The authors have declared that no competing interests exist.
Conceived and designed the experiments: HZ BZ CH E-BB JL. Performed the experiments: HZ. Analyzed the data: HZ X-MM. Contributed reagents/materials/analysis tools: HZ CH JL. Wrote the paper: HZ JL.
Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world without effective therapies
Phosphatase and tensin homolog (PTEN) is a plasma membrane lipid phosphatase that acts as a tumor suppressor and it dephosphorylates PIP3 to PIP2, inhibits the activation of the oncogene Akt and then negatively regulates the PI3K/Akt pathway
The histone deacetylases (HDACs) represent an ancient superfamily of enzymes conserved from yeast to human. The HDAC members of class I contains HDAC1, HDAC2, HDAC3 and HDAC8
Melittin is a water-soluble toxic peptide and the major component of bee venom
In light of the therapeutic potential of melittin in HepG2 cells, our study was performed to elucidate the biological mechanism by which melittin induces the inhibition of cell growth in HepG2 cells. Here, we hypothesized that melittin leads to inhibition of cell proliferation and up-regulation of PTEN gene which may be associated with the decrease of HDAC2 expression in HepG2 cell.
Melittin was purchased from Baichun Anhui co., Ltd, batch number: 20061216 (Anhui, china). Antibodies against CyclinD1, CDK4 were purchased from Boster (Wuhan, China) or β-actin was purchased from Santa Cruz Biotechnology (California, USA), Akt antibodies, phospho-Akt antibodies, H3 antibodies and ac-H3 antibodies were purchased from Cell Signaling (Beverly, MA, USA). HDAC2 antibody was purchased from anbo (San Francisco, CA, USA). HDAC2, cyclinD1, CDK4 and PTEN primers were produced from Shanghai Sangon Biological and Technological Company (Shanghai, China). Secondary antibodies for goat anti-rabbit immunoglobulin IgG horse radish peroxidase (HRP), and goat anti-mouse IgG HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA).
Hepatocellular carcinoma cell (HepG2) was obtained from Biopharmaceutical research institute, Anhui Medical University. HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA), supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mML-glutamine, and 10% fetal calf serum. Cell cultures were maintained at 37°C at an atmosphere of 5% CO2. Melittin was dissolved in dimethylsulfoxide (DMSO) and treated to cells. The final DMSO concentration was not exceeded 0.1% (v/v).
Cellular proliferation was measured using MTT assay. 5×103cells were seeded in 96-well plates at 37°C in a humid chamber with 5% CO2. The cells were treated with TSA and 1, 4 and 8 µg/ml of melittin in media containing 1% FBS for 0, 12, 24 and 48 h. 5 mg/ml MTT was then added to each well and incubated with cells at 37°C for 4 h. the medium was replaced and the formazan crystals were dissolved in 150 µl dimethylsuophoxide (DMSO). The optical density (OD) was determined with Thermomax microplate reader (Bio-TekEL, USA) at 490 nm wavelenth. All experiments were performed in triplicate and repeated at least three times.
HepG2 cells were seeded in 6-well plate. Cells were trypsinized and collected in ice-cold PBS. Cells were resuspended in 300 µL PBS and fixed by adding 700 µL of frozen 100% ethanol, then incubated overnight at −20°C. After fixation, cells were washed with cold PBS and stained with 0.5 ml of propidium iodide (PI) staining buffer, which contained 200 mg/ml RNase A, 50 µg/ml PI, at 37°C for 30 min in the dark. Analyses were performed on BDLSR flowcytometer (BD Biosciences). The experiments were repeated three times.
