Lobetyolin Suppresses the Proliferation of Hepatocellular Carcinoma through Activating DUSP1-ERK1/2 Signaling Pathway

Abstract

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer with limited treatment options. Lobetyolin (LBT), a polyacetylene glycoside mainly extracted from the roots of Codonopsis pilosula, has been reported to have anti-tumor efficancy in various cancers. However, the role of LBT as well as its underlying mechanisms in HCC remain unclear. Here we investigated the impact of LBT on the phenotype in HepG2 and Huh7 cells. We found that LBT significantly induced cell growth inhibition and mitochondria-dependent apoptosis in HCC cells. Moreover, LBT upregulated dual specificity phosphatase-1 (DUSP1) expression and knockingdown DUSP1 markedly attenuated those effects induce by LBT. Meanwhile, LBT decreased the phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2), a well-recognized downstream effector of DUSP1, and knockingdown DUSP1 partially recovered LBT-induced inactivation of ERK1/2. In conclusion, the present study indicated that LBT could induce cell death of HCC via promotion of DUSP1-mediated ERK1/2 inhibition. These data will help to establish the evidence of LBT to treat HCC.

INTRODUCTION

Hepatocellular carcinoma (HCC) is one of the most common malignancies which accounts for 80–90% of primary liver cancers. It remains a great threat to human health globally due to the high incidence rate and mortality.¹⁾ Despite extensive studies have led to an improved understanding in the molecular biological basis for HCC development, and the gradual advancement of modern medicine in HCC, the limitations of the efficacy and safety in HCC treatment still existed.²⁾ Therefore, it is urgently required to develop new treatment strategies to improve the outcomes for HCC.

Over the past few decades, the traditional Chinese medicine (TCM) and natural products have been applied as an effective complementary medicine in HCC due to its unique advantages including high cost performance, strong anticancer activity, less toxic and side effects.³⁾ Lobetyolin (LBT) is a polyacetylene glycoside mainly extracted from the roots of Codonopsis pilosula, and has been disclosed to exhibit anti-oxidative, anti-inflammatory, and xanthine oxidase inhibiting activities.⁴⁾ LBT has also shown great properties against several cancer cells and the molecular basis has been preliminarily elucidated. For example, LBT induces cell apoptosis through ASCT2 inhibition, thus leading to glutamine metabolism suppression in colon cancer cells and breast cancer cells.^5,6) In lung cancer, LBT largely reinforces the anti-cancer activity of cisplatin chemotherapy through the suppression of epithelial-mesenchymal transition.⁷⁾ However, neither the anti-tumor efficancy nor the potential mechanisms of LBT in HCC have not been clarified.

Dual specificity phosphatase-1 (DUSP1), also named mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), is a dual specificity phosphatase that dephosphorylates and inactivates 3 major MAPK subfamilies c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). MAPKs are critical regulators involved in multiple cellular responses and signal transduction.⁸⁾ Generally, the ERK signaling is predominantly responsible for promoting cell proliferation and differentiation, while the JNK and p38 signaling mediates the cell responses to stimulus.⁹⁾ Tumor expression of DUSP1 has been extensively studied as a predictive marker of response so far. DUSP1 plays different roles in different cancers. Increased expressions of DUSP1 in various human cancers such as prostate,¹⁰⁾ colon,¹¹⁾ and bladder,¹²⁾ has been demonstrated, and knocking down DUSP1 weakened the tumorigenicity of pancreatic cancer cells.¹³⁾ These findings indicated DUSP1 play a role of oncogene in those cancers. However, decreased expression of DUSP1 was observed in HCC¹⁴⁾ and human head and neck squamous cell carcinoma (SCC).¹⁵⁾ DUSP1 has been reported to repress HCC carcinogenesis through cooperating with Hcr1.¹⁶⁾ In addition, DUSP1 regulated p53 activation via the p38/HSP27 signaling pathway, and the upregulation of DUSP1 resulted in the suppression of HCC progression.¹⁷⁾

The present study aimed to determine whether LBT has an inhibitory effect on the cell growth of HCC cells and elucidate the potential underlying mechanisms. The associated mechanisms were probed in aspects of DUSP1/MAPKs signaling.

MATERIALS AND METHODS

Cell Culture

HepG2 and Huh7 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Logan, UT, U.S.A.) containing 10% (Gibco, Carlsbad, CA, U.S.A.) and 1% penicillin/streptomycin (Solarbio, Beijing, China) at 37 °C in an incubator with 5% CO₂.

