Study On Effects Of ASPS And PRPS On Biological Behavior Of Colorectal Cancer Cell Line CD133~+/CD44~+

Colorectal cancer is one of the clinical alimentary canal malignant tumors. Nearly50%-60%the patients who have radical resection will relapse and metastasis, and recurrence,infiltration and distant metastasis are the most important causes of death. The liver is the mostcommon distant metastasis organ of colorectal cancer patients.The cancer stem cells (CSCs) play a critical role in cancer recurrence, infiltration anddistant metastasis. CSCs are not sensitive to chemotherapy and radiotherapy, so traditionarytherapy is only to kill terminal differentiation tumor cells nor to CSCs. Amplification of relicCSCs induces cancer recurrence. A number of studies showed that polysaccharides iseffective to treat cancer and prevent recurrence. polysaccharides has many biologicalactivities including immunoregulatory activity and inhibiting tumor etc. Comparing withother chemotherapeutics, polysaccharides which has many benefits including multi-pathway,multi-target, hypotoxicity, efficacy, low price and not damaging immune system displayspotentially perspective in clinic.This research was about whether polysaccharides could control proliferation of CSCsby combination of CSCs and polysaccharides which can prevent cancer. And then, a fewbiological activities of polysaccharides such as treating and preventing cancer were studied.The experimental results showed ASPS and PRPS could inhibit tumor cells toproliferate and promote tumor cells apoptosis. So this experiment was based on biologicalbehavior of colorectal cancer stem cells. In addition, the effects of ASPS and PRPS onHT-29cell line and stem cells from colorectal cancer cells were studied, and mechanism ofactions was also initially studied. Further work is warranted to explore whether ASPS andPRPS may be a useful novel therapeutic drug for targeting colorectal cancer CSCs.1. Isolating and charaeterizing of Colorectal Cancer stem cellsMethods:120cases enrolled colorectal cancers were selected,80cases were withoutearly live metastases and40cases were with early liver metastases. Stem cells with CD133+and CD44+were isolated by immunofluorescent tests and then cultured. The ability offorming tumor on CD133~+/CD44~+cells is measured by NOD/SCTD. The expression of stemcell gene CD133and CD44were detected by Western blot in order to determine tumor markers of Colorectal Cancer with early liver metastases. Results: Of the120enrolledcolorectal cancers, of the80cases were without early liver metastases,12cases wereobserved CD133and CD44proteins co-expression, while36of the40cases with early livermetastases were found CD133and CD44proteins co-expression (15%vs.90%, P <0.05).The sensitivity and specificity of CD133and CD44proteins co-expression in predictingearly liver metastasis of colorectal cancer were75%and94.44%respectively.Mammosphere formation ability of CD133~+/CD44~+tumor cells is stronger than non-CD133~+/CD44~+tumor cells under CFCP culture.1×103CD133~+/CD44~+tumor cells couldsuccessfully form a tumor. With the increasing of CD133~+/CD44~+tumor cells, the rate offorming tumor is increased gradually. In addition, it was observed that the content of CD133and CD44protein decreased following growth2w4w and6w of the transplanted tumor.Conclusions: Comparing with non-CD133~+/CD44~+tumor cells, CD133~+/CD44~+tumor cellshave stronger abilities of reproduction and forming tumor. It confirms CD133~+/CD44~+tumorcells are colorectal cancer stem cells. The result of identification shows that CD133andCD44proteins were highly co-expressed in colorectal cancer with early liver metastases, andmay be a potential biomarker for the initiation and differentiation of colorectal tumors.2. The effects of ASPS and PRPS on HT-29from colorectal cancer stem cellsMenthods: Sera containing ASPS and PRPS were prepared by serum pharmacologicalmethod. MTT experiments were performed to detect whether ASPS and PRPS can inhibit theviability of HT-29cells. FCM assay was performed to detect the effects of sera containingASPS and PRPS on the apoptosis of HT-29cells. The effects of sera containing ASPS andPRPS on the invasion of HT-29cells was observed by Transwell. In addition, the effects ofASPS and PRPS on the expressions of Caspase-3,bαx,bcl-2in HT-29cells were measured.Results: ASPS and PRPS are ineffective on HT-29. High and middle concentration groupconcentration group sera containing ASPS and PRPS have shown inhibition of HT-29cellsproliferation, and inducing the apoptosis of HT-29cells in vitro. Sera containing ASPS caninhibit HT-29to invade Matrigel. The expression of bαx is increased, bcl-2down regulationand Caspase-3up-regulation. Conclusions: sera containing ASPS and PRPS can inhibit theabilities of proliferation and invasion on HT-29cells and induce apoptosis in vitro.3. The regulations of ASPS and PRPS on colorectal cancer stem cellsMethods: MTT experiments were performed to detect whether sera containing ASPSand PRPS can effect the reproduction and forming tumor of CD133~+/CD44~+tumor stem cells. At the same time, the experiment also study the effects of sera containing ASPS on theexpression of β-catenin from CD133~+/CD44~+tumor stem cells of Wnt signaling pathway.Results: sera containing ASPS can apparently inhibit CD133~+/CD44~+tumor stem cells toform cell colony. The cells were treated with different concentrations sera containing ASPSfor24h,48h and72h. Middle concentration sera containing ASPS has shown inhibition ofCD133~+/CD44~+tumor stem cells proliferation at24h (P<0.05). And high concentration seracontaining ASPS has shown inhibition of CD133~+/CD44~+tumor stem cells proliferation in24h,48h and72h respectively (P<0.05). But sera containing PRPS is ineffective toCD133~+/CD44~+tumor stem cells. A total expression of β-catenin from CD133~+/CD44~+tumorstem cells is low after sera containing ASPS intervention. Conclusions: Sera containingASPS can inhibit colorectal CSCs and decrease biological activities of CSCs of Wntsignaling pathway in vitro.