Figure 1.
Activations of MAP kinases were involved in DHA-induced apoptosis.
A. DHA induced apoptosis in liver cancer cells. Cells treated with either DMSO or 10 μM DHA for 24 and 48 h were stained with both Annexin V and Propidium Iodide (PI) for 45 min. Apoptosis induced by DHA was then assessed by flow cytometer analysis. B. The percentage of apoptotic cells were shown, after quantitative analysis of PI/Annexin V assay. Data are presented as mean±SD of three independent experiments. *P<0.05, versus the DMSO group. C. MAP kinases were activated by DHA. Cells were treated with 10 μM DHA for indicated time. The phosphorylation of p38, ERK and JNK was determined. D. Quantitative data from three independent experiments were shown to indicate the relative expression of p-p38, p-JNK, and p-ERK.
Figure 2.
Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis. A.
PD98059, an inhibitor of ERK phosphorylation, enhanced DHA-induced decrease of cell viability. Cells were pretreated with 10 μM PD98059 for 2 h, and then incubated with 10 μM DHA for another 24 h. The residual cell viability was determined by MTT assay. Data are mean ± SD of three independent experiments, *P<0.05. B. PD98059 treatment increased the production of apoptotic body. Cells pretreated with 10 μM PD98059 for 2 h, and then incubated with 10 μM DHA for another 24 h were stained with Hoechst 33342 dye. DNA fragmentation (indicated by asterisks) and nuclear condensation (denoted by arrows) were observed under a fluorescence microscope. C. PD98059 treatment enhanced DHA-induced apoptosis. TUNEL assay was performed to determine apoptosis. The number of apoptotic cells was determined and the percentage was indicated by histogram. *P<0.05. D. PD98059 plus DHA treatment led to cleavages of PARP and caspase 3. Proteins collected from cells treated with PD98059 and DHA for 24 h were subjected to western blot to detect the cleavages of PARP and caspase 3. Actin was used as a loading control.
Figure 3.
HDACi facilitated DHA-induced apoptosis. A.
Combination treatment with HDACi and DHA increased cell death. Cells were treated with 4 mM NaB, 1.25 μM SAHA, 10 μM DHA or combination of NaB/SAHA and DHA for 24 h. Cell viabilities were measured by MTT assay. B. The inhibitory effect of HDACi and DHA in combination on liver cancer cell growth was further confirmed by colony formation assay. One hundred of cells were seeded into 6-well plates for 7 d, and then cultured with either HDACi or DHA for another 7 d. Colonies were stained with 0.05% crystal violet. The number of colony in each well was counted and statistical analysis was performed. Data are presented as mean ± SD of three independent experiments. C. The effect of HDACi on DHA-induced apoptosis was measured by TUNEL assay, using in situ cell death detection kit. Hep G2 cells treated as described in A were subjected to TUNEL assay. Apoptotic cells were observed under fluorescent microscope. D. The effect of HDACi and DHA in combination was further confirmed by TUNEL assay, using flow cytometry. Percentage of apoptotic cells was calculated. E. Caspase 3 activation was involved in HDACi-mediated apoptosis in cells treated with DHA. The activity of caspase 3 in cells treated as described in A was determined and the relevant change was shown. For A, B, D and E, *P<0.05, **P<0.01, versus the DHA group.
Figure 4.
HDACi exposure in DHA-treated cells enhanced decreases of Mcl-1 and p-ERK. A.
Combination treatment with HDACi and DHA resulted in downregulation of Mcl-1 and upregulation of Bak. liver cancer cells were exposed to 4 mM NaB, 1.25 μM SAHA, 10 μM DHA or combination of NaB/SAHA and DHA for 24 h. Expression of Mcl-1, Bak and cleaved PARP was examined by western blot. Upper panel: a representative result was shown. Bottom panel: the relative expression of Mcl-1 and Bak normalized to Actin was indicated by histogram. B. The expression of p-ERK was reduced in cells treated with HDACi and DHA. Proteins collected from liver cancer cells treated with SAHA, DHA or the combined drugs were subjected to western blot to examine p-ERK expression. Upper panel: a representative result was presented. Bottom panel: the relative expression of p-ERK/ERK was shown. C. Reduction of mitochondrial membrane potential (MMP) was induced in HDACi/DHA-treated cells. Hep G2 and PLC/PRF/5 cells were treated as described in A. The MMP collapse (ΔψmL) was measured by flow cytometry after staining the cells with DioC6 and quantitative analysis of Δψm was shown. The data represented mean±SD of three independent experiments. D. Cytochrome c was released to cytosol in treated cells. Cells were treated as described in A. Fractions of cytosol were isolated to examine the distribution of cytochrome c. β-Actin was used as the marker for cytosol. E. HDACi pretreatment sensitized cells to DHA-induced apoptosis. liver cancer cells pretreated with 4 mM NaB or 1.25 μM SAHA for 2 h were further exposed to 10 μM DHA for another 24 h. TUNEL assays were performed to determine apoptosis. The percentage of TUNEL-positive cells was shown. For B, C and E, *P<0.05, versus the DHA group.
Figure 5.
SAHA enhanced antitumor effect of DHA on Hep G2 xenograft tumor in mice. A.
Combination of SAHA and DHA noticeably halted the growth of Hep G2 xenograft tumor. Nude mice were inoculated with 1×107 of Hep G2 cells. After the formed tumor was palpable, mice were randomly divided into four groups. DHA (5 mg/kg mouse body weight) was given to the ‘DHA’ group, SAHA (1 mg/kg mouse body weight) was given to the ‘SAHA’ group, combination of SAHA and DHA was given to 'DHA+SAHA' group, once daily for five consecutive days per week for 24 d. The tumor volumes were calculated every two days. Six xenografts were performed in each group. Data are mean±SD, *P<0.05, versus the DMSO group. B. Combination of SAHA and DHA treatments resulted in a dramatic decline of tumor weight. On day 24, mice were sacrificed, and the tumor weights were measured. C. Apoptosis was induced in vivo. Tumors were sectioned and apoptosis was determined using in situ cell death detection kit. Apoptotic cells were observed under fluorescent microscope. D. SAHA significantly increased apoptosis in DHA-treated mice. Percentages of apoptotic cells were measured by counting the number of green cells under five random fields.
Figure 6.
Decreased expression of Mcl-1 and increased levels of active PARP and cleaved caspase 3 were recorded in HDACi/DHA-treated mice.
SAHA enlarged the apoptotic region caused by DHA treatment in Hep G2 xenograft tumor. Tumors were excised and subjected to H&E staining for determination of pathological evaluation. On the other hand, tissues of xenografts were subjected to immunochemistry to detect the expression of Ki-67, p53, Mcl-1, p-ERK, and active PARP. Original magnification ×400.