Sorafenib-induced Aquaporin-3 Downregulation is Coupled With Proliferation Inhibition and Increased Apoptosis in Hepatocellular Carcinoma Cells


 Background

Sorafenib is the only targeted therapy promising to improve the prognosis of patients with advanced hepatocellular carcinoma (HCC), but its long-term clinical efficacy is limited due to chemotherapy resistance. The lack of a full understanding of the anti-tumor mechanism of sorafenib in HCC is attributed to the difficulties in understanding the mechanism of drug resistance. In recent years, a large number of preclinical and clinical data have confirmed the catalytic role of aquaporin-3 (AQP3) in a variety of tumors including HCC, but none of the studies reported the regulatory mechanism of AQP3 during sorafenib treatment. This study examined the effect of sorafenib on the expression of AQP3 in HCC cells and determined whether the effect is associated with cell proliferation inhibition, cell cycle arrest, and increased apoptotic.
Methods

mRNA and protein levels of AQP3 in hepatoma cell lines exposed to sorafenib or UO126 were detected via real-time quantitative polymerase chain reaction (qPCR) and western blotting, respectively. The effect of AQP3 expression changes on cell proliferation, cell cycle and apoptosis were determined by cell counting kit-8 (CCK-8) and flow cytometry. In addition, western blotting detected proteins involved in the regulation of proliferation and cell cycle progression.
Results

The results showed that AQP3 was down-regulated in all cell lines exposed to sorafenib or UO126 in a concentration dependent manner. The downregulation of AQP3 successfully inhibited cell proliferation, induced cell cycle arrest and increased cell apoptosis, while the reverse was true when AQP3 was overexpressed. Western blotting results showed that in AQP3 knockdown cells, the amounts of Erk, Akt, p53, p-Erk, p-Akt and cyclin-dependent kinase 2 (CDK2) decreased, while the amounts of cyclin-dependent kinase 4 (CDK4), p21 and p-p53 increased.
Conclusion

This study found that sorafenib may inhibit proliferation, induce cell cycle arrest, and increase apoptosis of HCC cells by regulating the expression of AQP3.


Introduction
The treatment of patients with hepatocellular carcinoma (HCC), especially those with advanced liver cancer, remains controversial, making HCC one of the deadliest cancers in the world 1-3 . HCC accounts for 90% of all primary liver cancers and is the fourth leading cause of cancer-related death worldwide; >80% of HCC cases occur in low-resource and middle-resource countries, particularly in Eastern Asia and sub-Saharan Africa 4 . Prognosis of HCC remains poor due to the fact that most of cases are diagnosed at late stages 1 , when there are no amenable curative therapies 1,5 . Systemic therapies are recommended, but the effectiveness of existing therapies is hampered by drug resistance [6][7][8] . Compelling data have emerged to improve the prognosis of patients with advanced HCC, and increased attention has been given to improving HCC chemosensitivity to the targeted therapy, recently [9][10][11] .
Sorafenib is a multi-kinase inhibitor and is currently the only targeted therapy promising to improve the prognosis of patients with advanced HCC. It blocks Raf signaling as well as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and KIT 1 . However, the initial response to treatment is heterogeneous and unsatisfactory during prolonged exposure, suggesting primary and acquired resistance [12][13][14][15] . So far, the exact mechanism behind the drug resistance remains a debatable aspect in the literature. Surprisingly, even anti-tumor mechanisms are not fully understood, which is thought to be the cause of the di culty in understanding the mechanisms of drug resistance. Extensive identi cation of tumor promoters and their regulation during treatment has been recommended as a strategy not only to understand drug resistance, anti-tumor mechanism but also to improve the sensitivity of HCC cells to drugs. In recent years, a large number of preclinical and clinical data have con rmed the catalytic role of aquaporins (AQPs) in a variety of tumors, including HCC [16][17][18][19][20][21][22][23][24][25] , but few studies have explored the regulatory mechanism of AQPs in sorafenib therapy.
Knockdown/knockout studies of AQP3 in various tumors have shown the opposite phenotypes 20,27,28 . In fact, studies have shown that the antitumor effects of sorafenib and the AQP3-related phenotypes are contradictory in HCC 16,18,22,24,29 . Then, we hypothesized that AQP3 was down-regulated in HCC cells that were highly sensitive to sorafenib.
To test this hypothesis, this study examined the expression of AQP3 in HCC cells exposed to sorafenib and determined whether it was associated with sorafenib-induced inhibition of cell proliferation, cell cycle arrest, and increased apoptotic effects.

