Article Info

Author Affiliations: Department of Anesthesiology, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Zhang J, Feng ZY and Zhu SM); Department of Gynaecology and Obstetrics, Hangzhou Cancer Hospital, Hangzhou 310002, China (Zhang D); and Department of Oncology, Zhejiang Provincial People’s Hospital, Hangzhou 310014, China (Wu GQ)

Corresponding Author: Sheng-Mei Zhu, PhD, Department of Anesthesiology, First Affiliated Hospital, Zhejiang University School of Medicine, 79 Qingchun Rd., Hangzhou 310003, China (Tel: 86-571-87236169; Email: smzhu20088@yahoo.com.cn)

Contributors: ZSM proposed the study. ZJ and ZSM performed the research and wrote the first draft. ZD, WGQ and FZY collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. ZSM is the guarantor.

BACKGROUND: Propofol is one of the extensively and commonly used intravenous anesthetics and has the ability to influence the proliferation, motility, and invasiveness of many cancer cells. In this study, the effects of propofol on hepatocellular carcinoma cells invasion ability were examined.

METHODS: We assessed the invasion ability of HepG2 cells in vitro by determining enzyme activity and protein expression of MMP-9 using gelatin zymography assay and Western blot. The real-time PCR was used to evaluate the effect of propofol on microRNA-199a (miR-199a) expression, and miR-199a-2 precursor to evaluate whether over-expression of miR-199a can affect MMP-9 expression. Finally, the effect of miR-199a on propofol-induced anti-tumor activity using anti-miR-199a was assessed.

RESULTS: Propofol significantly elevated the expression of miR-199a and inhibited the invasiveness of HepG2 cells. Propofol also efficiently decreased enzyme activity and protein expression of MMP-9. Moreover, the over-expression of miR-199a decreased MMP-9 protein level. Interestingly, the neutralization of miR-199a by anti-miR-199a antibody reversed the effect of propofol on alleviation of tumor invasiveness and inhibition of MMP-9 activity in HepG2 cells.

Hepatocellular carcinoma (HCC), one of the most common malignancy tumors worldwide, is particularly high in much of east Asia and sub-Saharan Africa.^[1] The vascular invasion propensity and high metastatic potential are the characteristics of HCC, which increase the difficulties for the treatment.^[2] It is well known that a lot of molecules are involved in tumor invasion and metastasis. Among these molecules, matrix metalloproteinases (MMPs) play an important role because they can degrade extracellular matrix (ECM) proteins.^[3] It has been reported that the expression and activity of MMPs are increased in HCC and the increases are related to advanced tumor stage and poor survival.^[4]

MicroRNAs (miRNAs) are a novel class of short, non-coding RNAs involved in post-transcriptional gene regulation by binding to the target site in the 3'-untranslated region (3'-UTR) of target mRNAs.^[5] miRNAs involve in invasion and metastasis of a variety of tumors, such as liver, lung, colon and pancreatic cancers.^[6] Microarray studies have determined a large number of miRNAs that are related to metastasis in HCC.^[7] Shen et al^[8] reported that microRNA-199a (miR-199a) was down-regulated in HCC. Decreased expression of miR-199a plays an important role in increasing cell invasion and metastasis in HCC.

Propofol (2, 6-diisopropylphenol) is one of the extensively and commonly used intravenous anesthetics. In recent years, more and more evidences indicate that propofol has the ability to influence the motility, proliferation, and invasiveness of cancer cells in vitro and in vivo.^[9-11] Therefore, propofol might be a better agent than other anesthetics for cancer surgery.^[12] However, the anti-tumor function of propofol on HCC cells has not been reported. The present study was to determine the effect of propofol on the invasion of HCC cells and the molecular mechanisms related with this activity.

Dulbecco's modified eagle medium (DMEM) was obtained from GIBCO (Invitrogen Company). Calf blood serum (CBS) was from Sigma Aldrich Chemical Co. (St. Louis, MO., USA). Propofol (2, 6-diisopropylphenol) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Cytomatrix human vitronectin-coated strips were obtained from Chemicon International Inc. (California, USA). MMP-9 ELISA kit was from R&D (Abingdon, UK). Polyclonal antibody against β-actin and MMP-9 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA., USA). The Detergent Compatible (DC) Protein Assay kit was purchased from Bio-Rad Laboratories (Hercules, CA., USA).

