Andrographolide inhibits breast cancer through suppressing COX-2 expression and angiogenesis via inactivation of p300 signaling and VEGF pathway
- 68 Downloads
Andrographolide (Andro), a diterpenoid lactone, has been used for treatment of various cancers with less adverse effects. However, the underlying mechanisms regarding its anti-tumor mechanism still remain unclear.
Cell viability and proliferation were measured by CCK8 and CFSE dilution assay. The localization of p50/p65 or cytochrome c was determined using confocal immunofluorescence. Streptavidin-agarose pulldown or ChIP assays were used to detect the binding of multiple transactivators to COX-2 promoter. The promoter activity was examined by a dual-Luciferase reporter assay. The functions of Andro on COX-2-mediated angiogenesis were also investigated using human HUVEC cells through tube formation and spheroids sprouting assay. The in vivo anti-tumor efficacy of Andro was analyzed in xenografts nude mice.
The results indicated that Andro could significantly inhibit the proliferation of human breast cancers, and suppress COX-2 expression at both protein and mRNA levels. Furthermore, Andro could dose-dependently inhibit COX-2-mediated angiogenesis in human endothelial cells. We have also found that Andro significantly promoted the activation of cytochrome c and activated caspase-dependent apoptotic signaling pathway. Our further explorations demonstrated that Andro inhibited the binding of the transactivators CREB2, C-Fos and NF-κB and blocked the recruitment of coactivator p300 to COX-2 promoter. Moreover, Andro could effectively inhibit the activity of p300 histone acetyltransferase (HAT), thereby attenuating the p300-mediated acetylation of NF-κB. Besides, Andro could also dramatically inhibit the migration, invasion and tubulogenesis of HUVECs in vitro. In addition, Andro also exhibited effective anti-tumor efficacy as well as angiogenesis inhibition in vivo.
In current study, we explore the potential effects of Andro in suppressing breast cancer growth and tumor angiogenesis, as well as the precise mechanisms. This work demonstrated the potential anti-cancer effects of Andro, indicating that Andro could inhibit COX-2 expression through attenuating p300 HAT activity and suppress angiogenesis via VEGF pathway, and thereby could be developed as an antitumor agent for the treatment of breast cancer.
KeywordsAndrographolide COX-2/NF-κB Angiogenesis p300 Breast cancer
Carboxyfluorescein succinimidyl ester
- cyt c
Nuclear Factor κB
- p300 HAT
Histone acetyltransferase p300
Vascular endothelial growth Factor
Breast cancer is the most frequently diagnosed cancer in women, occupying nearly 15% in China  and 30% in U.S . Because of its great invasiveness and malignancy, over 508,000 women might die of breast cancer a year in the world . The primary treatment strategy for breast cancer is surgery and the following adjuvant chemotherapy. However, surgery could cause great damage on patients physically and psychologically , especially for young women, and the recurrence often occur in spite of adjuvant chemotherapy . Current clinical management of breast cancer patients is also a great challenge due to the difficulty of designing individualized treatments, and drug resistance and dose-limiting toxicities could also hamper the therapeutic effect and prognosis of patients. Thus, it is very urgent to develop some novel therapeutic strategies and more effective agents to improve patients’ quality of life with less side effects.
Recently, the potential relationship between cyclooxygenase (COX-2) and cancer has become the hotspot in the anti-cancer research, and it has been demonstrated that COX-2 could play an important role in tumor promotion, especially breast cancer [6, 7, 8]. High expression of COX-2 can promote the proliferation, angiogenesis, invasiveness, metastasis, and also the inhibition of apoptosis and immuno- surveillance [9, 10, 11]. COX-2 expression could be transcriptionally regulated by the recruitment of trans-activators and co-activators to the corresponding sites of its promoters [12, 13]. Therefore, inhibition of COX-2 expression might be an effective way for the treatment of human cancer.
