Experimental Study Of The Anti-proliferation Effect And Its Mechanism Of Gossypol For Esophageal Cancer SEG-1 Cell In Vitro And In Vivo On Nude Mice Model

BackgroundsEsophageal cancer is currently a malignant tumor which has a serious threat to the life and health of human beings. Its mortality rate is ranked the sixth in all malignant tumors, and in some high-risk area of China is the first among all the malignancies. In recent years, the study of etiology and pathogenesis of esophageal cancer has made great progress, but it is still not fully elucidated and the morbidity and mortality rate has remained high in some high incidence area. Esophageal cancer is one of the highest malignant tumor among digestive tract tumors, and there are about 300 thousand people died of it. Until now, our country has carried out esophageal cancer surgery and chemotherapy for decades. Although chemotherapy plays an important role on the improvement of the patients' prognosis, the safe and reliable drug has not been yet found. So to find a safe and reliable drug is a hotspot in chemotherapy of esophageal cancer.Therefor this study conducted in vitro study of human esophageal carcinoma seg-1 cells with gossypol, in order to understand the gossypol's influence on cell proliferation and cell cycle of SEG-1 cell, explore gossypol's mechanism of esophageal cancer, and provide for experimental basis of gossypol's clinical applications in the treatment of patients with esophageal carcinoma.Part 1 The anti-proliferation effect of Gossypol on human esophageal cancer SEG-1 cell in vitro and its mechanismObjective and methods1.This experiment used trypan blue staining method to observe the gossypol's anti proliferation effect on esophageal carcinoma cell line SEG-1 cell.2. This experiment used several methods to observe the inducing apoptosis of gossypol on esophageal carcinoma cell line SEG-1 cell. It used Wright-Giemsa staining, Hoechst33258 fluorescence microscopy and transmission electron microscopy to observe the esophageal cancer cell morphology after using gossypol. It used agarose gel electrophoresis to observe ladder-type band after using gossypol and used flow cytometry cell to quantitatively analysize the DNA content.3. By determinating the activity of caspase-9 and caspase-3 and expression changes of Bcl-2 and Bcl-XL before and after treatment with gossypol this study explored the gossypol's mechanism on esophageal carcinoma cell line SEG-1 cells.4. This experiment used flow cytometric to analysize the cell cycle changes of esophageal carcinoma cell line seg-1 cells before and after gossypol's treatment and to explore the effects on SEG-1 cells cycle.ResultDifferent concentration of gossypol acetic acid and mono-aldehyde inhibited the growth of SEG-1 cells differently, and its inhibition had a close relation to drug concentration and acting time. The inhibition of 1 u M gossypol acetic acid on the growth of SEG-1 cells was weaker than others'. When the concentration of gossypol acetic acid and mono-aldehyde gossypol reached 10? M, SEG-1 cells growth was completely inhibited. When the cells were be incubated for 72 hours, they showed a negative growth state. When the concentration was less than 10 lM, under the same concentration the inhibitory effect of mono-aldehyde gossypol on SEG-1 cells growth was stronger than that of gossypol acetic acid (P<0.05); when the concentration was obviously higher than 10 ?M, under the same concentration the inhibition of mono-aldehyde gossypol on SEG-1 cells growth was weaker than that of gossypol acetic acid (P<0.05). The IC50 values of mono-aldehyde gossypol when the SEG-1 cells were cultured for 24h,48h and 72h were lower than those of gossypol acetic acid (P< 0.05).The SEG-1 cells were treated with 0.1%DMSO, stained by Wright-Giemsa and then observed through ordinary light microscope. We could see that the cells had varied shapes, clear edge, good morphology and good state. There was closely space among cells and the cells were neat and orderly growing. Only a few cells were apoptotic. With gossypol dose increased, the cell morphology changed obviously, the cell gap increased gradually, and cells gradually decreased; the cells were retracted and became round, and the cells increased which had intracytoplasmic vacuoles and floating like changes.Through hoechst33258 fluorescence microscope we could see that the normal cell nucleus was evenly dispersed and the blue fluorescence was weak. While the nucleus or cytoplasm had dense granule and the blue fluorescence was strong, and apoptotic bodies scattered in the cytoplasm after the cells were treated with mono -aldehyde gossypol for 12h.The normal cells were observed through transmission electron microscope that the