Method And Materials
Cell culture, reagents, and antibodies Human HCC cell lines Huh7, HepG2 and SK-Hep1 obtained from The Chinese Academy of Sciences in Shanghai-China were used. Cells were cultured in Dulbecco's Modi ed Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Shanghai, China) and 1% penicillin/streptomycin and maintained in an incubator with a humidi ed atmosphere of 5% CO 2 at 37°C 30,31 . Sorafenib and UO126 (Sigma-Aldrich, St. Louis, Missouri, USA) were purchased and dissolved in 100% DMSO to prepare a 10mM stock solution, which was then diluted with DMEM to the desired concentration, with a nal concentration of 0.1% DMSO, recommended for in vitro studies. Unless otherwise stated, all antibodies were obtained from Life Span BioSciences (LSBIO, Seattle, Washington, USA) 32 . The enhanced chemiluminescence detection kit was purchased from Amersham Pharmacia Biotech-UK. The primers for AQP3 and GAPDH were obtained from Sangon Biotech (Sangon Biotech, Shanghai, China). SYBR® Premix Ex Taq™ (Tli RNaseH Plus) was purchased from Takara Bio Inc (Takara, Beijing, China).

Cell proliferation assay
Cell count Kit-8 (CCK-8) was used to detect differences in cell proliferation (Sigma-Aldrich, St. Louis, Mo., USA). Brie y, cells were seeded in a 96-well plate at a density of 3000 cells/well and incubated for different time (0, 24, 48, and 72 h). After the indicated time, 10μl of CCK-8 solution was added to each well of the plate, followed by 2 h incubation before detecting absorbance at 450nm using a microplate reader. Cell viability (as a percentage) was determined in relation to the average absorbance of the untreated cells from three replicate samples.

Annexin V apoptosis assay
Annexin V apoptosis was used to detect apoptosis differences. Brie y, cells were seeded in 6-well plates at 2 ´ 10 5 per well for 24 h prior to treatment. After treatment/transfection, cells were harvested via trypsinization, washed twice with Phosphate buffered saline (PBS), and stained with a uorescein isothiocyanate Annexin V Apoptosis Detection kit (BD Pharmingen, Franklin Lakes, NJ, USA). Flow cytometry (Thermo Fisher Scienti c, Waltham, Massachusetts, USA) was used to determine the proportion of apoptosis.

Flow cytometry Cell cycle analysis
Flow cytometry was used to detect differences in cell cycle progression in accordance with Sigma-Aldrich CCK-8 protocol Number 96992. Brie y, cells were seeded in 6-well plates at 2 x 10 5 per well. After transfection, cells were harvested by trypsinization and washed with PBS 2x. Cells were xed in cold 70% ethanol for 24 h at 4 o C, washed with PBS 2x, resuspended in 1mL PBS; treated with 10μL RNase A (21 mg/mL); and then stained with 5 μL propidium iodide at 1mg/mL for 30 minutes at room temperature.
Finally, the stained cells were analyzed by ow cytometry (Thermo Fisher Scienti c, Waltham, Massachusetts, USA).

Cell transfection
Cells were plated in 6-well plates until 60% con uence and then infected with lentivirus-AQP3 (Lv-AQP3), lentivirus-AQP3RNAi (Lv-AQP3RNAi), or lentivirus-GFP (Lv-GFP)-control according to the company instructions (GenePharma Co. Ltd, Shanghai, China) 32 . Transfection e ciency was veri ed under uorescence microscope after 14 h. The cells with stable virus integration 48 h posttransfection were selected with puromycin and further incubated for 10 days. The effect of transfection on AQP3 expression was veri ed via qPCR and western blotting.
RNA extraction, reverse transcription, and qPCR Total RNA was extracted from cells using an RNAIsoPlus assay kit (Takara, Dalian, China) according to the manufacturer's instructions. After quanti cation, RNA was transcribed into cDNA using a two-step reverse transcription kit (Takara, Dalian, China). Subsequently, qPCR was used to detect target gene expression levels using the SYBR-Green qPCR master mix (Takara, Dalian, China). The thermocycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 35 cycles of a two-step PCR of 95°C for 14 s and 60°C for 1 min. The 2 ΔΔCq method was used to quantify the results; the relative expression level of AQP3 was normalized to that of GAPDH. The primers used were as follows: AQP3, forward (F), 5 -GGCTGTATTATGATGCAATCT-3 and reverse (R), 5 -ATATCCAAGTGTCCAGAGG-3 . GAPDH F, 5 -GATCATCAGCAATGCCTCCT-3 and R, 5 -GAGTCCTTCCACGATACCAA-3 . The data have been deposited in a publicly accessible database (GenBank) with accession number NM_004925.5 and NM_002046.7 for AQP3 and GAPDH, respectively 32 .

