Research On The Effects Of Anti-cancer Protein AC-01 On Colorectal Cancer Cells

BackgroundWith the development of people’s life standard, the incidence of colorectal cancer becomes more popular in citizens and the young. It’s reported that in our country, the average onset age of colorectal cancer is 10 years younger than in foreign countries, which has seriously affected people’s health. Thus, it’s urgent to find a new diagnostic method and treatment. The occurrence and development of colorectal cancer is a complex process of multiple steps, gene, multiple stage. And surgery, as the only treatment of zero order kinetics, plays an important role in tumor’s treatment. But its development is restricted by the improvement of operation instruments and technique. Though chemotherapy and radiotherapy have essential place in tumor treatment, their poisonous side efforts have restricted the patient’s tolerance. As the instability of gene and the heteromorphism of tumor cell, it’s a hard mission to investigate tumor from the aspect of gene and others. Therefore, to manufacture new anti-tumor drugs, and to change drugs’ effort on tumor, is a meaningful task most researchers confronting.Anticancer protein has become a heated tumor therapeutic strategy based on the development of molecular biology and immunology. Anti-tumor protein’s characteristics such as the molecular mass of small, easily penetrating the tumor cell, and having strong stability, can suppress tumor growth and metastasis by improving the immune response, inhibiting tumor angiogenesis, directly inducing the apoptosis of tumor cells or blocking tumor cell cycle mode. The research group has found a new human gene of unknown function through early experimental research. It has mitotic inhibition effect on colorectal cancer cell line （LoVo cells）, and there is a certain correlation among its coded protein, the unfolded protein response （Unfolded protein response, UPR） and endoplasmic reticulum stress （Endoplasmic reticulum stress, ERS）. Thus it is named AC-01, and had applied for the National Science Foundation of China. This experiment is one of divided subjects of the project. However, according to the present research results have been achieved, mechanism of AC-01 protein in colorectal cancer is not clear yet. Combining papers and the achievements during master, we considered AC-01 may inhibit colorectal cancer cell endoplasmic reticulum stress, activate the expression of its downstream signal transmission and related genes, thereby promoting tumor cell apoptosis and inhibiting tumor growth.The endoplasmic reticulum （ER） is a relatively sensitive membranous organelle in mammalian cells, it is a vital place of synthesis and modification for intracellular protein, lipid and carbohydrate. Endoplasmic reticulum regulates the intracellular calcium concentration though calcium storage and release. When a variety of physiological and pathological factors causing endoplasmic reticulum homeostasis, the unfolded protein and misfolded protein increase or calcium ion concentration changes in endoplasmic reticulum lumen, inducing endoplasmic reticulum stress （ERS）.ERS activates endoplasmic reticulum stress reaction and the unfolded protein response （UPR）. UPR eventually reduces endoplasmic reticulum stress and recovers homeostasis by reducing the synthesis of new proteins, promoting the folding of unfolded protein and increasing the degradation of unfolded protein. When endoplasmic reticulum stress is too strong or too long, the homeostasis cannot restore, then UPR can activate cell apoptosis inducing intracellular signal. So far, the apoptosis pathways mainly include death receptor activation （extrinsic pathway） pathway, mitochondrial damage pathway （intrinsic pathway） pathway and apoptosis induced by endoplasmic reticulum stress. Recent studies have found that cell apoptosis inducing by endoplasmic reticulum stress acts an important part in the pathogenesis of several diseases.There are 3 trans-membrane induced proteins in endoplasmic reticulum in mammalian cells, which can receive and transmit UPR signals. They are named inositol requiring enzyme-1（IRE-1）, activating transcription factors-6 （ATF-6）, and protein kinase R-like ER kinase （PERK） respectively. When the endoplasmic reticulum maintains steady, L ends of ATF-6 and PERK combine glucose-regulated protein78 （GRP78）to keep inactive state. When the endoplasmic reticulum is in stress state, large numbers of unfolded or misfolded proteins accumulate in the endoplasmic reticulum lumen, and