Caffeic acid attenuates the angiogenic function of hepatocellular carcinoma cells via reduction in JNK-1-mediated HIF-1α stabilization in hypoxia

Abstract

Hepatocellular carcinoma (HCC) is the third leading cause of tumor-related mortality worldwide. Angiogenesis plays a crucial role in HCC progression. Caffeic acid (CaA) is a novel anti-tumor agent, however, the functions of CaA in the regulation of angiogenesis in HCC, and the molecular mechanisms involved, remain largely uninvestigated. Here, we found that, in the presence of CoCl₂ (a hypoxia mimic), CaA attenuates the angiogenic function of HCC cells via reduction in JNK-1-mediated HIF-1α stabilization. Briefly, CaA attenuated the CoCl₂-induced autocrine vascular endothelial growth factor (VEGF) and angiogenesis in HCC cells. For the molecular mechanisms, CoCl₂ treatment increased the expressions of HIF-1α and phosphorylated signal transducers and activators of transcription-3 (p-STAT-3). Then, by directly binding to the promoter of the VEGF gene, HIF-1α effectively activated VEGF. However, CaA attenuated the CoCl₂-induced activation of HIF-1α likely by reducing JNK1 activation and reducing HIF-1α stabilization. Moreover, CaA decreased the CoCl₂-induced increased expression of p-STAT-3. These two functions resulted in an attenuated recruiting of the HIF-1α to the VEGF promoter. By understanding a novel mechanism whereby CaA inhibits the angiogenesis in HCC, our study expands the understanding of the molecular mechanisms involved in the anti-cancer potential induced by CaA.

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1. Introduction

Hepatocellular carcinoma (HCC), a highly vascularized tumor, is the most common liver malignancy worldwide.¹ In HCC, tumor cells recruit new blood vessels for their growth, maintenance, and metastasis; in fact, angiogenesis plays a crucial role in HCC progression.^1–3 For the past few years, the strategy of anti-angiogenesis for HCC treatments had been affirmed.⁴ However, the therapeutic effect of standard anti-angiogenesis treatments, sorafenib, is less than satisfactory because of toxic/side-effects and hypoxia-mediated sorafenib resistance.⁵ So the continued searches for novel agents, which exhibit less cytotoxic and have repressive effects on the angiogenesis in HCC are urgently needed.

Naturally occurring hydroxycinnamic acid derivatives are reported to have several biological and pharmacological properties.^6,7 Their natural origin and ubiquitous occurrence have prompted strong interest in the use of them as anticancer agents. Caffeic acid (3,4-dihydroxycinnamic acid, CaA), a naturally occurring hydroxycinnamic acid derivatives, is an active component in the phenolic propolis extract and also in a wide variety of plants.⁸ Studies indicate that CaA and its derivative, caffeic acid phenethyl ester, exert impactful anticancer effects. For example, CaA can decrease the viability of HCC cells;⁹ as well, it has also been identified as a nuclear factor κB (NF-κB) inhibitor, which in turn blocks the activity of matrix metalloproteinase-9 (MMP-9), leading to the attenuations of tumor growth and metastasis.¹⁰ Further, CaA and its derivative, caffeic acid phenethyl ester, suppress the angiogenic ability of human renal carcinoma cells by blocking the secretion of vascular endothelial growth factor (VEGF).¹¹ However, the effects of CaA on the angiogenesis in HCC, and the molecular mechanisms underlying in remain largely unclear.

Up to date, two major mechanisms regulating the angiogenesis in human cancers have been identified, that are the hypoxia-regulated signaling and the NF-κB signaling, both of which synergize in the regulation of VEGF expression.^12,13 On one hand, as a classical angiogenesis inducer, hypoxia-inducible factor-1 alpha (HIF-1α) stimulates the tumor cells growth via transcriptional up-regulation of VEGF;¹³ on the other hand, NF-κB elevates the VEGF expression by IL-6/STAT-3 cascade.¹² In fact, NF-κB, IL-6, and STAT-3 constitute a positive feedback loop.¹⁴ Our previous study found that, by attenuating the autocrine IL-6, CaA blocked the NF-κB–IL-6–STAT-3 feedback loop in HCC cells.¹⁴ Here, to provide a better understanding of CaA-caused anti-angiogenesis in HCC, to investigate the relationship between CaA and HIF-1α, and to further indicate the potential molecular mechanisms, we treated HCC cells in both in vitro and in vivo models to examine the early molecular changes.

2. Experimental

2.1. Ethics statement

According to a management of experimental animals promulgated by Jiangsu province, China, this study was reviewed and approved by Nanjing Medical University Institutional Animal Care and Use Committee (the permit number: NJMU-IACUC-1403024), and animals were treated humanely and with regard for alleviation of suffering.

