| Objective:Gastric cancer is really a serious threat to people's health. It accounts the second incidence and cancer mortality of worldwide, while in our country its incidence ranks first in all kinds of tumors,and the number of people die from it each year occupies nearly 1 / 4 of all cancer deaths.Etoposide (etoposide, VP-16) is the common cancer chemotherapy drug in modern clinical,which is used for a variety of tumors and displays very good effect. VP-16 is one of the derivatives of podophyl- lotoxin and belongs to DNA topoisomerase II (Topo II) inhibitor. The inhibition of Topo II is known to be a major mechanism for the action of VP-16 by stabilizing the cleavable complexes and inhibit the rejoin of DNA so that result in the damage of DNA that trigger cell to death. In clinical practice,the joint application of HLFP and VP-16 for those inoperable or recurrent gastric cancer patients after surgery has achieved good curative effect. Therefore, the in-depth understanding of the targets of VP-16 is helpful to reduce the drug resistance and increase the treatment of gastric cancer.SIRT1 is a member of the nicotine adenine dinucleotide (NAD)-dependent deacetylase --sirtuin family,which has the homologization with yeast SIR2 gene. SIRT1 has the protein deacetylase activity, through which it takes part in the pathophysiology and physiology process such as cell metabolism,cell aging and the development of tumor.E2F is a family able to encode a series of transcription factor genes,and it is an important regulating factor in cell cycle. Among which E2F1 is an important positive cell cycle regulatory factor, which plays an important role in the process of cell circle from G0 / G1 phase to S phase. Meanwhile, E2F1 shows a key function during cell proliferation and cell apoptosis progress . There are rare reports in the expression of SIRT1 and E2F1 during gastric cancer cells apoptosis progress in current literature. In this study, we use human gastric cancer BGC823 cells as study object , with the application of MTT, Hoechst 33258 staining and flow cytometry techniques, we observed the growth inhibition and promotion of apoptosis to BGC823 cells in vitro due to VP-16. Meanwhile, with the application of immunocytochemistry,RT-PCR and Western blot,we detected the expression changes of SIRT1 and E2F1 during VP-16 inducing apoptosis of gastric cancer cells , to further explore the relationship between SIRT1,E2F1 and tumor cells , and provide help in increasing the awareness of targets of VP-16 during the treatment of tumor.Methods:1 Cell culture1.1 cell resuscitationBGC823 cells were removed from the liquid nitrogen canister, into the warm water of 37℃quickly, dissolved and mixed in RPMI 1640 medium which plus 10 times the mix, by 500 ~ 1000 rpm / min centrifugal removal of frozen liquid, and washed once with culture medium. Subsequently, cells were transferred to culture bottles, added with the medium (containing 10% fetal calf serum (FCS), penicillin 100 U/ml, streptomycin 100μg/ml)and cultured at 37℃in humidified 5% CO2 incubator.1.2 cell passageWhen the cells covered the full bottom of culture flask surface, absorb the culture medium, add 0.04% EDTA, 37℃digesting for 8 minutes. Discard the digestion, wind and boast the cells with the culture medium again and again . Based on cell concentration, divide the cells into 2-3 culture bottles and continue to culture.2 Cell inhibition effect measured by MTT assay of BGC823 cells due to VP-16:2.1 VP-16 dilution and concentration selected:VP-16 was diluted with serum-free medium, in accordance with the different concentrations the cells were added into the culture bottles in different holes, so that the final concentration of drugs were 10ug/ml, 20ug/ml, 40 ug / ml, 80 ug / ml, 160 ug / ml, 320 ug / ml.2. 2 Cell inhibition rate measured by MTT assay:The log-phase growth of BGC823 cells of different groups were detachated with 0.04% ethylenediamine tetraacetic acid (EDTA) and seeded in 96-well tissue culture plates respectively at 200μl/well of 1×105 /mL cell concentration, incubated at 37℃in humidified 5% CO2 incubator. Then in accordance with the experimental groups were added by the serum-free RPMI 1640 diluted drugs, each compound were made six holes, Amomg the totle, the blank control group was only added by the medium .While the control group added by DMSO. The cells were cultured for 24 hours and 48 hours separately . Then 20μl/well of 5 mg/ml MTT solution was added and incubated for 4 hours. After centrifugalization the MTT (Methyl thiazolyl tetrazolium) solution 1000 r / min for 10 minutes and cell supernatant was removed and the cells and dye cristals were dissolved by adding 150μl/well of dimethylsulfoxide (DMSO) to terminate reaction