Histone acetyltransferase p300, known as p300 HAT or p300, is an enzyme that is encoded by the EP300 gene in humans , and could be a transcriptional coactivator. P300 could be of great effect in regulating cell growth and division, and preventing the cancer growth. And p300 is usually expressed in relatively higher levels in cancer cells, compared with normal cells. P300 HAT activity regulates transcription of genes by binding to transcription factors , such as NF-κB, thereby enhancing transactivator binding . Besides, p300 HAT has been demonstrated to be very essential for COX-2 promoter activation, which could significantly enhance the binding of transactivators [16, 17], and its downregulation could abrogate the stimulatory effect of various pro-inflammatory mediators on COX-2 expression. Thus, to find more effective agents especially natural compound, suppressing p300 HAT activity is regarded as an useful approach to promote the curation of COX-2-mediated diseases.
In recent years, increasing amounts of studies have focused on the natural products that exist in fruit and vegetables, due to their beneficial roles for human health. Andrographolide (Andro), a natural diterpenoid lactone which is isolated and identified from Andrographis paniculata that was used as traditional herbal medicine in many Asian countries for thousands of years. Andro has been reported to be of various biological activities, such as anti-inflammatory properties [18, 19] and anti-cancer properties [20, 21]. Previous studies have shown that Andro inhibited NF-κB activation and DNA binding activity [22, 23]. These studies strongly supported that Andro could be used as an effective agent for treatment of chronic inflammation-related disease including cancer.
Therefore, in present work, we aim to investigate the effect of Andro on COX-2 suppression and angiogenesis in human breast cancer cells in vivo and in vitro, and to explore whether Andro could target p300 signaling pathway to regulate COX-2 expression. Our findings fully indicated that Andro could serve as a potential candidate targeting p300 signaling pathway to suppress NF-κB activation for treatment of COX-2- mediated breast cancer.
Reagents and antibodies
Andrographolide (Andro) was isolated from Andrographis paniculata by our laboratory with its purity of 98.7%. In present study, Andro was dissolved in dimethyl sulfoxide (DMSO) as a 100 mM stock solution and stored at − 20 °C. Andro was diluted to obtain the desired concentration in cell culture medium, where the final concentration of DMSO was less than 0.1%. Control cultures received the carrier solvent (0.1% DMSO). The primary antibodies for COX-2, p-Cofilin, F-actin, cleaved-caspase 3/9, NF-κB p65 and p-p65, and all the secondary antibodies were obtained from Cell Signaling Technology (Cell Signaling Technology, Inc., USA). The primary antibodies for GAPDH, p300, and NF-κB p50 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies for Bax, Bcl-2, CD31, β-actin and cytochrome c were obtained from Proteintech Group (Proteintech Group, Inc., USA). Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640 and fetal bovine serum (FBS), trypsin were obtained from HyClone Laboratories (HyClone Laboratories Inc.). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified.
Cell lines and cell culture
Human breast cancer cell lines MDA-MB-231, MCF-7, T47D, MDA-MB-361, and BT549 were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA). These cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) or 1640 medium supplemented with 10% bovine serum albumin (FBS), 100 μg/ml penicillin and 100 μg/ml streptomycin. Primary human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical vein as described . HUVECs were cultured in M199 containing 10% fetal bovine serum (FBS), 25 U/mL heparin, 5 ng/mL bFGF and 10 ng/mL EGF. The cells were cultured in a humidified atmosphere of 5% CO2 at 37 °C.
Cell viability assay
Cell viability was determined using the CCK-8 assay. In brief, 5 × 103 cells were seeded into 96-well culture plates allowed to adhere for overnight, and then the cells were changed to fresh medium containing various concentrations of Andro (5, 10, 25 and 50 μM) dissolved in DMSO (final concentration, less than 0.1%). After incubation for 48 h, CCK-8 was added, and the absorbance was measured at 450 nm by EnSpire® Multimode Plate Reade (Perkin Elmer, USA). Cell viability in vehicle control groups was considered 100%. Each assay was carried out at least in triplicate.
