DNA microarray assay and analysis
HepG2 cells were treated different doses of LC for 24 h, and then cells were collected and extracted with TRIzol agents. RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. DNA microarray was performed by KangChen biotech company (Shanghai) in compliance to MIAME guidelines. The Human 126135K Gene Expression Array was manufactured by Roche NimbleGen. Briefly, About 5 mg total RNA of each sample was used for labeling and array hybridization as the following steps: (1) Reverse transcription with by Invitrogen Superscript ds-cDNA synthesis kit; (2) ds-cDNA labeling with NimbleGen one-color DNA labeling kit; (3) Array hybridization using the NimbleGen Hybridization System and followed by washing with the NimbleGen wash buffer kit; (4) Array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation). Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Fold increases of gene expression were calculated compared to the vehicle control.
HDAC activity assay in vitro and in cultured cells
HDAC activity in vitro and in cultured cells was detected with The HDAC-GloTM I/II Assay and Screening System (Promega, USA) following the standard protocol. HDAC assay was performed in white 96-well plate. For in vitro HDAC activity assay, cell lysate (optimal protein content: 1 mg) was incubated with various agents in a HDAC assay buffer (100 ml) at 37uC for 60 mins and then 100 ml HDACTM I/II reagent was added, after 30 mins, luminescence was measured. For HDAC assay in cultured cells, cells (10000 cells/well) were treated with LC or positive control agents for 6 h or 12 h, then cell medium was changed to serum-free medium plus HDAC buffer (50 ml+50 ml). After 15 min incubation, HDACTM I/II reagents (100 ml) were added to the cells, luminescence was measured after 30 mins.
Statistical methods
Unless indicated otherwise, Mean+SD are presented where applicable. Unpaired Student’s t-test or one way ANOVA is used where appropriate for determining statistic probabilities. P value less than 0.05 is considered significant.Results LC treatment selectively inhibits cancer cell growth in vivo and in vitro
It has been well known that most cancer cells depend on glycolysis for ATP generation. To further confirm that in our cell systems, multiple cancer cell lines were used to test whether oligomycin could inhibit ATP production. The result in representative cancer cells was shown. It was found that in the presence of L-glucose in the culture medium, oligomycin quickly depleted ATP production while in the presence of D-glucose in the culture medium oligomycin did not affect ATP generation (Fig. 1a). Since L-glucose can not be used to produce ATP production [10], this result implies that cancer cells mainly rely on D-glucose glycolysis for ATP production, consistent to previous reports [4,5]. We next investigated whether LC treatment could increase intracellular ATP production in cancer cells. Consistent to our prediction, LC treatment could not further increase ATP production in both hepatic HepG2 and SMMC-7721 cancer cells (Fig. 1b). Quantitative real-time PCR
Total RNAs were extracted from HepG2 cells with TRIzol reagent. Reverse transcription of purified RNA was performed using superscript III reverse transcription according to the manufacturer’s instructions (Invitrogen). Quantification of all gene transcripts was done by quantitative using the TaKaRa SYBR Premix Ex Taq kit with Applied Biosystems 7500 Fast Real-Time PCR system. in contrast to the effect in cancer cells, oligomycin, when added in mouse thymocytes cultured with D-glucose, time-dependently inhibited ATP generation (Fig. 1c) while LC efficiently increased intracellular ATP content at different time points under this condition (Fig. 1d). These results confirm that LC is able togenerate ATP in normal mouse thymocytes, but not in hepatic HepG2 and SMMC-7721 cancer cells. Next we compared the effects of LC on normal tissue and cancer growth in vivo. Normal Balb/c mice or Balb/c nude mice inoculated with HepG2 cancer cells were i.p. injected with LC (400 mg/kg) for 15 days except day 8, followed by termination of the experiment. This dose schedule is a tolerated dose for Balb/c nude mice. Organ weight and tumor weight of each mouse treated by LC or control were compared. It was found that LC treatment inhibited more than 70% of cancer growth, while the same treatment decreased less than 20% of the normal organ development and body weight (Fig. 2a). We then investigated the effects of LC on cell proliferation by MTS assay. It was found that LC dose-dependently decreased HepG2 cell viability (Fig. 2b), associated with the cell cycle arrest at the G0/G1 phase (Fig. 2c) and low levels of cell death (Fig. 2, d and e). These results implied that LC predominantly induced cell proliferation inhibition but slightly induced cell death in cancer cells. Finally, in normal mouse thymocytes, LC had little effect on thymocyte viability and cell count within 24 h treatment (Fig. 2f). These results confirm that LC selectively induced cancer cell cytotoxicity in vitro and in vivo.
LC treatment induces the expression of p21cip1 gene, mRNA and protein in cancer cells
We next determined the molecular mechanism for how LC could selectively inhibit tumor growth. To do so, the gene expression profile was investigated in HepG2 cells after three doses of LC treatment (2.5, 5, and 10 mM) for 24 h. All the up-regulated and down-regulated genes (at least 2 fold increase or decrease at all the three doses) after LC treatment were summarized (data not shown). Among the up-regulated genes, p21cip1 gene but not p27kip1 gene, two important genes associated with cell cycle regulation, was found to be highly and dose-dependently expressed (Fig. 3a). The effects of LC treatment on p21cip1 and p27kip1 mRNA levels were next determined by real-time PCR. HepG2 cells were cultured with or without LC (2.5, 5, 10 mM) for either 12 h or 24 h, RNA were prepared from the cells. After treatment with LC, the p21cip1 mRNA level dose-dependently increased both at 12 h and 24 h time points, and p21cip1 mRNA level is relatively higher after 12 h treatment than 24 h treatment, while p27kip1 mRNA did not show much change at these two time points (Fig. 3b). To determine the protein levels of p21cip1 and p27kip1, Western blot assay was performed in the treated HepG2 cells. The results showed that after treatment with various doses of LC for 48 h, p21kip1 protein levels increased in a dose-dependent manner in HepG2 cancer cell lines (Fig. 3c, left). Further dynamic study in HepG2 cells showed that after treatment with LC (5 mM) for 12, 24, 36, and 48 h, p21cip1 time-dependently increased (Fig. 3c, middle). In SMMC7721 cells LC treatment also increased p21 protein expression in a dose-dependent manner similar to in HepG2 cells (Fig. 3c, right). Unexpectedly, p27 protein increased in both dose- and time-dependent manners after LC treatment (Fig. 3c) even though p27kip1 mRNA level is stable, suggesting that LC might inhibit the degradation of p27 protein. To further investigate the effect of LC on the downstream effects of p21cip1 and p27kip1, levels of unphosphorylated Rb and phosphorylated Rb (phos-Rb) were detected. It was found that LC treatment slightly decreased Rb protein but dramatically decreased Phos-Rb protein (Fig. 3d). These results demonstrated that LC selectively induced p21cip1 expression.
