OBJECTIVESTo establish a gemcitabine-resistant pancreatic cancer cell line SW1990/GZ, and to explore the relationship between drug-resistant cell line SW1990/GZ and pancreatic cancer stem cell.

METHODSGemcitabine-resistant pancreatic cancer cell line SW1990/GZ was obtained by treating parental cell line SW1990 in vitro with increasing dosage of gemcitabine in culture medium intermittently for 24 weeks. Stable cultures were obtained which were 77.2-fold increased in resistance relative to parental cells. Gene expressions of ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP were determined by real-time PCR. Tumorigenic potential was performed by nude mice xenograft transplant experiments. Side population analysis and CD24CD44 positive cells explore were determined by flow cytometry to examine cancer stem cell proportion.

RESULTSGemcitabine-resistant cell line SW1990/GZ underwent obvious morphological and functional changes. Compared with the parental cell line, SW1990/GZ cell was small and turned into round shape. SW1990/GZ had a higher gene expression level of ABCB1/MDR1, ABCC1/MRP and ABCG2/BCRP than SW1990 (P < 0.01). Nude mice xenograft transplant experiments showed that only 1 × 10(5) SW1990/GZ cells were sufficient for tumor formation, whereas an injection of 1 × 10(5) SW1990 cells did not initiate tumors. Flow cytometry analysis showed that SP proportion in SW1990/GZ was (11.0 ± 1.0)%, whereas in parental SW1990 it was (4.6 ± 0.9)%, CD44CD24 positive cells was (8.73 ± 0.81)% in SW1990/GZ, whereas (1.1 ± 0.4)% in SW1990.

CONCLUSIONSGemcitabine-resistant cell line SW1990/GZ has a higher proportion of pancreatic cancer stem cells compared to its parental cell line SW1990. CD44 is mainly responsible for acquired drug resistance, which can be a potential target to overcome acquired drug resistance in pancreatic cancer.

Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Drug Resistance, Neoplasm ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplastic Stem Cells ; drug effects ; pathology ; Pancreatic Neoplasms ; drug therapy ; metabolism ; pathology ; Xenograft Model Antitumor Assays

OBJECTIVETo explore the action mechanism of action of Qingyi Huaji Formula (QYHJ), whether its inhibition on the growth of the tumor is by way of down-regulating Ski expression.

METHODSSW1990 tumor cell with low Ski expression was created by lenti-virus mediated RNA interfering technique. Tumor cells with different levels of Ski expression were subcutaneously transplanted to nude mice, and the response of cancer cells to QYHJ treatment was detected.

RESULTSAfter QYHJ treatment, the tumor growth slowed-down significantly, with the Ski mRNA and protein expressions in tumor reduced by 39.6% and 41.3% of that in untreated tumor respectively (P < 0.05). Ski mRNA and protein expressions in the created high and low Ski expression tumor cells were 105%, 123% and 46%, 30% respectively of that in parental cells (P < 0.05). The tumor weight inhibitory rates of QYHJ on high Ski expression cells were 29.6% and 32.2%, while on low Ski expression cells, it was 16.0% to 17.8% (P < 0.05).

CONCLUSIONSki acts as an important therapeutic target of QYHJ in treating SW1990 pancreatic cancer, and its expression status mediates different response of tumor cells to QYHJ treatment.

Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA-Binding Proteins ; genetics ; metabolism ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Pancreatic Neoplasms ; drug therapy ; pathology ; Phytotherapy ; Proto-Oncogene Proteins ; genetics ; metabolism

OBJECTIVETo investigate the effect and mechanism of NF-kappaB P65 gene silencing by small interference RNA on the apoptosis of human pancreatic cancer cells induced by gemcitabine in vitro and in vivo.

METHODSHuman pancreatic cancer cells (BxPC-3 and PANC-1) were cultured and respectively divided into five groups: blank control group, negative control siRNA group, gemcitabine group, NF-kappaB P65 siRNA group and gemcitabine + P65 siRNA group. The ability of cell proliferation was analyzed by MTT; the expression of NF-kappaB P65 and the apoptosis related proteins were examined by Western blot assay; the apoptosis was evaluated by the flow cytometry and laser scanning confocal microscopy analysis stained with Annexin V-FITC/PI; the DNA binding activity of NF-kappaB was examined by electrophoretic mobility shift assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors. The tumor volume was monitored and TUNEL assay was used to assess the apoptosis index in tumor tissue after treatment.

RESULTSAt 72 h after transfection, the combination with gemcitabine and p65 siRNA significantly decreased the cell viability index (P < 0.05), and down-regulated the expression of Bcl-2 and procaspase-3 and up-regulated the expression of Bax compared with other groups. The combined treatment significantly increased the rate of apoptosis compared with other groups (P < 0.05). EMSA assay indicated that the DNA binding activity of NF-kappaB significantly decreased in NF-kappaB P65 siRNA group and gemcitabine+P65 siRNA group compared with Control group. The combined therapy inhibited the growth of pancreatic xenograft tumors by apoptosis induction in nude mice (P < 0.01).

