| IntroductionPancreatic cancer is a common malignant tumor in digestive system. There has been a significant increasing trend in the incidence of pancreatic cancer in recent years. The prognosis of advanced pancreatic cancer is depressed, and the whole 5-year survival is less than 5%. Due to pancreatic cancer with liver metastases, there has got incurative effect on pancreatic cancer, and 80% of patients are already in local infiltration and distant metastasis when diagnosed, among which the incidence of liver metastasis is more than 50%. At present effective medication and drugs is lacking for the treatment of pancreatic cancer with liver metastases, which resist most of chemotherapeutic agents and radiation. Former researches indicated that QYHJ traditional Chinese medicine (TCM) had showed some advantages in treating this malignancy. Many biological factors secreted by pancreatic cancer are related with tumor growth and immigration, and also demonstrated abundant around pancreatic cancer tissue. Some biological factors related to the proliferation and invasion for pancreatic cancer has been identified as a target for therapies.ObjectiveTo observe the effects of QYHJ on liver metastases from human pancreatic cancer; and to explore the potential mechanism of QYHJ in the treatment of liver metastases from human pancreatic cancer through detecting the expression of biological factors associated with metastasis of pancreatic cancer.MethodsPart I:The animal model with metastatic liver tumors were produced by intrasplenic inoculation of SW1990 HM cells in male BALB/C mice, mice with intransplenic tumor inoculation were randomly assigned to one of the following four groups: normal saline group(NS), QYHJ high dose group(QYHJ-H), QYHJ middle dose group(QYHJ-M), QYHJ small dose group(QYHJ-S). The effect of QYHJ on the liver metastases from pancreatic cancer was evaluated by means of mice weight, incidence of liver matastasis, liver colonization and total clone volumes. PartⅡ:the potential mechanism research of QYHJ treatment on pancreatic cancer with liver metastases was carried out as follows:1. RT-PCR detection:Fresh tissues of liver metastasis was ground into powder in liquid nitrogen, after total mRNA isolated, RT-PCR semi-quantitative detection was performed to observed the expression changes of MMPs, VEGF, cyr61, TGF-β1, bFGF and CTGF mRNA in each group.2. immunohistochemistry(IHC) SP method was applied to measure the factors significant changes in protein level:positive expression of cells was counted to assess the expression changes of the factors above in the cancer tissue, and the optical density of positive region was computed to judge the factors changes in tumor tissue. 3. Based on the factors expression changes and liver clone volume in each group, correlation was analyzed between these factors and clone proliferation, and furthermore, Regression analysis has been conducted on the relationship between significant factors and clone growth.4. Using Western blot method to measure the protein expression changes on molecular level about those factors close-relative with liver clone proliferation in each group. In this way, it is to confirm the results of 1HC array and reveal the therapeutic targets of QYHJ inhibition of liver metastasis from pancreatic cancer.4. ELISA allay to examine the concentration of CEA,CA19-9 in blood serum。