Colony formation assay
To analyze the effects of Andro on colony formation, single cells (1 × 103 per well) were seeded in six-well plate containing 2 ml growth medium with 10% FBS and cultured for 24 h. Then, removed the culture medium, and cells were treated with various concentrations of Andro. After 24 h, cells were washed with PBS and supplemented with fresh growth medium, and cells were routinely incubated for about 2 weeks until colonies were large enough to be visualized. Then colonies were stained with 0.1% crystal violet counted.
Flow cytometry analysis
To determine the proportion of apoptotic cells and CFSE dilution, we performed flow cytometry analysis using a flow cytometer (BD FACS Accuri C6, CA, USA). For the apoptosis examination, the cells were washed with PBS, and stained using the Annexin V-FITC Apoptosis Detection Kit in the dark at room temperature for 15 min. The cell cycle distribution and the fraction of apoptotic cells were determined using a FACS analysis system. For CFSE dilution assay, CFSE (carboxyfluorescein succinimidyl ester) dilution was usually used to detect cell proliferation. Briefly, cells were stained with 5 μM of CFSE at 37 °C for 15 min followed by adding 5-fold medium with FBS to terminate the reaction. CFSE-stained cells were cultivated with Andro for 48 h, and then the cell division was analyzed by flow cytometry. Each experiment was performed in triplicate.
Western blot analysis
Protein lysates were separated by electrophoresis in a 10% sodium dodecyl sulfate-polyacrylamide minigel (SDS-PAGE), electrophoretically transferred to a PVDF membrane, and immunoblotted with specific antibodies. The protein bands were detected by enhanced chemiluminescence. The protein concentrations were determined using a BCA protein assay kit (Beyotime Biotechnology, China). Similar experiments were carried out at least three times.
In briefly, the cells grown on chamber slides with indicated Andro treatment were fixed with 4% paraformaldehyde and permeabilized with 0.2% TritonX-100. The samples were probed with specific antibodies against Cytochrome c (cyt c), p50 or p65 (Santa Cruz) and fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies. Subsequently, the stained samples were stained with DAPI to counterstain cell nuclei. The samples were examined under a Leica DM14000B confocal microscope.
Streptavidin-agarose pulldown assay to detect DNA protein binding
Binding of NF-κB p65/p50 to COX-2 promoter probes was determined by a streptavidin-agarose pulldown assay. A 478-bp biotin-labeled double stranded probe corresponding to COX-2 promoter sequence (− 30 to − 508) was synthesized. Briefly, 400 ul mixture solutions containing nuclear extract proteins (400 μg) biotinylated DNA probe (4 μg), streptavidin-conjugated agarose beads (40 μl) and supplemented with PBSi (PBS buffer with 1 mM EDTA, 1 mM DTT and protease inhibitor cocktail complete) buffer at room temperature for 5 h on a rotating shaker. After washing with PBSi buffer, the beads were resuspended with the SDS-PAGE loading buffer and boiled at 100 °C. The supernatant was analyzed by Western blotting.
The chromatin immunoprecipitation (ChIP) assay was performed as previously described. In brief, after treatment with Andro for 24 h, MDA-MB-231 cells were fixed with 1% paraformaldehyde for 10 min, and scraped in lysis buffer (1% SDS, 10 mM Tris–HCl, pH 8.0, with 1 mM phenylmethylsulfonyl fluoride, pepstatin A, and aprotinin). P50-specific antibody or control IgG antibodies were added into samples overnight at 4 °C. The protein A/G plus agarose beads were added into samples for DNA enrichment. DNA segment enrichment of COX-2 promoter (Forward primer: ACGTGACTTCCTCGACCCTC, and Reverse primer: AAGACTGAAAACCAAGC CCA) was examined by PCR.