Sorafenib inhibits cell proliferation and cell cycle, and induces apoptosis of HCC cells
In this study, the anti-proliferation effect of sorafenib on HCC cells was con rmed via CCK-8 assay and ow cytometry. CCK-8 results showed that sorafenib successfully inhibited the viability of Huh7, HepG2 and SK-Hep1 cells in a dose-dependent manner after 24 h (Fig. 1A). The results also showed that sorafenib inhibited cell proliferation in a time-dependent manner (Fig. 1B) Fig. 1C and D). In addition, ow cytometry results showed that sorafenib increased the proportion of apoptotic cells from 23.48 ± 6.13 to 32.46 ± 2.98 in Huh7, 17.62 ± 3.74 to 48.12 ± 17.00 in HepG2, and 21.20 ± 4.05 to 35.51 ± 3.59 in SK-Hep1 cells (Fig. 1E). Together, the results con rmed that sorafenib inhibited proliferation, induced cell cycle arrest and increased apoptosis of HCC cells.

Sorafenib treatment coupled with AQP3 down regulation in HCC cells
Previous studies have shown that the AQP3-related phenotypes and the antitumor effects of sorafenib are contradictory in different cancer types 16,18,22,24,29 . Here, we determined the effect of sorafenib on the expression of AQP3 in Huh7, HepG2 and SK-Hep1 cells. mRNA and protein levels of AQP3 were detected by RT-qPCR and western blotting, respectively. RT-qPCR results showed that after sorafenib treatment for 24 h, the levels of AQP3 mRNA in Huh7, HepG2 and SK-Hep1 cells decreased signi cantly in a dosedependent manner. The level decreased by 27% and 47% in the 5 µM and 10 µM sorafenib groups of Huh7, 18% and 46.8% in the 5 µM and 10 µM sorafenib groups of HepG2, 20.7% and 49.3% in the 5 µM and 10 µM sorafenib groups of SK-Hep1 respectively ( Fig. 2A). RT-PCR results were con rmed by western blotting. At the protein level, western blotting also demonstrated trends similar to mRNA levels (Fig. 2B). In together, the results suggest that sorafenib down-regulated AQP3 in HCC cells.

Sorafenib down-regulates AQP3 in HCC cells via Raf/Mek/Erk signaling pathway
To investigate the mechanism of sorafenib-induced AQP3 downregulation in HCC cells, we detected the level of AQP3 in cells after UO126 (Mek1/2 inhibitor) inhibited Raf/Mek/Erk signaling pathway. Huh7, HepG2, and SK-Hep 1 cells were incubated with either DMSO, 5 µM, 10 µM sorafenib or 10 µM UO126 in a 6-well plate for 24 h prior protein extraction. Consistently with the previous results in Fig. 2, we observed the same trend, importantly, we noted that AQP3 protein level decreased in a similar manner in 10 µM sorafenib and 10 µM UO126 (Fig. 3A and B). Western blotting results revealed that treatment with either 5 µM, 10 µM sorafenib or 10 µM UO126 successfully inhibited phosphorylation of Erk protein as evidenced by decreased values of p-Erk/Erk ratio in those groups (Fig. 3C). Together, the results con rmed that Sorafenib down regulated AQP3 expression in HCC cells via Raf/Mek/Erk signaling pathway.
Changes of AQP3 expression in uence the proliferation, cell cycle progression, and apoptosis of HCC cells Here, we further investigated whether the downregulation of AQP3 in HCC cell lines can inhibit cell proliferation, induce cell cycle arrest, and increase apoptosis. Huh7 and HepG2 cells were transfected with lentivirus carrying either AQP3RNAi (Lv-AQP3RNAi), AQP3 (Lv-AQP3) or GFP (Lv-GFP). The transfection e ciency was con rmed via RT qPCR and western blotting results. Cell proliferation was detected by CCK-8 and cell cycle progression and apoptosis rate were detected by ow cytometry. In addition, western blotting was used to investigate the expression of proteins involved in the regulation of proliferation and cell cycle. Compared with the control group (GFP), the proliferation of Huh7 and HepG2 cells transfected with Lv-AQP3RNAi decreased, while the proliferation of Lv-AQP3 group increased over time (Fig. 4A). Flow cytometry showed that cell cycle arrest was enhanced after transfection with Lv-AQP3RNAi. The proportion of Huh7 and HepG2 cells increased by 30% and 23% respectively in the resting stage (G1 phase). The proportion of cells in S-phase decreased by 50% in Huh7 and 35% in HepG2 cells. Opposite results were observed in cells transfected with Lv-AQP3 (Fig. 4B). Flow cytometry showed that the proportion of apoptotic cells transfected with Lv-AQP3RNAi increased, whereas that of Lv-AQP3 transfected cells decreased. The proportion of late apoptotic cells increased from 29.50 ± 2.24 (GFP) to 44.84 ± 3.44 (Lv-AQP3RNAi) in Huh7 and 27.01 ± 2.40 (GFP) to 35.28 ± 3.86 in HepG2 cells (Fig. 4C). Western blotting results showed that in Lv-AQP3RNAi transfected cells, the amounts of Erk, Akt, p53, p-Erk, p-Akt and CDK2 decreased, while the amounts of CDK4, p21 and p-p53 increased (Fig. 4D). The results suggested that the proliferation inhibition, cell cycle arrest and increased apoptosis of sorafenib in HCC cells were the result of AQP3 down-regulation.

