Correlational Studies Of Drug-resistance And Anti-apoptosis Of Colorectal Cancer Stem Cell And Its Hypoxia Microenvironment

Background and aimsThe theory of cancer stem cell insists that there are a small number of cancer cells among the tumor tissue called cancer stem cells (CSCs) which possess peculiar biological functions, such as self-renewal, certain multiplex differentiation potential, excellent ability of tumorigenicity in immunodificient animals and differentiation into others constituent part of tumor tissue. At present, cancer stem cells have been found in variety of solid tumors. The identification and separating technology for CSCs depend on the specifically different surface markers which express on them and sorted by flow active cell sorting (FACS) and magnetic active cell sorting after the special surface marker of CSCs labeled in vitro. Furthermore, the subpopulation cells sorted through efflux special dry Hochest 33342 also have the stem cell characteristics and thus it’s another method of sorting CSCs.The tumor recurrence is very common and troublesome during the clinical therapeutic process and more and more researchs have focused on cancer stem cells and related hypoxia microenviorment. First of all, cancer stem cell is in the stationary phase—G0 phase of cell cycle, while a large proportion of chemotherapeutics could only act on cellular proliferative stage. In the second place, the feature of self-renewal and undifferentiation is maintained and modulated jointly through tumor mesenchymal cells, vascular endothelial cells and related microenvironment. Also, cancer stem cell could tolerate chemotherapy drugs and resist radiation therapy owning to high level of drug-resistantal protein and powful ability of repair after DNA injury. Howerer, the research on cancer cell line seems not enough. So our experiment has focused on the characters of drug resistance and anti-apoptosis and the possible mechanism.Besides, some tumor cells and cancer stem cells are in the ischemic and hypoxia microenvironment because of overgrowth of tumor tissue and asymmetry of internal vascular distribution. Meanwhile, the hypoxia microenvironment might change the biological characters of cancer cells and cancer stem cells that result in drug resistance, metastasis and recurrence. Besides, cobalt chloride could imitate the anoxia microenvironment of cancer cells in vitro through preventing hydroxyl of HIF-la of cancer cells in turn the reaction to anoxia so that the expression level of HIF-la could indicate anoxia degree of cells. Therefore, we regard the hypoxia as the initiation in order to reach the widely research.In our study, human colon cancer cell lines SW480 was selected, sorted by MACS labeling surface marker CD133 and required CD133+ SW480 stem cell subsets. The sensibility to chemotherapeutics used clinically and the gene expression level of drug-resistant and anti-apoptosis were investigated in vitro regarding the common SW480 as the control group. At the same time, hypoxia microenvironment was brought in our study. The biological characteristics change of common SW480 and CD133+ SW480 subsets and the gene expression level of surface marker, invasion and metastasis of cancer cells resulted from anoxia microenvironment were probed in order to lay the fundation of the research and treatment in colorectal cancer and colorectal cancer stem cells. Materials and methods1. Cell culture and magetic active cell sorting (MACS)The human colon cancer cell line, SW480, were maintained in RPMI 1640 medium containing 10% fetal bovine serum,1×105U/L penicillin G and streptomycin at 37℃in a 5% humidified CO2 atmosphere and 2.5% of trypsin digestion to go down the future generation. SW480 cells in the exponential phase were digested, collected and resuspended in sorting buffer. FcR retardant and CD 133 micro magnetic beads were added and incubated in dark then washed and resuspended the cells with sorting buffer. The cell suspension were added to MS sorting column and the negative cells flowed, then added the sorting buffer to the column and push the plunger to obtain the positive cells.2. CCK-8 cell cytotoxicity testTreated cells were seeded at concentrations of 5×103 contained 90μl medium per well in 96-well plates and each group set five repeated wells. Then 5-FU (10μl)and L-OHP (10μl) with four concentration gradient were added to each group. After 24 hours, CCK-8 (10μl)was added to each well and incubated at 37℃for 2 hours and absorbance at 450nm was measured, following the manufacturer’s instructions. Then the curve graph of the two groups of cells and two drugs was pictured based on the results. This experiment repeated three times and the results were recorded mean±SD.3. Detection of apoptosis1×106 CD133+ SW480 cells or SW480 cells were grown to confluence in 6cm culture dish, then IC20 of 5-FU and L-OHP were added into in order to induce apoptosis. About 1～5×105 cells were collected, added Annexin V-FITC binding buffer 195μl and Annexin V-FITC 5μl, then incubated in dark place for 10min in room temperature. The cells were centrifuged at 1000g for 5min, discarded the supernatant and added Annexin V-FITC binding buffer 190μl and PI ice-bath in dark place for FCM.4. The hypoxia enviorment