)-conjugated anti-rabbit (BL003A) and anti-mouse (BL001A) immunoglobulin glucose were purchased from Biosharp (Anhui, China). DCFH-DA ROS detection kit (S0033), NAC and SP600125 (S1876) were obtained from Beyotime (Haimen, China). BCA protein assay kit (23227) and Pierce ECL western blotting substrate (34095) were obtained from Thermo Scientific (Waltham, MA). Primary antibodies POLG (ab128899), EF4 (GUF1, ab171161), β-actin (ab8226) were obtained from abcam (HKSP, New Territories, HK). Phospho-SAPK/JNK Thr183/Tyr185 (#4668), JNK (#9252), E-cadherin (#8834S), N-cadherin (#13116S), cleaved Caspase-3 (#9664S), LATS1 (#3477), MOB1 (#13730), p-MOB1 (#8699), MST1 (#3682), MST2 (#3952), SAV1 (#13301), Nrf1 (#69432), Nrf2 (#12721), YAP (#4912), Bcl-2 (#2870), p-Aktser473 (#4060), Cyclin B1 (#4138), Akt (#9272) and GAPDH (#5174) were obtained from Cell Signaling Technology (USA). MMP-2 (sc13594) and MMP-9 (sc21736) were purchased from Santa Cruz Biotechnology, Inc. P21 (10355–1-AP) were obtained from Precision Technologies Group (Chicago, USA).
Twenty two primary breast cancer specimens and their adjacent tissue counterparts were obtained from the First Affiliated Hospital of Wenzhou Medical University and informed consents were obtained from the patients. All studies and procedures involving human tissues were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Cell culture and transfection
Human breast cancer cell lines, MDA-MB-231 and Hs578T cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. BEAS-2B cells were obtained from American Type Culture Collection (CRL-9609). Cells were cultured with dulbecco’s modified eagle medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified incubator with 5% CO2. The MCF10A cells were purchased from the ATCC (CRL-10317). Cells were grown in DMEM/F12 supplemented with 5% horse serum, 0.5 mg/ml hydrocortisone, 20 ng/ml EGF, 10 μg/ml insulin, 100 ng/ml cholera toxinand and cultured at 37 °C with 5% CO2. The si-YAP and YAP overexpressing vector were transfected into MDA-MB-231 cells with lipofectamine 2000.
Cell proliferation assay
Cell proliferation was evaluated by the CCK8 assay. MDA-MB-231, Hs578T, BEAS-2B and MCF10A cells were initially plated in 96-well plates at 5 × 103 cells per well, cultured overnight. MDA-MB-231 and Hs578T cells were treated with curcumin or WZ35 with concentrations of 5, 10 and 20 μg/mL. For cytotoxicity assay, BEAS-2B and MCF10A cells were treated with WZ35 with concentrations of 0.5, 1, 2.5, 5, 7.5, 10 μg/mL and 0.25, 0.5, 1, 2.5, 5, 7.5 and 10 μg/mL respectively. MDA-MB-231 cells were used as control group. After 24 h or 48 h, 10 μl CCK8 reagent was added into each well and incubated for 3 h, followed by measurement of the optical density (OD) at a wavelength of 450 nm using a microplate reader (Bio-Rad). Curcumin, WZ35 and SP600125 were dissolved in 0.03% DMSO; NAC was dissolved in PBS and diluted with a complete medium containing 10% FBS to the final concentration. After treating with drug for 24 h, 48 h or 72 h, Cell proliferation was evaluated by the CCK8 assay.
Flow cytometry analysis for cell cycle, apoptosis and ROS determination
MDA-MB-231 cells were plated in 6-well plates at 1 × 105 cells per well. After treatment with curcumin (10 μg/mL) and WZ35 (10 μg/mL) for 24 h, cells were harvested with trypsin and washed with PBS, then resuspended with 70% prechilled ethanol, and stored at − 20 °C overnight. After 24 h, the fixed cells were collected and stained with propidium iodide using Cycle Test Plus DNA Reagent Kit (Becton Dickinson, San Jose, CA). The stained cells were then analyzed for DNA content using FACS caliber (BD, Franklin Lakes, NJ). To analyze cell apoptosis, the drug pre-treated cells were washed with ice-cold PBS twice and harvested with trypsin, then resuspended with 5 μl Annexin V-FITC/PI (BD, San Jose, CA) mixture. After incubated at room temperature for 20 min in the dark condition, the cells were measured by BD Accuri TM C6 flow cytometer (BD, Franklin Lakes, NJ). To detect the total intracellular ROS generation, cells were collected and washed with pre-warmed PBS, then stained with 10 μM DCFH-DA in DMEM at 37 °C for 30 min in the dark condition. Cells were collected and flow cytometer was used to measure the DCFH-DA fluorescence.