Discussion
Extensive exploration of sorafenib's role in tumor growth and progression promoters is critical not only to understanding the mechanisms of drug resistance, but also to sensitizing HCC cells to drugs. Data from preclinical and clinical studies suggest that AQP3 plays a key role not only in promoting invasion, migration, angiogenesis, and EMT protein activation, but also in cell proliferation and cell cycle progression in a variety of tumors, including HCC [16][17][18][19][20][21][22][23][24][25] . This study investigated the effect of sorafenib on the expression of AQP3 in HCC cells and determined whether it was associated with sorafenib-induced proliferation inhibition, cell cycle arrest, and increased apoptotic effects. For the rst time, this study has demonstrated that sorafenib could inhibits proliferation, induces cell cycle arrest, and increases apoptosis of HCC cells by down-regulating the expression of AQP3. Sorafenib down-regulated AQP3 expression in concentration dependence via Raf/Mek/Erk signaling pathway. Similar effects of proliferation inhibition, cell cycle arrest, and increased apoptosis were observed in the AQP3 knockdown group, suggesting a potential target for sorafenib in HCC.
The Raf/Mek/Erk signaling pathway is one of the most famous, regulating a variety of proteins, including those that promote tumorigenesis and development 1 . Sorafenib has been shown to inhibit the signaling pathway by blocking Raf-1 and B-Raf, leading to downstream inhibition of mitogen-activated protein kinase/extracellular signaling regulated kinase (ERK). The inhibitory effect of sorafenib on this pathway is related to the down-regulation of cyclin D1 and anti-apoptotic factor ml-1, and the reduction of the phosphorylation level of eIF4E protein, thus inhibiting cell proliferation, cell cycle arrest, and increasing the apoptosis of liver cancer cells 5 . In recent years, this pathway has been found to be involved in the regulation of aquaporins, especially the aquaporin-3 subtype, which is a known promoter of tumor proliferation, invasion, angiogenesis, and EMT activation in many cancer types, including HCC 16-18,22,29,33−35 . Activation of the signaling pathway stimulates overexpression of constitutive-transcriptional activity of the AQP3 promoter, leading to up-regulation of AQP3 expression and vice versa 25 36 . This study investigated the effect of sorafenib on the expression of AQP3 in HCC cells and determined whether the observed effect was related to the antitumor effect of sorafenib.
The qPCR and western blotting results of this study con rmed that the mRNA and protein levels of AQP3 were down-regulated in the HCC cells exposed to sorafenib, respectively. The use of UO126 (Mek1/2 inhibitor) con rmed that the inhibition of Raf/Mek/Erk signaling pathway is involved in the downregulation of AQP3 in HCC. In addition, in the HCC cells transfected with lentivirus carrying AQP3RNAi (Lv-AQP3RNAi), cell proliferation and cell cycle progression were signi cantly reduced and apoptosis was increased. These results suggest that the observed effects of sorafenib in HCC cells may be the result of AQP3 downregulation. Consistent with previous studies, Li and colleagues found that AQP3 de ciency impaired proliferation of gastric cancer cells 20 . Chen and colleagues found that silencing of AQP3 induces apoptosis of gastric cancer cells via downregulation of glycerol intake and downstream inhibition of lipogenesis and autophagy 28 . These results also suggest a further discussion in the literature on the mechanisms by which metabolic reprogramming may be a therapeutic target for treatment of HCC 39 .

Conclusion
This study found that sorafenib may inhibit proliferation, induce cell cycle arrest, and increase apoptosis of HCC cells by regulating the expression of AQP3. Sorafenib down-regulates AQP3 expression in a concentration-dependent manner through Raf/Mek/Erk signaling pathway, leading to proliferation inhibition, cell cycle arrest and increased apoptosis of HCC cells. Further in vivo studies are needed to con rm our ndings.

Abbreviations AQP3
Aquaporin-3 Availability of data and materials The data for primer sequences used have been deposited in a publicly accessible database (GenBank) with accession number NM_004925.5 and NM_002046.7 for AQP3 and GAPDH, respectively.