CONCLUSIONSThe effect of gemcitabine inducing cell apoptosis may be potentiated through inhibiting the DNA binding activity of NF-kappaB and regulating the expression of apoptosis related proteins by NF-kappaB P65 siRNA, which can activate the mitochondria apoptosis pathway in pancreatic cancer in vitro and in vivo.

Animals ; Apoptosis ; drug effects ; genetics ; Cell Line, Tumor ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Humans ; Mice ; Mice, Inbred BALB C ; Pancreatic Neoplasms ; drug therapy ; metabolism ; pathology ; RNA, Small Interfering ; genetics ; Transcription Factor RelA ; genetics ; metabolism ; Transfection ; Xenograft Model Antitumor Assays

PURPOSE: This study was aimed to investigate the effect of pseudolaric acid B (PAB) on proliferation, invasion and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cells and to explore the possible mechanism. MATERIALS AND METHODS: The pancreatic cancer cell line SW1990 was cultured and treated with PAB dose- and time-dependent manners. Cell proliferation and invasion ability were measured by MTT assay and Matrigel/Transwell test, respectively. Semi-quantitative real-time polymerase chain reaction and Western blotting were conducted to detect the expression of EMT markers and the key molecules. Finally, nude mice subcutaneous transplantation tumor model was used to confirm the therapy efficacy of PAB. RESULTS: PAB could inhibit SW1990 cell proliferation and invasion in time- and dose-dependent manners. Vimentin, fibronectin, N-cadherin, Snail, Slug, YAP, TEAD1, and Survivin were down-regulated (p < 0.01), while E-cadherin, caspase-9, MST1, and pYAP were up-regulated (p < 0.05). Combined PAB and gemcitabine treatment markedly restricted the tumor growth compared with gencitabin or PAB alone groups. CONCLUSION: PAB could inhibit the proliferation and invasion ability of pancreatic cancer cells through activating Hippo-YAP pathway and inhibiting the process of EMT.
Animals ; Antineoplastic Agents/pharmacology ; Antineoplastic Agents/therapeutic use ; Biomarkers, Tumor/metabolism ; Cadherins ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation/drug effects ; Cytokines ; Deoxycytidine/analogs & derivatives ; Deoxycytidine/pharmacology ; Deoxycytidine/therapeutic use ; Diterpenes/pharmacology ; Diterpenes/therapeutic use ; Epithelial-Mesenchymal Transition/drug effects ; Female ; Humans ; Mice, Nude ; Neoplasm Invasiveness ; Pancreatic Neoplasms/diet therapy ; Pancreatic Neoplasms/pathology ; Real-Time Polymerase Chain Reaction ; Signal Transduction/drug effects ; Vimentin/metabolism

Pancreatic ductal adenocarcinoma is one of the most dreaded malignancies and the 5th leading cause of cancer-related death in Korea. Late diagnosis and unfavorable response to both chemotherapy and radiotherapy result in exceptionally poor prognosis. Recently, the rapid advances of molecular biology allowed an in-depth understanding of pancreatic carcinogenesis, and there are many attempts to modulate signal pathway using specific targeted agent. However, the most of them have so far failed to improve survival significantly except erlotinib. The real challenge is now how these impressive advances of molecular biology could be successfully integrated into better clinical implications. Herein, we summarize the latest insights into the carcinogenesis, and their repercussions for novel targeted agents for pancreatic cancer, and provide a review of recent clinical trials using molecular targeted therapy.
Antineoplastic Agents/*therapeutic use ; Epigenesis, Genetic ; Humans ; Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism ; Molecular Targeted Therapy ; Pancreatic Neoplasms/*drug therapy/metabolism/pathology ; Poly(ADP-ribose) Polymerases/antagonists & inhibitors/metabolism ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptor, IGF Type 1/antagonists & inhibitors/metabolism ; Vascular Endothelial Growth Factor A/antagonists & inhibitors/metabolism

Pancreatic ductal adenocarcinoma is one of the most dreaded malignancies and the 5th leading cause of cancer-related death in Korea. Late diagnosis and unfavorable response to both chemotherapy and radiotherapy result in exceptionally poor prognosis. Recently, the rapid advances of molecular biology allowed an in-depth understanding of pancreatic carcinogenesis, and there are many attempts to modulate signal pathway using specific targeted agent. However, the most of them have so far failed to improve survival significantly except erlotinib. The real challenge is now how these impressive advances of molecular biology could be successfully integrated into better clinical implications. Herein, we summarize the latest insights into the carcinogenesis, and their repercussions for novel targeted agents for pancreatic cancer, and provide a review of recent clinical trials using molecular targeted therapy.
Antineoplastic Agents/*therapeutic use ; Epigenesis, Genetic ; Humans ; Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism ; Molecular Targeted Therapy ; Pancreatic Neoplasms/*drug therapy/metabolism/pathology ; Poly(ADP-ribose) Polymerases/antagonists & inhibitors/metabolism ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptor, IGF Type 1/antagonists & inhibitors/metabolism ; Vascular Endothelial Growth Factor A/antagonists & inhibitors/metabolism

OBJECTIVETo investigate the anti-tumor activity of combined gemcitabine with dihydroartemisinin, and the mechanism of the anti-tumor effect of gemcitabine enhanced by dihydroartemisinin on pancreatic cancer.