Establishment of Stable Transfected Cell Lines

The DUSP1 knockdown and negative control lentiviruses were purchased from Genechem (Shanghai, China). Cells were infected with the lentiviruses for 48 h and selected with 2 µg/mL puromycin for 14 d.

Cell Counting Kit-8 (CCK-8) Assay

Cells (8 × 10³ cells/well) were seeded in 96-well plates and cultured overnight. After indicated treatments, the medium was replaced with 100 µL fresh medium containing 10% CCK-8 solution (Beyotime, Beijing, China). After 2 h of incubation at 37 °C, the absorbance was read at 450 nm on a microplate reader.

Colony Formation Assay

Cells (500 cells/well) were seeded in 12-well plates and cultured overnight. Three days later, LBT was added and incubated for another 3 d. After a 10-d growth period in fresh medium, colonies were stained with 0.1% crystal violet (Solarbio) in methanol for 20 min. Colonies consisting of approximately 50 cells were calculated.

Western Blotting Analysis

Harvested cells were lysed by RIPA Lysis Buffer (Solarbio) on ice for 30 min, and the supernatant proteins were determined using a BCA protein assay kit (Thermo Fisher, Waltham, MA, U.S.A.). The proteins were separated by 10–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, St. Louis, MO, U.S.A.), followed by sealing with 5% nonfat milk in TBST. After that, the membranes were incubated with primary antibodies overnight. They were Ki67 (A11005, Abclonal, Wuhan, China), Bcl-2 (12789-1-AP, Proteintech, Wuhan, China), Bax (50599-2-Ig, Proteintech), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (60004-1-Ig, Proteintech). p38 (8690, Cell Signaling Technology, Danvers, MA, U.S.A.), p-p38 (4511, Cell Signaling Technology), JNK1/2 (9252, Cell Signaling Technology), p-JNK1/2 (9255, Cell Signaling Technology), ERK1/2 (4695, Cell Signaling Technology), p-ERK1/2 (4370, Cell Signaling Technology). The secondary antibodies (Cell Signaling Technology) were then incubated for another 1 h at room temperature. An ECL detection system (Thermo Fisher) was employed to visualize the signals. Finally, the protein bands were captured by Amersham Imager 680 (GE, Boston, MA, U.S.A.) and quantified by densitometry using the Image J software.

Quantitative (q)RT-PCR

RNA extraction was performed using RNA purification kit (Qiagen, Germantown, MD, U.S.A.) according to the manufacturer’s instruction. cDNA was obtained using PrimeScript™ RT Master Mix (TaKaRa, Shiga, Japan) and qRT-PCR was performed in 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.) using SYBR^® Premix Ex Taq™ II (TaKaRa). GAPDH was used as the mRNA internal control. Relative RNA levels were calculated using the 2^−ΔΔCt method. The primer sequences were listed as follows: DUSP1: F, 5′-GCC ACC ATC TGC CTT GCT TAC C-3′; R, 5′-ATG ATG CTT CGC CTC TGC TTC AC-3′, GAPDH: F, 5′-CAG GAG GCA TTG CTG ATG AT-3′; R, 5′-GAA GGC TGG GGC TCA TTT-3′.

5-Ethynyl-2′-deoxyuridine (EdU) and Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling (TUNEL) Incorporation Assays

Cells (8 × 10³ cells/well) were seeded into 12-well plates with glass coverslip and cultured overnight. BeyoClick™ EdU-488 kit (Beyotime) was used to detect the cell proliferation. In brief, cells were incubated with EdU (10 µM) for 2 h and fixed with 4% paraformaldehyde for 15 min, followed by permeabilized with 0.3% Triton X-100 for another 15 min. Afterwards, cells were stained with Click Reaction Mixture for 30 min. One Step TUNEL Apoptosis Assay Kit (Beyotime) was used to determine the cell apoptosis. Cells were fixed and permeabilized with the same procedures as EdU. And then incubated with TUNEL reaction mixture for 1 h. The slides were sealed with anti-fluorescence quenching tablets (including 4′-6-diamidino-2-phenylindole (DAPI)) and observed under a fluorescence microscope (DM4000B, Leica, Wetzlar, Germany).