normal cells had much normal mitochondria and nuclear chromatin was floc point and dispersed. After being treated by 25 ?M/ml mono-aldehyde gossypol for 12 hours, most of the mitochondrial became swelling and round, cristae structure disappeared, internal structures were disordered, and the chromatin of nucleus was agglutinated and part of it became block. After being treated by 25 ?M/ml mono-aldehyde gossypol for 24 hours, most of mitochondria were vacuolated, the cristae and internal structure disappeared, nucleus deformated and the bulk chromatin was closer to the nuclear membrane.By nectar electrophoresis analysis we could observe that cells in the DMSO treatment group had no DNA ladder and the DNA concentrated in the vicinity of the sample adding hole without diffusion which was genomic DNA performance. And after gossypol treatment the cells had DNA ladder which indicated that gossypol could lead to DNA of esophageal cancer cell fragmentation and induce those cells apoptosis.Determination of cellular DNA content:After being treated by 5 ?M/ml,10 ?M,25 ?M/ml gossypol acetic acid and mono-aldehyde gossypol for 24 and 48 hours, the proportion of diploid cells were determinated. The results showed that gossypol acetic acid and mono-aldehyde gossypol, along with the increase of concentration, the cell apoptosis rate increased gradually (P< 0.05). At the same concentration and at the same processing time, the apoptosis rate of momo-aldehyde gossypol was significantly higher than that of gossypol acetic acid (P< 0.05).The enzymatic analysis of cell apoptosis:two kinds of gossypol could cause the activity of caspase-9 and caspase-3 of SEG-1 cells increase. After treatment the activity increased significantly in 6 hours, and then decreased gradually. After treatment with gossypol acetic acid the activity of caspase-9 was significantly higher than that of mono-aldehyde gossypol in 6 hours, and it was significantly lower than that of the mono-aldehyde gossypol in 18 and 24 hours after treatment. This declared that in early stage after treatment with gossypol acetic acid the activity of caspase-9 increased rapidly and after 6 hours it also dropped fast. While after treatment with mono-aldehyde gossypol for 6 hours the activity of caspase-9 decreased slowly. After treatment with gossypol acetic acid the activity of caspase-3 increased significantly and was higher than that of mono-aldehyde gossypol within 12 hours. Then it decreased gradually and was significantly lower than that of the mono-aldehyde gossypol after 18 hours.Analysis of Bcl-2 family proteins showed that after treatment with gossypol acetic acid and mono-aldehyde gossypol the expression level of Bcl-2 and Bcl-XL was significantly reduced. The Bcl-2 and BCL-XL expression after treatment with DMSO was set to 100%. The bcl-2 expression of gossypol acetic acid treatment group decreased to 69.4% and that of mono-aldehyde gossypol treatment group decreased to70.8%. The BCL-XL expression of gossypol acetic acid treatment group decreased to 54.0% and that of mono-aldehyde gossypol treatment group decreased to 39.5%.Analysis of cell cycle showed that the SEG-1 cell cycle treated with different concentrations of gossypol changed a little within 24 hours. However when the treatment time was extended to 48 hours, among SEG-1 cells the G1 phase cells had increased at different degrees, the S phase cells decreased and the G2 phase cells changed little. This indicated gossypol acetic acid could cause esophageal cancer cells arrest in Gl phase and DNA synthetic speed slow down. Mono-aldehyde gossypol could cause G2 phase cells increase, Gl phase cells decrease and S phase cells change little, which indicated that mono-aldehyde gossypol mainly caused esophageal cancer cells arrest in G2 phase. There was no significant difference in the cell cycle distribution between the mono-aldehyde gossypol treatment of 25?M/l and the DMSO control group for 48 hoursConclusion1. Gossypol could effectively inhibit the proliferation of SEG-1 cells in esophageal cancer cell lines, and its effect had a dose-time effect. But the effect of mono-aldehyde gossypol was stronger than that of gossypol acetic acid.2. Gossypol could induce the apoptosis of SEG-1 cells, and its effect had a dose-time effect. The effect of mono-aldehyde gossypol is stronger than that of gossypol acetic acid, which might be related to the activation of caspase-9 and caspase-3 and the decrease of Bcl-2 and Bcl-xL expression.3. Gossypol had a regulatory role on the cell cycle of SEG-1 cell line. Gossypol acetic acid and mono-aldehyde gossypol could respectively arrest the cells in G1 phase and G2 phase.Part 2 The research of the gossypol's effect in vivo on Nude mice model of SEG-1 cellObjective