METHODSFor cultured cells, cell growth was determined by the MTT assay and apoptosis was evaluated by flow cytometry analysis and confocal laser scanning microscope stained with Annexin V-FITC/PI. The nuclear extract for determining NF-kappaB DNA-binding activity was analyzed by EMSA, while nuclear P65 and its downstream gene expression was determined by Western blot assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors and the tumor volume was monitored after exposure to agents. TUNEL assay was used to assess tumor cell apoptosis in tumor tissue.

RESULTSAfter combination of gemcitabine and dihydroartemisinin treatment, the proliferative inhibition rates of pancreatic cancer cells BxPC-3 and Panc-1 reached up to (81.1 +/- 3.9)% and (76.5 +/- 3.3)%, and the apoptosis rates were up to (53.6 +/- 3.8)% and (48.3 +/- 4.3)%, the differences were significantly (P < 0.01) compared with gemcitabine [(24.8 +/- 2.9)% and (21.8 +/- 3.5)%]. All the treatment groups inhibited the growth of pancreatic xenograft tumors in nude mice. The tumor volume and apoptosis index were (262 +/- 37) mm(3) and (50 +/- 4)% respectively in the combined treatment, compared to those of [(384 +/- 56) mm(3) and (25 +/- 3)%] in gemcitabine, the differences were significantly (P < 0.05). EMSA showed that gemcitabine alone obviously enhanced its DNA-binding activity compared to control. However, dihydroartemisinin significantly reduced its DNA-binding activity, so that abrogated the inducing effect of gemcitabine on NF-kappaB activation. Western blot assay indicated that dihydroartemisinin downregulated expression of nuclear P65, and combined treatment not only downregulated the expression of Cyclin D1, Bcl-xL and Bcl-2 while upregulated Bax, thus reduced the Bcl-2/Bax ratio, but also increased the caspase-3 activation, all of which increased apoptosis in both BxPC-3 and Panc-1 cells.

CONCLUSIONDihydroartemisinin significantly abrogated the inducing effect of gemcitabine on NF-kappaB activation and downregulated the expression of NF-kappaB targeted gene products, which may be one possible mechanism by which dihydroartemisinin augments the anti-tumor effect of gemcitabine on pancreatic cancer.

Animals ; Antimetabolites, Antineoplastic ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Apoptosis ; drug effects ; Artemisinins ; therapeutic use ; Cell Line, Tumor ; Deoxycytidine ; analogs & derivatives ; therapeutic use ; Humans ; Male ; Mice ; Mice, Nude ; NF-kappa B ; metabolism ; Pancreatic Neoplasms ; drug therapy ; metabolism ; pathology ; Xenograft Model Antitumor Assays

OBJECTIVETo study the expression and prognostic significance of ERG and SPINK1 expression in endocrine-treated prostatic cancer.

METHODSProstatic needle biopsies from 118 prostatic cancer patients primarily treated with endocrine therapy were reviewed. Immunohistochemical study for ERG and SPINK1 protein was carried out.

RESULTSCo-expression of ERG and SPINK1 was not observed. The frequency of ERG protein expression in the 118 biopsies studied was 9.3% (11/118). The positive expression correlated with T stage (P=0.04) but not with patient age at diagnosis, prostatic specific antigen level, Gleason's score, M stage, tumor area and progression-free survival. Positive expression of SPINK1 was demonstrated in 11.0% (13/118) of the biopsies. SPINK1-positive cases carried a significantly shorter progression-free survival, as compared with SPINK1-negative cases (P=0.022). The expression was not associated with any other clinicopathologic variables. The following expression pattern showed statistically significant correlation with the clinical progress (P=0.029): ERG+/SPINK1- (11/118, 9.3%), ERG-/SPINK1+ (13/118, 11.0%) and ERG-/SPINK1- (94/118, 79.7%).

CONCLUSIONSERG and SPINK1 proteins are mutually exclusive.SPINK1 expression is associated with more aggressive clinical behavior and can serve as a prognostic biomarker in prostatic cancer.

Aged ; Aged, 80 and over ; Antineoplastic Agents, Hormonal ; therapeutic use ; Carrier Proteins ; metabolism ; Disease-Free Survival ; Follow-Up Studies ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neoplasm Grading ; Neoplasm Staging ; Prognosis ; Prostatic Neoplasms ; drug therapy ; metabolism ; pathology ; Trans-Activators ; metabolism ; Transcriptional Regulator ERG ; Trypsin Inhibitor, Kazal Pancreatic