Colony formation assay
MDA-MB-231 and Hs578T cells were seeded in 6-well plates at low density 4 × 103 cells/well and cultured with curcumin (0.1 μg/mL) or WZ35 (0.5 μg/mL) for 14 days till visible colonies appeared. The cells were then stained with crystal violet. Colony number was calculated by Image J software. The test was repeated 3 times.
Transwell migration and invasion assays
In vitro cell migration and invasion assays were performed using a 24-well transwell (Coring, USA) with an 8-μm pore polycarbonate membrane as previously described . Briefly, for the invasion assay, the top portion of the chambers were precoated with Matrigel (BD Biosciences) diluted with FBS free media (1: 20). The chambers without Matrigel were used for migration assay. Twenty thousand cells were added into the top chamber with 200 μL serum-free medium, after incubation at 37 °C for 2 h, curcumin (10 μg/mL) or WZ35 (10 μg/mL) was added to the top compartment of the chambers. Then 500 μL complete medium were filled in the lower chambers. After being incubated at 37 °C and allowed to migrate for 24 h, non-migratory or non-invasive cells above the upper chambers were removed with cotton swabs. The cells migrated or invade stuck to the lower transwell surfaces were fixed with 4% paraformaldehyde and stained with crystal violet for 3 min. The cells were imaged and counted in five fields of vision observed using a microscope with 20x magnification.
Measurement of oxidative phosphorylation
The Seahorse XF96 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA, USA) was used to detect real time integrated cellular oxygen consumption rate (OCR) according to the manufacturer’s protocol. In brief, MDA-MB-231 cells were treated with or without drugs for 12 h and 1 × 103 cells were plated into the seahorse customized cell plates. After baseline measurements, the OCR was detected with sequential injection of oligomycin (ATP synthase inhibitor; 1 μM), FCCP (uncoupler; 0.5 μM), rotenone (complex I inhibitor; 1 μM), and antimycin A (complex III inhibitor; 1 μM).
Total RNA was extracted with Trizol reagent (Invitrogen) from MDA-MB-231 cells pre-treated with curcumin and WZ35 according to the manufacturer’s instructions. RNA integrity was confirmed using spectrophotometry and formaldehyde/agarose gel electrophoresis. 1000 ng RNA was reverse transcribed into cDNA using the Prime Script TM RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Quantitative real-time PCR assays were performed on a CFX connect TM real-time system (Bio-Rad) using SYBR Green (Bio-Rad) according to the manufacturer’s protocol. Each sample was replicated for three times. All results are expressed as means ±SD.
Western blot analysis
Cells and tissue samples were washed with PBS and lysed in RIPA lysis Buffer (Beyotime, Jiangsu, China) supplemented with protease inhibitors (Complete, EDTA-free; Roche, USA) and then centrifuged at 12000 rpm for 10 min. Equal amounts of protein lysates (50 μg each) were separated by 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore, Burlington, MA, USA). The Membranes were blocked with 5% non-fat milk in PBS with 0.05% Tween 20 (PBST) at room temperature for 1.5 h and incubated overnight with primary antibodies (1: 1000) at 4 °C. After wash with PBST for 5 min three times, the membranes were incubated with corresponding secondary antibodies (1:2000) for 1 h at room temperature. After extensive washing, the protein bands were detected using ECL kit (Bio-Rad, Hercules, CA).
Tissue sections were initially deparaffinized with xylene, rehydrated, and antigen retrieval was performed in 0.01 M citrate buffer (PH = 6.0) for 3 min at 95 °C. Followed by incubation with primary antibody at 4 °C overnight, the tissue sections were incubated with secondary antibody at room temperature for 2 h. Finally, after DAB staining and a neutral gum sealing, immunohistochemical signals were photographed and observed under a microscope (Olympus Corporation, Tokyo, Japan) with magnification of 200 × .
