ABSTRACT
Recent population studies suggest that the use of artemisinin is associated with reduced incidence and improved prognosis of certain cancers. In the current study, we assessed the effect of artemisinin on gastric cancer cells (AGS and MKN74 cells). We found that artemisinin inhibited growth and modulated expression of cell-cycle regulators in these cells. Treatment with artemisinin was also associated with induction of p27 kip1 and p21 kip1, two negative cell-cycle regulators. Furthermore, we revealed that artemisinin treatment led to an increased expression of p53. Taken together, these results provide evidence for a mechanism that may contribute to the antineoplastic effects of artemisinin suggested by recent population studies and justify further work to explore potential roles for it in gastric cancer prevention and treatment.
Subject(s)
Artemisinins/administration & dosage , Gene Expression Regulation, Neoplastic/drug effects , Stomach Neoplasms/drug therapy , Tumor Suppressor Protein p53/biosynthesis , Apoptosis/drug effects , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Tumor Suppressor Protein p53/geneticsABSTRACT
BACKGROUND/AIMS: To investigate ER stress-mediated CHOP-signaling pathway of gastric cancer apoptosis in vitro and in vivo. METHODOLOGY: Based on the dose-and time-response experiments about tunicamycin (TM),gastric cancer cell line BGC823 was treated with 10tg/mL of TM for 24h. BGC823 apoptosis was detected with TUNEL assay and ultrastructural changes in BGC823 cells under ER stress were observed with transmission electron microscopy (TEM). RT-PCR and western blot-ting were used to determine the expression of ERS-related proteins, glucose-regulated protein 78 (GRP78) and CHOP and apoptosis-associated protein B-cell lympho-ma 2 (Bcl-2). After the knockdown of CHOP, the changes were also observed in vitro and in vivo. RESULTS: TEM assay showed that after treatment with TM, BGC823 cell size became smaller with ER dilation, vacuolization and karyopyknosis. RT-PCR and western blotting indicated that TM up-regulated GRP78 and CHOP expression and down-regulated Bcl-2 expression. The knock-down of CHOP could convert Bcl-2 expression and reduce BGC823 apoptosis caused by ERS in vitro and in vivo, but failed to influence GRP78. CONCLUSIONS: TM can induce ESR and regulate downstream molecules CHOP up-regulation and Bcl-2 down-regulation which lead to BGC823 apoptosis. This study may provide a new theoretical basis for the pathogenesis of gastric cancer.
Subject(s)
Apoptosis , Endoplasmic Reticulum Stress , Signal Transduction , Stomach Neoplasms/metabolism , Animals , Apoptosis/drug effects , Blotting, Western , Cell Line, Tumor , Dose-Response Relationship, Drug , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Gene Expression Regulation, Neoplastic , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , In Situ Nick-End Labeling , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Microscopy, Electron, Transmission , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Stomach Neoplasms/genetics , Stomach Neoplasms/ultrastructure , Time Factors , Transcription Factor CHOP/genetics , Transcription Factor CHOP/metabolism , Transfection , Tumor Burden , Tunicamycin/pharmacologyABSTRACT
AIM: To investigate the copy number variation of NACO3 gene in colorectal cancer (CRC) and its correlation with tumor progression. METHODS: A total of 142 samples of case-matched CRC tissues and adjacent normal tissues were obtained from patients undergoing bowel resection. Quantitative real-time polymerase chain reaction method was used to investigate the copy number variations of NCOA3 as well as gene expression in the collected tissues. RESULTS: Copy number gains of NCOA3 were detected in 39 CRC samples (27.5%) and were correlated with tumor progression (χ2 = 6.42, P = 0.0112). Moreover, there was a positive correlation between copy number gain and mRNA over-expression of NCOA3 in CRCs (P = 0.0023). Expression level of NCOA3 mRNA was also enhanced in the CRC samples with unaltered copy numbers (3.85 ± 1.23 vs. 2.71 ± 0.64, P < 0.01). CONCLUSION: Sporadic colorectal cancers exhibit different mechanisms of NCOA3 regulation.
