| Background:Pancreatic carcinoma has become one of the most common malignant tumors in the digestive tract. In recent years, the incidence rate of pancreatic cancer showed a clear upward trend in the world, While the survival rate did not significantly increased, 5-year survival rate of <5%. Chemotherapy is still the most important adjuvant therapy among the Comprehensive treatment of pancreatic cancer. Gemcitabine is the first-line chemotherapy agent approved by the U.S. Food and Drug administration (FDA), but the clinical effect is still to be improved. Inherent and acquired drug resistance is the main reason for the failure of the treatment. Cancer stem cell theory provides a new insight into the awareness of pancreatic cancer development and resistance to chemotherapy.Objective: 1. To separate the cancer stem cell-like cell subsets by means of side population approach in human pancreatic cancer cell lines and research the relationship between side population cells and cancer stem cells. 2. To compare the different tolerance ability of gemcitabine among the different cell subsets from human pancreatic cancer cell line. 3. To explore the relationship between gemcitabine- resistant pancreatic cancer cells and cancer stem cells, To compare the expression level of hedgehog signaling pathway members between gemcitabine-resistant cells and parental cells. 4. To investigate whether inhibition of hedgehog signaling pathway can reverse acquired gemcitabine- resistance in pancreatic cancer cells.Methods: 1. Hoechst33342 staining and flow cytometry assay were used to analyze the proportion of side population in BxPC-3,CFPAC-1,Mia PaCa-2,PANC-1, SW1990 pancreatic cancer cell lines, and SP and non-SP subsets were separated. Cell growth curve were determined by methyl thiazoly tetrazolium (MTT) assay. Cell cycle distribution of SP and non-SP cells were determined by flow cytometry. The tumorigenicity of SP and non-SP cells from SW1990 were compared by nude mice xenograft transplant experiments. 2. Hoechst 33342 staining and CD44, CD24 antibodies label were used to separate the cancer stem cell-like cells from SW1990 cells. After treated with gemcitabine in different concentration, IC50 to gemcitabine in different cell subsets were determined by MTT method. Cell apoptosis and death were analyzed by Annexin V and PI double stain. Fluorescence quantitative real time PCR and western blot were used to determine the expression of ABCB1, ABCG2, Shh and Gli-1 in mRNA and protein levels, respectively. 3. human pancreatic gemcitabine-resistant SW1990 cell line were established by intermittently exposure to increasing concentration gemcitabine. The percentage of side population, CD44+CD24+ and CD133+ cells were analyzed by flow cytometry assay. Different expression of ABCB1, ABCG2, SMO and Gli-1 between drug resistant cells and parental cells at mRNA and protein levels were compared by quantitative RT-PCR and western blot, respectively. 4. Before and after treated with hedgehog inhibitor, cyclopamine, the percentage of CD44+ and CD133+ cells were analyzed. Quantitative RT-PCR and western blot were performed to compare the changes of ABCB1, ABCG2, SMO and Gli-1 at mRNA and protein level, respectively. Results:1. The proportion of side population in BxPC-3,CFPAC-1,Mia PaCa-2,PANC-1,SW1990 pancreatic cancer cell lines were 0.79±0.11%,2.59±0.19%,0.03±0.02%,7.57±1.87%,4.19±0.98%, respectively. SP cells showed no significant difference in cell growth curve, and had a lower percentage of cells in the S phase (21.63±3.09% vs. 29.22±1.75%, P=0.02), and a higher percentage of cells in the Gl phase (70.02±2.68% vs. 63.58±1.24%, P=0.019), when compared to non-SP cells. SP cells can generate SP and non-SP cells in vitro and in vivo, higher tumorigenicity of SP cells were also observed. 2. SP and CD44+CD24+ subsets in pancreatic cancer cell line SW1990 are more resistant to gemcitabine, The SP cells and CD44+CD24+ demonstrated a higher IC50 than the non-SP cells and CD44+CD24+, (23.59±9.30 vs. 4.38±1.59, P=0.024, 12.30±0.43 vs. 3.97±0.86, P=0.008, respectively.) SP and CD44+CD24+ cells are more tolerance to gemcitabine induced apoptosis. SP and CD44+CD24+ cells highly expressed ABCB1, ABCG2, Shh and Gli-1 at mRNA level, and the expression of ABCB1, ABCG2 and Gli-1 in protein level were also higher expressed, when compared to non-SP and CD44-CD24- cells, repectively. 4. after treatment of 2 uM cyclopamine for 7 days, the percentage of CD44+ and CD133+ in gemcitabine-resistant cells decreased from 93.16±2.46%to 36.68±2.44%, and from 95.87±1.47% to 62.76±1.28%, respectively. Expresson of ABCB1, ABCG2, SMO and Gli-1 at both mRNA and protein level were down-regulated. Aftertreatment with cyclopamine, gemcitabine-resistant cells were re-exposed to high dose of gemcitabine, a significant increased cell apoptosis and death were observed by flow cytometry assay.Conculsion: 1.Side population cells in SW1990 possess the property of cancer stemcells. 2. Cancer stem cell-like subsets in SW1990 cells are more tolerance to gemcitabine, high expressed some members of hedgehog signaling pathway. 3. gemcitabine- resistant pancreatic SW1990 cells were enriched with cancer stem cells and high expressed hedgehog member SMO and Gli-1. 4. Inhibition of hedgehog signaling pathway by cyclopamine can reduce the proportion of cancer stem cell in gemcitabine-resistant cells, down-regulate SMO and Gli-1 level, restore thesensitivity to gemcitabine and reverts the acquired drug resistance. |
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