Five-week-old, athymic BALB/c nu/nu female mice (16-19 g, totally n = 24) were purchased from Vital River Laboratories (Beijing, China). The mice were randomly divided into three groups. MDA-MB-231 cells were subcutaneously inoculated into the right flank of mice (1 × 107 cells in 100 ul PBS per mouse). After tumor volume reached 50 mm3, the mice were intraperitoneally injected Castor oil, curcumin and WZ35 (0.2 mL, 25 mg/kg for each) for 15 days. The tumor size and body weight of nude mice were measured and recorded once every other day. The tumor volumes were determined by measuring length (L) and width (W) and calculating volume (V = 0.5 × L × W2) at the indicated time points. At the end of experiment, the animals were sacrificed and the tumors were harvested for use in proteins expression and histology studies. To investigate the effects of curcumin or WZ35 on lung metastasis in vivo, 4 × 105 of MDA-MB-231 cells in 100 ul of PBS were intravenously injected into the mice. After 24 days, 24 nude mice were randomly divided into four groups (normal saline, castor oil, curcumin, WZ35) and treated mice for 21 days. The lung metastases were evaluated by microscope after HE staining.
All experiments were conducted in triplicate (n = 3) and the data are presented as the mean ± SD. All statistical analyses were processed with GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Two-sided student’s t-test was performed to analyze the differences between two groups of data. P value< 0.05 means statistically significant.
WZ35 exhibits stronger anti-tumor activities than curcumin in the breast cancer cells
WZ35 suppresses MDA-MB-231 xenograft tumor growth and metastasis in vivo
YAP and JNK pathways are involved in WZ35 mediated breast cancer cell growth inhibition
WZ35 induced ROS generation is responsible for YAP and JNK activation
ROS-YAP-JNK pathway is involved in WZ35 induced mitochondrial dysfunction
YAP expression is down-regulated in breast cancer and correlated with the prognosis of certain breast cancer patients
Currently, the mainstay of systemic treatment for breast cancer is endocrine therapy. However, chemotherapy still is indispensable especially for patients they are lacking hormone receptors . Thus, discover novel drugs and therapeutic targets become a matter of great interest. WZ35, an analog of curcumin, has been demonstrated to exhibit superior anti-tumor effects in gastric cancer and hepatocellular carcinoma [13, 14]. However, anti-tumor effects of WZ35 in breast cancer and its underlying anti-tumor mechanisms are still unclear. Here, we demonstrated that WZ35 exhibits superior anti-tumor effects compare to the curcumin on breast cancer cells both in vitro and in vivo with no evident side effects. Further analysis showed that ROS mediated YAP and JNK activation were involved in WZ35 induced anti-tumor activities. In addition, we found that ROS-YAP-JNK pathway is implicated in mitochondria dysfunction in breast cancer cells (Fig. 6f).
Of note, curcumin has been reported to stimulate immune systems to exert anti-cancer activity [10, 40]. In our animal study, curcumin showed less in vitro anti-tumor activities, however, it is quite effective in vivo mice xenograft model suggesting possible immune stimulatory effect of curcumin. Although we used immune compromised mice to compare anti-tumor efficacy of WZ35 and curcumin, considering that NK cells and macrophages do still exist in these mice, WZ35 may have had some immune stimulatory activity which might contribute to its anti-tumor activities in vivo although further study needs to validate this proposal.