Dual-luciferase reporter assay
MDA-MB-231 cells in a 6-well plate, were co-transfected with pRL-TK plasmid (0.07 μg/well) and pGL3-NFκB plasmid (1.5 μg/well) or pGL3-basic plasmid (1.5 μg/well) (as negative control), which was encapsulated by Lipofectamine 2000 Reagent (3.75 μl/well) (Invitrogen, Grand Island, NY, USA). Meanwhile, cells were treated with Andro. After 48 h treatment, the dual-luciferase reporter assay system (Promega, Madison, WI) was used to measure the changes in luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity. All values are shown as mean ± SD of triplicate samples.
In vitro transfection assay
The MDA-MB-231 cells (1 × 106) were plated in 60 mm dishes 1 day before transfection. The relevant plasmids encoding Flag-p300 (3 μg/dish) was transfected with the Lipofectamine 2000 transfection reagent (7.5 μl/dish) (Invitrogen), following the manufacturer’s instructions. After transfection, the cells were cultured for another 24 h. Subsequently, the transfected cells were trypsinized and seed in 60 mm dishes, followed by culturing with fresh medium containing indicated doses of Andro for 48 h, and then collected for indicated experiments.
HAT activity assay
The HAT Activity was detected using the HAT Activity Colorimetric Assay Kit (Biovision,USA) following the manufacturer’s instructions. The nuclear extracts were prepared from the indicated cells treated with or without Andro.
In vitro migration assay
Scratch assay (wound healing assay) was performed to detect cell migration ability. The HUVECs were grown to full confluence in six-well plates. Cell monolayers were wounded with a sterile 200 μl pipette tip and then washed with PBS after 6 h-starvation. The cells were changed to fresh medium (5% FBS) containing indicated doses of Andro, with or without 50 ng/mL VEGF. After 48 h, medium was replaced with PBS, the wound gap was observed, and cells were photographed using a Leica DM14000B microscope fitted with digital camera and the distance of the wound gap was measured.
Transwell invasion assay
The motility of HUVECs was performed in 24-well transwell plates as previously described . Briefly, the upper surface of polycarbonate filters with 8 μm pores was coated with 75 μL Matrigel (Matrigel: M199 = 1:3, without growth factors) and incubated for 0.5 h at 37 °C for gelling. Then, 5 × 104 cells were seeded into the upper chambers and the bottom chamber were filled with 600 μL M199 with 10% FBS supplemented with VEGF (50 ng/ mL) or vehicle. Both top and bottom chamber contained the same concentrations of Andro. After 24 h incubation, non-invasive cells on the upper membrane surfaces were removed by wiping with cotton swabs. Invaded cells were fixed with methanol and stained with 0.1% Crystal Violet Staining Solution. The membrane was dried in the air. Images were taken using a Leica DM 14000B microscope. Cell invasion counted in five independent areas per membrane. The results were the means calculated from five replicates of each experiment.
Tube formation assay
HUVECs [1 × 104 in 50 μL M199 medium with 0.1% BSA containing Andro (0, 10, 20 μM), with or without 50 ng/mL VEGF] were seeded on Ibitreat angiogenesis slides (Ibidi, Martinsried, Germany) pre-coated with 10 μL Matrigel, and the formation of tubular like structure was recorded by a Leica DM14000B microscope at 6 h.
Spheroids sprouting assay
Spheroids were generated by gravity as described . Briefly, spheroids were embedded into a collagen gel, and then rapidly transferred into a 24-well plate and allowed to polymerize at 37 °C incubators for 30 min, M199 medium with or without VEGF and Andro was applied on top of the gel. After cultured for 24 h, spheroid sprouts were evaluated by measuring the cumulative length of all capillary like sprouts using a Leica DM14000B microscope. At least 5 randomly selected spheroids per experimental group and experiment were analyzed. And sprout length was measured with Image J software.