2.2. Cell culture and reagents

The HCC cell line HepG2, and human umbilical vein endothelial cell line (HUVECs), were obtained from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The HCC cell line, MHCC97H was obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University (Shanghai, China). These cells were identified by China Center for Type Culture Collection (Wuhan, China). Cells were maintained in a 37 °C humidified incubator with 5% CO₂. HepG2 and MHCC97H cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies/Gibco, Grand Island, NY), while HUVECs were cultured in ECM medium (Invitrogen, Carlsbad, USA). The mediums were supplemented with 10% fetal bovine serum, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin (Gibco), 100 μg ml⁻¹ heparin, and 30 μg ml⁻¹ endothelial cell growth supplement (for HUVECs, Sigma-Aldrich, MO, USA). To mimic hypoxia in HCC cells, CoCl₂ (purity ≥ 99.5%, Sigma Chemical Co, St. Louis, MO, USA) was added at a final concentration of 100 μM, and this dose of cobalt has been previously used to induce hypoxia with non-toxic effects.¹⁵ Phenol red was added into the medium to reflect the pH. A mycoplasma stain assay Kit (Beyotime Co. Ltd, Haimeng, China) was used for mycoplasma testing. The caffeic acid (CaA, purity ≥ 99.5%) was purchased from Sigma Chemical Co, and was re-suspended in dimethyl sulfoxide (Sigma Chemical Co.) at a stock concentration of 180 mg ml⁻¹, and stored at −20 °C. The proteasome inhibitor, MG132, was purchased from Calbiochem Co. Ltd (Darmstadt, Germany).

2.3. Animals, xenografts, and immunohistochemistry

The BALB/c nude mice were obtained from SLRC laboratory animal center (Shanghai, China), and kept in a temperature-controlled environment (20–22 °C) with a 12 hour light dark cycle and with free access to drinking water and chow. For xenograft studies, 5 × 10⁶ MHCC97H cells were injected subcutaneously into the right armpit of the mice (6 mice per group). Three weeks later (after the establishment of the tumors), CaA (0 or 10 mg kg⁻¹ BW) was administered intraperitoneally (i.p) twice per week as we described previously.¹⁶ Tumor volumes were measured weekly and tumor size was calculated using the formula: V = 1/2(width² × length). After 11 week, the mice were sacrificed, and tumor tissues were removed for further investigation.

For immunohistochemistry (IHC), sections mounted on silanized slides were dewaxed in xylene; dehydrated in ethanol; boiled in 0.01 M citrate buffer, pH 6.0, for 20 min in a microwave oven; and then incubated with 3% hydrogen peroxide for 5 min. After washing with PBS, sections were incubated in 10% normal bovine serum albumin for 5 min, followed by incubation with a rabbit-anti-HIF-1α (Novus, Littleton, CO, USA), rabbit-anti-STAT-3 (Cell Signaling Technology, Beverly, MA, USA), rabbit-anti-VEGF, or rabbit-anti-CD31 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody, respectively, at 4 °C overnight. The dilution used was 1 : 50. Then the slides were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (Beyotime Co. Ltd, the dilution was 1 : 300) at room temperature for 30 min. The staining was visualized using diaminobenzadine, and the sections were counterstained with hematoxylin, dehydrated, cleared, mounted, and photographed under a Pannoramic-scan digital slice scanning system (3DHISTECH Co. Ltd, Budapest, Hungary). The quantitation of immunostaining was performed by two independent researchers who were blinded regarding experimental details. The immunostaining score of HIF-1α, STAT-3, or VEGF in tumors was semi-quantified by Q-score based on intensity and heterogeneity.^17,18 The positive rates were scored as 0 point (0%), 1 point (1–25%), 2 points (26–50%), 3 points (51–75%), and 4 points (76–100%). The score of the staining intensity was presented as 0 point (none), 1 point (low), 2 points (medium), and 3 points (high). The Q-score was the sum of heterogeneity and intensity. For the angiogenesis quantification, microvessel density (MVD) was evaluated by counting CD31-positive immunostained cells as described previously.¹⁹ The average count of five vision fields was recorded as the final MVD.