5. Flow Cytometry (FCM) to examine the cell cycle and the apoptosis of the liver metastases from pancreatic cancer in each group.ResultsPart I:The mice in QYHJ groups weighed more than NS group on the end of 6th week when mice were killed(vs NS, p<0.05) and especially the QYHJ-H group was more significant(vs NS, P＜0.01). The number of mice with liver metastases is 11 from 15(11/15),9 from 15(9/15),6 from 14(6/14) and 8 from 14(8/14) in NS group, QYHJ-S group, QYHJ-M group and QYHJ-H group respectively. The inhibition rate of liver metastasis occurrence in mice is 18.1%,41.6% and 22.1% in QYHJ-S group, QYHJ-M group and QYHJ-H group respectively, with contrast to NS group. The liver metastatic lesions in QYHJ groups is significantly less than NS group(P<0.01); there was no significance in QYHJ groups(P>0.05). QYHJ groups inhibited the growth and proliferation of liver lesions more obviously than NS group (P＜0.05). The inhibition rate is respectively 24.6%,45.6% and 45.5% in QYHJ-S group, QYHJ-M group and QYHJ-H group. Among the QYHJ groups, the inhibition rate of QYHJ-M group and QYHJ-H group is less than QYHJ-S group(P<0.05) and there was on significance between the QYHJ-M group and QYHJ-H group(P>0.05).PartⅡ:1. After QYHJ treatment, the expression of bFGF mRNA in the liver lesions was up-regulated by 21.8%,47.1% and 39.2% in QYHJ-H group, QYHJ-M group and QYHJ-S group respectively, to contrast with NS group (P＜0.05); the expression of CTGF mRNA was down-regulated in QYHJ-H group by 9.61% and increased 11.5% in QYHJ-M gruop,9.63% in QYHJ-S group (VS NS, P>0.05); the expression of MMP-1 mRNA was up-regulated by 28.3%,12.2%,19.6% in QYHJ-H group, QYHJ-M group and QYHJ-S group respectively, to contrast with NS group (VS NS, P>0.05); the expression of MMP-7 mRNA was up-regulated by 18.2%,12.9% and 1.3% in QYHJ-H group, QYHJ-M group and QYHJ-S group respectively, to contrast with NS group (VS NS, P>0.05), The expression changes of MMP-9 mRNA was 7.4%,13.8%,11.2% respectively(VS NS, P>0.05). However, the expression of cyr61 mRNA was down-regulated by 57.9%,69.5% and 29.5% in QYHJ-H group, QYHJ-M group and QYHJ-S group respectively, to contrast with NS group (VS NS, P＜0.05); the expression of MMP-2 mRNA was down-regulated by 42.1%,28.1% and 62.3% in QYHJ-H group, QYHJ-M group and QYHJ-S group respectively, to contrast with NS group (VS NS, P＜0.05); the expression of TGF-β1 mRNA was down-regulated by 78.6%,69.9% and 33.1% in QYHJ-H group, QYHJ-M group and QYHJ-S group respectively, to contrast with NS group (VS NS, P＜0.05); the expression of VEGF mRNA was down-regulated by 78.9%,64.9% and 43.8% in QYHJ-H group, QYHJ-M group and QYHJ-S group respectively, to contrast with NS group (VS NS, P＜0.05). 2.The results of IHC showed that MMP-2, VEGF, cyr61,bFGF and TGF-βimmunoreactivity was in most cases located in the cytoplasm of the tumor cells and Positive expression of factors showed yellow-brown in the cancer cell cytoplasm of pancreatic cancer tissue, some distribution in the stroma around tumor. The number of positive tumor cells of cyr61 expression is 39±10,21±3,23±4 and 32±6 in NS group, QYHJ-H group, QYHJ-M group and QYHJ-S group respectively, QYHJ groups is less than NS group(P＜0.05); The number of positive tumor cells of MMP-2 expression is 38±11,28±5,24±4 and 29±3, QYHJ groups is less than NS group(P＜0.05); The number of positive tumor cells of bFGF expression is 45±8,65±5,64±9 and 56±6, QYHJ groups is more than NS group(P＜0.05); The number of positive tumor cells of TGF-β1 expression is 47±10,31±4,29±3 and 36±6, QYHJ groups is less than NS group(P＜0.05); The number of positive tumor cells of VEGF expression is 42±7,30±4,29±5,32±5, QYHJ groups is less than NS group(P<0.05).3. Meanwhile, through the computation of optical density about positive staining region, the distribution of factors secreted by cancer cell in tumor tissue and ECM in NS group and QYHJ groups. The results were shown as follows: optical density of cyr61 was 508.82±6.69,319.92±2.96,323.46±4.12,435.29±4.83; of VEGF for 447.6±7.54,337.3±5.44,317.8±5.28,360.6±5.42;of TGF-β1 for 748.66±10.12,435.23±4.77,418.17±5.26,658.41±6.33; of bFGF