Female athymic nude mice (BALB/c nu/nu, 4 weeks old, 18–20 g) were purchased from the SPF Laboratory Animal Center of Dalian Medical University (Dalian, China), and maintained in pathogen-free conditions at animal facility. MDA-MB-231 cells (1 × 107 in 100 μL PBS) were injected subcutaneously near the axillary fossa of each nude mouse. Two weeks later, when the formed tumor reached about 30 mm3 after cell inoculation, the animals were divided randomly into three groups with 5 mice in each group, followed with Andro treatment (0, 5, 10 mg/kg) by intraperitoneal injection in 100 μl solution(PBS: Propylene Glycol: DMSO = 7:2:1, v/v)/per mouse, once every 2 days. Meanwhile, tumors were measured with a caliper once every 2 days, and the tumor volume was calculated using the formula V = 1/2 (width2 × length). Body weights were also recorded. After treatment for 15 days when the growth of tumor tissue in high dose (10 mg/kg) group has entered platform-phase, all experimental mice were sacrificed and the tumors from each mouse were excised and their weight was measured. To determine indicated proteins’ expression, the tumor tissues were fixed with 10% neutral formalin and embedded in paraffin. The sections (4 μm, from each tumor) were stained with COX-2 (1:100) antibody, and examined under a light microscope. To determine the microvessel density in tumor tissues, the sections (4 μm) were stained with CD31 (1:100) antibody, and examined under immunofluorescent microscope. The images were examined under a Leica DM 4000B fluorescence microscope equipped with a digital camera. The other parts of tumors were used to prepare tumor tissue lysates for Western blot analysis.
All animals were given free access to sterilized food and water and were habituated for a week before the experiments. All procedures were carried out in strict accordance with the recommendations established by Animal Care and Ethics Committee of Dalian Medical University as well as the guidelines by U.S. National Institutes of Health Guide for Care and Use of Laboratory Animals. The protocol was approved by the Animal Care and Ethics Committee of Dalian Medical University.
All experiments were repeated at least three times. Data are represented as mean ± standard deviation (SD). One way analyses of variance or two-tailed Student’s t-test were used to compare the values of the test and control samples in vitro and in vivo. P < 0.05 was considered to be a statistically significant difference. SPSS 17.0 software was used for all statistical analysis.
Andro suppressed the proliferation of human breast cancer cells
Andro promoted cell apoptosis by modulating cytochrome c and caspase signaling
Andro suppressed COX-2 expression through NF-κB signaling
Andro suppressed COX-2 promoter activation by inhibiting the binding of multiply trans-activators to COX-2 promoter
By acting on the promoter region, COX-2 expression has been reported to be regulated by several transcription factors and transcriptional coactivator, including NF-κB , CREB-2, C-Fos and p300 . To further determine whether Andro-mediated inhibition of COX-2 could be mediated by inhibiting the binding of functionally important transactivators to its promoter in human breast cancer cells, we next performed a streptavidin-agarose pulldown assay by using a 478-bp biotin-labeled double-stranded oligonucleotide probe containing COX-2 promoter sequence from − 30 to − 508. As shown in Fig. 4d and e, treatment of Andro dramatically inhibited the binding of these transactivators (CREB-2, C-Fos and NF-κB p50/p65) to COX-2 promoter region in a dose-dependent manner. However, Andro treatment could exert no effect on intracellular expression of all trans-activators (Fig. 4d and e). All these indicated that the inhibitory effect of Andro on breast cancer cells is partially mediated by inactivating the COX-2 signaling via suppressing the binding of muliple transactivators to the COX-2 promoter.
