| BackgroundIn western countries, the morbidity and mortality of colorectal cancer are both in the third in malignant tumor. In China, the morbidity of colorectal cancer is in the third,and the mortality of colorectal cancer is in the fifth of malignat tumor. And,the morbidity and mortality of colorectal cancer have both showed an upward trend in recent years. The methods of treatment of colorectal cancer have surgery, chemotherapy, radiotherapy, and molecular targeted therapy and so on. Among them, the most effective treatment of choice is surgical excision. But because the early diagnosis of colorectal cancer is very low in current clinical, so most patients are diagnosed in the late stages and lose the opportunity of surgery. So most patients of colerectal cancer need chemotherapy.Since5-fluorouracil(5-FU) has been listed in the1950s, it has always been the most widely used drug and the first choice of the chemotherapy of colorectal cancer. While the development of resistance to5-FU and sever side effects appears to be major obstacles to the successful chemotherapy. And the objective of monotherapy efficiency of5-FU is about20%. Therefore, the study on how to improve the sensitivity of5-FU on colorectal cancer is very significant in clinic, and thus can prolong the lives of patients of colorectal cancer and improve the quality of life of patients of colorectal cancer.A proliferation-inducing ligland (APRIL) is a new number in the superfamily of the tumor necrosis factor. APRIL can take part in promoting the proliferation of various tumor cells, the extension of the tumor cell survival and accelerating the process of tumor formation and so on by binding with its specific receptor. In normal human tissues, the expression level of APRIL is very low. But in many tumor tissues and tumor cell lines (particularly in digestive system cancer), the expression level of APRIL is in a high stage.Our previous studies have found, the expression of APRIL has different levels in different colorectal cancer cell lines. In LOVO cells and HCT116cells, the expression level is in relatively high expression and in SW480cells, SW620cells, and HT29cells,the expression level is in relatively low expression. And the expression of level is correlated with the resistance to5-FU. Therefore, our experiment choose LOVO cells in the late study.Polypeptide antineoplastic agents have became a new focus in the study of tumor molecular target therapy. Polypeptide antineoplastic agents have many advantages, such as easy to large-scale synthesis, simple to purification, low biological immunogenicity, high penetrability in tissue and so on. According to the research of biological characteristics of APRIL and inhibition research based on molecular biological characteristics of APRIL, we have not yet found the report of the use of Polypeptide antineoplastic agents to suppress the activity of APRIL, and then to inhibit tumor cell growth, and to improve the sensitivity of5-FU. In our previous study, we had successfully use Phage Screening Technigues to synthesized sAPRIL-binding peptide, with high affinity of APRIL. And we had already verified the inhibition effectives of colorectal cancer by the experimental study in vivo and in vitro. But we have not completed verification that sAPRIL-binding peptide can enhance the sensitivity of5-FU on colorectal cancer.Therefore, this experimental study which is based on our previous experimental studies have verified sAPRIL-binding peptide can enhance the sensitivity of5-FU on colorectal cancer and slow the growth rate of colorectal cancer in vivo and in vitro.Part1Study on sAPRIL-binding peptide can improve the sensitivity of5-FU on colorectal cancer LOVO cells in vitroObiectiveVerify that sAPRIL-binding peptide can improve the sensitivity of5-FU on colorectal cancer LOVO cell by experimental study in vitro.Method1) Cell proliferation assay:LOVO cells in the logarithmic phase were seeded in96well plates. On the second day, Group A was treated with PBS, group B was treated with5-FU, group C was treated with sAPRIL-binding peptide and group D was treated with5-FU and sAPRIL-binding peptide. After various treatments for48hours, we used Cell Counting Kit-8to decect anti-proliferative effects on LOVO cells.2) Cell cycle assay:LOVO cells in the logarithmic phase were seeded in6well plates. On the second day, Group A was treated with PBS, group B was treated with5-FU, group C was treated with sAPRIL-binding peptide and group D was treated with5-FU and sAPRIL-binding peptide. After various treatments for48hours, we used Cell Cycle and Apoptosis Analysis Kit to detect cell cycle of LOVO cells in each group.3) Cell apoptosis assay:LOVO cells in the logarithmic phase were seeded in6well plates. On the second day, Group A was treated with PBS, group B was treated with5-FU, group C was treated with sAPRIL-binding peptide and group D was treated with5-FU and sAPRIL-binding peptide. After various treatments for48hours, we used Annexin V-FITC Apoptosis Detection Kit to detect apoptosis of LOVO cells in each group.4) Western blot assay:LOVO cells in the logarithmic phase were seeded in12well plates. On the second day, Group A was treated with PBS, group B was treated with5-FU, group C was treated with sAPRIL-binding peptide and group D was treated with5-FU and sAPRIL-binding peptide. After various treatments for48hours, we used western-blotting to detect the expression levels of relevant cell-cycle proteins and cell-apoptosis