Establishment Of Human Pancreatic Carcinoma SW1990 Cell Line With Highly Metastatic Potential In The Liver And Interventional Research Of Traditional Chinese Drugs

Backgound: The liver metastasis of pancreatic carcinoma in the later period isextremely common, the liver metastatic rate has above 50%, still not effectivetreatment method. The liver metastasis of pancreatic carcinoma is a complicatedprocess, the establishment of pancreatic carcinoma liver metastasis model is a keystep of studies about the liver metastasis mechanism and anti-metastasis medicinescreening. Vezeridis used human pancreastic carcinoma cell line COLO357 by in vivoscreening to establish highly liver metastasis cell line L3.5. Shishido used humanpancreastic carcinoma cell line HPC-3 by in vivo screening to establish highly livermetastasis cell line HPC-3H4. These two cells have long medium- survey -time andshortcoming of few liver metastasis numbers. There still did not have the report ofpancreatic carcinoma with highly liver metastasis animal model in our country.Screening and establishing human pancreatic carcinoma cell line with highly livermetastasis is a great meaning of the research of liver metastasis mechanism andanti-metastasis Traditional Chinese Drugs screening.Objective: To establish a human pancreatic carcinoma SW1990 cell line with HighlyMetastatic Potential in the Liver and liver metastatic nude model, then to interfere it using QYHJ and Huachansu and to understand its stabilization and credibility.Methods:PartⅠEstablishment of Human Pancreatic Carcinoma SW1990 cell Line withHighly Metastatic Potential in the LiverHuman pancreatic carcinoma clone cells SW1990 were injected into the spleenof nude mice. The liver metastatic lesions were harvested from the moribund animalsand then re-injected into the nude mice for the second round of selection. The sameprocedure was repeated six times. New cell line (SW1990HM) from the sixth roundof liver metastasis was thus established. To research the difference betweenSW1990HM and SW1990 was tested by CCKS, flow cytometry, Transwell, PCR,metastatic-gene PCR microarray as well as Antibody Microarray.PartⅡInterventional Research of Traditional Chinese drugs on liver metastasisnude model1. To replicate liver metastasis model, human pancreatic carcinoma cellsSW1990HM were injected into the spleen of nude mice. At the day of modeling, allrats were randomly divided into five groups: NS group, Gemcitabine group, QYHJhigh, median or low dose group. Rats in NS control group were administrated orally0.4ml NS once a day for 28 days and injected 0.2ml NS into the abdominal cavity d_1,5,9; rats in Gemcitabine groups were administrated orally 0.4ml NS once a day for 28days and injected 0.2ml Gemcitabine(120mg/kg)into the abdominal cavity d_1,5,9; ratsin QYHJ high, median or low dose groups were administrated orally 0.4ml QYHJonce a day for 28 days and injected 0.2ml NS into the abdominal cavity d_1,5,9; In these day of experimental therapy completed, animals were killed, spleens and liverwere taken out; tumor nodules in spleens incised, and tumor inhibitory rate calculated;then, counted number of metastatic clones on livers. Lastly, MMP-2, VEGF mRNA intumor tissues of metastases were detected by immunohistochemistry.2. To replicate liver metastasis model, human pancreatic carcinoma cellsSW1990HM were injected into the spleen of nude mice. At the day of modeling, allrats were randomly divided into five groups: NS group, Gemcitabine group,Huachansu high, median or low dose group. Rats in NS control group were injected0.2ml NS into the abdominal cavity d_1,5,9; rats in Gemcitabine groups were injected0.2ml Gemcitabine (120mg/kg) into the abdominal cavity d_1,5,9; rats in Huachansuhigh, median or low dose groups were injected 0.2ml Huachansu into the abdominalcavity once a day for 28 days; In these day of experimental therapy completed,animals were killed, spleens and liver were taken out; tumor nodules in spleensincised, and tumor inhibitory rate calculated; then, counted number of metastaticclones on livers. Lastly, MMP-2, VEGF mRNA in tumor tissues of metastases weredetected by immunohistochemistry.Results:(1)By in vivo selection of SW1990 