Inhibition of LGR5/β-Catenin Axis and Activation of miR134 Are Critically Involved in Apoptotic Effect of Sanggenol L in Hepatocellular Carcinoma

Abstract

Although Sanggenol L (SL), derived from the root bark of Morus alba, has hepatoprotective, neuroprotective, and antitumor effects, the antitumor mechanism of SL remains unclear to date. Thus, in the current work, the apoptotic mechanisms of SL were investigated in HepG2 and Huh hepatocellular carcinoma (HCC) cells in relation to leucine-rich repeat containing G protein-coupled receptor 5 (LGR5)/β-catenin and miR134 signaling axis. Herein, SL significantly incremented cytotoxicity, sub-G1 population, and the number of terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) positive apoptotic bodies and also inhibited proliferation in HCCs. Consistently, SL activated pro-Caspase7 and pro-Caspase3 and induced the cleavage of Poly ADP-ribose polymerase (PARP) in HCCs. Of note, the pivotal role of LGR5/β-catenin signaling was verified in SL-induced apoptosis in LGR5 overexpressed AML-12 cells and LGR5 depleted HepG2 cells. Furthermore, SL upregulated miR134 expression levels in HepG2 cells, while miR134 inhibitors disturbed the capacity of SL to cleave PARP and pro-Caspase3 in HepG2 cells. Taken together, our findings highlight evidence that inhibition of the LGR5/β-catenin axis and upregulation of miR134 play critical roles in SL-induced apoptosis in HCCs.

INTRODUCTION

Liver cancer is globally known the fourth most common cause of cancer death among both sexes.¹⁾ Since 2007, sorafenib has been applied for treatment of advanced hepatocellular carcinoma (HCC), thereby prolonging median survival.²⁾ Nonetheless, a number of molecular targeted agents failed to prolong overall survival rate in many clinical trials.³⁾ Thus, recently, natural compounds, chemotherapeutics, immunotherapies, and combination therapy are attractive for the treatment of liver cancers via molecular target therapy.⁴⁾

Accumulating evidence reveals that the leucine-rich repeat containing G protein-coupled receptor 5 (LGR5)/R-spondin complex promotes Wnt/β-catenin signaling by suppressing β-catenin degradation in normal intestinal stem cells.⁵⁾ Without Wnt signaling, degradation of cytoplasmic β-catenin is activated by a destruction complex comprised of adenomatous polyposis coli (APC) and Axin.^6,7) RNA level of LGR5 was significantly upregulated in HCCs,⁸⁾ since LGR5 increases HCC cell survival, regulates epithelial cell phenotype⁹⁾ and promotes metastasis through inducing epithelial-to-mesenchymal transition (EMT).¹⁰⁾ Hence, a high level of LGR5 indicates poor prognosis of HCC patients.¹¹⁾

It is well documented that microRNAs (miRNAs) play pivotal roles in the modulation of cell cycle checkpoint, metastasis, angiogenesis, and mitochondria-mediated apoptosis in HCCs.¹²⁾ Especially, miR134 is downregulated in glioblastomas, breast cancer, renal cell carcinoma, colorectal cancer, non-small cell lung cancer (NSCLC), and HCCs, since miR134 is significantly involved in invasion, metastasis, cell proliferation, apoptosis, and drug resistance.¹³⁾

Saggenol L (SL), an ipresonylated flavonoid isolated from Morus alba, was reported to have hepatoprotective and neuroprotective effects,¹⁴⁾ antidiabetic,¹⁵⁾ antitumor effects in melanoma,¹⁶⁾ prostate cancer,¹⁷⁾ and ovarian cancer cells.¹⁸⁾ Nevertheless, the antitumor pathogenesis of SL has not been fully understood in HCCs. Thus, in the current study, the apoptotic mechanisms of SL were explored in Huh7 and HepG2 cells in relation to LGR5/β-catenin and miR134 signaling axis.