and then oscillationed by oscillation apparatus for 10 minutes. The absorbance numerical value was measured at 490 nm wavelength with ELX800 microplate reader. According to 24 hours and 48 hours of A value , we drew the growth curve and calculated the IC50 of 24- hour and 48-hour. The assays were repeated for six times.3 cell nuclear morphological changes measured by Hoechst 33258 fluorescence staining :The log-phase growth of BGC823 cells of each group were collected, and made into a single cell suspension,washed with PBS and mixed with fluorescence reagent (Hoechst 33258) 0.5ml for 10 minutes. Then, cells were examined under high power lens of fluorescence microscope to observe morphological changes. The assays were repeated for three times.4 The distribution of cell cycle and cell apoptosis rate detected by flow cytometry (FCM) :The cell cycle: Experimental groups the same as above . The log-phase growth of BGC823 cells of each group were collected, and made into a single cell suspension, PBS washed two times , 70% ethanol fixed for 24 hours at 4℃, and stained using EB for 30 minutes. The cell cycle was detected using flow cytometer. The cell cycle was analyzed by the software ModFit LT 2.0 , Calculated the G0/ G1,S,G2/M phase distribution percentages. The experiment was repeated for 3 times.The cell apoptosis : Experimental groups the same as above . The log-phase growth of BGC823 cells of each group were digested with 0.25% trypsin, and collected, PBS washed twice, each added 1 mL of PBS, re-suspension the cells and counted to about 1×106 / mL single-cell suspension for 0.5 mL . Quickly added with 4 mL cold 70% ethanol and mixed thoroughly, 4℃fixed for 24 hours , PBS washed once, using flow cytometry to detect the 24-hour cell apoptosis rate of each group. The experiment was repeated for 3 times.5 The expression of SIRT1 and E2F1 proteins analysed by immunocyt-ochemistryExperimental groups the same as above. They were seeded onto glass-coverslips in 6 well culture plates. After confluence, medium was removed and the cells were fixed with 4% paraformaldehyde for 15 min followed by treatment with 3% H2O2 for 10 min, 0.3% Triton-X-100 for 10 min and 10% normal goat serum for 15 min. The rabbit primary antibody against human SIRT1 and E2F1 were used overnight at 4℃, primary antibody was replaced by PBS as negative control. Goat anti-rabbit secondary antibody and peroxydase labeled strepto-avidin were incubated respectively for 40 min at 37℃. Cells were stained with 0.05% DAB, then dehydrated with alcohol gradually, sealed with neutral gum after transparence with xylol. After all, cells were observed under light microscope. The experiment was repeated for 3 times.6 The expression of SIRT1 mRNA and E2F1 mRNA detected by RT-PCR :Primers of human SIRT1,E2F1 andβ-actin were designed with Premier 5.0 software according to their nucleotides sequence. Total RNA was extracted from each groups of BGC823 cells with Trizol reagent. 2 microgram of RNA was reversely transcribed using random hexamer primers in a thermocycler (37℃for 60 minutes and 95℃for 5 minutes). After predenaturation at 95℃for 10 minutes, cDNA amplification was performed. PCR amplification products were separated in a 1.5% agarose gel, photographed and analysed with gel image analysis system. Data was presented as the ratio of the integrated optical density (IOD) of SIRT1 and E2F1 to that ofβ-actin acting as an internal standard. The experiment was repeated for 3 times.7 The expression of SIRT1 and E2F1 protein detected by western blot-ting assay :The protein was abstracted on ice from each groups of BGC823 cells lysed in buffer. Protein concentration was determined using coomassie brilliant blue G250 protein assay kit. Samples (50μg) were separated by SDS-PAGE and were transferred to a nitrocellulose filter (NC filter). The membrane was blocked for 1 hour at room temperature with blocking solution containing 5% nonfat milk and then incubated with primary antibody overnight at 4℃. The membrane was washed and incubated with secondary antibody (goat anti rabbit IgG-horseradish peroxidase) at 37℃for 1 hour and then washed again and developed using the ECL ( enhanced chemiluminescence ) kit according to the supplier's instructions. The membrane was photographed and then analysed with gel image analysis system expressed as the ratio of the integrated optical density (IOD) of SIRT1 and E2F1 to that ofβ-actin acting as an internal standard. The experiment was repeated for 3 times.8 Statistical analysesThe results were expressed as mean±standard deviation and evaluated with SPSS 13.0 solftware by analysis of variance (One-Way ANOVA).P<0.05 was consided statistically significant.Results:1 Cell growth inhibition effect measured