Flow Cytometric Analysis of Apoptosis

Fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit I (BD Biosciences, San Jose, CA, U.S.A.) was used to evaluate the cell apoptosis. Cells were stained with Annexin V-FITC (5 µL) and 7-AAD (5 µL) for 15 min in the darkness and examined by flow cytometry (BD FACSVerse™, BD Biosciences). The apoptosis rate was analyzed using FlowJo software (v10, LLC, Bethesda, MD, U.S.A.).

Statistical Analysis

Data were presented as mean ± standard deviation (S.D.). GraphPad Prism software (v8, La Jolla, CA, U.S.A.) was employed for statistical analyses. Comparisons between two groups were assessed using Student’s t-test while comparisons among more than two groups were performed using one-way ANOVA. A p-value less than 0.05 was considered statistically significant.

RESULTS

LBT Suppressed Cell Proliferation in HCC

The antitumor activities of LBT (Fig. 1A) and its structural analogs, lobetyol (Fig. 1B) and lobetyolinin (Fig. 1C), were investigated in HCC cells. Data from CCK-8 assay revealed that LBT led to a dose-dependent reduction in the cell viability of HepG2 and Huh7 cells (Fig. 1D). However, lobetyol showed weak toxicity against HCC cells than LBT at the equivalent concentrations (Fig. 1E). In addition, lobetyolinin brought little toxic effects on HCC cells even at the highest tested concentration (Fig. 1F). LBT was then selected for subsequent analysis. The colony formation capacity of the HCC cells was also observed decreased after LBT treatment (Fig. 2A). In consistency, EdU staining assay disclosed that LBT was able to inhibit cell proliferation of HCC cells (Fig. 2B). The proliferation marker Ki67 was also measured. Data from Western blot assay showed that LBT led to a decrease in Ki67 protein expressions in HCC cells (Fig. 2C). Collectively, these data suggested that LBT had an anti-proliferation effect on HCC.

Fig. 1. LBT Suppressed Cell Viability in HCC

A–C, Molecular structural formula of LBT (A), lobetyol (B) and lobetyolinin (C). D–F, Cell viability detected by CCK-8 assay. Cells were incubated with LBT (D), lobetyol (E) and lobetyolinin (F) for 24 h. Values were presented as mean ± S.D. of three independent experiments. * p < 0.05 vs. control group.

Fig. 2. LBT Suppressed Cell Proliferation in HCC

A, Colony formation ability. Cells were treated with LBT (20, 40, 80 µM) for 72 h. B, Cell proliferation measured by EdU staining. Green, EdU; blue, DAPI; scale bar, 100 µm. Cells were incubated with LBT (20, 40 µM) for 24 h. C, Western blot analysis for Ki67. GAPDH provided a loading control. Cells were treated with LBT (20, 40 µM) for 24 h. Values were presented as mean ± S.D. of three independent experiments. * p < 0.05 vs. control group.

LBT Provoked Cell Apoptosis in HCC

Annexin V-7-AAD double staining assay demonstrated that LBT treatment resulted in an increase of early (AnnexinV+/7-AAD−) and late (AnnexinV+/7-AAD+) apoptotic cells in HCC in a dose-dependent manner (Fig. 3A). Consistent with this, TUNEL-positive cells were significantly increased upon LBT administration (Fig. 3B). Bax and Bcl-2 are two central regulators of mitochondrial apoptosis which elicit opposite effects. Bax (proapoptotic) formed heterodimers with Bcl-2 (antiapoptotic) to suppress Bcl-2 activity and induce cell apoptosis. As shown in Fig. 3C, LBT increased the ratio of Bax/Bcl-2 in HCC. These data collectively indicated that LBT induced mitochondrial-dependent apoptosis in HCC.

Fig. 3. LBT Induced Cell Apoptosis in HCC

A, Flow cytometer for cell apoptosis. Apoptosis rate was calculated as the percentage of cells with early (Annexin-FITC+ and 7-AAD−) and late apoptotic cells (Annexin-FITC+ and 7-AAD+) to total cells. B, TUNEL assay for apoptosis. Red, TUNEL; blue, DAPI; scale bar, 100 µm, Apoptosis rate was expressed as percentage of TUNEL positive cells to total nuclei. C, Western blot analyses for Bax and Bcl-2. GAPDH provided a loading control. Cells were treated with LBT (20, 40 µM) for 24 h. Values were presented as mean ± S.D. of three independent experiments. * p < 0.05 vs. control group.