2.4. Enzyme-linked immunosorbent assay (ELISA)

A total of 1 × 10⁵ HepG2 or MHCC97H cells were seeded in 6-well plates for 24 h. After then, such cells were serum starved for 8 h and treated by DMEM medium supplemented with 2% FBS and 100 μM CoCl₂, in the presence or absence of 0 or 20 μM CaA for 24 h, respectively. Then the conditioned mediums were collected, cleared by centrifugation, and stored at −80 °C. To analyze VEGF secretion, we performed ELISA using the human VEGF Quantikine kit (R&D Systems, MN, USA). Briefly, 0.5 μg ml⁻¹ VEGF antibody was added to 96-well polyvinyl microplates (R&D Systems) at 4 °C overnight. Samples (50 μl) or standard protein (Recombinant human VEGF, R&D Systems) were added to the wells. After incubation for 1 h at 37 °C, the plates were washed with phosphate buffer saline (PBS) for 3 times, and then, 50 ng ml⁻¹ biotinylated VEGF antibody (R&D Systems) was added for 1 h at 37 °C. The plates were then washed with PBS for 3 times, and incubated with streptavidin-HRP for 1 h at 37 °C. After washing, 0.2 mM 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonicacid)-diammonium salt (ABTS, Sigma) was added to the wells, and after 10 min, the colorimetric reaction was measured at 450 nm with a multi-well plate reader (Model 680, Bio-Rad, USA).

2.5. Tube formation assay

For the tube formation assay, the HUVECs cells were trypsinized and seeded at 2 × 10⁴ cells per well in a 96-well plate on matrigel (BD Bioscience, San Jose, CA, USA) that had polymerized for 30 min at 37 °C. After then, such cells were incubated in the conditioned mediums as described above for 6 h, respectively. Capillary morphogenesis was evaluated by using a phase-contrast microscope (Olympus, Tokyo, Japan). Quantification of tube formation was assisted by S.CORE, a web-based image analysis system (S.CO BioLifescience, Munich, Germany) as described previously.¹⁵ Briefly, tube formation indices represent the degree of tube formation. The indices were calculated using the following formula: (mean single tube index)² × (1−confluent area) × (number of branching points/total length skeleton). The values of the variables used in the equation were obtained automatically by S.CORE.

2.6. Quantitative real-time polymerase chain reaction (qRT-PCR)

HepG2 or MHCC97H ells were treated as the same as ELISA for 8, 16, or 24 h, respectively. Then, after be washed with ice-cold PBS for 3 times, total RNA was isolated using Trizol (Invitrogen, Carlsbad, USA) according to the manufacturer's recommendations. For the detection of HIF-1α and VEGF mRNAs, total RNA (2 μg) was transcribed into cDNA using AMV Reverse Transcriptase (Promega, Madison, USA). The qRT-PCR was performed using the Applied Biosystems 7300HT machine and MaximaTM SYBR Green/ROX qPCR Master Mix (Fermentas, Waltham, MA, USA). Primers used were listed in ESI Table S1.† The amplification conditions used were: 95 °C for 30 s; 40 cycles of 95 °C for 5 s; 60 °C for 30 s; and melting at 95 °C for 5 s, followed by 60 °C for 1 min, and cooling at 50 °C for 30 s. The PCR reaction was evaluated using melting curve analysis. The Tubulin was amplified to ensure cDNA integrity and to normalize expression. Fold changes in expression of each gene were calculated by a comparative threshold cycle (C_t) method using the formula 2^−(ΔΔC_t) as described previously.¹⁴

2.7. Western blots assay

HepG2 or MHCC97H ells were treated as described above for 8, 16, or 24 h, respectively. Then, total protein was extracted by lysing cells in RIPA buffer (Beyotime Co. Ltd) for 30 min on ice. Then the protein concentrations were measured with the BCA kit (Beyotime Co. Ltd). Afterwards, proteins (20 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by transferring to polyvinylidene fluoride membranes (Millipore, Billerica, USA). After blocking with 10% non-fat milk in TBST for 1 h the room temperature, membranes were incubated with the primary antibody (the dilution was 1 : 1000), including rabbit-anti-VEGF and mouse-anti-JNK1 or phosphorylated-JNK1 (Santa Cruz Biotechnology), rabbit-anti-HIF–1α (Novus), rabbit-anti-phosphorylated-STAT-3 (p-STAT-3, Cell Signaling Technology), or mouse-anti-Tubulin (Beyotime Co. Ltd), at 4 °C overnight. Then, the membranes were washed 5 min with TBST (Tris–HCl + NaCl + Tween20, Beyotime Co. Ltd) for 3 times, followed by incubating with horseradish peroxidase-conjugated secondary antibodies (Beyotime Co. Ltd, the dilution was 1 : 2000) for 1 h at the room temperature. After being washed 10 min with TBST for 5 times, the immune complexes were detected by using an enhanced chemiluminescence kit (Cell Signaling Technology).