by MTT assay of BGC823 cells due to VP-16:The results showed that, VP-16 inhibited the growth of BGC823 cells, in a dose-dependent and time-dependent way, we calculated the cell proliferation rate of different drug concentrations of BGC823 cells according to the absorbance value (Tab.1). with the application of IC50 calculation software we get half of the inhibition concentration of BGC823 cells to VP-16 for 24 hour is 81.30ug/ml, 48 hours is 64.54ug/ml. According to the IC50 value ,we divided the cells into one control group (without adding VP-16) and three experimental groups : 40ug/ml VP-16 group, 81.30ug/ml VP-16 group and 120ug/ml VP-16 group. The experiments below all followed these groups. According to the A values of 24 hours and 48 hours, we drew the growth curve (Fig.1)2 Hoechst 33258 staining to detect ratio of nuclear damage:Ratio of nuclear damage in the control group,40ug/ml VP-16 group, 81.30ug/ml VP-16 group, 120ug/ml VP-16 group were separately 4.52±0.56 %,38.34±2.35%, 52.47±3.67%, 68.62±4.22%. With the increase in drug concentration, the percentage of nuclear damage increased significantly compared with the control group which were significantly different (P <0.05). (Fig.2 A,B,C,D; Table 2)3 The distribution of cell cycle and cell apoptosis rate by FCMThe G0/G1 phase cell rate of control group, 40ug/ml VP-16 group, 81.30ug/ml VP-16 group, 120ug/ml VP-16 group were 56.1±3.12%, 26.9±2.14%, 19.1±2.03% and 5.96±1.87%; the S phase cell rate were 33.2±2.98%, 60.4±3.09%, 65.9±3.29%, 75.8±3.42%; the G2/M phase cell rate were 10.8±2.17%, 12.7±2.28%, 15.0±2.31% and 18.2±2.42%. With the increase in drug concentration, the S phase cells increased significantly compared with the control group which were significantly different (P <0.05). showing there was S phase arrest. (Fig.3,Table 3)There was no obvious peak of apoptosis before the diploid peak of the control group. Its apoptosis ratio was 0.98±0.09%. There were obvious peaks of apoptosis before the diploid peaks of the drug groups, the apoptosis ratio of 40ug/ml VP-16 group, 81.30ug/ml VP-16 group, 120ug/ml VP-16 group were 7.21±0.38%,11.13±0.51% and 15.46±0.62%. With the increase in drug concentration, the apoptosis ratio of cells got significantly higher compared with the control group which were significantly different (P <0.05). (Fig.4,Table 4)4 Expression of SIRT1 and E2F1 protein analysed by immunocytochemistry The immunopositive SIRT1 staining were most located in cytoplasm. There was also a small amount of immunopositive staining in nucleus. Positive experimental group cells were fewer and light brown.(Fig.5 A,B;)The immunopositive E2F1staining were most located in nucleus. Positive experimental group cells were more numerically and dark brown. (Fig.5 C,D;)The SIRT1 protein expressions of experimental group cells were reduced in compare with control group cells. While the E2F1 protein expressions of experimental group cells were increased in compare with control group cells.5 Expression of SIRT1mRNA and E2F1mRNA detected by RT-PCR : The integrated optical density (IOD) ratios between SIRT1 andβ-actinin control group, 40ug/ml VP-16 group, 81.30ug/ml VP-16 group, 120ug/ml VP-16 group cells were 1.33±0.05,1.02±0.03,0.84±0.03和0.61±0.02 respectively; those between E2F1 andβ-actin were 0.85±0.04,1.10±0.05,1.38±0.06和1.69±0.08 respectively; The results showed:With the increase in drug concentration, the expression of SIRT1 mRNA got apparentely lower, compared to the control group, there were significant differences (P <0.05); While the expression of E2F1 mRNA got apparentely higher, compared to the control group, there were significant differences (P <0.05). (Fig.6,Table 5)6 Expression of SIRT1 and E2F1 protein detected by Western blot :The ratios of IOD between SIRT1 andβ-actin protein in control group, 40ug/ml VP-16 group, 81.30ug/ml VP-16 group, 120ug/ml VP-16 group cells were 1.42±0.06,1.19±0.08,0.98±0.06和0.76±0.04;Those between E2F1 andβ-actin were 0.79±0.03,1.04±0.04,1.27±0.05和1.51±0.07;The results showed : With the increase in drug concentration, the expression of SIRT1 protein got lower apparentely, compared to the control group, there were significant differences (P <0.05); While the expression of E2F1 protein got higher apparentely, compared to the control group, there were significant differences (P <0.05). (Fig.7,Table 6)Conclusions :1 VP-16 can inhibit the proliferation of BGC823 cells, through a dose-dependent and time-dependent way.2 VP-16 can block BGC823 cells at S phase, and induce cell apoptosis.3 VP-16 inhibiting the growth of BGC823 cells and inducing cell apoptosis are related to its reducing SIRT1 expression and increasing E2F1 expression.
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