Human hepatoma cell line HepG2 was purchased from the American Type Culture Collection (Manassas, VA). The cells were cultured in DMEM, containing 4 mmol L-glutamine, 10% CBS, 100 units/mL penicillin, 65 units/mL streptomycin, 1.5 g/L sodium bicarbonate, and 4.5 g/L glucose. Cultured cells were maintained in a humidified incubator containing 5% CO₂ at 37 �� and were stimulated with propofol [dissolved in dimethyl sulfoxide (DMSO)] in complete DMEM medium. As a control, cultured cells were incubated in complete DMEM medium containing DMSO at a final concentration of less than 0.1%.

Cytomatrix human vitronectin-coated strips were used to perform the activity of cell adhesion. Under serum-free conditions, the strips were incubated with 100 µL of HepG2 cells (10⁵/mL) with control or different concentrations of propofol (1, 5, 10 µg/mL) at 37 �� for 45 minutes. Washed with phosphate buffer solution (PBS) for three times, the adhered cells were stained for 5 minutes with 0.2% crystal violet in 10% ethanol at 25 ��. After PBS washing the excess stain for five times, the stained cells were dissolved in 100 µL of solubilization buffer (1:1 mixture of 50% ethanol and 0.1 mol NaH₂PO₄, pH 4.5). The absorbance was read at 540 nm. The absorbance of dye in the control (without propofol treatment) cells was regarded as 100% adherence and the percentage adherence of propofol-treated cells was compared with that of the control cells.

Gelatin zymography assays were used to detect the activity of MMP-9 in the medium according to the method described previously.^[13] Briefly, 20 µL of collected media were mixed with sodium dodecyl sulfate (SDS) sample buffer and subjected to SDS-PAGE electrophoresis without boiling or reduction. Washed with 2.5% Triton X-100, the gels were incubated in a reaction buffer (10 mmol/L CaCl₂, 0.01% NaN₃, and 40 mmol/L Tris-HCl, pH 8.0) at 37 �� for 12 hours. At last, Coomassie brilliant blue R-250 was used to stain the gels.

Approximately 5×10⁶ cells were treated without (Control) or with propofol for 24 hours. miRNAs were isolated and purified using Trizol reagent (Invitrogen, USA), according to the manufacturer's protocol. The miR-199a level was measured by Q-PCR using TransStartTM SYBR Green qPCR Supermix (TransGen Biotech, Beijing, China). The U6 small nuclear RNA was used as an internal normalized reference. For miR-199a, the primers were as follows: forward, 5'-ACA CTC CAG CTG GGC CCA GTG TTC AGA CTA C-3' and reverse, 5'-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG GAA CAG GT-3'. For U6, the primers were as follows: forward, 5'-CTC GCT TCG GCA GCA CA-3' and reverse, 5'-AAC GCT TCA CGA ATT TGC GT-3'. The comparative threshold cycle (Ct) method was used to analyze fold change. The change of miRNA expression was calculated as fold-change, i.e. relative to control.

HepG2 cells were lysed with ice-cold lysis buffer containing: 150 mmol/L NaCl; 50 mmol/L Tris-HCl, pH 7.4; 1 mmol/L EDTA; 1% NP-40; and 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentration in the cell lysate was detected using the DC protein assay kit (Bio-Rad). After protein content was determined, Western blot analysis was performed.

miR-199a was overexpressed or knocked down by transfection with 100 nmol/L of miR-199a-2 precursor or anti-miR-199a (Ambion) with lipo-fectamine 2000 (Invitrogen, USA) in antibiotic-free medium, at 30% to 50% confluency, in 60-mm dishes. Twenty-four hours after transfection, the cells were treated with or without propofol.

Statistical analysis was performed with SPSS software, version 13.0. Statistical analyses were performed using either an analysis of variance (ANOVA) or Student's t test. Results are presented as means±SD. A P value <0.05 was considered statistically significant, and a P value <0.01 was highly significant.

HepG2 cell adhesion was significantly inhibited by propofol at the concentration of 5 µg/mL after incubation for 24 hours. The inhibition was dose-dependent (29.5% decrease at 5 µg/mL and 43.2% at 10 µg/mL; P<0.01 compared with the control (Fig. 1).

We next examined the effect of propofol on MMP-9 activity in HepG2 cells. Gelatin zymography showed that MMP-9 activity was significantly inhibited by propofol at the concentration of 5 µg/mL (P<0.01, Fig. 2A), and the inhibitory effect of propofol on the activity of MMP-9 was dose-dependent.