GRP78 dissociates with induction-proteins of ATF-6 and PERK, then GRP78 combines with the unfolded protein, which can reactivate IRE-1 directly. Activated IRE-1, ATF-6 and PERK activate their downstream signal transmissions and the expressions of and related genes respectively, which is referred as UPR. Most studies have showed that PERK pathway activates ATF-4, CHOP and GADD34, which have played important roles in apoptosis. Inhibiting protein synthesis through PERK pathway is beneficial to reduce the stress of endoplasmic reticulum, making the recovery of endoplasmic reticulum homeostasis. PERK pathway is also capable of promoting phosphorylation of elF-2α, inhibiting translation and synthesis of protein in order to relive the pressure of ER. And the above pathway can also induce apoptosis by STAT3 signal pathway, control expression of cyclinD1, CDK2, MMP2 and MMP9, therefore regulating apoptosis and angiogenesis of colorectal cancer cells.Stat 3 is one of an important members of STAT, and STAT3 signal transmission pathway has a close relationship with proliferation, differentiation and apoptosis of cells. The continuous activation of the pathway can lead to abnormal cell proliferation and malignant transformation, currently defined as cancer gene. My research "Construction of STAT3 short hairpin RNA and its effect to human colorectal cancer HCT116 cells proliferation and invasion" during master stage used RNAi to specifically silent STAT3 in human colorectal cancer cells HCT116, and observed the growth, proliferation and apoptosis of colorectal cancer cells after RNA interfered STAT3. It was confirmed that inhibiting the expression of STAT3mRNA can effectively depress cancer cell proliferation, then block cancer cells at GO,G1 phase and induce cell apoptosis. At the meanwhile, invasive ability of tumor cells decreased significantly.Therefore, we predict that AC-01 protein modulates UPR signal pathway through regulating stress of ER in colorectal cancer cells, then further affected its survival, progression, angiogenesis and apoptosis in order to inhibit the occurrence and development of tumor. This study expects to deeply explore how anti-tumor protein AC-01 affects the characteristics of colorectal cancer cells and to know its biological mechanism via vitro and vivo experiments, with the hope to provide experimental evidence for colorectal cancer’s biological treatment.ContentThe whole project includes five parts:Part I The expressions of AC-01 in human colorectal cancer and the relationship between it and STAT3ObjectivesTo study the expressions of AC-01 and its role in human colorectal cancerMethodsBased on the prophase research that suggested that STAT3 is a promoter tumor gene, to study the relationship between the expression of AC-01 of human colorectal cancer and their adjacent tissues by immunohistochernical method.Samples of human colorectal cancer were divided into 4 groups by using randomgrouping test method.Each group was devided into human colorectal cancer and their adjacent tissues. The expressions of AC-01、STAT3 and pSTAT3 in four groups were examined by western blot and compared.ResultsThe expression of AC-01 in human colorectal cancer was lower than that in adjacent tissues. The detection and analysis by western blot showed that the expression of AC-01 in human colorectal cancer was lower than that in adjacent tissues in each group and showed obvious difference （P<0.05）.The expression of STAT3 in human colorectal cancer was higher than that in adjacent tissues in each group and showed obvious difference （P<0.05）. The expression of pSTAT3 in human colorectal cancer was higher than that in adjacent tissues in each group and showed obvious difference（P<0.05）. There was a significant negative correlation between the expression of AC-01 and the expression of STAT3 in human colorectal cancer. ConclusionsThe experimental study showed that the expression of AC-01 in human colorectal cancer was lower than that in normal tissues. The expression of STAT3 in human colorectal cancer was higher than that in normal tissues.. The expression of pSTAT3 in human colorectal cancer was higher than that in normal tissues. There was a significant negative correlation between the expression of AC-01 and the expression of STAT3 in human colorectal cancer. Based on the prophase research which suggested that STAT3 is a promoter tumor gene, we speculated that AC-01 could be an anticancer proteinPart II Construction and identification vector and stable expression cell strainExperiment 1 