Subject(s)
Asian People/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , DNA Copy Number Variations , Gene Amplification , Nuclear Receptor Coactivator 3/genetics , Disease Progression , Gene Expression Regulation, Neoplastic , HumansABSTRACT
We investigated the role of endoplasmic reticulum (ER) stress response and p38 MAPK pathways in the resistance of gastric cancer cells to chemotherapy. Pretreatment of the gastric cancer cells with the ER stress inducer drastically decreased the apoptotic rate induced by cisplatin or doxorubicin. Induction of ER stress also led to the activation of p38. Inhibition of p38 activity abrogated the effects of ER stress-induced resistance to apoptosis induced by cisplatin- and doxorubicin treatment. Thus, ER-stress response in gastric cancer cells causes resistance to cisplatin- and doxorubicin-induced apoptosis, and ER-stress induced chemo-resistance can be overcome by blocking p38 activity.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Drug Resistance, Neoplasm , Endoplasmic Reticulum/physiology , MAP Kinase Kinase Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Stomach Neoplasms/drug therapy , Unfolded Protein Response , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cisplatin/pharmacology , Cisplatin/therapeutic use , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Endoplasmic Reticulum/drug effects , Enzyme Activation , Humans , MAP Kinase Kinase Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Stomach Neoplasms/pathology , Stress, PhysiologicalABSTRACT
Heterozygosity for the Pax6 allele is associated with impaired glucose tolerance in humans. With a Pax6 mutant mouse model, we found many of the metabolic abnormalities were consistent with the effects of down-regulating the expression of glucagon-like peptide 1 (GLP-1). In addition to impaired glucose tolerance, adult heterozygous mutant mice (Pax6(m/+)) secreted less insulin responding to glucose and arginine administration compared with control mice. Moreover, Pax6(m/+) mice showed increased food intake compared with control mice, although they were resistant to diet-induced fat accumulation. Indeed, levels of circulating GLP-1 and intestinal transcription of Gcg/Proglucagon were dramatically reduced in Pax6(m/+) mice. Mutated Pax6 also failed to activate the Gcg/Proglucagon promoter by in vitro transfection assay. Finally, administering the GLP-1 receptor agonist exendin-4 to Pax6(m/+) mice largely reversed their abnormal food intake, glycemic excursion, and insulin secretion. Our studies suggested that disruption of metabolic homeostasis mainly caused by Pax6 haploinsufficiency was mainly mediated by down-regulation of GLP-1. Administration of exendin-4 may be a useful therapy in humans with a similar mutation.
Subject(s)
Eye Proteins/genetics , Glucagon-Like Peptide 1/genetics , Homeodomain Proteins/genetics , Homeostasis/genetics , Loss of Heterozygosity/physiology , Metabolic Diseases/genetics , Paired Box Transcription Factors/genetics , Repressor Proteins/genetics , Age Factors , Animals , Down-Regulation/genetics , Down-Regulation/physiology , Embryo, Mammalian , Eye Proteins/physiology , Gene Expression Regulation, Developmental/physiology , Glucagon-Like Peptide 1/metabolism , Homeodomain Proteins/physiology , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Islets of Langerhans/physiology , Male , Metabolic Diseases/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Organ Size/genetics , PAX6 Transcription Factor , Paired Box Transcription Factors/physiology , Repressor Proteins/physiologyABSTRACT
AIM: To study the relationship between the abnormal differentiation thymocytes and the pathogenesis of myasthenia gravis (MG). METHODS: The gene expression of some ILs, IFN and their receptors was examined by cDNA array. The percentage of CD45RO(+) and D45RA(+) thymocytes was measured by flow cytometry (FCM). The expression and distribution of CD45RA and CD45RO in the samples of thymus tissues were observed by immunohistochemistry. RESULTS: The levels of IL-1R, IL-4R, IFNgammaR1, IFNgammaR2, IL-6 and IL-8 in the patients with MG were lower than those of normal controls and there were significant differences between them. But the levels of IL-1, IL-2, IL-4, IL-10, IL-7, IFN-alpha, IFN-beta and IFN-gamma showed no significant changes in patients with MG in comparison with normal controls. The percentage of CD56(+) thymocytes in MG patients 0.56+/-0.33 was significantly lower than that in normal controls(1.78+/-0.69).The CD45RO(+) and CD1a(+) in MG patients increased more significantly those in normal controls. The immunohistochemistry and RT-PCR showed the same result. CONCLUSION: Aberrant transformation from CD45R0(+)T to CD45RA(+)T has been found in the development of thymocytes of the patients with MG.
