Mechanism Of Inhibition Of "Yiqi-Huayu-Jiedu" Recipe On Colorectal Cancer Metastasis

Aims:Metastasis of colorectal cancer is a biological feature of malignant tumor,which is an important subject in medical research.From the existing international and domestic research progress,the mechanism of metastasis is related to cancer stem cells and/or the body’s immunity.We sought to observe the effect of Chinese Medicine-"Yiqi Huayu Jiedu" recipe on colorectal cancer metastasis.We also explored its possible mechanism from both cancer stem cell and the immune cells of nude mice with transplanted tumor.Methods:(1)Establish the HPLC method to verify the "Yiqi Huayu Jiedu" recipe and to lay the foundation for Chinese herbal recipe’s quality control.(2)Six colorectal cancer cell lines including CaCo-2,HT-29,SW480,SW620,LoVo,HCT-116 were tested on their expression of CD133 and CD44 for clarify the percentages of the CD133highCD44high cells among these different colorectal cancer cells.(3)CD133highCD44high cells and CD133lowCD44low cells were sorted by flow cytometer respectively for compare their nucleic acid levels of stemness and differentiation markers,cell cycle differences,stem cells sphere forming ability under serum-free culture conditions,cell differentiation ability after the recovery of serum,colony formation ability on soft agar in vitro.In vivo,the isolated CD133highCD44high cells and CD133lowCD44low cells were inoculated in nude mice.After 3 weeks,the tumor forming ability,the expression of CD44 and CD133 of tumor tissues were detected.(4)The culture medium was directly dosed "Yiqi Huayu Jiedu".decoction for testing its effect on colon cancer stem cells in vitro.After intragastrically administration of "Yiqi Huayu Jiedu"decoction for 3 weeks,the proportion of CD133highCD44high cells was tested in nude mice,whose spleens were modeled by in situ inoculating colon cancer cells.(5)The effect of "Yiqi Huayu Jiedu" recipe on the transplanted tumor in nude mice was observed and the size and other general situation of nude mice with transplanted tumor after 3 weeks of intervention were compared.(6)After in situ colon transplanted of the same volume of fluorescent labeled colon cancer tissue block,the colorectal cancer implantation metastasis ratio were compared among the "Yiqi Huayu Jiedu" Group,5-Fu group and the combination of traditional Chinese and Western medicine group and the negative control group.(7)By injecting the same number of fluorescent labeled colon cancer cells in nude mice spleen,the liver metastasis of colorectal cancer was compared among the group of "Yiqi Huayu Jiedu," Group,5-Fu group and combination of traditional Chinese medicine and Western medicine group and the negative control group.(8)The effects of "Yiqi Huayu Jiedu" Recipe on the innate immune cells such as NK cells,macrophages and neutrophils in peripheral blood and spleen of nude mice were analyzed by flow cytometry.(9)Through the high throughput screening of cytokine microarray chips,the effects of "Yiqi Huayu Jiedu" Recipe on plasma cytokines in nude mice after intervention were compared.(10)The effects of "Yiqi Huayu Jiedu" Recipe on the macrophages were compared by flow cytometry among the whole recipe group,"Fuzheng" group and "Quxie" group.Results:(1)The HPLC method was established.Allantoin,calycosin glycoside,liquiritin and glycyrrhizic acid were successfully detected in traditional Chinese medicine recipe,which laid the foundation for the quality control of Chinese herbal compound.(2)Among human colon cancer cell lines such as CaCo-2,HT-29,SW480,SW620,LoVo,HCT-116,only HCT-116 this cell is a high proportion of CD133highCD44high subsets.3(3)In vitro,CD133highCD44high cells and CD133lowCD44low cells were sorted respectively by flow cytometer.Compared to CD133lowCD44low cells,CD133highCD44high cells have higher expression of stemness markers(CD133,ALDH2 and dry Lgr5)and low expression of differentiation marker CK20(p<0.05).Cell cycle analysis showed that more cells of CD133highCD44high are in G0/G1 phase.The serum-free medium and soft agar test showed the stronger clone formation ability of stem cell sphere.After the recovery of serum,cells of sphere recovered to the polymorphic adherent cells.In vivo,subcutaneous injection of the sorted CD133highCD44high cell showed that this kind of cells have more subcutaneous tumor forming ability.When injected 105 and 104 cells,100%tumors formed while injected 103 cells still showed 4/9 tumor formed,double of whose of CD133l0WCD44low cells.At the same time,the weight of tumors is heavier too.After regrinding the tumor tissue,the proportion of CD133highCD44high cell in 105 group is 6.8%±1.8%(v.s.2.6%± 0.8%,p<0.05).All of these verify the CD133highCD44high cells are cancer stem cells in HCT-116 cell line.