Andro attenuated the activity of p300 HAT and p300-mediated acetylation of NF-κB
Previous studies have shown that p300 could acetylate the NF-κB p50, thereby increasing NF-κB-mediated transactivation [16, 33]. To further confirm the inhibitory effect of Andro on p300 HAT activity in human breast cancer cells, we thus evaluated the effect of Andro on NF-κB acetylation in p300-tranfected MDA-MB-231 cells. As shown in Fig. 5d, Andro significantly inhibited the p300 HAT-mediated acetylation of NF-κB p50, resulting in a marked reduction in acetyl-p50 protein levels. However, treatment of Andro had no significant effect on the expression of intracellular p50. Furthermore, by using streptavdin-agarose pulldown (Fig. 5e) and ChIP assay (Fig. 5f), we have also evaluated the effect of Andro on the binding activity of NF-κB p50 to COX-2 promoter in p300-tranfected MDA-MB-231 cells. Besides, compared with C646 (p300 selective inhibitor) treatment, Andro exerted more prominent inhibitory effect on the acetylation and binding activity of NF-κB p50 to COX-2 promoter (Fig. 5d, e and f). Above all, these results indicated that p300 HAT could be an important mediator for transcriptional regulation of Andro. And Andro could significantly inhibit p300 HAT activity and attenuate the acetylation and binding of trans-activators and suppress the expression of COX-2.
Andro inhibited angiogenesis
Andro inhibited tumor growth and tumor angiogenesis in vivo
Andrographolide (Andro) has been reported to be of therapeutic effect for the treatment of various diseases [38, 39]. It has been reported that Andro possessed various biological activities, such as hepatoprotective , anti-HIV virus , antimalarial  antibacterial , anti-inflammatory properties [18, 19]. The anti-cancer effect of Andro attracts more and more attention in recent years [44, 45, 46], and previous studies have demonstrated that Andro exerted inhibitory effect on human breast tumor growth in vivo and in vitro [20, 21]. In addition, inhibiting HIF-1 and its upstream PI3K/AKT signaling, and inducing G1 arrest and cell apoptosis could all contribute to Andro-induced inhibition of the growth of breast tumor [21, 47]. However, whether there are other signals that involved in the inhibitory effect of Andro still remain unclear.
In China, Andro are prepared as tablets in clinic for anti-bacterial, anti-inflammatory, upper respiratory tract infection and bacterial dysentery. For adult, 2–3 tablets (0.1–0.15 g) were taken at one time, 3–4 times/day, which is about 6.43 mg/kg. The 12.33 fold of human dose equals to the dose of mice, which is a simple method for converting the dosage from human to mice . Converting to dose in mice is about 79.3 mg/kg in oral. And previous study showed that the oral bioavailability of Andro in mice was 9.27 ± 1.69% . Thus, with this conversion and calculation from the oral human dosage, the i.p. dosage should be 7.37 mg/kg. For the security, we set 5 and 10 mg/kg for the animal studies according to clinical use, which is for anti-bacterial, anti-inflammatory, upper respiratory tract infection and bacterial dysentery, and there were no obvious toxic effects were detected in mice treated with Andro.
In current study, we have evaluated the response of human breast cancer cells to Andro treatment. Our results indicated that Andro significantly suppressed breast cancer cells growth and induced cell apoptosis through down-regulating COX-2 expression, as well as the activation of COX-2 promoter. Besides, we also found that Andro effectively inhibited COX-2-mediated angiogenesis in human HUVEC cells through suppressing the survival, migration, tube formation and endothelial cell sprouting, which are key steps of angiogenesis. Further explorations showed that Andro-mediated suppression of COX-2 was mainly mediated via inhibiting p300 HAT activity, thereby abrogating the acetylation of trans-activators as well as their binding to COX-2 promoter region in human breast cancer cells. To our knowledge, we are the first to find that Andro could target p300 signaling pathway to regulate COX-2 expression and COX-2-related angiogenesis in human breast cancer cells.
Recently, the carcinogenic properties of chronic inflammation have been recognized as an emerging hallmark of various cancers. COX-2, an inducible form of the COX enzymes, had been reported to be increased in a variety of human cancers [10, 50, 51], including human breast cancer. In current study, we have revealed the precise molecular mechanisms by which Andro inhibited COX-2 expression and COX-2-mediated angiogenesis in human breast cancer. Unlike previous studies, our results indicated that Andro-mediated COX-2 suppression was specifically via targeting p300 signaling pathway. Our study demonstrated that Andro specifically inhibited p300 HAT activity, and abrogates the acetylation of NF-κB p50 and their binding to COX-2 promoter region in human breast cancer.