Subject(s)
Cytokines/genetics , Leukocyte Common Antigens/metabolism , Myasthenia Gravis/metabolism , Thymus Gland/metabolism , Adolescent , Adult , Child , Child, Preschool , Female , Flow Cytometry , Humans , Immunohistochemistry , Interferon-alpha/genetics , Interferon-beta/genetics , Interferon-gamma/genetics , Interleukin-1/genetics , Interleukin-10/genetics , Interleukin-2/genetics , Interleukin-4/genetics , Interleukin-6/genetics , Interleukin-7/genetics , Interleukin-8/genetics , Male , Middle Aged , Myasthenia Gravis/genetics , Oligonucleotide Array Sequence Analysis , Receptors, Interferon/genetics , Receptors, Interleukin-1/genetics , Receptors, Interleukin-4/genetics , Reverse Transcriptase Polymerase Chain Reaction , Young Adult , Interferon gamma ReceptorABSTRACT
OBJECTIVE: The B7-H1/PD-1 co-signaling pathway has recently been found to play a pivotal role in the immune evasion of tumor cells from host immune system. The aim of this study was to examine the B7-H1 and PD-1 expression and TILs status in gastric cancer and to elucidate the clinical relevance of B7-H1 and PD-1 to the pathogenesis of gastric carcinoma. METHODS: Immunohistochemistry and ANAE histochemical staining were used to investigate the in situ expression of B7-H1 and PD-1 and TILs status in the gastric tissues. RT-PCR was used to explore B7-H1 and PD-1 expression at the transcriptional level. The B7-H1 expression at protein level was detected by Western blot. RESULTS: Expression of B7-H1 and PD-1 was found to be increased in gastric carcinoma, but absent in normal gastric tissue. B7-H1 expression in gastric carcinoma was inversely correlated with TILs infiltration. B7-H1 but not PD-1 expression in tumor tissue was significantly correlated with some clinicopathhological variables including depth of invasion, lymph node metastasis and distant metastasis. CONCLUSION: B7-H1 and PD-1 expressions are increased in gastric carcinoma. This signaling pathway may inhibit antitumor immune responses in gastric carcinoma. B7-H1 expression plays a critical role in the pathogenesis of human gastric carcinoma,and might be a promising prognostic marker and therapeutic target in the treatment of this disease.
Subject(s)
Antigens, CD/metabolism , Apoptosis Regulatory Proteins/metabolism , Lymphocytes, Tumor-Infiltrating/immunology , Stomach Neoplasms/immunology , Adult , Aged , Antigens, CD/genetics , Apoptosis Regulatory Proteins/genetics , B7-H1 Antigen , CD4-Positive T-Lymphocytes/immunology , Female , Humans , Lymphatic Metastasis , Lymphocyte Subsets/immunology , Male , Middle Aged , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Staging , Programmed Cell Death 1 Receptor , RNA, Messenger/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/pathologyABSTRACT
OBJECTIVE: To investigate the mRNA and protein expression of nucleostemin (NS) in human esophageal squamous cell carcinoma. METHODS: The mRNA and protein expression of NS were detected in 31 mucosal atypical hyperplasia specimens, 62 esophageal squamous cell carcinoma specimens and the matched normal esophageal mucosa samples by RT-PCR and immunohistochemistry method, respectively. RESULTS: The positive expression rate of NS protein in normal esophageal mucosa, atypical hyperplasia and esophageal squamous cell carcinoma was 17.7% (11/62), 41.9% (13/31) and 69.4% (43/62), respectively. There was a significant difference among the above three groups (chi2 = 33.676, P < 0.01). The expression levels of NS mRNA in esophageal squamous cell carcinoma (0.971 +/- 0.121) was significantly higher than that in the atypical hyperplasia (0.913 +/- 0.085) and also in the normal esophageal mucosa (0.866 +/- 0.103; F = 14.829, P < 0.01). The expression level of both NS protein and mRNA was positively correlated with histological grade, infiltration depth, and lymph node metastasis (P < 0.05), but not with age, gender or pathological type (P > 0.05). CONCLUSION: Our results indicate that nucleostemin mRNA and protein are over-expressed in human esophageal squamous cell carcinoma, and it may be related with its oncogenesis.