MATERIALS AND METHODS

SL and Reagents

SL was bought from Chem Faces (Wuhan, China; CAS No. 329319-20-2). LGR5, β-actin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Sigma-Aldrich (St. Louis, MO, U.S.A.). Also, specific antibodies for pro-Caspase3, pro-Caspase 7, cleaved poly ADP-ribose polymerase (PARP) were bought from Cell Signaling Technology (Danvers, MA, U.S.A.), and Bcl2 antibody was purchased from Abcam (Waltham, MA, U.S.A.).

Cell Culture

Human HepG2 (HB-8065^TM) cells purchased from ATCC (Manassas, VA, U.S.A.) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics. Also, Huh7 cells supplied by Korean Cell Line Bank (Seoul, Korea) were grown in RPMI1640 in a humidified atmosphere of 5% CO₂ at 37°C. AML-12 hepatocytes isolated from the normal liver of a mouse (ATCC, CRL-2254) were grown in DMEM/F-12 Medium with 10% FBS and 1% 100 μg/mL streptomycin and penicillin at 37°C.

Cell Viability Assay

As previously described,¹⁹⁾ the cytotoxicity of SL was examined in Huh7 and HepG2 cells by MTT assay. Huh7 and HepG2 cells were exposed to MTT solution (1 mg/mL) at 37°C for 2 h. The cytotoxicity was determined with optical density values at 570 nm by a microplate reader.

Colony Formation Assay

As previously described,²⁰⁾ Huh7 and HepG2 cells (3 × 10³/well) were grown in 6 well plates at 37°C for 24 h. The cells were exposed to the concentrations (0, 30 μM) of SL for 24 h. Ten days after medium replacement, the cells were stained with Diff quick solution from Sysmex (Kobe, Japan). Then, the number of colonies on the dried plates was counted using light microscopy.

Cell Cycle Analysis

As previously described,²¹⁾ Huh7 and HepG2 cells exposed to SL (0, 15, and 30 μM) for 24 h were incubated overnight at –20°C. The cells exposed to RNase A (1 mg/mL) were subjected to staining with propidium iodide (PI; 50 μg/mL). The DNA contents for each cell cycle phase were determined using FACS Calibur Flow Cytometer (BD Bioscience, U.S.A.).

Terminal Deoxynucleotidyl Transferase-Mediated Deo-xyuridine Triphosphate Nick-End Labeling (TUNEL) Assay

As described previously,²²⁾ cell death was evaluated using the deadend^TM TUNEL system kit (Abcam, Waltham, MA, U.S.A.). Huh7 and HepG2 cells treated with SL for 1 d were exposed to 4% paraformaldehyde, permeabilization solution, and TUNEL assay mixture. Then, the number of TUNEL-stained apoptotic bodies was photographed and counted through a FLUOVIEW FV10i Confocal Microscope (Olympus, Irvine, U.S.A.).

Real-Time Quantitative PCR (RT-qPCR)

As previously described,²³⁾ total RNAs were isolated from Huh7 and HepG2 cells using QIAzol Lysis Reagent. After the cDNAs were synthesized with oligodTand M-MLV reverse transcriptase, qRT-PCR was carried out with the primers of miR134. miR134:5′-GGTGTGACTGGTTGACCA-3′ (forward) and 5′-TGCGTGTCGTGGAGTC-3′ (reverse), miR134 mimic:5′-UGUGACUGGUUGACCAGAGGGG-3′; and antisense, 5′-CCUCUGGUCAACCAGUCACAUU-3′, miR134 inhibitor :5′-CCCCUCUGGUCAACCAGUCACA-3′ and negative control: 5′-CAGUACUUUUGUGUAGUACAA-3′ that were supplied by Bioneer (Daejeon, Korea).