Construction of targeting AC-01 expression vectorObjectivesTo culture human colorectal cancer cell line LOVO in vitro, extract plasmid and construct the pBabe-puro-AC-01 over expression vector. Meantime, to design and construct the pSUPERretro-puro/AC-01/RNAi expression vector with AC-01 as the target gene, and to construct stable expression cell strain finally.MethodsHuman colorectal cancer cell line LOVO was cultured in vitro and analyzed according to the AC-01 gene sequence provided by NCBI （NCBI accession number XM₀06714717）. With the plasmid carrying AC-01 gene as template, to amplify coding sequence of AC-01 gene by PCR and construct the pBabe-puro-AC-01 over expression vector. According to the design principle of the shRNA, to choose select specific sequences as target for interference effects by reference to many relevant documents, and design and synthesis of one sequence, which can be disturbed. Also, to design a non-specific sequence which has no significant homologous with human gene sequences in present gene library by Basic Local Alignment Search Tool （BLAST） retrieval as negative control sequence, construct the pSUPERretro-puro/AC-01/RNAi expression vector. The vectors were identified by enzyme digestion and gene sequencing. Finally, to extract the plasmid.ResultsHuman colorectal cancer cell line LOVO was cultured in vitro. To design and synthesis of one sequence with AC-01 gene which can be disturbed and the negative control sequence. To successfully construct the pBabe-puro-AC-01 over expression vector and pSUPERretro-puro/AC-01/RNAi expression vector. Using the alkaline lysis method completed successfully purified plasmid extraction. The plasmid were identified by enzyme digestion with BamH I and PstI respectively, and the recombinant vector can be incised by BamH I, but not by Pst I. At the same time, positive clones which identified by PCR were sequenced in order to verify that interference plasmid included the sequence of the target gene, the recombinant plasmid was successfully constructed.Experiment 2 Construction and identification stable expression cell strainObjectsTo screen the most efficient sequence AC-01/RNAi, transfect into human colorectal cancer cell line LOVO with retroviral vector, screen and reserve the stable expression cell strain, identify stable expression cell strain, and detect the AC-01 gene expression.MethodspSUPERretro-puro/AC-01/RNAi expression vector was transfected into the human colorectal cancer cell line LOVO by liposome method. Transfected cells were divided into 4 groups:nterference-control group, AC-01/RNAi-1 interference group, AC-01/RNAi-2 group and AC-01/RNAi-3 group. After transfection of 72h,the expression of AC-01 protein was examined by western blot and compared. To screen the most efficient sequence AC-01/RNAi.To prepare the retrovirus by calcium phosphate method, pBabe-puro/AC-01 plasmid and pSUPERretro-puro/AC-01/RNAi plasmid has virus as a vector to LOVO cells to infection. After infection, to add puromycin and screen positive clones. To collect stable expression cell strain and divided into four groups:LOVO-vector, LOVO-AC-01, LOVO-scramble, and LOVO-AC-01/RNAi. The RNA of stable expression cell strain was amplified by RT-QPCR and examined. The AC-01 of stable expression cell strain was examined and compared. To analyze the effect after transfection.ResultsAC-01 RNA interference recombinant successfully was transfected into LOVO cells, Western blot test showed that the effect of AC-01/RNAi-1 group was the best sequence. AC-01 plasmid and AC-01/RNAi-1 plasmid were transfected into LOVO cells mediated by retrovirus. RT-PCR assay used FTC-2000A to examine each sample Ct value. There were no significant differences on the expression of AC-01 mRNA between LOVO-vector group and LOVO-scramble group （P>0.05）. AC-01mRNA expression in LOVO-AC-01 group was significantly higher, which were significantly different from other three groups （P<0.05）,especially higher than that in LOVO-AC-01/RNA group. Accordingly, AC-OlmRNA expression in LOVO-AC-01/RNA group was significantly lower, which were significantly different from other three groups （P<0.05）, especially lower than that LOVO-AC-01/RNA group. Western blot test showed that the development of internal reference β-actin expression was consistent in all groups. The expression of AC-01 protein between LOVO-vector group and LOVO-scramble group had no significant difference （P>0.05）. AC-01mRNA expression in LOVO-AC-01 group was significantly higher, which were significantly different from