pancreatic carcinoma cell line, we established apancreatic tumor cell line (SW1990HM) which had dramatically high liver metastaticcapabilities. The SW1990HM cells were in round-shuttled shape. The cell populationdoubling time was 56 hours, which was significantly faster than the wild type (64hours) (P＜0.05). The ratio of SW1990HM cells in S phase was significantly higher than that of the wild type (36.76% vs 31.89%, P＜0.05). The invasive capabilitywas nearly as twice as that of the wild type (P＜0.05). In inoculated nude mice,100% clones of SW1990HM metastasized consistently to the livers in four weeks,while less than 40% liver metastasis in wild type(SW1990) (P＜0.05). SW1990HMand SW1990 mean number of liver metastasis was 36 and 9 respectively (P＜0.05.This high metastatic capability was characterized by up-regulation of MMP-2,VEGF, bFGF, E-eadherin mRNA and down-regulation of MMP-9 mRNA expression.The result of gene microarray showed 59 pieces of metastastic genes changed in theSW1990HM HM cell line, The result of protein microarray showed 40 pieces ofmetastastic proteins changed in the SW1990HM HM cell line.(2) In vivo experiment show: The tumor inhibitory rate in high, median or low doseQYHJ group 21.59%,44.87% and 20.63 % respectively; median dose QYHJ groupwere significantly higher than the other groups(p＜0.05). The experimental resultsconfirmed that QYHJ can markedly inhibite transplanted tumor growth in spleen.Their metastatic inhibitory rate in high, median or low dose group reached 29.09%,38.16% and 21.31% respectively; It indicated that QYHJ exerted an effectiveinfluence on liver metastasis. The results from hybridization in situ of metastasistumor tussues showed that:①The MMP-2 immune histochemistry index of high,median or low dose QHJ group compared to the control group the differences weresignificant(p＜0.05); The difference between every therapy groups was insignificant(p＞0.05).②The VEGF immune histochemistry index of high or median dose QYHJgroup compared to the control group the differences were significant(p＜0.05); The VEGF immune histochemistry index of low dose QYHJ group compared to thecontrol group the differences were insignificant(p＞0.05); The difference betweenevery therapy groups was insignificant(p＞0.05).In vivo experiment show: The tumor inhibitory rate in high, median or low doseHuachansu group 55.16%, 21.63% and 12.56% respectively; high dose Huachansugroup were significantly higher than the control groups(p＜0.05). The experimentalresults confirmed that Huachansu can markedly inhibite transplanted tumor growth inspleen. Their liver metastasis inhibitory rate in high, median or low dose Huachansugroup reached 47.06%, 35.29% and 23.52% respectively; It indicated that Huachansuexerted an effective influence on liver metastasis. The results from hybridization insitu of metastasis tumor tussues showed that:①The MMP-2 immune histochemistryindex of high, median or low dose Huachansu group compared to the control groupthe differences were significant(p＜0.05); The difference between every therapygroups was insignificant(p＞0.05).②The VEGF immune histochemistry index ofhigh or median dose Huachansu group compared to the control group the differenceswere significant(p＜0.05); The VEGF immune histochemistry index of low doseHuachansu group compared to the control group the differences were insignificant(p＞0.05); The difference between every therapy groups was insignificant(p＞0.05).Conclusion:(1)The new human pancreatic carcinoma line can provide an accessible model for theidentification of genes involved in the multistep process of pancreatic tumormetastasis and anti-metastasis medicine screening. Manipulation of candidate genes and proteins in these cells will permit evaluation of their functional significance in theliver metastasis mechanism of pancreatic cancer and target of anti-metastasismedicine.(2)QYHJ and Huachansu can inhibit not only spleen graining neopasms, but it maybecome one of effective treatments for liver metastasis. What is more, atdown-regalate MMP-2 expression and VEGF expression may be its mechanisms. Itmeans that liver metastatic model is a good model of anti-metastasis TraditionalChinese Drugs screening.