| BackgroundThe morbidity and mortality of gastric cancer are both the first place in our country.Because most of the patients are in middle-late stages due to the lack of special symptoms in the early stage,the survival rate of gastric cancer is not yet satisfactory.Chemotherapy is one of the main means for gastric cancer,but there are few drugs effective for gastric cancer despite of the fact that more and more anticancer drugs are brought into clinical practices.5-Fluorouracil(5-Fu)keeps as one of the first place and main drugs for gastric cancer to updates.The common way to increase the sensitivity of drugs is to increase the dose of drug or to combine different drugs,but they don't work well in gastric cancer for the low sensitivity of gastric cancer to most of the drugs.The higher dose of chemotherapy drugs could increase the effects to some degree,but the toxicity to normal tissue or organs(such as bone marrow,heart,liver,et al)increases even more.It is the hot point for the treatment of gastric cancer to combine different drugs of different mechanism so as to decrease the dose and side effects of respective drug.Thalidomide is an antiangiogenic and immunomodulatory drug that was first used for the treatment of pregnancy-associated morning sickness,It subsequently was withdrawn for the birth of children with severe malformations in the limbs and internal organs.The return of Thalidomide as a therapy agent in cancers and infections stems from its broad array of anti-inflammatory and anti-angiogenic effects,as demonstrated in diseases such as HIV,Crohn's disease and leprosy,and cancers.The cell target and mechanism of the action of Thalidomide are poorly understood yet.This study aimed to investigate the proliferation inhibition and apoptosis induction effect of Thalidomide,5-Fu and their joint effect on MGC-803 cell.We also studied the corresponding presentation changes of apoptosis-related genes in MGC-803 cell.MethodsMGC-803 cell of different density 1×10~3,1×10~4,5×10~4,1×10~5,1×10~6/ml were plated in 96-well plates,the volume in each well is 100μl,100μl culture media as the simulation of drug were added into every well the next day after plating,MTT analysis were performed on 24,48,72,96,120 hours beginning from the next day of plating,standard cell proliferation curves of different density were made according to the OD value at different time.The suitable original plating density for corresponding checking period could be selected resulting from the curve. According to the result of above,MGC-803 cells of 4×10~4/ml density were plated in 96-well dishes for following test groups,and drugs were added next day which was defined as the beginning time.MTT assay was conducted at 24 and 48h.Test groups were divided into three parts:Five Thalidomide groups which concentration of Thalidomide in the culture system was 6.25,12.5,25,50,100μg/ml respectively (the maximal concentration of DMSO which was the solvent of Thalidomide was 0.1%).Four 5-Fu groups with 6.25,12.5,25,50μg/ml 5-Fu in the culture system for each group.Four combined groups inclduded 5-Fu 25μg/ml+Thalidomide 50μg/ml,5-Fu 25μg/ml+Thalidomide 100μg/ml,5-Fu 12.5μg/ml+Thalidomide 50μg/ml,5-Fu 12.5μg/ml+Thalidomide 50μg/ml.The control group was added RPMI medium with 0.1%DMSO in it instead of the drugs.There was a vehicle control group which was just added RPMI media with 0.1%DMSO in it without cells. The morphological changes of MGC-803 cells could be found by converted phase contrast microscope for liable cells,ordinary light microscope for HE stain and fluorescence microscope which needed the AO/EB stain in advance.MTT analysis and FACs were used to estimate the proliferation inhibition effect and apoptosis induction effect of two drugs alone or jointly.The expression of apoptosis-related genes—peroxisome proliferator-activated receptors(PPAR-γ)and Bcl-2 was checked by immunocytochemical stain(ICC).ResultMGC-803 cells of 1×10~3—5×10~4/ml density showed linear proliferation corresponding to the time within 72 hours and they were all suitable for the following test.4×10~4/ml was selected as the original plating density of 96-well plate for the convenience of calculation when the cells were resuspended before plating.The cells of 1×10~3/ml density showed linear proliferation even on 120h time point,and could be used as suitable original plating density for longer period test.The MGC-803 cells in all test groups showed morphological characteristic of apoptosis despite of converted phase-contrast microscope,ordinary light microscope or fluorescence microscope.By using converted phase-contrast microscope,the cell number decreased conversely with the increase of the concentration of Thalidomide or 5-Fu.It became significantly less in combined groups and there were many floating cell fragments in the supernatants.The attached cells were less bright compared with control cells.Some of the cells showed the apoptosis characteristics:contracted cytoplasm in apparently smaller round or irregular cells,and