other three groups（P<0.05）,especially higher than that in LOVO-AC-01/RNA group. Accordingly, AC-OlmRNA expression in LOVO-AC-01/RNA group was significantly lower, which were significantly different from other three groups （P<0.05）,especially lower than that LOVO-AC-01/RNA group.Part Ⅲ Effect of AC-01 on biological characteristics of human colorectal cancerObjectivesTo explore the effect of AC-01 on proliferation, differentiation, apoptosis and invasiveness of human colorectal cancer HCT116 cell proliferation.MethodsAC-01 plasmid and AC-01/RNAi-1 plasmid were transfected into LOVO cells mediated by retrovirus. The LOVO cells were divided into four groups: LOVO-vector,LOVO-AC-01,LOVO-scramble and LOVO-AC-01/RNAi. ①To observe transfected positive cells and transfected negative cells by using laser confocal scanning microscopy, and compare its nucleus, morphology, and cell number. ②To examine the inhibitory effect of AC-01 on the growth of LOVO cells by MTT assay. After interference by RNA, to compare the changes. ③To analyze invasiveness of cancer cell in vitro invasion assay.④cratch adhesion test:Taking four horizons from each hole after scratching respectively in Oh,12h,24h,36h to observe scratching healing ability. The healing rate represented the healing ability. To observe cells healing ability of four groups. ⑤With 3D-Culture experiments on four groups of cells, morphological changes were observed and compared.ResultsAC-01 plasmid and AC-01/RNAi-1 plasmid were successfully transfected into LOVO cells mediated by retrovirus. The LOVO cells were divided into four groups: LOVO-vector, LOVO-AC-01, LOVO-scramble and LOVO-AC-01/RNAi.①Observing transfected AC-01 positive cell by using laser confocal scanning microscopy, its nucleus was significantly decreased compared with surrounding-control cells. AC-01 transfected cells manifestation showed that differentiation has more processes. With the increase of AC-01 recombinant protein concentration, colorectal cancer cell line LoVo cell count decreased significantly.②The MTT assay showed that:There were no significant differences on the proliferation of LOVO cells between LOVO-vector group and LOVO-scramble group（P>0.05）.The proliferation of LOVO cells was significantly inhibited in LOVO-AC-01 group, which were significantly different from other three groups（P<0.05）, especially weaker than group LOVO-AC-01/RNA group. The proliferation of LOVO cells in LOVO-AC-01/RNA group were significantly increased, which were significantly different from other three groups（P<0.05）, especially obvious stronger than the LOVO-AC-01/RNA group.③The vitro invasion assay showed that there were no significant differences on the invasive cell numbers between LOVO-vector group and LOVO-scramble group（P>0.05）, The invasive cell numbers was significantly inhibited in LOVO-AC-01 group, which were significantly different from other three groups（P<0.05）, especially weaker than group LOVO-AC-01/RNA group. The invasive cell numbers in LOVO-AC-01/RNA group were significantly increased, which were significantly different from other three groups （P<0.05）, especially obvious more than the LOVO-AC-01/RNA group.④Scratch adhesion test show that there were no significant differences on the cell-healing rate between LOVO-vector group and LOVO-scramble group （P>0.05）. The cell-healing rate was significantly lower in LOVO-AC-01 group, which were significantly different from other three groups （P<0.05）. The cell healing rate in LOVO-AC-01/RNA group were significantly higher which were significantly different from other three groups （P<0.05）.⑤3D-Culture experiments showed that LOVO cells in LOVO-vector group and LOVO-scramble group had little change. The antennas on the LOVO cells surface in LOVO-AC-01 group were obviously shrinkage, and the surface becomes smooth. The antenna on the LOVO cells surface in LOVO-AC-01 group were obviously prolong, and the surface becomes more rugged.ConclusionsThis study showed that:The AC-01 protein can specifically inhibit the proliferation and invasive ability of cancer cells, and promote apoptosis of cancer cells, moreover the effect is remarkable. The specific silencing of AC-01 gene expression can promote the proliferation of cancer cells, enhancing the ability of invasion, and the effect is remarkable. AC-01 could inhibit the proliferation of colorectal cancer cells line LoVo cells in a dose-dependent manner. The AC-01 protein was a kind of anticancer protein, might be a molecular marker for colorectal cancer clinical poor prognosis, and even become