In order to determine the MMP-9 protein level in cells after treatment with propofol, Western blot analysis was made (Fig. 2B). Being consistent with the observed alteration of its activity, MMP-9 protein expression was significantly reduced in HepG2 treated with propofol at 5 µg/mL (P<0.01 compared with control). The inhibitory effect of propofol on MMP-9 protein in HepG2 cells was dose-dependent, with a 46% inhibition at 5 µg/mL and a 68.4% decrease at 10 µg/mL (Fig. 2C).

Compared with the control, propofol increased miR-199a expression in HepG2 cells in a dose-dependent manner (Fig. 3). Propofol at 5 µg/mL increased the miR-199a expression in HepG2 cells to 2.23-fold. Propofol at 10 µg/mL elevated the miR-199a level to 3.53-fold.

To determine whether miR-199a regulates MMP-9 expression in HepG2 cells, we determined the protein level of MMP-9 after transfection of miR-199a-2 precursor. The result of Q-PCR revealed that miR-199a-2 precursor significantly increased the expression of miR-199a in HepG2 cells (Fig. 4A), suggesting that miR-199a-2 precursor is efficiently introduced into the cells and induces miR-199a over-expression. Furthermore, transfection of miR-199a-2 precursor significantly decreased MMP-9 expression (Fig. 4B).

We used anti-miR-199a antibody to evaluate the role of miR-199a in the effect of propofol on the inhibition of HepG2 cells adhesion anti-miR-199a antibody significantly reduced the expression of miR-199a, suggesting that anti-miR-199a antibody efficiently penetrated into HepG2 cells (Fig. 5A). Furthermore, anti-miR-199a antibody reversed the inhibitory effect of propofol on cell adhesion (Fig. 5B) and neutralized the inhibitory effect of propofol on MMP-9 activity in HepG2 cells (Fig. 5C).

HCC has become one of the most common malignancies worldwide, and has high metastatic potential.^{[2, 14]} In this study we investigated the effect of propofol on invasion ability of hepatoma cell line HepG2. It is well known that cell adhesion is essential in tumor metastasis and the changing adhesive preferences of cancer cells to fibroblasts play an important role in the metastatic process.^[15] We evaluated the effect of propofol on HepG2 cells invasion ability using cell adhesion assay and found that propofol mitigated the cell invasiveness (Fig. 1). Our results were consistent to the study of propofol on breast cancer cells invasion.^[16]

Increasing evidence showed that propofol perfectly fits anesthesia in tumor surgery.^{[16, 17]} In clinical anesthesia, the target plasma propofol concentrations for general anesthesia are 3-8 µg/mL^[18] and these concentrations of propofol effectively inhibited invasion of HCC. We found that 5 µg/mL propofol significantly inhibited invasion ability of HepG2 cells. To further explain the molecular mechanism involved in the suppression of invasion of these cells, we investigated the effect of propofol on the expression and activity of MMP-9. MMP-2 and MMP-9 which degrade ECM components and play an important role in HCC invasion.^{[3, 4]} Miao et al^[11] reported that propofol treatment significantly decreased the expression of MMPs and subsequently inhibited the invasive activity of colon carcinoma cells. We also found that propofol significantly inhibited the activity of MMP-9 in HepG2 cells but had no effect on MMP-2 expression (data not shown).

It has been reported that the expression of miR-199a was often down-regulated in HCC.^{[19, 20]} When the expression of miR-199a was up-regulated, it could reduce the invasion ability of HepG2 cells.^[8] Thus, miR-199a may be useful as a new effectively therapeutic target for HCC. In this study, the over-expression of miR-199a inhibited MMP-9 protein expression, indicating that miR-199a played a role in cancer invasion through down-regulation of MMP-9. Besides, propofol stimulated the expression of miR-199a in HepG2 cells. More importantly, neutralizing miR-199a with anti-miR-199a antibody reversed the effect of propofol on the alleviation of the invasiveness of HepG2 cells and on the inhibition of MMP-9 expression. These results suggested that the propofol suppression of HCC cell invasion was partly due to the miR-199a down-regulation of MMP-9. However, the mechanism of miR-199a regulation of MMP-9 expression should be clarified in future.

In conclusion, the concentrations of propofol in clinical application inhibited the invasion of HCC cells effectively and miR-199a possibly contributes to this antitumor action of propofol. Further studies are needed to validate its clinical relevance.

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