Total RNA was extracted from human HepG2 cell using TRIzol reagents (Invitrogen). The first strand cDNA was synthesized from total RNA using Thermoscript RT-PCR System (Takara) according to the manufacturer's instructions. RT-PCR was carried out understanding protocol using the primers (
Target RNA | Accession No. | Primer | Sequence |
HDAC2 | NM001527.3 | forward | |
reverse | |||
PTEN | NM000314.4 | forward | |
reverse | |||
CyclinD1 | NM053056.2 | forward | |
reverse | |||
CDK4 | NM000075.3 | forward | |
reverse | |||
β-actin | NM001101.3 | forward | |
reverse |
Relative levels of specific mRNA were determined with a SYBR Green using quantitative real-time PCR detection system (Themal 5100) according to the manufacturer's instruction. PCR was performed at follows: 95°C for 5 min, then 40 cycles of 95°C for 15 s, 60°C for 30 s, 72°C for 30 s, and then a final extension at 72°C for 30 s. Relative expression levels were calculated according to the standard 2−ΔΔCT method using β-actin gene as endogenous control for normalization. Quantitative real-time PCR was carried out with standard protocol using the primers (
The whole cell extracts were prepared, and protein concentration of samples was determined by using a BCA protein assay kit (Boster, China). Whole-cell extracts were then fractionated by electrophoresis through a 12% sodium dodecyl sulf-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were run at a 120 V for 2 h before being transferred on to a PVDF membrane (Millipore Corp., Billerica, MA, USA). After blockade of nonspecific protein binding, nitrocellulose blots were incubated for 1 h with primary antibodies diluted in TBS/Tween20 (0.075%) containing 3% Marvel. Anti-phospho-Akt, anti-Akt, H3 and ac-H3 were diluted 1∶1000. Human monoclonal antibodies directed against PTEN, HDAC2 or β-actin were used at 1∶300 and 1∶500. Following incubation with primary antibodies, blots were washed four times in TBS/Tween-20 before incubation for 1 h in goat anti-mouse or anti-rabbit horse radish peroxidase conjugate antibody at 1∶10000 dilution in TBS/Tween-20 containing 5% skim milk. After extensive washing in TBS/Tween-20, the blots were processed with distilled water for detection of antigen using the enhanced chemilumin escence system. Proteins were visualized with the ECL-chemiluminescent kit (ECL-plus, Thermo Scientific).
RNAi experiments in HepG2 cells were performed by forward transfection in 24 h cultured HepG2 using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's protocol. Small interfering RNA (siRNA) oligonucleotides against HDAC2 genes or scrambled sequences were synthesized by the Shanghai GenePharma Corporation. Transfection was allowed to proceed for various times and cells were processed for different assays. The siRNA transfection efficiency of Lipofectamine RNAiMax in cells was determined by the BlocK-iT Alexa Fluor Red Fluorescent Oligo protocol (Invitrogen). All experiments were performed in triplicate and repeated at least three times. The siRNA sequences were shown in
Sense | Antisense | |
HDAC2siRNA | 5′-CCCAUAACUUGCUGUUAasdfAATT-3′ | 5′-UUUAACAGCAAGUUAUGGGTT-3′ |
Negative control | 5′-UUCUCCGAACGUGUCACGUTT-3′ | 5′-ACGUGACACGUUCGGAGAATT-3′ |
Target gene HDAC2 was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The amplified PCR products were electrophoresed on a 1% agarose gel and were visualized under UV light. The gel-purified PCR segment was cut with both Xho I and BamH I restriction enzymes, and then the segment was introduced into the pEGFP-C2 vector (stored in our laboratory) to form the pEGFP-C2-HDAC2 plasmid. The pEGFP-C2-HDAC2 plasmid was transformed into Tag 1 cells, and the correct fragment was identified by digestion with both Xho I and BamH I and was verified as the correct clone through sequence analysis. The constructed plasmid was transfected into HepG2 cells and observed under fluorescence microscopy assays. All experiments were performed in triplicate and repeated at least three times.
Data are represented as mean±SE. Statistical analysis was performed by using ANOVA followed by Student's test. For changes in mRNA or protein levels, ratios of mRNA (relative expression) and protein (densitometric values) to respective house-keeping controls were compared. Significance was defined as p<0.05 or p<0.01.
To investigate whether melittin affected proliferation of HepG2 cells, HepG2 cells were treated with melittin and TSA for 0, 24, 48 and 72 h and the cell viability was measured. As shown in
Cell viability was assessed for 0, 24, 48 and 72 h by MTT (a), and melittin reduced cell viability in HepG2 cells in a dose-dependent manner. Results (mean ± SE) were calculated as percent of corresponding control values. Representative images of three independent experiments are shown. *P<0.05, **P<0.01, are significant.
Next, to study the potential mechanisms by which melittin suppressed HepG2 cells growth, the effect of melittin on the cell cycle progression was evaluated by flow cytometry. HepG2 cells were also treated with 1, 4 and 8 µg/ml of melittin as well as TSA for 24 h, and cell cycle distribution was then analyzed. As shown in
In order to investigate the mechanisms by which melittin regulated the G0/G1 cell cycle arrest, the effects of melittin on the expression of G0/G1 cell cycle regulatory moleculars were examined by Western blot and RT-PCR analysis. CyclinD1 and CDK4 (
(a and b)Total cellular proteins and RNA were prepared and the expressions of CyclinD1 and CDK4 proteins and mRNA were analyzed using Western blot and RT-PCR. β-actin was used as an internal control. Representative blots and images of three independent experiments are shown. *P<0.05, **P<0.01 vs Control. Statistical analysis was performed by ANOVA.