The Hippo pathway is associated with cell proliferation, tissue homeostasis and tumorigenesis [19, 41]. The components of Hippo pathway (Mst1/2, Lats1/2 etc.) have been reported to play tumor suppressive roles in cancers. YAP is a key downstream effector of Hippo pathway, which has been reported to act as either an oncogene or tumor suppressor in breast cancer . Moreover, YAP was reported to act as a tumor-suppressor in lung SCC via disruption of intracellular ROS homeostasis . In our study, we demonstrated that YAP exerts tumor suppressive role in breast cancer cells. WZ35 induced YAP activation was involved in inhibition of breast cancer cell growth and migration. We also found that YAP mRNA and protein levels were markedly down-regulated in breast cancer tissue specimens and reduced YAP expression was associated with poor prognosis of certain type of breast cancer patients. Accumulated ROS generation in cancer has been reported to inhibit cell proliferation , induce DNA damage , autophagy [46, 47], cellular injury , cell death [43, 46] and drug resistance [48, 49]. In addition, previous research demonstrated that elevated ROS production could activate JNK signaling pathway, resulting in cell apoptosis [50, 51]. Cancer cells with increased oxidative stress are likely to be more vulnerable to the damage. Therefore, elevated ROS production in cancer cells that do not cause significant toxicity to normal cells might be a potential therapeutic method. Our study showed that WZ35 induced ROS generation in breast cancer cells and NAC effectively attenuated the anti-tumor activities of WZ35. We also found for the first time that WZ35 mediated ROS generation is involved in YAP and JNK activation in breast cancer cells. Several studies have highlighted that activation of JNK signaling pathway induces mitochondria-dependent cell apoptosis via activating downstream signaling molecules of BCL-2 family proteins and caspase-3 [52, 53]. We demonstrated that YAP activation exerts anti-tumor activities via JNK phosphorylation and activation, and subsequent BCL-2 downregulation and cle-caspase3 upregulation.
Mitochondria plays critical role in the cells through regulation of energy metabolism, ATP generation, and calcium homeostasis [54, 55]. Accumulated evidences have indicated that mitochondrial metabolism is an attractive target for cancer therapy. Emerging studies have begun to demonstrate that mitochondria takes part in the activation of signaling pathways including the PI3K pathway, and activation of oncogenes such as MYC and KRAS, which result in promoting cell proliferation . The mitochondrial ATP is mainly generated by glycolysis and mitochondrial oxidative phosphorylation. It has been reported that both glycolytic and mitochondrial functions were decreased by either KRAS suppression or ERK inhibition . In this study, we further investigated relationship between ROS-YAP-JNK pathway activation and mitochondrial dysfunction, and found that WZ35 inhibits mitochondrial oxidative phosphorylation by activating ROS-YAP-JNK pathway. Activation of ROS-YAP-JNK pathway not only induced apoptosis, but also accelerated mitochondrial dysfunction by possibly inhibiting ATP generation in breast cancer cells resulting in inhibition of tumor growth. Thus, activating ROS-YAP-JNK pathway might increase the sensitivity of breast cancer cells to anticancer drug. Further study needs to prove this matter.
Altogether, our study shows that WZ35 exhibits stronger anti-tumor activities than curcumin by activating ROS-YAP-JNK signaling pathway. The activated ROS-YAP-JNK pathway involved in anti-tumor activities of breast cancer cells by inducing mitochondrial dysfunction and apoptosis. These results suggest that novel therapeutic strategies for breast cancer by targeting ROS-YAP-JNK pathway.
Our study shows that WZ35 exhibits stronger anti-tumor activities than curcumin by activating ROS-YAP-JNK signaling pathway. Our study revealed novel ROS-YAP-JNK pathway which involved in anti-tumor activities of breast cancer cells by inducing mitochondrial dysfunction and apoptosis. These results suggest that novel therapeutic strategies for breast cancer by targeting ROS-YAP-JNK pathway.
TC, RC and GL conceived the idea and designed the research. LW, CW, ZT, LZ, ZZ and WW performed in vitro experiments. LW, CW, ZT, LZ, YH, HC and BZ performed mice xenograft and metastasis experiments. LW, CW, XH and YY analyzed patient samples. TC, RC, GL, LW, ZT and LZ analyzed the data. RC, TC and CW wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by National Natural Science Foundation of China (81672305, 81622043, 81770926), The national key Basic Research Development Program of China (2016YFC1101200), The key Basic Research Development Program of Zhejiang Province (2018C03G2090634), The Natural Science Foundation of Zhejiang Province (LQ17H120009).
Ethics approval and consent to participate
All animal studies were performed with an approved protocol by the Institutional Animal Care and Use Committee of Wenzhou Medical University. Patient’s biopsy samples were obtained from the First Affiliated Hospital of Wenzhou Medical University, based on the Medical Research Ethics Committee and the Institutional Review Board of Wenzhou Medical University approved research protocol.
Consent for publication
The authors declare that they have no competing interests.
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