(4)"Yiqi Huayu Jiedu" Recipe has little influence on colon cancer stem cells in vitro,for the proportion of CD133highCD44high cell subsets changed little.In vivo in the models of colorectal cancer cells spleen injection with liver metastasis,after treatment of "Yiqi Huayu Jiedu" Recipe,the proportion of CD133highCD44high cell subsets of spleen in situ tumor still changed limited(14.97%± 8.08%v.s.13.75%± 6.93%,p=0.790).(5)In the subcutaneous xenograft model,after 3 weeks treatment of "Yiqi Huayu Jiedu" Recipe,compared to the negative control group,5-Fu group and small dose Chinese medicine combined with 5-Fu group,large dose group and small dose group of Chinese medicine have no significant change in nude mice body weight(p>0.05).Although the qualities of the subcutaneous transplantation tumor decreased,there were no statistically significant difference(p>0.05).(6)Using the model of fluorescence labeled tissue transplanted intestinal tissue in situ,after 3 weeks intervention,the metastasis rate was 33.3%in the negative control group,28.6%in 5-Fu control group,0%in the Chinese medicine group,0%in Chinese medicine combined with 5-Fu group.But due to the small number of cases,Chi square test showed p value was more than 0.05.(7)The spleen injection fluorescent labeled HCT-116 colorectal cancer cells for liver metastasis model was established.After 3 weeks of "Yiqi Huayu Jiedu" Recipe,there is no obvious difference between the body weights of each group;the survival rate of 5-Fu group was the lowest(66.7%).The liver metastasis rate was 90.9%in negative control group(10/11),71.4%(5/7)in 5-Fu control group,44.4%(4/9)in Chinese medicine group and 30%(3/10)in combined with traditional Chinese medicine and 5-Fu group.Compared with the negative group,Chinese medicine group and combination group had statistically significant difference(p<0.05).(8)Based on the model of liver metastasis of colorectal cancer,using flow cytometry to detect"Yiqi Huayu Jiedu" Recipe’s effect.The rate of activated NK cell,macrophage content and neutrophils in peripheral blood and spleen of nude mice were analyzed.The data showed that the content of neutrophils in CD45 positive cells in the granulocyte gate of the peripheral blood of "Yiqi Huayu Jiedu" Recipe group was significantly higher than that of the negative control group(77.09%± 12.55%v.s.61.76%±11.51%),the difference was statistically significant(p<0.05).(9)The peripheral blood plasma of nude mice was detected by cytokines microarray chip.After the intervention of "Yiqi Huayu Jiedu" Recipe,the results showed that the levels of GM-CSF,γ-IFN,IL-1a,IL-3,IL-6,IL-13,and IL-17 were more than 2 times of those of the negative control group.(10)By using the tumor-free model of direct intervention of traditional Chinese medicine(TCM)in nude mice,after the intervention of "Yiqi Huayu Jiedu" Recipe for 2 weeks,compared with the negative group,it was found that the proportion of spleen macrophages increased significantly(2.09%± 0.42%v.s.1.28%± 0.26%,p<0.05).At the same time,neither"Fuzheng" group nor "Quxie" group could achieve the statistical difference.Conclusions:"Yiqi Huayu Jiedu" Recipe could active the innate immune system of the body,especially by increasing the content of neutrophils and macrophages,quickly kill the wandering cells detached from primary colon cancer in the blood system,reduce colon cancer implantation metastasis and hepatic metastasis,which reflects the ideology of "Tonifying Qi" in traditional Chinese medicine theory.The CD 13 3highCD44high is a marker of cancer stem cells in human colon cancer,but the effect of "Yiqi Huayu Jiedu" Recipe on them is limited,suggesting that its anti-tumor mechanism may not through inhibiting the caner stem cells.The research shows the prescription of recipe has its particularity,for "Fuzheng" and "Quxie" are both indispensable,reflecting the unique effect of traditional Chinese medicine.