DUSP1 Contributed to LBT-Induced Cell Growth Inhibition of HCC

DUSP1, a well-recognized tumor suppressor gene in HCC, was upregulated both in mRNA (Fig. 4A) and protein (Fig. 4B) levels after LBT treatment. Thus, we investigated whether DUSP1 played a role in the cell growth inhibition induced by LBT. The efficiencies of sh-DUSP1 were then evaluated by qRT-PCR (Fig. 5A) and Western blot (Fig. 5B) assays, which showed a remarkably reduction in mRNA and protein levels of DUSP1 in HCC cells. DUSP1 knockdown per se caused no change in cell viability and colony formation capacity of HCC cells, but restored LBT-induced suppression of those effects (Figs. 5C, 5D), Likewise, the decreased expression of Ki67 induced by LBT was markedly reversed after sh-DUSP1 transfection (Fig. 5E). Taken together, the upregulation of DUSP1 was the key that mediating LBT-induced cell growth inhibition of HCC.

Fig. 4. LBT Induces DUSP1 mRNA and Protein Expressions in HCC

A, qRT-PCR analyses for DUSP1 mRNA level. GAPDH provided a loading control. B, Western blot analyses for DUSP1. GAPDH provided a loading control. Cells were treated with LBT (20, 40 µM) for 24 h. Values were presented as mean ± S.D. of three independent experiments. * p < 0.05 vs. control group.

Fig. 5. DUSP1 Was the Key Mediator Controlling LBT-Induced Cell Growth Inhibition of HCC

A, qRT-PCR analyses for DUSP1 mRNA level. GAPDH provided a loading control. B, Western blot analyses for DUSP1. GAPDH provided a loading control. Cells were infected with the sh-DUSP1 and sh-negative control (sh-NC) lentiviruses for 48 h and selected with puromycin. C, Cell viability detected by CCK-8 assay. D, Colony formation ability. E, Western blot analyses for Ki67. GAPDH provided a loading control. Stable transfected cells were treated with LBT (40 µM) for 24 h. Values were presented as mean ± S.D. of three independent experiments. * p < 0.05 vs. sh-NC group. #p < 0.05 vs. sh-NC + LBT group.

DUSP1 Mediated LBT-Induced Cell Apoptosis of HCC

As depicted in Fig. 6A, DUSP1 knockdown had no apparent effect on the cell apoptosis, but DUSP1 knockdown could partially prevent LBT-induced apoptosis. Meanwhile, the elevated Bax/Bcl-2 ratio in LBT-treated HCC cells was largely reversed by DUSP1 knockdown (Fig. 6B). These data suggested that DUSP1 is involved in LBT-induced apoptosis of HCC.

Fig. 6. DUSP1 Mediated LBT-Induced Cell Apoptosis of HCC

A, Flow cytometer for cell apoptosis. B, Western blot analyses for Bax and Bcl-2. GAPDH provided a loading control. Stable transfected cells were treated with LBT (40 µM) for 24 h. Values were presented as mean ± S.D. of three independent experiments. * p < 0.05 vs. sh-NC group. # p < 0.05 vs. sh-NC + LBT group.

DUSP1–ERK1/2 Pathway Is Responsible for LBT-Induced Cell Death

We further investigated the influence of DUSP1 knockdown on MAPK signaling in LBT-treated HCC cells. LBT resulted in a reduced phosphorylation of ERK1/2 without affecting the phosphorylation levels of p38 and JNK1/2 (Fig. 7A). More importantly, LBT-induced the inactivation of ERK1/2 was recovered by DUSP1 knockdown (Fig. 7A). U0126, an ERK1/2 inhibitor, were then utilised to determine the impact of ERK1/2 in the antitumor activity of LBT. U0126 caused a significant decrease in the cell viability of HCC cells (Fig. 7B). Furthermore, U0126 treatment markedly enhanced the antitumor property of LBT (Fig. 7B). The results revealed that the antitumor effect of LBT in HCC may be probably through DUSP1-ERK1/2 pathway.

