| Part I Glucose Deprivation and Formation of Multidrug Resistanceof Hepatocellular CarcinomaObjective To investigate the influence of glucose deprivation on the formation of multidrug resistance of hepatocellular carcinoma and to clarify its mechanism.Methods HepG2 cells were exposed to glucose-free DMEM medium for 24h, 48h, 72h respectively. Apoptosis Index of the HepG2 cells exposed to low glucose was analyzed by Annexin/PI method using Flow Cytometry after administration of 5-Fu. Real-time fluorescent quantitative PCR and Western-blot technique were respectively used to determine the expressions of mdr1, MRP1, and LRP gene at the mRNA and the protein level.Results The resistance of HepG2 cells to 5-Fu was gradually enhanced and the apoptosis peak was delayed as the exposed time extended under the surroundings of low glucose. In HepG2 cells subjected to low glucose, the expressions of the mdr1, MRP1 and LRP at mRNA and protein level were stepped up as the exposed time increased and LRP made the most notable changes.Conclusion Glucose starvation is one of the causes that facilitate the development of MDR of HCC. Low glucose induces MDR phenotype of HCC by upregulating the expressions of a cohort of the MDR related genes.Part II Role of hypoxia-inducible factor-1-α in formation of multidrug resistance induced by low glucose microenvironment in hepatocellular carcinomaObjective To explore the status and role of hypoxia-inducible factor-1-α (HIF-1α) in formation of multidrug resistance (MDR) induced by microenvironment of low glucose and to find a new and effective molecular target on preventing and reversing MDR in hepatocellular carcinoma.Methods In HepG2 cells exposed to low glucose, the expression of HIF-1α at mRNA and protein level was respectively analyzed using real-time fluorescent quantitative PCR and Western-blot technique and its expression localization was investigated by immunohistochemistry. Plasmid pcDNA3/HIF-1α was transfected into HepG2 cells and the expression of mdr1, MRP1 and LRP in transfected cells was determined by the same mothods.Results In HepG2 cells exposed to low glucose, HIF-1α was overexpressed at mRNA and protein level in varying degrees and translocated into nucleus. The gene expression amounts of mdr1, MRP1 and LRP in HepG2 cells transfected by plasmid pcDNA3/HIF-1α were respectively increased 2.4±0.2*, 2.2±0.3# and 2.3±0.4& folds than those of non-transfected HepG2 cells (*t =3.75, P <0.01; #t =3.42, P <0.01; &t =3.26, P <0.01 ) and similar changes were witnessed in protein level.Conclusions Microenvironmental factors around HCC could modulate the transcription of the MDR related genes by HIF-1α of nuclear transcript factor, thereby conferred MDR of HCC. Upregulation of HIF-1α expression could hold a central position in the formation of MDR of HCC induced by microenvironment. HIF-1α probably becomes a new and effective molecular target on preventing and reversing MDR in hepatocellular carcinoma.Part III Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance of hepatocellular carcinoma induced by low glucose microenvironmentObjective To elucidate intracellular signal pathway in formation of MDR of HCC induced by microenvironment and to explore the potential role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in this process. Methods Activity of ERK/MAPK was examined by Western blot technique through comparing the ratio of phosphorylation of ERK/MAPK to total ERK/MAPK protein in HepG2 cells exposed to low glucose. After being treated by the specific ERK/MAPK pathway inhibitor U0126, RT-PCR and Western blot technique were respectively used to analyze the alterations of the expression of mdr1, MRP1, LRP and HIF-1α at mRNA and protein level. Cellular location of HIF-1α protein was determined by immunocytochemistry after being treated by U0126.Results The activations of ERK/MAPK determined by the ratio of phosphorylated ERK/MAPK to the total ERK/MAPK were increased in varying degrees in HepG2 cells respectively exposed to low glucose. After being treated by U0126 for 12h, the expressions of mdr1, MRP1, LRP genes and protein in those cells were decreased to some extent. However, the gene expression of HIF-1α was not influenced and only its protein was decreased. Details see figures and tables in the context. By the results of immunocytochemistry, HIF-1α protein was reversely translocated into cytoplasm from nucleus after being treated by U0126.Conclusions ERK/MAPK pathway is involved in the course of the formation of MDR of HCC induced by microenvironment. Stimulate from microenvironmental factors around HCC could be diverted into cytoplasm via ERK/MAPK pathway, which could phosphorylate HIF-1α protein and make it translocate into nucleus to accelerate the transcription of the MDR related genes and synthesis of their corresponding drug transporter proteins, thereby conferred MDR of HCC.Part IV Enhancing Sensitivity of Hepatocarcinoma Cells to Chemotherapeutic Drugs by Small Interference RNA of HIF-1αObjective To explore the effect of specific small interference RNA (siRNA) of HIF-1α on enhancing sensitivity of HCC to chemotherapeutic drugs.Methods Double strand oligonucleotide with complementary sequence and encoding short hairpin RNA (shRNA) was synthesized and then was inserted into eukaryotic expression vector PSilencer2.1 to generate a PSilencer2.1/HIF-1α-siRNA plasmid, the product of which could form a siRNA specifically targeted HIF-1α gene. Liposome carrying PSilencer2.1/HIF-1α-siRNA plasmid was stably transfected into C28 hepatocarcinoma cell line resistant to multiple chemotherapeutic drugs. The whole experiments were divided into three groups: experiment group with C28 cells transfected by plasmid PSilencer2.1/HIF-1α-siRNA, negative control group with C28 cells containing PSilencer2.1 empty vector, empty control group with C28 cells. RT-PCR and Western-blot technique were respectively used to analyze at mRNA and protein level the expression of mdr1, MRP1 and LRP in the above three group cells. Flow cytometry was used to determine apoptosis index (AI) of those cells after being administrated by 5-Fu. Randomly, every group of cells was subcutaneously inoculated into 10 nude mice. After subcutaneous neoplasms having grown, their sensitivities to ADM were compared among these three groups.Results HIF-1α-siRNA could specifically knocked out HIF-1α gene and inhibit its expression. Moreover, HIF-1α-siRNA made the amounts of mRNA and protein of mdr1, MRP1, LRP decrease in C28 hepatocarcinoma cell line resistant to multiple chemotherapeutic drugs. At 24h, 48h and 72h after administration of 5-Fu, AI of experiment group was respectively 2.88, 3.56 and 3.87 folds of that of negative control group, which suggested the sensitivity of experiment group to 5-Fu was significantly increased. In experiment group, the average size of subcutaneous neoplasms of nude mice was remarkably reduced after being treated by ADM and tumor inhibiting rate was 41.35%, which was significantly different from other two groups (P<0.01).Conclusion SiRNA specifically targeted HIF-1α gene could enhance the sensitivity of hepatocarcinoma cells to chemotherapeutic drugs. And effective therapeutic strategy for HCC could possibly be realized by united administration of conventional chemotherapeutic agents and specific siRNA of HIF-1α...
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