Subject(s)
Bone Neoplasms/pathology , Osteosarcoma/pathology , Animals , Cell Line, Tumor , Cell Movement , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Osteosarcoma/secondaryABSTRACT
BACKGROUND & OBJECTIVE: Chemokine receptors express on many tumor cells, and closely correlate with migration and metastasis of tumor cells. This study was to investigate expressions of chemokine(C-X-C) receptor 4 (CXCR4) and chemokine (C-X-C motif) ligand 12 (CXCL12) in human ovarian epithelial tumor cells, and their effects on migration of tumor cells. METHODS: Expression of CXCR4 mRNA and protein in 15 specimens of epithelial ovarian cancer tissue, ovarian cancer cell line CAOV3, endothelial cell line HUVEC, and 10 specimens of normal ovary tissue were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. Expression of CXCL12 mRNA in retroperitoneal lymph nodes, and smooth muscle of fallopian tube from the same 15 epithelial ovarian cancer patients was tested by RT-PCR, quantity of CXCL12 in ascites of 15 patients was assayed using ELISA. Boyden Transwells was used to analyze effects of CXCL12, and cancerous ascites on chemotaxis of CAOV3, and HUVEC cells. RESULTS: (1) Expression levels of CXCR4 mRNA in ovary cancer tissues, CAOV3 cells, and HUVEC cells were 2.30+/-1.12, 1.89+/-1.20, and 1.68+/-1.11, respectively; those of CXCR4 protein were 1.35+/-0.14, 1.86+/-0.34, and 1.96+/-0.23, respectively; CXCR4 mRNA and protein can't be detected in normal ovarian tissues. (2) In 15 ovarian cancer patients, concentrations of CXCL12 in ascites were 632-9 326 pg/ml, and CXCL12 mRNA level in retroperitoneal lymph nodes was 1.14+/-0.87, CXCL12 mRNA can't be detected in smooth muscle of fallopian tube. (3) Recombinant human CXCL12 induced migration of CAOV3, and HUVEC cells, the chemotactic indices (CI) were 3.9+/-1.2, and 4.1+/-1.6, significantly higher than those of control (1.0+/-0.4, and 1.1+/-0.7) (P<0.05)u cancerous ascites induced migration of CAOV3 cells with CI of 1.9+/-0.8, significantly higher than that of control (P<0.05). CONCLUSION: CXCR4 and CXCL12 may play roles in metastasis of epithelial ovarian cancer by promoting migration of tumor cells and endothelial cells.
Subject(s)
Cell Movement , Chemokines, CXC/biosynthesis , Ovarian Neoplasms/metabolism , Receptors, CXCR4/biosynthesis , Adult , Aged , Ascitic Fluid/metabolism , Cell Line, Tumor , Chemokine CXCL12 , Chemokines, CXC/genetics , Chemokines, CXC/physiology , Chemotaxis , Epithelial Cells/metabolism , Female , Humans , Lymph Nodes/metabolism , Middle Aged , Neoplasm Metastasis , Ovarian Neoplasms/pathology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, CXCR4/genetics , Receptors, CXCR4/physiologyABSTRACT
BACKGROUND & OBJECTIVE: Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) is a newly discovered enzyme, which plays a key role in tumor metastasis. This study was to observe inhibitory effect of MT1-MMP antisense nucleotide on proliferation and invasive potential of human highly metastatic ovarian carcinoma cell line SW626. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify MT1-MMP cDNA fragments with 2 different restriction sites at its 5c-end. RT-PCR products were cloned into plasmid pcDNA3.1 in antisense direction. The recombinant pMMP14as was transfected into SW626 cells. Changes of cell proliferation, MT1-MMP protein expression, activities of MMP-2 and MMP-9, and cell invasion ability were detected by MTT assay, Western blot, optimized gelatin zymography, and matrigel in vitro invasion assay, respectively. RESULTS: Antisense MT1-MMP eukaryotic expression vector pMMP14as was constructed successfully. After 48-h transfection with pMMP14as, proliferation of pMMP14as-transfected SW626 cells was significantly lower than that of control cells. Compared with control cells, the expression of endogenous MT1-MMP protein in pMMP14as-transfected cells was decreased with a inhibition rate of 65.8%. The activation of proMMP-2 was remarkably inhibited, and the mean invasive cell percentage was (63.3+/-5.8)% in pMMP14as-transfected cells, which was far less than (97.6+/-7.5)% in control cells (P< 0.05). CONCLUSION: Both cell proliferation and invasive potential of SW626 cells were inhibited effectively by antisense MT1-MMP, suggesting that MT1-MMP may be a proper molecular target of anti-invasion therapy for human ovarian cancer.
Subject(s)
Metalloendopeptidases/biosynthesis , Neoplasm Invasiveness , Oligonucleotides, Antisense , Ovarian Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Female , Humans , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/genetics , Ovarian Neoplasms/metabolism , Plasmids , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , TransfectionABSTRACT
OBJECTIVE: To investigate the expression of RhoA, RhoC and their effector ROCK-1 in four ovarian cancer cell lines in vitro and their correlation with invasiveness. METHODS: Expression of RhoA, RhoC and ROCK-1 mRNA and protein in four ovarian cancer cell lines SW626, Skov-3, A2780 and Caov-3 was detected by RT-PCR and Western blot assay. Invasion assay was done in Boyden chamber. RESULTS: The expression levels of RhoA, RhoC and ROCK-1 mRNA and protein varied in the four different cell lines examined. The expression level of RhoC, but not RhoA and ROCK-1, was significantly correlated with the invasive capability of these cells in vitro (r = 0.95, P < 0.01). Expression of RhoA at the level of transcription was not correlated with that at the translation level. The expression of RhoA and RhoC did not correlate with that of ROCK-1. CONCLUSION: Expression level of RhoC may serve as an independent parameter in evaluating metastasis and become a new target in inhibiting ovarian cancer metastasis.