Western Blotting

As previously described,²⁴⁾ Huh7 and HepG2 cells (2 × 10⁵/well) treated with SL (0, 15, and 30 μM) for 24 h were lysed with RIPA lysis buffer and spinned at 13000 × g. Isolated proteins were loaded onto a nitrocellulose transfer membrane at 300 mA. After blocking with 5% non-fat skim milk, it was probed with the antibodies of PARP, pro-Caspase 3, 7, cleaved Caspase 3, LGR5 from Sigma, and Bcl2 from Abcam (Waltham, MA, U.S.A.).

RNA Interference and Plasmid Transfection

AML-12 cells were transfected with the LGR5 overexpression plasmid and small interfering RNA (siRNA) control vector. In contrast, HepG2 cells were transfected with LGR5 siRNA plasmid and pcDNA 3.0 control vector from Addgene (Watertown, MA, U.S.A.) using lipofetamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.) according to manufacturer’s protocol. Also, HepG2 cells were transfected with miR134 mimic or miR134 inhibitor and miR control. The transfected cells were incubated for 48 h for the next experiment.

Statistical Analysis

All data are represented as means ± standard deviation (S.D.). To evaluate statistical significance, Student’s t-test was applied for comparison between SL and control groups with Sigmaplot version 12 software (Grafiti, Palo Alto, CA, U.S.A.). The values of p < 0.05 were accepted significant difference. All experiments were performed in triplicates and repeated 3 times.

RESULTS AND DISCUSSION

SL-Enhanced Cytotoxicity and Inhibited Proliferation in Huh7 and HepG2 Cells

To evaluate the cytotoxicity of SL (Fig. 1A), an MTT assay was performed in Huh7 and HepG2 cells treated with different concentrations of SL (0, 12.5, 25, and 50 μM) for 24 h. As shown in Fig. 1B, SL significantly reduced the viability of Huh7 and HepG2 in a concentration-dependent fashion. Furthermore, colony formation assay showed that SL reduced the number of colonies in Huh7 and HepG2 cells compared to untreated control (Fig. 1C), implying the cytotoxic and anti-proliferative effect of SL in HCCs.

Fig. 1. Effect of SL on Cytotoxicity and Proliferation in Huh7 and HepG2 Cells

(A) Chemical structure of SL. (B) Cytotoxic effect of SL in Huh7 and HepG2 cells by MTT assay. ** p < 0.01, *** p < 0.001 vs. untreated control. (C) Anti-proliferative effect of SL in Huh7 and HepG2 cells by colony formation assay.

SL-Regulated Apoptosis Related Proteins and Incremented Sub G1 Portion in Huh7 and HepG2 Cells

To examine whether or not the cytotoxicity of SL is due to apoptosis, Western blot assay and cell cycle analysis were conducted in SL treated-Huh7 and HepG2 cells. Herein, flow cytometry analysis reveals that SL increments sub-G1 population in Huh7 and HepG2 cells (Fig. 2A). It is well documented that apoptosis is usually regulated by the Bcl2 or Caspase family proteins through the cell death process. Caspase3 and Caspase6, Caspase7 as executioner Caspases are responsible for initiating the degradation phase of apoptosis along with the features of cell shrinkage, DNA fragmentation and membrane blebbing.²⁵⁾ Notably, Caspase7 activation requires Caspase1 inflammasomes under inflammatory condition, whereas Caspase3 cascade proceeds apoptosis independently of Caspase1.²⁶⁾ Indeed, SL-induced activation of Caspase7, Caspase3 and PARP in Huh7 and HepG2 cells (Fig. 2B), indicating apoptotic effect of SL in HCCs.

Fig. 2 Effect of SL on Sub G1 Portion and Apoptosis-Related Proteins in Huh7 and HepG2 Cells

(A) Effect of SL on sub-G1 portion in Huh7 and HepG2 cells. (B) Effect of SL on apoptosis-related proteins in Huh7 and HepG2 cells.

SL Increased the Number of TUNEL-Positive Apoptotic Bodies in HepG2 and Huh7 Cells

To confirm the apoptotic effect of SL, a TUNEL assay was executed in HepG2 and Huh7 cells treated by SL. Here, SL significantly incremented the number of TUNEL-positive HepG2 and Huh7 cells compared to the untreated control (Figs. 3A, 3B), demonstrating the apoptotic feature of SL in HCCs.