and methodsObjective:To investigate the effect of gossypol on SEG-1 cells in nude mice model of esophageal carcinoma. Methods:30 healthy SPF female BALb/c nude mice were divided into 6 groups, namely gossypol acetic acid 40mg/kg group, gossypol acetic acid 60mg/kg group,mono-aldehyde gossypol 40mg/kg group,mono-aldehyde gossypol 60mg/kg group,blank control group and negative control group. There were 5 rats in each group. The blank group was without inoculation and gossypol treatment. The remaining 5 groups were inoculated with SEG-1 cells and then given corresponding concentration gossypol in the third days after tumor inoculation. Then we observed the variation of tumor volume and weight, calculated the tumor growth delay and tumor growth inhibition rate,observed the body weight changes after inoculation,and at last measured the weigh change of liver and spleen.Result:General:Before inoculation the nude mice were normal in appetite and behavior. After 1 x 107 SEG-1 cells were subcutaneously inoculated, in the first day the mice was dispirited, inappetent, and then sluggish until death. In the fourth days the subcutaneous mass in nude mice was obviously visible and then gradually increased. The survival time of nude mice was 13±1 days. After death the mice were dissected. We found that the tumor adhered to surrounding tissue and part of nude mice had ascites and hepatosplenomegaly. The mass was made into cell suspension, stained and then observed by the microscope. It was tested that the morphological structures of the mass was similar to that of SEG-1 cells.Pathological examination:The tumor specimens of gossypol acetic acid 40 mg/kg group, which was on behalf of tumor drug group, were contrasted pathology with those of negative control group. The size and invasion of the tumor drug group was less than that of the negative control group. Its tumor differentiation of the tumor drug group was slightly better than that of the negative control group. Its tumor angiogenesis was worse than that of the negative control group.Changes in tumor growth:All subcutaneous mass with drug treat reduced in different levels and the growth rate of the mass slowed down (Figure 2-1). Inhibition rate of tumor growth of gossypol acetic acid 40 mg/kg, gossypol acetic acid 60mg/kg group, mono-aldehyde gossypol 40mg/kg group, mono-aldehyde gossypol 60mg/kg group was respectively 14.3%,31.2%,33.7%,32.9%(P< 0.05). The tumor growth delay days of gossypol acetic acid 40 mg/kg group was less one day than that of gossypol acetic acid 60mg/kg group, mono-aldehyde gossypol 40mg/kg group, mono-aldehyde gossypol 60mg/kg group. The nude mice of the negative control group after inoculation of SEG-1 cells weighed more than the mice without inoculation of SEG-1 cells after 13 days. The nude mice in gossypol acetic acid 40mg/kg group and gossypol acetic acid 60mg/kg group with intraperitoneal injection of gossypol acetic acid act less and became thinner from the 1 st day. At the 5th days after treatment the nude mice weight of gossypol acetic acid 60mg/kg group was significantly lower than that of negative control group. And at the 6th days the nude mice weight of gossypol acetic acid 40mg/kg group was significantly lower than that of negative control group. The nude mice weight of mono-aldehyde gossypol 40mg/kg group and mono-aldehyde gossypol 60mg/kg group was almost similar to that of the negative control group.Gossypol effects on the weight of liver and spleen and survival of the nude mice:at the 10th days after tumor inoculation and death nude mice were dissected and it was found that the liver of negative control group mice was obviously heavier than the normal liver. That declared that liver metastases of SEG-1 tumor cells occurred. The liver weight in acetic acid gossypol 40mg/kg was significantly lower than that of the negative control group, while the nude mice liver of mono-aldehyde gossypol 40 or 60 mg/kg group did not reduce compared with the negative control group. The survival time of gossypol acetic acid 40mg/kg and 60mg/kg group was significantly shorter than that of the negative control group. Although gossypol acetic acid had a good anti-tumor effect, at the same time it shortened the survival time of mice. It explained that the toxic effect of gossypol acetic acid was larger than the mono-aldehyde gossypol. Mono-aldehyde gossypol had strong anti-tumor effect without significant side effects and could prolong the survival time of nude mice.Conclusion:1. Gossypol acetic acid and mono-aldehyde gossypol could inhibit the tumor growth of esophageal carcinoma in nude mice.2. The toxic effects of mono-aldehyde gossypol on esophageal cancer in nude mice model was less than that of gossypol acetic acid.