Furthermore, it is known that COX-2 expression could be mediated by its binding with multiple trans-activators to enhancer elements of its promoter, such as CREB-2, AP-1 (C-Fos) and NF-κB. Besides, p300, the transcriptional coactivator, could increase the transcriptional activity of the NF-κB through directly promoting the acetylation of NF-κB p50 to augment COX-2 transcription . In current study, we used a biotin-labeled DNA probe corresponding to COX-2 promoter region, to investigate the effects of Andro on the transcription activation of COX-2. Our results demonstrated that Andro could siginificantly suppress the binding of muliple trans-activators to the COX-2 promoter, including the CREB-2, C-Fos and NF-κB, thereby inhibiting the transcriptional activation of COX-2. Besides, the promoter activity was also inhibited by Andro in MCF-7 cells, which showed low-level expression of COX-2. That’s because although MCF-7 cells had low-level expression of COX-2, COX-2-related transcription factors, such as NF-κB p65/p50, C-Fos and CREB2, could still express in MCF-7 cells (Fig. 4e). When we transfected an exogenous luciferase expression vector containing COX-2 promoter, these factors could also bind to COX-2 promoter and promote its activity. It is similar for the streptavidin-agarose pulldown assay. In addition, the results also suggested that Andro could inhibit p300 recruitment to the COX-2 promoter.
In the present study, our results indicated that Andro-induced toxicity to breast cancer cells is associated with COX-2. However, we also found Andro-induced apoptosis in MCF-7. The responsible reason was that Andro suppressed COX-2 expression through inhibiting p300 HAT activity and attenuating the acetylation and binding of trans-activators. Although MCF-7 cells had low-level expression of COX-2, Andro could still suppress these targets, such as NF-κB. Studies have shown that NF-κB signaling was related to apoptosis. That is to say, high activity of NF-κB signaling could cause anti-apoptosis in cancer cells [52, 53, 54]. Therefore, suppression of NF-κB signaling was probably responsible for Andro-induced apoptosis in MCF-7, which could also inhibit COX-2 expression.
It is known that COX-2 is an important regulator of angiogenic milieu, and it stimulates tumor angiogenesis by elevating the expression of vascular endothelial growth factor (VEGF) [34, 55]. Inhibiting COX-2 could lead to angiogenesis suppression, cell growth suppression and apoptosis induction . Results from previous studies have revealed that COX-2 inhibitors could effectively suppress angiogenesis and tumor growth, prevent metastasis, and increase overall survival . In our study, for the first time, we demonstrated the effect of Andro on COX-2 expression and angiogenesis. These results suggested that Andro effectively inhibited the angiogenesis of human HUVECs, including the survival, migration and invasion, tube formation and endothelial cell sprouting.
In the present study, our results fully demonstrated that Andro could suppress the proliferation and angiogenesis of breast cancer cells through suppressing the p300/COX-2 and VEGF signaling pathway. Our findings have highlighted the potential inhibitory effects of Andro on human breast cancer and have also revealed its precise molecular mechanisms of anti-cancer. Above all, Andro could be used as an effective and safe agent for breast cancer therapy in near future.
We thank the NSFC (81622047, 81503201, 81473334 and 21572029), and the State Key Laboratory of Fine Chemicals (KF1603), Distinguished professor of Liaoning Province, Liaoning BaiQianWan Talents Program for financial support.
This work is supported by the National Natural Science Foundation of China (No. 81622047, 81503201, 81473334 and 21572029), and the State Key Laboratory of Fine Chemicals (KF1603), Distinguished professor of Liaoning Province, Liaoning BaiQianWan Talents Program.