Fig. 3. Effect of SL on the Number of TUNEL Positive Cells in HepG2 and Huh7 Cells

(A, B) The cells treated with SL (0, 30 μM) were analyzed by TUNEL assay. Apoptotic TUNEL-labeled bodies (green) and DAPI-stained cell nuclei (blue) were observed using a Delta Vision imaging system (Abcam, Waltham, MA, U.S.A.). Scale bar = 100 μm.

SL Attenuated the Expression Level of LGR5 and β-Catenin in Huh7 and HepG2 Cells

LGR5, a receptor for R-spondins, is known to be a member of the G protein receptor super-family, while the Wnt/β-catenin signaling pathway^27,28) was also reported as molecular targets in colorectal cancer⁷⁾ and cancer biology.²⁹⁾ Here, LGR5 was highly expressed in HepG2 cells while lowly expressed in AML-12 normal hepatocyte cells (Fig. 4A). To examine the effect of SL on LGR5 and β-catenin, Western blotting was carried out in Huh7 and HepG2 cells. Here, SL abrogated the expression of LGR5 and β-catenin in Huh7 and HepG2 cells (Fig. 4B).

Fig. 4. Effect of SL in LGR5/β-Catenin Signaling in Hepatocellular Carcinoma Cells

(A) Endogenous expression level of LGR5 in AML-12, Huh7, and HepG2 cells. (B) Effect of SL on LGR5 and β-catenin in AML-12, Huh7, and HepG2 cells. Western blotting was performed for LGR5, β-catenin, and β-actin in Huh7 and HepG2 cells.

LGR5 Overexpression Disturbed Apoptotic Effect of SL in AML-12 Cells, while LGR5 Depletion Enhanced Apoptosis of SL in HepG2 Cells

To investigate the critical role of LGR5 in SL-induced cytotoxic and apoptotic effects, Crystal violet assay and Western blotting were executed in LGR5-overexpressed AML-12 cells. Overexpression of LGR5 incremented the viability of AML-12 cells, which was reversed by SL (Fig. 5A). Similarly, overexpression of LGR5 disturbed the capacity of SL to lessen the expression of LGR5 and β-catenin in LGR5 overexpressed AML-12 cells (Fig. 5B), indicating that SL induces apoptosis through LGR5/β-catenin signaling in HCCs. Conversely, LGR5 depletion reduced the viability of HepG2 cells compared to untreated control, which was synergistically suppressed by SL in HepG2 cells (Fig. 5C). Likewise, LGR5 depletion enhanced the capacity of SL to diminish the expression band of LGR5, β-catenin, pro-PARP, and pro-Caspase3 in HepG2 cells (Fig. 5D). Interestingly, LGR5 overexpression or depletion affected the expression band of β-catenin in HepG2 cells in our work, implying LGR5 is an upstream target of β- catenin, which was supported by the correlation between LGR5 and β-catenin³⁰⁾ (Fig. 5E) and Kawasaki et al.’s paper³¹⁾ that LGR5 induces β-catenin activation.

Fig. 5. Effect of LGR5 Overexpression or Depletion on SL-Induced Cytotoxicity and Apoptosis in Hepatocellular Carcinoma Cells