initated by cobalt chloride in vitroThe mother soltion of cobalt chloride is preparated by SFM. Dilute to different contrations for 50,100,150,200 and 250μmol/L respectively. Then add to 6cm culture dish of SW480 cells and CD133+ SW480 subsets to imitate hypoxia.5. Real-time fluorescence quantitative PCRRNA was extracted through TriZol and the purity and concentration were measured by spectrophotometer.500ng RNA was reverse transcripted by reverse transcription kit and the reactive system were composed of primer, cDNA formwork, enzyme, SYBR and ROX. ABI 7500 real-time PCR instrument collect the fluorescent signal constantly and 95℃for 30s as the pre-degeneration, then 95℃5s and 60℃34s for 40 cycles as the PCR reactions. The results were conversion on the basis of Ct value.6. Western blottingTotal cell protein was extracted and the concentration was measured by BCA.10% electrophoresis gel and 5% stacking gel were prepared. Protein (30μg) was mixed with loading buffer and separated by running in SDS-polyacrylamide gel. Proteins were then transferred to PVDF membrane and then blocked for lh at room temperature in TBS-T (50mmol/L Tris-HCl,150mmol/L NaCl,0.1% Tween 20 buffer containing 5% nonfat milk). Membranes were then incubated overnight at 4℃with the respective primary antibodies (1:500-1000). Then wash the membrane in TBS-T and incubate secondary antibody (1:2000-5000) and the blots were developed with a ECL western blotting subsrate. The blots were investigated under transilluminator and the results were analysed by Quantity one software. 7. Statistical analysisStatistical analysis was performed with SPSS 13.0 software. Descriptive statistics were calculated with means and standard deviation. The proportion of positive cells got from MACS, the inhibition ratio of growth detected by CCK-8 tests and the proportion of early apoptosis were calculated with mean value for at least three times. The western blotting results were analyzed by statistics after grayscale value scanning. The results in terms of real-time PCR, the Ct values in experimental group were showed that of relative multiples in control group, since the gene expression was regarded as basic value 1. Before the comparation of parameters, Levene’s homogeneity test of variances was performed. If the variance was equal, independent sample t-test was adopted. One-way ANOVA was used in multiple comparison. If the variance was equal, we applied LSD method. If the variance was unequal, Brown-Forsythe or Welch approximate variance analysis was adopted. Difference was statistically significant when P value<0.05.Results1. MACS is an effective method for cell sorting:the cells which express CD133 in the CD133+ SW480 subsets separated by MACS account for 85.56±0.89%.2. The inhabition ration of CD133+ SW480 cells is lower than that of common SW480 cells in the same contration of 5-FU and L-OHP, respectively.3. The apoptisis ration of CD133+ SW480 cells is lower than that of common SW480 cells when exposed in the same concentration of 5-FU and L-OHP to induce apoptosis.4. The expression of HIF-la protein depends on the concentration and time of cobalt chloride acting to SW480 cells.200μmol/L cobalt chloride for 8h is the optimum condition for HIF-la protein expressed.5. In the oxygen deficit condition, the number of CD133+ SW480 spherical cells is more than that in the normal condition.6. The expression of HIF-la protein of common SW480 is higher than that of CD133+ SW480 when exposed in cobalt chloride with the optimum concentration and time.7. The expression of PROM1, survivin and VEGF is high than that of SW480 cells untreated by cobalt chloride.ConclusionMACS is the good method to sorting CSCs since lower pollution rate, higher ration of aimed cells, more activited cells and more convenient operation. In our study, CD133+ SW480 subsets were drug-resistant and anti-apoptosis through CCK-8 cell cytotoxicity test and detection of apoptosis. Meanwhile, some genes related to drug-resistant and anti-apoptosis may have high level expression. It’s convinced that this is one of the reasons for the biological characters of CSCs.COCl2 could imitate the anoxia condition of cancer cells in vitro and the HIF-la protein expression in high level under the most appropriate concentration and action times of COCl2. CD133+ SW480 subsets have higher sphere formation ration when imitate anoxia by COCl2 than common cultural conditions. The expression of PROM 1 is higher in SW480 cells which were induced by COCl2 than the cells cultured in normal condition. We can infer that hypoxia is more or less connected with the transformation from common cancer cells to cancer stem cells. We also find that the HIF-la protein is higher expressed in SW480 than that in CD133+ SW480 subsets which induced by COCl2 for the most appropriate concentration and action times. It’s indirectly reflect the characters of CD133+SW480 subsets that they are not sensitive to the anoxia microenvironment. We found that the genes related anti-apoptosis, tumor invasion and metastasis increasing express in SW480 induced by COCl2. Therefore, it must exist indiscerptible connection between anoxia microenvironment and drug-resistance, anti-apoptosis, invasion, metastasis and tumor recurrence.