PTEN is well known to regulate cell proliferation and survival. To validate whether the melittin-mediated cell proliferation is associated with PTEN expression, PTEN expression was examined by Real-time PCR and Western blot. As shown in
The PTEN mRNA were analyzed by real-time PCR (a), and proteins expression was analyzed by Western blot(b). Melittin restores PTEN activity. All gels were representative of at least three independent experiments. *P<0.05, **P<0.01 vs Control.
HDAC inhibitors have been reported to trigger cell cycle arrest. To investigate whether administration of melittin induces cell cycle arrest and inhibits cell proliferation in HDAC-dependant mechanisms, HepG2 cells was treated with melittin in the range from 1 to 8 µg/ml as well as TSA. As shown in
HepG2 cells were treated with 1, 4, 8 µg/ml of melittin and 0.5 µmol/ml TSA for 24 h. The HDAC2 mRNA expression were analyzed by real-time PCR (a). The HDAC2 and ac-H3 proteins expression were analyzed by Western blot (b) and (c). Representative images of at least three independent experiments are shown. *P<0.05, **P<0.01 vs Control.
HepG2 cells were treated with pEGFP-C2, pEGFP-C2-HDAC2 plasmid, pEGFP-C2-HDAC2 plasmid and melittin, pEGFP-C2-HDAC2 plasmid and TSA for 24 h. The mRNA expression of HDAC2 was analyzed by real-time PCR (a). The HDAC2 and ac-H3 proteins expression were analyzed by Western blot (b) and (c). Relative HDAC2 mRNA and protein levels are presented as mean ± standard of the mean (S.E.) of optical densities from three separated experiments. **P<0.01 vs control and empty pEGFP-C2 group. #P<0.05 vs pEGFP-C2-HDAC2 plasmid group.
To examine the function of PTEN in the HepG2 cells. The results of PTEN expression was intensified under TSA. At the same time, it was demonstrated that (
HepG2 cells were transfected with HDAC2 siRNA for 24 h. The HDAC2 and PTEN mRNA expressions were analyzed by RT-PCR (a). The HDAC2, PTEN and ac-H3 proteins expression was analyzed by Western blot (b) and (c). Representative images from 3 to 4 independent experiments are shown. **P<0.01 vs control and scrambled siRNA.
HepG2 cells were treated with pEGFP-C2, pEGFP-C2-HDAC2 plasmid, pEGFP-C2-HDAC2 plasmid and melittin, pEGFP-C2-HDAC2 plasmid and TSA for 24 h. The PTEN mRNA expression was analyzed by real-time PCR (a). The PTEN protein expression was analyzed by Western blot (b). Relative PTEN mRNA and protein levels are presented as mean ± standard of the mean (S.E.) of optical densities from three separated experiments. **P<0.01 vs control and vector. #P<0.05 vs pEGFP-C2-HDAC2 plasmid group.
Akt is a downstream receptor of PI3K that is known to mediate survival signaling. To determine whether melittin-inhibited cell proliferation is closely related to an Akt signal, we examined the phosphorylation levels of Akt in cells following melittin treatment for 24 h. It was demonstrated that (
HepG2 cells were treated with 1, 4, 8 µg/ml of melittin and 0.5 µmol/ml TSA for 24 h. The p-Akt and Akt proteins expression were analyzed by Western blot (a). *P<0.05, **P<0.01 compared to cells without melittin treatment. Silencing of HDAC2 genes in HepG2 cells was performed and phosphorylation of Akt was checked by Western blot (b) as described in the
Mutation or other inactivation of PTEN is a common feature of many types of tumor
Acetylation of histones and non-histone proteins pivotally modulates gene expression and signaling. Acetylated histones contribute to an epigenetic mechanism marking transcriptionally active regions of chromatin
Date from study has been demonstrated that PTEN inhibits cell growth and induces cell cycle arrest through the PI3K/Akt pathway
In summary, melittin may exert anti-proliferation activity. Our experiment results tested the hypothesis that melittin may have inhibitory effects in HepG2 cell cycle progression through HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways (
Melittin downregulates the expression of HDAC2. Then, inhibition of HDAC2 leads to an increased levels of PTEN and promotes Akt inactivation, inhibition of the PI3K/Akt signaling pathways, thereby inhibits cell proliferation.
This project was supported by the National Science Foundation of China (NO 81072686, 81273526) and the funder had designed the study, analyzed the data and wroted the manuscript.