for 661.24±8.13,685.74±5.32,721.56±5.38,771.41±6.24; of MMP-2 for 538.42+6.47,312.84±4.47,445.75±5.73,239.43±4.32 in NS group, QYHJ-H group, QYHJ-M group and QYHJ-S group respectively.4.With a combination between number of positive cells and liver lesions volume, correlation analysis showed that the expression changes of the factors of cyr61,MMP-2,VEGF,TGF-β1 and bFGF had a positive relation with the liver clone growth. And then multiple linear regression analysis indicated that expression of VEGF, bFGF and cyr61 influence the liver metastasis growth ultimately. The regression formula is y^=-0.954+0.026x1+0.023 x2+0.027x3.5. Using western blot method in protein molecular level to confirm the results acquired with RT-PCR and IHC examination, in QYHJ groups the protein expression of VEGF, TGF-β1, cyr61 and MMP-2 was significantly less than NS group (P＜0.05), and that of bFGF was markedly more than NS(P<0.05).6. ELISA allay results showed the average concentration of CEA in QYHJ-H, QYHJ-M and QYHJ-S group is 12.51±2.53ng/Ml,13.67±2.72ng/mL and 15.83±3.16ng/mL, lower obviously than NS group of 23.26±4.62ng/mL(P<0.05); The average concentration of CA19-9 in QYHJ-H,QYHJ-M and QYHJ-S group is 318.26±42.13 U/mL,355.47±44.29 U/mL and 362.37±45.08 U/mL respectively, less than NS group of 423.72±45.43 U/mL (P<0.05); 7. Flow Cytometry examination showed that the proliferation index (PI) of QYHJ-H group, QYHJ-M and QYHJ-S group was 19.28%, 21.14％and 24.63%, less than NS group of 30.72%(P<0.05). In the comparison of cell cycle, G0/G1 cells of QYHJ groups got more than NS group(P<0.05), yet G2/M cells in QYHJ groups grew less than NS group(P<0.05). The apoptosis rate of QYHJ groups,15.29%,15.42% and 11.89% respectively, was higher than NS group of 7.47%(P<0.05). CONCLUSIONPart I:QYHJ could reduce the formation of liver clone, but the inhibition effect didn't increase with the dosage escalation of QYHJ. After QYHJ treatment, the growth and proliferation of liver metastases in the QYHJ groups became slow, compared with NS group, and the QYHJ-H and QYHJ-M group effect was better than QYHJ-M group, no obvious differences between QYHJ-H and QYHJ-M groups. According to the weight changes, which was taken as criteria to assess the quality of life, QYHJ could enhance the quality of life, QYHJ-H group was better than QYHJ-M and QYHJ-S groups, no differences between QYHJ-M group and QYHJ-S group. As for the experimental results, QYHJ treatment could inhibit the liver metastases from pancreatic cancer to some extent.Part II:QYHJ treatment could reduce the expression of VEGF, MMP-2, cyr61 and TGF-β1 mRNA and decrease the expression of VEGF, MMP-2, cyr61 and TGF-β1 at protein level in liver metastases tissue; meanwhile, QYHJ could up-regulate the expression of bFGF at mRNA and protein level, which revealed the QYHJ inhibition effect on the liver metastases from pancreatic cancer was resulted from complicated action among many factors regulated by QYHJ. On the basis of correlation and regression analysis of relationship between factors and metastases size, we came into a conclusion that after QYHJ treatment, VEGF, bFGF and cyr61 maybe is the therapeutic targets of QYHJ inhibition effect on liver clone growth and proliferation. QYHJ could down-regulate the expression of TGF-β1, MMP-2, VEGF and cyr61 in SW 1990 HM, as well as reduce these factors concentration in ECM around tumor tissue, to suppress the growth and proliferation of liver metastases. QYHJ could reduce the concentration of CEA and CA19-9 in blood serum of mice with liver metastases of pancreatic cancer, meanwhile, enhance cancer cells apoptosis and block the cancer cell into the G0/G1 cycle, to inhibit the liver metastases growth.
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