2.8. Co-immunoprecipitation assay (Co-IP)

A total of 1 × 10⁶ HepG2 or MHCC97H cells were seeded in 6 cm-diameter plates for 24 h. After then, such cells were serum starved for 8 h and treated by DMEM medium supplemented with 2% FBS and 100 μM CoCl₂. Simultaneously, the plates were incubated with proteasome inhibitor, MG132 (0 or 10 μM), CaA (0 or 20 μM), or a combination of these two reagents for 24 h, respectively. The extraction and quantitation of total protein were the same as described in western blots. Then, a total of 500 μg total protein was subjected to immunoprecipitation with a rabbit-anti-HIF-1α anti-body (Novus, the dilution was 1 : 200) at 4 °C overnight. Subsequently, a Protein G Sepharose (Beyotime Co. Ltd) was added and incubated continued overnight at 4 °C. After centrifugation (5000 round per min × 3 min) at 4 °C, the precipitates were washed three times with ice-cold RIPA buffer, resuspended in RIPA buffer containing sodium dodecyl sulfate sample buffer (20% of the total volume), boiled for 10 min to remove proteins from the beads, and analyzed by western blots with antibodies for ubiquitin (Novus) and p-STAT-3 (Cell Signaling Technology), both the dilution used was 1 : 1000. Meanwhile, the total protein was directly subjected to western blots assay as an “input control”.

2.9. Chromatin-immunoprecipitation (ChIP) assay

HepG2 or MHCC97H ells were treated as described above for 24 h. Then, to crosslink proteins to DNA, such cells were treated with 1% formaldehyde in PBS by gentle agitation at room temperature for 10 min. Next, cells were washed, resuspended in lysis buffer, and sonicated on ice for 15 s × 10 times at a 25% output in an EpiShear probe sonicator (Active Motif, Carlsbad, CA, USA). For the immunoprecipitation of protein-DNA complexes, 5 μl of specific magnetic Dynal bead (Invitrogen)-coupled antibody against HIF-1α or an isotype IgG (an negative control) was added at 4 °C overnight, followed by a centrifugation (3000 rpm × 1 min). Then, the immunoprecipitated DNA and input DNA (total DNA) were cleaned by RNase A (0.2 mg ml⁻¹, Beyotime Co. Ltd) and proteinase K (2 mg ml⁻¹, Beyotime Co. Ltd) before phenol/chloroform-purification. The specific sequences from immunoprecipitated and input DNA were determined by PCR analysis. Amplifications of the VEGF gene sequences among the pull of DNA were performed with specific primers flanking from −1041 to −750 in the VEGF promoter upstream regions (ESI Table S1†). The PCR conditions for were 94 °C for 1 min; 35 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for45 s; followed by 72 °C for 10 min, and cooling at 4 °C. The PCR products were then analysed by an agarose gel electrophoresis.

2.10. Cell transfection

Commercial specific JNK1-siRNA was purchased from Santa Cruz Biotechnology (http://datasheets.scbt.com/sc-29380.pdf). Cells were transiently transfected using the Lipofectamine 2000 reagent (Invitrogen). Briefly, cells were seeded in 6-well plates at a density of 1 × 10⁵ per well for 24 h, then they were transfected with 20 nM siRNA for 12 h. After transfection, such cells were cultured in fresh medium supplemented with 10% FBS for another 24 h before being used for other experiments.

2.11. Statistical analysis

Data sets were compared using GraphPad-6.0 (GraphPad Software, Inc, La Jolla, CA, USA). Data were presented as the means ± SD. The difference between two groups was analyzed using a two-tailed Student's t test. For the repeated measures data, a one- or two-way ANOVA followed by a Sidak's multiple comparisons test was used. The p values < 0.05 were considered statistically significant.

3. Results

3.1. CaA attenuated the hypoxia-mediated autocrine VEGF in HCC cells

The VEGF plays a critical role in promoting the formation of blood vessels, by inducing proliferation, migration, network formation, and branching of endothelial cells.¹³ Studies indicate that, VEGF production is regulated by two major mechanisms, hypoxia (low oxygen supply) and various cytokines/signals, such as IL-6/STAT-3.^12,15 We previously found that, a concentration of 20 μM CaA blocked the autocrine IL-6 effectively, and had no significant cytotoxicity in HepG2 and MHCC97H cells.¹⁴ Here, these cells were treated with 100 μM CoCl₂ (this dose of cobalt has been previously used to induce hypoxia in HCC cells with non-toxic effects¹⁵), in the presence of absence of 20 μM CaA for 8, 16, or 24 h, respectively. As expectant, hypoxia induced a time-dependent increased levels of VEGF mRNA; however co-treated with CaA attenuated this phenomenon, which reached a maximum inhibition at 24 h (Fig. 1A). So, the 24 h time point was chosen for further experiments. As shown in Fig. 1B and C, CaA also significantly decreased the hypoxia induced increased expression/secretion of VEGF protein. These results suggested that CaA blocked the hypoxia induced autocrine VEGF by the transcriptional inhibition of endogenous VEGF in HCC cells.