Fig. 7. DUSP1-ERK1/2 Pathway Is Responsible for LBT-Induced Cell Death

A, Western blot analyses for p-p38, p-JNK1/2 and p-ERK1/2. p38, JNK1/2 and ERK1/2 provided a loading control respectively. Stable transfected cells were treated with LBT (40 µM) for 24 h. B, Cell viability detected by CCK-8 assay. Cells were treated with LBT (40 µM) and U0126 (5 µM) for 24 h. Values were presented as mean ± S.D. of three independent experiments. * p < 0.05 vs. Control or sh-NC group. # p < 0.05 vs. sh-NC + LBT group.

DISCUSSION

DUSP1 shows a moderate decline in HCC especially in patients with poor prognosis, suggesting that DUSP1 functions as a tumor suppressor in HCC.¹⁴⁾ The DUSP1 protein increased while the cell growth of HCC decreased along with elevated LBT concentrations, this was coincided with the role of DUSP1 as a tumor suppressor in HCC. Further investigation observed that knockdown of DUSP1 significantly reversed the inhibitory effect of LBT, so we concluded that DUSP1 mediated the anti-tumor effect of LBT in HCC. This was the first time to demonstrate the critical role of DUSP1 in the anti-tumor effect of LBT. DUSP1 might be a promising therapeutic target for LBT treatment in HCC.

Although the anticancer effects of LBT have been widely reported, no in-human studies have yet been confirmed, only one was with animals, revealing that LBT dramatically enhances the efficacy of cisplatin in lung cancer mice through the inhibition of epithelial-mesenchymal transition.⁷⁾ Applications beyond the antitumor activity of LBT are also under exploration. LBT protects LPS-induced sepsis through inhibition of inflammatory cytokines in mice.¹⁸⁾ LBT attenuates muscle injury induced by chronic kidney disease via inhibiting hedgehog-mediated ferroptosis.¹⁹⁾ LBT exerts a cardioprotective effect in cardiorenal syndrome type 4 through osteopontin inhibition via activation of JNK signaling pathway.²⁰⁾ The above results were corroborated in animal studies. The antitumor efficiency and the underlying mechanisms of LBT in HCC needs to be further elucidated in vivo.

DUSP1 was reported to be regulated by ERK/SKP2/CKS1-dependent ubiquitination in HCC cells.²¹⁾ The Ser296 phosphorylation of DUSP1 rendered the DUSP1 protein susceptible to proteasomal degradation by the ubiquitin ligase complex SKP2/CKS1.²¹⁾ In addition, DUSP1 expression was directly regulated by E2F6 in HCC, which acted as a transcriptional repressor to inhibit DUSP1 transcriptional activity through binding to the DUSP1 promoter region.²²⁾ Wild-type p53 could also bind to DUSP1 gene and transcriptionally activated DUSP1 in HCC. In turn, the increased DUSP1 expression could induce p53 activation via the p38/HSP27 pathway.¹⁷⁾ Further study on the ubiquitination modification and transcriptional regulation of DUSP1 will provide intensive understanding of the molecular mechanisms for the anti-HCC effect of LBT.

The MAPK signaling pathway (p38, ERK1/2 and JNK1/2), which is negatively regulated by DUSP1, plays a major role in tumor progression including cell proliferation, cell apoptosis, and cell survival.⁹⁾ Only ERK1/2 signaling showed a decreasing trend after LBT treatment in HCC, suggesting ERK1/2 was implicated in the anti-HCC effect of LBT. Further investigation revealed that knockdown of DUSP1 dramatically reversed the inhibitory effect of LBT on ERK1/2. Nevertheless, DUSP1 might not the main causative event responsible for ERK1/2 activation in HCC. Besides, the prolonged activation of ERK1/2 promoted the phosphorylation of DUSP1 at Ser296, thus leading to DUSP1 degradation.²¹⁾ Thus, the anti-tumor effect of LBT might be augmented via the formation of a negative feedback loop between DUSP1 and ERK1/2.

In addition to MAPK, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is another important signaling to regulate tumor development and survival.²³⁾ Strategies to disrupt PI3K/AKT signaling through enhancing phosphatase activity result in blunted proliferation and metastasis in HCC.²⁴⁾ DUSP1 negatively modulates AKT activation, and this effect is under control of USP22-E2F6 axis in HCC cells.²²⁾ Whether DUSP1 mediated AKT inhibition played a role in LBT-induced cell growth suppression of HCC needs to be further explored.

Acknowledgments

We thank Professor Jie Deng for her help in the experiment.

Conflict of Interest

The authors declare no conflict of interest.

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