proteins.5) Caspase-3activity assay:LOVO cells in the logarithmic phase were seeded in6well plates. On the second day, Group A was treated with PBS, group B was treated with5-FU, group C was treated with sAPRIL-binding peptide and group D was treated with5-FU and sAPRIL-binding peptide. After various treatments for48hours, we used Caspase-3Activity Assay Kit to detect the activity of csspase-3of LOVO cells in each group.6) Caspase-9activity assay:LOVO cells in the logarithmic phase were seeded in6well plates. On the second day, Group A was treated with PBS, group B was treated with5-FU, group C was treated with sAPRIL-binding peptide and group D was treated with5-FU and sAPRIL-binding peptide. After various treatments for48hours, we used Caspase-9Activity Assay Kit to detect the activity of csspase-9of LOVO cells in each group.7) Statistical analysis:we used SPSS13.0software to do statisital analysis.First we tested homogeneity of variance, if results had constant variance, differences among group were analysed with one way ANOVA, and differences between two groups were analysed with LSD method. If results did not have constant variance, differences among group were analysed with Brown-Forsythe, and differences between two groups were analysed with Dunnett’s C method.Results1) Cell proliferation assay:Inhibition rate in group B(5-FU group) was (37.62±7.07)%, in group C(sAPRIL-binding peptide group) was (31.45±4.87)%, and in group D(5-FU+sAPRIL-binding peptide group) was (63.05±2.15)%. The three experiment groups had significant differences (F=32.262, P=0.001).More effective inhibition of cell growth was induced in (5-FU+sAPRIL-binding peeptide) group compared with5-FU group alone (P=0.001, compared with5-FU).2) Cell cycle assay:The cell cycle was arrested in S phase in group B(5-FU group). The cell cycle was arrested in G0/G1phase in group C(sAPRIL-binding peptide group). The cell cycle was arrested in G0/G1and S phase in group D(5-FU+sAPRIL-binding peptide group). And, compared with group B, the S phase was arrested more obviously in group D.3) Cell apoptosis assay:The Early apoptosis rate was respectively (0.21+0.09)%,(12.8+0.30)%,(0.40+0.26)%,(17.1±0.66)%in A(PBS、B(5-FU)、 C(sAPRIL-binding peptide)、D(5-FU+sAPRIL-binding peptide) each group. The four groups had significant differences (F=32.262, P=0.001), and D(5-FU+sAPRIL-binding peptide) group induced more LOVO cells apoptosis than B(5-FU) group(P=0.000).4) Western blot assay:CyclinA and CDK2were high expressed in group B and group D, and was higher in group D. CyclinD1and CDK4was low expressed in group C and group D. XIAP, Survivin and Bcl-2were low expressed in group B and group D and XIAP was lower in group D. BAX was high expressed in group B and group D.5) Caspase-3activity assay:The activity of caspase-3was respectively (0.70±0.06) unit,(2.41±0.26) unit,(0.85±0.04) unit,(3.74±0.12) unit in A(PBS)、 B(5-FU)、C(sAPRIL-binding peptide)、D(5-FU±sAPRIL-binding peptide) each group. The four groups had significant differences (F=201.259, P=0.000), and D(5-FU±sAPRIL-binding peptide) group induced LOVO cells to produce more caspase-3than B(5-FU) group(P=0.000).6) Caspase-9activity assay: The activity of caspase-9was respectively (0.62±0.06) unit,(1.99±0.12) unit,(0.74±0.06) unit,(3.31±0.18) unit in A(PBS)、 B(5-FU)、C(sAPRIL-binding peptide)、D(5-FU±sAPRIL-binding peptide) each group. The four groups had significant differences (F=344.655, P=0.000), and D(5-FU±sAPRIL-binding peptide) group induced LOVO cells to produce more caspase-9than B(5-FU) group(P=0.000).ConclusionsAPRIL-binding peptide can improve the sensitivity of5-FU on colorectal cancer LOVO cell by experimental study in vitro.Part2Study on sAPRIL-binding peptide can improve the sensitivity of5-FU on colorectal cancer in vivoObjectiveVerify that sAPRIL-binding peptide can improve the sensitivity of5-FU on colorectal cancer by experimental study in nude mice.Method1) Transplant tumor in nude mice:LOVO cells were passaged by typsin digestion and were cultured to logarithmic phase. Cell viability was confirmed over 95%by typan blue. LOVO cells were prepared a2×107/ml suspension with PBS.4-6weeks female nude athymic(BALB/c-nu/nu) mice tegother20were breeded in specific pathogen free (SPF) conditions. About3weeks, tumor volume reached100-200mm3,2) Experimental groups and treatments:Nude mice with transplantation tumor were randomly divided into four groups (n=5). Group A was injected with0.1ml PBS both intraperitoneally and intratumorally. Group B was in injected with0.1ml5-FU intraperitoneally and0.1ml PBS intratumorally. Group C was in injected with0.1ml sAPRIL-binding peptide intratumorally. and0.1ml PBS intraperitoneally. Group D was in injected with0.1ml sAPRIL-binding peptide intratumorally. and0.1ml5-FU intraperitoneally. Treat once in every2days and altogether7times. The tumor volume was calculated before each time of injection. Then curve tumor growth. Seven days after the final injection, tumors were excised and weighed to calculate tumor inhibition.ResultsWeight of rumors in the four groups were (346.55±26.34)mg,(238.16±19.11)mg,(257.66±18.47)mg, and (170.53±19.58)mg respectively. The calculated tumor inhibitive rate was31.28%in the5-FU treated group,25.65%in the APRIL-binding peptide treated group, and50.79%in the combination treated group.ConclusionIn nude mice, sAPRIL-binding peptide can improve the sensitivity of5-FU on colorectal cancer. |
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