Fig. 1 CaA attenuated the hypoxia-mediated autocrine VEGF in HCC cells (a) HepG2 or MHCC97h cells were treated with 100 μm CoCl₂ in the presence of absence of 20 μm CaA for 8, 16, or 24 h, respectively, qRT-PCR analyses in triplicate of VEGF mRNA expression, a two-way ANOVA followed by a Sidak's multiple comparisons test was used for statistical analysis. (b and c) HepG2 or MHCC97h cells were treated 100 μm CoCl₂ in the presence of absence of 20 μm CaA for 24 h, respectively, and then the conditioned mediums were collected. (b) Western blots analyses of the VEGF protein expression. (c) ELISA analyses in triplicate of VEGF protein secretion, a one-way ANOVA followed by a Sidak's multiple comparisons test was used for statistical analysis.

3.2. CaA attenuated the hypoxia-mediated angiogenesis in HCC cells

To further confirm the CaA anti-angiogenic activity suggested by the induced the decrease of VEGF levels under hypoxia, the tube formation assay was performed. Compared with conditioned medium collected from HepG2 or MHCC97H cells treated by CoCl₂ alone, the conditioned medium collected from HCC cells treated by CoCl₂ plus CaA could not induced HUVECs to display their typical morphology and phenotype of endothelial cells, and the degree of tube formation (calculated by tube formation index, S.CORE) was attenuated (Fig. 2A and B). Then we determined if the CaA-induced anti-angiogenic activity was VEGF dependent. As shown in the ESI Fig. S1A and B,† we used 10 ng ml⁻¹ VEGF neutralization antibody (Abcam Co, Ltd, Cambridge, UK) to attenuate the autocrine of VEGF in the medium, and found that no significant difference of tube formation between CaA-treated HCC conditioned medium and untreated HCC-conditioned medium, which were all in the presence of the VEGF neutralization antibody. Further, in order to avoid the addition of CaA and/or CoCl₂ to the endothelial cells in tube formation assay, these two chemicals in the conditioned mediums should be washed out. Nevertheless, the removal of CaA and/or CoCl₂ after the collection of conditioned mediums is technologically formidable. So in our present study, we have performed two independent experiments to avoid the addition of CaA or CoCl₂ to the endothelial cells in tube formation assay as we described previously.¹² As shown in the ESI Fig. S2A and S2B,† no detectable change of tube formation was observed after HUVECs were directly exposed to CaA or CoCl₂ alone. Collectively, based on these results (Fig. 1 and 2, S1, and S2†), we suggested that, in HCC cells, CaA could attenuate the hypoxia-induced VEGF-dependent angiogenesis, and that the transcriptional inhibition of endogenous VEGF gene, which in turn decreased the autocrine of VEGF might be involved.

Fig. 2 CaA attenuated the hypoxia-mediated angiogenesis in HCC cells HUVECs were exposed to the conditioned mediums as described above in Fig. 1c for 6 h, and the formations of tube were detected. Bars = 250 μm. Quantification of tube formation was assisted by S.CORE a two-tailed Student's t test was used for statistical analysis.

3.3. CaA increased HIF-1α ubiquitination under hypoxia

We then investigated the effects of CaA on HIF-1α expression under hypoxia in HCC cells. As shown in Fig. 3A and B, CoCl₂ treatment induced a time-dependent increased levels of HIF-1α mRNA and protein. Interestingly, co-treated with CaA attenuated the CoCl₂-induced increased expression of HIF-1α protein, however, there was no significant effects of CaA on HIF-1α mRNA expression under CoCl₂ treatment. These results indicated that the inhibition of HIF-1α accumulation upon CaA exposure possibly mainly occurred at the post-transcriptional level. Therefore, the potential function of CaA in the modification of HIF-1α protein was considered.

Fig. 3 CaA decreased the HIF-1α via an ubiquitin-dependent degradation (a and b) HepG2 or MHCC97h cells were treated as described in Fig. 1a, (a) qRT-PCRanalyses in triplicate of HIF-1α mRNA expression, a two-way ANOVA followed by a Sidak's multiple comparisons test was used for statistical analysis; (b) western blots analyses of the HIF-1α protein expression. (c and d) HepG2 or MHCC97h cells were treated by CoCl₂ (0 or 100 μm), MG132 (a proteasome inhibitor, 0 or 10 μm), and CaA (0 or 20 μm) for 24 h, respectively. HIF-1α was immunoprecipitated with its specific antibody. Western blots and densitometry analyses in triplicate of the expressions of HIF-1α and/or ubiquitin in immunoprecipitated proteins or in input control. Note: in (d), compared with Con group, the ratio of HIF-1α's ubiquitination in CoCl₂-treatment group was 37.8 ± 6.2 (%), while the ratio of HIF-1α's ubiquitination in (CoCl₂ plus CaA)-treatment group was 71.4 ± 8.1 (%). Then we compared these two ratios of HIF-1α's ubiquitination via a two-tailed Student's t test (p = 0.00029).