Subject(s)
Ovarian Neoplasms/metabolism , Protein Serine-Threonine Kinases/biosynthesis , rho GTP-Binding Proteins/biosynthesis , rhoA GTP-Binding Protein/biosynthesis , Cell Line, Tumor , Cell Movement , Female , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins , Neoplasm Invasiveness , Neoplasm Metastasis , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Phenotype , Protein Biosynthesis , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transcription, Genetic , rho GTP-Binding Proteins/genetics , rho-Associated Kinases , rhoA GTP-Binding Protein/genetics , rhoC GTP-Binding ProteinABSTRACT
OBJECTIVE: To study the mechanism of topotecan (TPT) resistance in ovarian cancer cell line. METHODS: A TPT-resistant ovarian cancer cell line A2780/TPT established in this laboratory was used in this study. Intracellular rhodamine fluorescence intensity of the TPT-resistant cells and parental cells were measured by flow cytometry. The gene expression of membrane protein transporter such as transporter P-glycoprotein (P-gp), multidrug resistance associated protein (MRP), breast cancer resistance protein (BCRP) was evaluated by RT-PCR. The antisense-phosphorothioate oligonucleotide (ASODN) including a translation initiation site of BCRP mRNA was transferred into resistant cells by liposome. RESULTS: Intracellular rhodamine fluorescence intensity of the resistant cells was 31.19% of that in the parental cells (P < 0.01). No expression of P-gp was demonstrated, and that of MRP was very weak in the TPT-resistant cells (relative expression value = 0.057). BCRP was overexpressed in the TPT-resistant cells (relative expression = 0.66), but not in the parental cells. Transfer of ASODN into resistant cells resulted in a 59.42% reduction of BCRP gene expression (P < 0.05) and an obviously increased intracellular rhodamine fluorescence intensity from 5.42 to 16.63 (P < 0.05). CONCLUSION: The overexpression of BCRP which mediated drug efflux may play an important role in the induction of TPT-resistance in ovarian cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Ovarian Neoplasms/drug therapy , Topotecan/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm , Female , Humans , Neoplasm Proteins/genetics , Ovarian Neoplasms/pathology , RNA, Messenger/analysisABSTRACT
BACKGROUND & OBJECTIVE: Breast cancer resistance protein (BCRP) was overexpressed in topotecan (TPT)-selected human ovarian cancer cell line A2780/TPT, strongly suggesting BCRP to be responsible for the drug-resistance of ovarian cancer. The current study was designed to investigate the reversal effect of BCRP antisense oligonucleotide (ASODN) on topotecan- resistant A2780/TPT cells. METHODS: The antisense-phosphorothioate oligonucleotide including the translation initiation site of BCRP mRNA was artificially synthesized, and the sense oligonucleotide (SODN) corresponding to the ASODN was also synthesized as control. Lipofect-2000 (LF) was used for the transfer of either ASODN or SODN into A2780/TPT cells. The changes of BCRP mRNA expression, intracellular fluorescence intensity of rhodamine and resistance index to topotecan of in vitro transfected A2780/TPT cells were detected respectively by reverse transcription-polymerase chain reaction (RT-PCR),flow cytometry (FCM),and methyl thiazolyl tetrazolium (MTT) assay. RESULTS: The transfer of ASODN/LF into A2780/TPT cells resulted in:(1)a 59.42% reduction of BCRP mRNA level (P< 0.05); (2)an obviously increased intracellular rhodamine fluorescence intensity from 5.42 to 16.63(P< 0.05); (3)a decreased resistance index to topotecan from 25 to 5 indicating sensitivity to topotecan in A2780/TPT cells recovered, as compared with non-transfected cell. But after transfecting SODN, no significant change could be measured. CONCLUSION: ASODN transfection may partly reverse BCRP-mediated drug- resistance of ovarian cancer cells.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Neoplasm Proteins/antagonists & inhibitors , Oligonucleotides, Antisense/pharmacology , Topotecan/pharmacology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Drug Interactions , Drug Screening Assays, Antitumor , Female , Humans , Neoplasm Proteins/genetics , Ovarian Neoplasms/pathology , RNA, Messenger/biosynthesis , RNA, Messenger/drug effects , Transfection , Tumor Cells, CulturedABSTRACT
OBJECTIVE: To study the role of T lymphoma invasion/metastasis gene 1 (Tiam1) and protein in ovarian tumor cells. METHODS: Expressions of Tiam1 mRNA, Rac1 mRNA, and Tiam1 protein in four ovarian tumor cells A2780, Caov3, Skov3, and SW626 were studied by using RT-PCR and Western blot, respectively. The cell migration ability was analyzed by in vitro invasion assay. RESULTS: Expressions of Tiam1 mRNA and protein, as well as Rac1 mRNA were detected in all four ovarian tumor cells. There was a strong direct correlation between the levels of Tiam1 and Rac1 mRNA expression and migration potentials of all four ovarian cancer cells in vitro experiments. The increased expressions of Tiam1 mRNA were coincident with those of Rac1 mRNA, with a parallel relationship (P = 0.003, r = 0.874). Levels of Rac1 mRNA expression were significantly correlated with the potentials of tumor cell migration (P = 0.042, r = 0.814). CONCLUSION: Tiam1-Rac1 signaling pathway plays a positive role in assessing tumor cell invasion and metastasis and provides a new target for gene therapy of ovarian cancer.