a target for treatment of colorectal cancer.]Part Ⅳ The role of AC-01 and its mechanisms on colorectal cancer ObjectivesTo explore the mechanism that AC-01 influences survival, tumor progression, angiogenesis and apoptosis of LOVO cells through the regulation of endoplasmic reticulum stress of colorectal cancer.MethodsOwing to the distribution of this protein, we hypothesized that the expression of the protein may be associated with colorectal cancer cells related to endoplasmic reticulum stress.Therefore, we used the classical endoplasmic reticulum stress model induced by tunicamycin, observed the expression characteristics of the proteins under stress conditions and detect the AC-01 protein and endoplasmic reticulum stress marker GRp78, GADD expression in order to understand the relationship between the endoplasmic reticulum stress and AC-01 protein.The AC-01 plasmid and AC-01/RNAi-1 plasmid was transfected into LOVO cells by retro virus, divided into four groups:LOVO-vector group, LOVO-AC-01 group, LOVO-scramble group, LOVO-AC-01/RNAi group.By real-time quantitative PCR and Sybrgreen dye method, to detect the expression of makers GRp78, GADD, XBP1, ATF6, eIF2 alpha, p-e1F2 alpha, cyclinD1, CDK2 in the endoplasmic reticulum stress path in different groups through housekeeping gene acting as an internal calibration.And thereby to affect the expression of its downstream gene proteins STAT3, p-STAT3, MMP2, MMP9, and compare the data.ResultsBiacore 3000 detection showed that there existed binding activity ofAC-01 fusion protein to Ca2+.In cell endoplasmic reticulum stress condition which tunicamycin induced colorectal cancer cell line LoVo cells. The expression of GRp78 and GADD increased with the concentration of tunicamycin increased, while the expression of AC-01 protein decreased with the concentration of tunicamycin concentrations increased.With real-time fluorescence quantitative RT-PCR assay, there was no no significant difference on markers of endoplasmic reticulum stress and the effect of its downstream gene protein between LOVO-vector group and LOVO-scramble group （P>0.05）.The expressions of GRp78, GADD, XBP1, ATF6, eIF2, p-eIF2, cyclinD1, alpha, alpha CDK2, MMP2, MMP9 in LOVO-AC-01 group were down-regulated, and STAT3, p-STAT3 expression were significantly increased, which were significantly different from other three groups（P<0.05）, especially weaker than LOVO-AC-01/RNA group.The expressions of GRp78, GADD, XBP1, ATF6, eDF2, p-eIF2, cyclinD1, alpha, alpha CDK2, MMP2, MMP9 in LOVO-AC-01/RNA group were up-regulated, and STAT3, p-STAT3 expression was significantly decreased, which were significantly different from other three groups（P<0.05）, especially stronger than LOVO-AC-01/RNA group.ConclusionsThis study showed that:the AC-01 protein related to endoplasmic reticulum, and it may be a protective protein for inhibition of endoplasmic reticulum stress injury. AC-01 influence UPR signal transduction pathway by endoplasmic reticulum stress,and the related three pathways were as following:PERK-eIF2A pathway, IRE1-XBP1 pathway and ATF6 pathway, thus affecting the mechanism of survival, tumor progression and apoptosis in the colorectal cancer LOVO cells. The AC-01 further inhibiti STAT3, p-STAT3 and its associated protein MMP2, MMP9, thereby affecting the proliferation and tumor angiogenesis formation in colorectal cancer LoVo cells.Part V Effect of AC-01 on colorectal cancer cells in miceObjectivesTo explore the effect of AC-01 on colorectal cancer cells through animal experiments.MethodsXeno-transplantation of tumor cells experiments in nude mice:male nude mice BALB/c vaccinate target cells via Tail intravenous or subcutaneous injection,10mg, 3/day. Then divided into LOVO-AC-01 vaccination group and LOVO vaccination group（control group）, gross tumor volume is cultivated by withxlength x0.5, and used Slide caliper to measure and cultivate Lx×0.5. The nude mice is killed in euthanasia, then weigh and cultivate tumor index, （tumor index=tumor weigh/nude mice weigh×1000）.ResultsCompared with the control group,the speed of tumor growth in LOVO-AC-01 injected group decreased obviously. What’s more, the average volume, number and weight of LOVO-AC-01 vaccinated group were all lower than LOVO vaccinated group. The tumor index also had shown that, after vaccination of AOVO-AC-01,the proliferation of LOVO cells in nude mice obviously attenuated.ConclusionsThis study has made a conclusion that AC-01 recombination protein could depress tumor’s formation and growth in colorectal in nude mice.