Ubiquitination is the common mechanism leading to proteasome-dependent degradation.²⁰ We next determined whether CaA inhibited HIF-1α accumulation by enhancing its ubiquitination. HepG2 or MHCC97H cells were treated by CoCl₂ (0 or 100 μM), MG132 (a proteasome inhibitor, 0 or 10 μM), and CaA (0 or 20 μM) for 24 h, respectively. HIF-1α was immunoprecipitated with its specific antibody, and its ubiquitination-binding status was analyzed with ubiquitin antibody. Here, compared with respective Con group, the ubiquitination of HIF-1α was decreased in CoCl₂-treated group; however, CaA-treatment increased the extents of HIF-1α ubiquitination compared with CoCl₂-treated group (Fig. 3C and D). These results suggested that CaA increased HIF-1α ubiquitination in HCC cells under CoCl₂-induced hypoxia.

3.4. CaA inhibited HIF-1α stabilization by reducing JNK1 activation

Previous studies indicate that knockdown of JNK1 causes the ubiquitin-dependent degradation of HIF-1α,²¹ and that CaA is a regulator of mitogen-activated protein kinases (MAPKs).^8,22 So we hypothesized that the JNK1 might be involved in the CaA-induced degradation of HIF-1α. To confirm this hypothesis, we used RNA interference to knockdown of JNK1. Here, Con-siRNA- or JNK1-siRNA-transfected HepG2 cells were treated by CoCl₂ (0 or 100 μM) and CaA (0 or 20 μM) for 24 h, respectively. As shown in Fig. 4A, under the treatment of CaA, compared with respective Con group, the inhibition ratio of the HIF-1α expression in si-Con group was 68.5 ± 13.6 (%), while the inhibition ratio of HIF-1α expression in si-JNK1 group was 10.7 ± 4.6 (%). Then we exposed Con-siRNA- or JNK1-siRNA-transfected MHCC97H cells to CoCl₂ (0 or 100 μM), MG132 (0 or 10 μM), and CaA (0 or 20 μM) for 24 h, respectively. As shown in Fig. 4B, under the treatment of CaA, compared with respective Con group, the fold of HIF-1α's ubiquitination in si-Con group was 4.58 ± 0.41, while the fold of HIF-1α's ubiquitination in si-JNK1 group was 1.47 ± 0.34. Collectively, these data suggested that CaA increased HIF-1α ubiquitination, and given the abrogation of this effect upon knockout of JNK-1, indicating that CaA inhibited HIF-1α stabilization by reducing JNK1 activation.

Fig. 4 JNK1 was involved in the CaA-induced degradation of HIF-1α. (a) si-Con- or si-JNK1-transfected HepG2 cells were treated with 100 μm CoCl₂ in the presence of absence of 20 μm CaA for 24 h, respectively. Western blot analyses of the expressions of HIF-1α, p-JNK1, and JNK1. (b) si-Con- or si-JNK1-transfected HepG2 cells were treated with 100 μm CoCl₂, 10 μm of MG132, 0 or 20 μm CaA for 24 h, respectively. HIF-1α was immunoprecipitated with its specific antibody. Western blots analyses of the expressions of ubiquitin in immunoprecipitated proteins. Note: in (a), under the treatment of CaA, compared with respective Con group, the inhibition ratio of HIF-1α in si-Con group was 68.5 ± 13.6 (%), while the inhibition ratio of HIF-1α in si-JNK1 group was 10.7 ± 4.6 (%). Then we compared these two inhibition ratios via a two-tailed Student's t test. Similarly, in (b), under the treatment of CaA, compared with respective Con group, the fold of HIF-1α's ubiquitination in si-Con group was 4.58 ± 0.41, while the fold of HIF-1α's ubiquitination in si-JNK1 group was 1.47 ± 0.34. Then we compared these two extents of HIF-1α's ubiquitination (fold) via a two-tailed Student's t test.