XM, CL initiated the work, designed the experiments. YP, YW, and NT performed the experiments. YL, ZY, XZ and LF analyzed the data. ZY, DS, BZ, LJ and FY prepared the figures. YP, ZY, XM wrote the paper. All authors reviewed the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors agree for publication.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 5.Early Breast Cancer Trialists’ Collaborative G, Peto R, Davies C, Godwin J, Gray R, Pan HC, Clarke M, Cutter D, Darby S, McGale P, Taylor C, Wang YC, Bergh J, Di Leo A, Albain K, Swain S, et al. Comparisons between different polychemotherapy regimens for early breast cancer: meta-analyses of long-term outcome among 100,000 women in 123 randomised trials. Lancet. 2012;379(9814):432–44.CrossRefGoogle Scholar
- 10.Yu Z, Guo W, Ma X, Zhang B, Dong P, Huang L, Wang X, Wang C, Huo X, Yu W, Yi C, Xiao Y, Yang W, Qin Y, Yuan Y, Meng S, et al. Gamabufotalin, a bufadienolide compound from toad venom, suppresses COX-2 expression through targeting IKKbeta/NF-kappaB signaling pathway in lung cancer cells. Mol Cancer. 2014;13:203.CrossRefGoogle Scholar
- 12.Park E-J, Cheenpracha S, Chang LC, Kondratyuk TP, Pezzuto JM. Inhibition of lipopolysaccharide-induced Cyclooxygenase-2 and inducible nitric oxide synthase expression by 4-[(2′-O-acetyl-α-L-Rhamnosyloxy)benzyl]Isothiocyanate fromMoringa oleifera. Nutr Cancer. 2011;63(6):971–82.CrossRefGoogle Scholar
- 13.Asting AG, Carén H, Andersson M, Lönnroth C, Lagerstedt K, Lundholm K. COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status. BMC Cancer. 2011;11:238.Google Scholar
- 30.Moodie FM, Marwick JA, Anderson CS, Szulakowski P, Biswas SK, Bauter MR, Kilty I, Rahman I. Oxidative stress and cigarette smoke alter chromatin remodeling but differentially regulate NF-kappaB activation and proinflammatory cytokine release in alveolar epithelial cells. FASEB J. 2004;18(15):1897–9.CrossRefGoogle Scholar
- 34.Mohankumar K, Sridharan S, Pajaniradje S, Singh VK, Ronsard L, Banerjea AC, Somasundaram DB, Coumar MS, Periyasamy L, Rajagopalan R. BDMC-A, an analog of curcumin, inhibits markers of invasion, angiogenesis, and metastasis in breast cancer cells via NF-kappaB pathway--a comparative study with curcumin. Biomed Pharmacother. 2015;74:178–86.CrossRefGoogle Scholar
- 48.Huang JH, Huang XH, Chen ZY, Zheng QS, Sun RY. Dose conversion among different animals and healthy volunteers in pharmacological study. Chin J Clin Phannacol Ther. 2004;9:1069.Google Scholar
- 51.Yang WT, Lewis MT, Hess K, Wong H, Tsimelzon A, Karadag N, Cairo M, Wei C, Meric-Bernstam F, Brown P, Arun B, Hortobagyi GN, Sahin A, Chang JC. Decreased TGFbeta signaling and increased COX2 expression in high risk women with increased mammographic breast density. Breast Cancer Res Treat. 2010;119(2):305–14.CrossRefGoogle Scholar
- 53.Rashidi M, Bandala-Sanchez E, Lawlor KE, Zhang Y, Neale AM, Vijayaraj SL, O'Donoghue R, Wentworth JM, Adams TE, Vince JE, Harrison LC. CD52 inhibits toll-like receptor activation of NF-kappaB and triggers apoptosis to suppress inflammation. Cell Death Differ. 2018;25(2):392–405.CrossRefGoogle Scholar
- 56.Xu L, Stevens J, Hilton MB, Seaman S, Conrads TP, Veenstra TD, Logsdon D, Morris H, Swing DA, Patel NL, Kalen J, Haines DC, Zudaire E, St Croix B. COX-2 inhibition potentiates antiangiogenic cancer therapy and prevents metastasis in preclinical models. Sci Transl Med. 2014;6(242):242ra284.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.