(A) Effect of LGR5 overexpression on the viability of SL-treated AML-12 cells. The viability was evaluated in AML-12 cells transfected with LGR5 overexpression plasmid in the presence or absence of SL by MTT assay, while LGR5 overexpression alone control and negative intact control were set. ** p < 0.01 vs. untreated control. (B) Effect of LGR5 overexpression on LGR5 and β-catenin in SL-treated AML-12 cells. Western blotting was performed for LGR5 and β-catenin in AML-12 cells transfected with LGR5 overexpression plasmid in the presence or absence of SL compared to untreated intact control. (C) Effect of LGR5 depletion on the viability of SL-treated HepG2 cells. The viability was evaluated in HepG2 cells transfected with LGR5 depletion plasmid in the presence or absence of SL by MTT assay, while LGR5 depletion alone control and negative intact control were set. *** p < 0.001 vs. untreated control. (D) Effect of LGR5 depletion on LGR5, β-catenin, pro-PARP, and pro-Caspase3 in SL-treated HepG2 cells. Western blotting was performed for LGR5, β-catenin, pro-PARP, and pro-Caspase3 in HepG2 cells transfected with LGR5 depletion plasmid in the presence or absence of SL compared to untreated intact control. (E) A strong correlation between LGR5/β-catenin with Spearman correlation of 0.38 by cBioportal database.

The Critical Role of miR134 in SL-Induced Apoptosis in HepG2 Cells

Accumulating evidence reveals that microRNAs are associated with apoptosis,³²⁾ proliferation,³³⁾ invasion, migration,¹⁵⁾ and metastasis³⁴⁾ in hepatocellular carcinoma cells. Herein, SL upregulated mRNA expression level of miR134 in HepG2, Huh7, and AML-12 cells, and miR134 mimics enhanced cytotoxicity and apoptosis via inhibition of procaspse3, pro-PARP and Bcl2 in SL-treated HepG2 cells (Figs. 6A–6C). Conversely, the miR134 inhibitor reduced the cytotoxicity of SL treatment in HepG2 cells (Fig. 6D), while it also suppressed the apoptotic effect of SL to reduce the expression of procaspse3, pro-PARP, and Bcl2. Our findings provide scientific evidence that SL induces apoptosis in hepatocellular carcinoma cells via inhibition of the LGR5/β-catenin axis and upregulation of miR134. Nonetheless, further study is required in the future by targeting p53/P21/MDM2 and NF-kb/cMyc signaling axis, since these signalings are critically involved in apoptosis in several types of HCCs, including p53 wild-type HepG2 cells, non-sense p53 mutant Hep3B cells, inframe p53 gene deleted SNU423 cells, and p53 point mutated Huh7 and SNU449 cells.

Fig. 6. The Critical Role of miR134 in SL-Induced Apoptosis in HepG2 Cells

(A) Effect of SL on miR134 level in HepG2, Huh7, and AML-12 cells. (B) Effect of miR134 mimic on the viability of the HepG2 cells. The viability was evaluated in HepG2 cells transfected by control miRNA mimic or miRNA134 mimic plasmid (C) Effect of miR134 mimic on PARP, pro-Caspase3, and Bcl2 in the HepG2 cells. (D) Effect of miR134 inhibitor on the viability of the HepG2 cells with or without SL. ** p < 0.01 vs. untreated control. (E) Effect of the miR134 inhibitor on PARP, pro-Caspase3, and Bcl2 in HepG2 cells with or without SL.

CONCLUSION

In the current work to elucidate the apoptotic mechanisms of SL, SL significantly incremented cytotoxicity, sub-G1 population, and TUNEL positive cells, inhibited proliferation, activated Caspase7 and Caspase3 and cleaved PARP in Huh7 and HepG2 cells. Of note, LGR5 overexpression disturbed cytotoxicity and apoptosis of SL, while LGR5 depletion enhanced cytotoxicity and apoptosis of SL in HepG2 cells. Also, SL upregulated miR134 expression levels in HepG2, Huh7, and AML-12 cells, while miR134 inhibition reduced the capacity of SL to cleave PARP and Caspase3 in HepG2 cells. Overall, our findings highlight evidence that SL induces apoptosis via inhibition of the LGR5/β-catenin axis and upregulation of miR134 as a potent candidate for liver cancer treatment. However, further experiments are required in animals and humans in the future.

Funding

This work was funded by the National Research Foundation of Korea (NRF) Grant funded by the Korean government (MSIT) (No. 2021R1A2C2003277).

Conflict of Interest

The authors declare no conflict of interest.

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