3.5. CaA attenuated the accumulation of HIF-1α to VEGF promoter

As well as HIF-1α, STAT3 also behaves as an angiogenesis inductor involved in VEGF expression.¹¹ Activated via phosphorylation, it enhances HIF-1α stability and acts as a co-activator; actually, hypoxia increases p-STAT3 and HIF-1α recruitment as a transcriptional complex within the VEGF promoter.¹⁵ We previously found that, via an epigenetic silencing manner, CaA blocked the STAT-3 signaling in HCC cells.¹⁴ Here, HepG2 or MHCC97H cells were treated with 100 μM CoCl₂ in the presence of absence of 20 μM CaA for 24 h, respectively. Then, HIF-1α was immunoprecipitated with its specific antibody, and its p-STAT-3-binding status was analyzed with p-STAT-3 antibody. As shown in Fig. 5A, CoCl₂ treatment resulted in elevated expressions of HIF-1α and p-STAT-3, in addition, Co-IP data suggest that HIF-1α formed a complex with p-STAT-3. However, CaA attenuated the CoCl₂-induced increased expressions of HIF-1α and p-STAT-3. Furthermore, compared with medium control cells, CoCl₂-treatment resulted in the enhanced accumulation of HIF-1α to VEGF promoter, however, this effect was also suppressed by CaA treatment (Fig. 5B). Collectively, these results suggested that, via decreasing the expressions of HIF-1α and p-STAT-3, CaA attenuated the accumulation of HIF-1α and p-STAT-3 (possibly) to VEGF promoter in HCC cells under hypoxia.

Fig. 5 CaA attenuated the accumulation of HIF-1α to VEGF promoter HepG2 or MHCC97h cells were treated 100 μm CoCl₂ in the presence of absence of 20 μm CaA for 24 h, respectively. (a) HIF-1α was immunoprecipitated with its specific antibody. Western blots analyses of the expressions of HIF-1α and p-STAT-3 in immunoprecipitated proteins or in input control. (b) Effect of CaA on HIF-1α binding to VEGF promoter region analysed by chip.

3.6. Effects of CaA on the tumor growth, angiogenesis, and the expressions of JNK1, HIF-1α, STAT-3, and VEGF in vivo

We previously showed that, a concentration of 10 mg kg⁻¹ BW CaA inhibited the in vivo growth of MHCC97H cells in a mice xenograft model.¹⁶ Here, we further found that, compared with the vehicle control (dimethyl sulfoxide, DMSO), CaA treatment resulted in a significant reduction in tumor weight (Fig. 6A); in addition, CaA significantly attenuated the angiogenesis in the xenograft tumor as determined by the CD31 staining and MVD (Fig. 6B and C). Then we further investigated the effects of CaA on the expressions/activations of JNK1, HIF-1α, STAT-3, and VEGF. Compared with vehicle control, reductions of p-JNK1, HIF-1α, STAT-3, and VEGF expression, and attenuations of HIF-1α and STAT-3 nuclear location were observed in CaA-treated group (Fig. 6D and E). Collectively, these results suggested that CaA down-regulated the JNK1, HIF-1α, STAT-3, VEGF, and blocked the angiogenic ability of HCC cells in an in vivo model.

Fig. 6 Effects of CaA on the tumor growth, angiogenesis, and the expressions of JNK1, HIF-1α, STAT-3, and VEGF in vivo after MHCC97h cells were injected subcutaneously into the right armpit of the mice (6 mice per group) for 3 weeks, CaA (0 or 10 mg kg⁻¹ BW) was administered (i.p) twice per week. After 11 week, the mice were sacrificed, and the tumor tissues were removed for further investigation. (a) tumor weight. (b) IHC analyses of the expression of CD31. (c) Angiogenesis quantification by MVD. (d) Western blots analyses of the expressions of HIF-1α, p-JNK1, STAT-3, and VEGF. (e) IHC analyses and (f) quantification scores of VEGF, HIF-1α, and STAT-3. Data were presented as the means ± SD (n = 6), a two-tailed Student's t test was used for statistical analysis.

4. Discussion

As a classical plant chemical, the natural origin and ubiquitous occurrence have prompted strong interest in developing CaA as a potential anticancer agent. For example, CaA directly targets ERK1/2 to attenuate solar UV-induced skin carcinogenesis; in addition, it attenuated the invasive ability and cancer stem cells-like properties in skin and liver cancer cells via the inhibition of NF-κB and TGFβ signaling; moreover, it inhibited the growth of colon cancer by blocking the activation of PI-3K and AMPK signal pathways.^16,23–26 Here, we used a relative lower pharmacological concentration of CaA (20 μM, as described previously¹⁴) to investigate the functions of CaA on the progression of HCC, and found that CaA effectively blocked the hypoxia-induced endogenous VEGF autocrine and angiogenesis in HCC cells in vitro and in vitro, which was associated with the ubiquitin-dependent degradation of HIF-1α.