chromatin in nucleolus condensed or broken.As for HE stained cells under the ordinary light microscope,the cells became sparse with contracted cytoplasm in apparently smaller round or irregular cells,and chromatin in nucleolus was stained strongly or formed the chromatin mass gathered around the edge of nucleolus forming crescent figure.Some cells were broken into fragments.Part of the cell membrane bulged and fall off into apoptotic bodies.By using fluorescence microscope,chromatin was stained orange gathered around the edge of nucleolus in the condensed cells.Part of the cell membrane bulged and fall off into apoptotic bodies.The results of MTT array were as below:For Thalidomide groups,Thalidomide of all testing concentrations could inhibit the proliferation of MGC-803 cell,and the proliferation inhibition rate increased accompanying the increasing of drug concentration and extending of the time.The inhibition rate of each group at 24h is 0.7%,1.4%,5.6%,15%and 18.1% respectively,and those at 48h are 0.8%,2.7%,7.3%,14.7%and 18.9%respectively. The difference was significant between Thalidomide groups of 25μg/ml or above concentration and the control group(P＜0.05,P＜0.01=.Thalidomide groups of 6.25μg/ml and 12.5μg/ml concentration didn't differ significantly from the control group(P＞0.05),and there was also no significant difference between these two groups(P＞0.05).There was also no significant difference between 50μg/ml and 100μg/ml group at 24 h(P＞0.05),but the difference became significant when the cells were incubated in drug media for 48 hrs(P＜0.05).For 5-Fu groups,5-Fu of all testing concentrations showed significant difference compared with the control group no matter the drug incubation time was 24 or 48 hrs(P＜0.01).There was no significant difference between 6.25μg/ml and 12.5μg/ml group as well as between 25μg/ml and 50μg/ml group at 24h(P＞0.05),but the result was quite different at 48h,there was significant difference regardless of any two groups(P＜0.01).For combined groups with the same concentration of 25μg/ml 5-Fu,combined groups showed distinct difference from the corresponding 5-Fu group(P＜0.01),but two combined groups showed no distinct difference with each other(P＞0.05).For combined groups with the same concentration of 5-Fu 12.5μg/ml,5-Fu+50 or 100μg/ml Thalidomide showed no distinct difference from 5-Fu group of 25μg/ml(P＞0.05).With FACs study,the concentration of two Thalidomide groups is 12.5,50μg/ml respectively,the concentration of 5-Fu group is 25μg/ml,the concentration of combined group is 5-Fu 25μg/ml+Thalidomide 50μg/ml,the control group is the culture medium with 0.05%DMSO in it instead of drug,the apoptosis rate of above groups is 3.31%,6.7%,13.3%,19.2%,1.05%variably.All test groups showed significant difference compared with the control group(P＜0.01).There was also significant difference between two test groups arbitrarily(P＜0.01=.With IHC analysis,Thalidomide 12.5,50μg/ml or 5-Fu 25μg/ml,5-Fu 25μg/ml+Thalidomide 50μg/ml significantly increased P P A R—γprotein expression and decreased Bcl—2 protein expression compared with control group in MGC-803 cells(P＜0.01).There was significant difference between combined group and single drug group,too(P＜0.01).ConclusionBoth Thalidomide and 5-Fu could inhibit the proliferation and induce the apoptosis of MGC-803 cells.The effect was reinforced with the combination significantly.Combination with Thalidomide could decrease the concentration of 5-Fu but have the similar effect.Increased PPAR—γprotein expression and decreased Bcl—2 protein expression was maybe relative with the proliferation inhibition and the apoptosis induction caused by Thalidomide and 5-Fu.SignificanceThere are more and more evidences that Thalidomide is a anti-angiogenic and immunomodulatory drug as well as a apoptosis induction drug.It become a hot point for the research of anti-tumor effect.Uptate preclinical and clinical practices approve the fact that Thalidomide can inhibit effectively the proliferation,invasion and metastasis of malignant tumors,and has achieved some inspiring results.Thalidomid can be combined with chemotherapy drugs to improve the effects without the aggravation of bone marrow inhibition.Although the effect of Thalidomide couldn't be consistent in various kinds of tumors,it is believed more sorts of tumors can be confirmed to response to Thalidomide.It needs much efforts to understand the mechanism,applicability and the coordination with other drugs of course.We used MTT array,ICC stain,FACs to study the effects of the proliferation inhibition and apoptosis effect of Thalidomide,5-Fu and their joint effect on MGC-803 cell and to study the presentation of apoptosis-related genes in MGC-803 Cell.It demonstrated the relationship of Thalidomide and MGC-803 cell,and explained whether the mechanism of Thalidomide is relative with P P A R—γ.It will fill up the blank of this field,and open further our mind in the treatment of solid tumors with Thalidomide.
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