Classically, under hypoxia, HIF-1α is stabilized and translocate to the nucleus, where it induces the expression of several genes such as VEGF; however, normoxia leads to the ubiquitination and subsequent proteasomal degradation of HIF-1α.¹⁵ Recently studies indicate that the stabilization of HIF-1α is not only induced by hypoxia, but is activated response to various kinases cascade, for example, mitogen-activated protein kinases (MAPKs).²⁷ Our previous study found that the activation of c-Jun N-terminal kinases (JNKs, a member of MAPKs family) played an important role in the stabilization of HIF-1α.²⁷ In human cancer cell lines, hypoxia results in an accumulation of reactive oxygen species (ROS), which can lead to JNKs activation through several mechanisms, including the oxidation and inhibition of MAPK phosphatases.²⁸ Interestingly, in both cell and mouse models, UVB (an oxidative stress inducer) caused a significantly increased activation of JNKs, these effects, however, was blocked by the treatment of CaA or its derivative, chlorogenic acid.²⁵ So, based on these findings, we hypothesized that in our present study, the inhibition of JNKs might be involved in the CaA-induced de-stabilization of HIF-1α.

JNKs are members of the superfamily of MAPKs, which are involved in the regulation of various mammalian physiological events.²⁹ While JNK1 and JNK2 are extensively expressed in mammalian tissues, expression of JNK3 is restricted to certain tissues, such as the brain and testis.³⁰ Previous studies indicate that JNK1 can affect the expression and the functions of the Hsp90/70 chaperone proteins, thereby preventing the degradation of HIF-1α protein, while JNK2 can increase the stability of HIF-1α mRNA via enhancing the binding of nucleolin protein to the latter.^21,31 In our present study, CaA reduced JNK1 phosphorylation, decreased HIF-1α accumulation, and increased HIF-1α ubiquitination, and these effects were abrogated in the JNK1 knockout cells. Based on the literature^21,31 and our present findings, we suggested that, under hypoxia, CaA reduced the phosphorylation of JNK1, which attenuated the stabilization of HIF-1α. Further, we hypothesized that the CaA-increased ubiquitination of HIF-1α might be though another mechanism, which likely involved the von Hippel-Lindau (VHL).

In human cancers, STAT-3 has been shown to be a potential partner of HIF-1α, which synergistically mediates the VEGF expression. Although several studies report that either the activation of HIF-1α or STAT-3 alone can transcriptionally elevates the expression of VEGF gene,^11,15,32 recent evidence suggest that a maximal induction is reached when both transcription factors bind to the VEGF promoter, where they are presumably linked within the same transcriptional complex together with CBP/p300 co-activator.^15,33 Our previous study found that CaA could enhance the expression of microRNA-124 (miR-124) by inducing the DNA de-methylation. MiR-124, which targeted the 3′-UTR regions of STAT-3 mRNAs, decreased the expressions/activations of this protein.¹⁴ Here we further found that, via an ubiquitin-dependent manner, CaA effectively induced a degradation of HIF-1α. As a result, CaA attenuated the expression of HIF-1α/p-STAT-3 complex, and decreased the accumulation of HIF-1α to VEGF promoter.

5. Conclusions

Our present study revealed that, CaA blocked the CoCl₂-induced VEGF autocrine and angiogenesis in HCC cells in vitro and in vitro. For the molecular mechanisms, on one hand, CaA attenuated the CoCl₂-induced activation of HIF-1α likely by reducing JNK1 activation and reducing HIF-1α stabilization; on another hand, CaA decreased the CoCl₂-induced increased expression of p-STAT-3. These two functions resulted in an attenuated recruiting of the HIF-1α and p-STAT-3 (possibly) to VEGF promoter.

Authors' contributions

The conception and design: Yuan Li and Huibin Dong; contributed the reagents/materials/analysis tools: Yuan Li and Huibin Dong; performed the experiments: Weiting Gu, Chi Zhang, Ye Yang, Lijun Chen, Jian Shen, and Guiying Li; analysis of the data: Ye Yang, Huibin Dong, Weiting Gu, and Yuan Li; wrote and revised the manuscript: Chi Zhang, Ye Yang, Yuan Li and Weiting Gu. Note: Weiting Gu, Ye Yang, and Chi Zhang contributed equally to this work.

Conflict of interest

The authors have declared that no competing interests exist.

Acknowledgements

The authors are grateful to the National Natural Science Foundation of China (81402667), the Collegiate Natural Science Foundation of Jiangsu Province (14KJB330003), the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD-2014), the Research Fund for the Doctoral Program of Jiangsu Province (1302050C), the Technology Development Fund of Nanjing Medical University (2013NJMU021), and the science and technology support program funded by Changzhou Municipal Science and Technology Bureau (CE20145046).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra07703j

‡ These authors contributed equally to this work.

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