Subject(s)
Carcinoma, Squamous Cell/metabolism , Carrier Proteins/biosynthesis , Esophageal Neoplasms/metabolism , Esophagus/pathology , Nuclear Proteins/biosynthesis , Carcinoma, Squamous Cell/pathology , Carrier Proteins/genetics , Esophageal Neoplasms/pathology , Female , GTP-Binding Proteins , Gene Expression Regulation, Neoplastic , Humans , Hyperplasia , Lymphatic Metastasis , Male , Middle Aged , Mucous Membrane/metabolism , Neoplasm Invasiveness , Neoplasm Staging , Nuclear Proteins/genetics , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , RNA, Messenger/metabolismABSTRACT
OBJECTIVE: To investigate the mRNA expression levels of nucleostemin (NS) in human esophageal squamous cell carcinoma tissue. METHODS: Real-time PCR was used to quantify the mRNA expression of NS in the samples of esophageal squamous cell carcinoma tissue and their matched normal esophageal mucosa tissue from 62 patients, 36 males and 26 females, aged (61 +/- 10) (38-75). The relationship between NS mRNA expression level and clinical pathological features was analyzed. RESULTS: The NS mRNA expression level of the 62 cases of esophageal squamous cell carcinoma tissue was(4.5 +/- 2.1), significantly higher than that of the matched normal esophageal mucosa tissue [(2.1 +/- 1.3), t = -5.045, P = 0.000]. The mRNA expression level of NS was associated with tumor grade, depth of infiltration, and lymph node metastasis (all P < 0.05), but not with gender, age, and pathological type (all P > 0.05). Multiple linear regression analysis revealed that clinical and pathological features influenced the NS mRNA expression level (P = 0. 000), and the depth of infiltration and lymph node metastasis were important influencing factors for NS mRNA expression level(both P < 0.05). CONCLUSION: NS may play an important role in the progression and proliferation of esophageal squamous cell carcinoma.
Subject(s)
Carcinoma, Squamous Cell/pathology , Carrier Proteins/genetics , Esophageal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Nuclear Proteins/genetics , Adult , Aged , Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Female , GTP-Binding Proteins , Humans , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Staging , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
OBJECTIVE: To construct a eukaryotic expression vector for the small interfering RNA (siRNA) targeting nucleostemin (NS) gene. METHODS: The siRNA targeting NS gene was designed according to the sequence of NS mRNA available in GenBank. Three siRNA sequences were obtained, and the corresponding cDNAs were synthesized and inserted into plasmid pRNAT-U6.1 for constructing the recombinant plasmids, which were transformed into E.coli DH5alpha strain. The plasmids, after identification by PCR and DNA sequencing, were transfected into EC9706 cell line via liposome, and the mRNA and protein expressions of NS gene in the cells were determined by RT-PCR and Western blotting, respectively. RESULTS: Three recombinant plasmids were identified by PCR and sequence analysis, the results of which showed correct insertion of the designed sequences in the plasmids. RT-PCR and Western blotting showed substantially decreased mRNA and protein expressions of NS gene in the transfected cells. CONCLUSION: The recombinant plasmid expressing the siRNA targeting NS gene has been successfully constructed, which provides the basis for studying RNA interference of the NS gene.
Subject(s)
Carrier Proteins/genetics , Genetic Vectors/genetics , Nuclear Proteins/genetics , RNA Interference , RNA, Small Interfering/genetics , Base Sequence , Blotting, Western , Carrier Proteins/biosynthesis , Cell Line , Cloning, Molecular , Eukaryotic Cells/metabolism , GTP-Binding Proteins , Humans , Molecular Sequence Data , Nuclear Proteins/biosynthesis , Oligonucleotide Array Sequence Analysis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
OBJECTIVE: To investigate the effect of hTERT antisense oligodeoxynucleotide (ASODN) on the oncogenicity and the inductive apoptosis of HL-60 cells. METHODS: Apoptosis of HL-60 cells was detected by flow cytometry (FCM) and agarose gel electrophoresis. Both treated and untreated HL-60 cells were collected and transplanted into 5 BALB/c nude mice respectively, the formation of transplanted neoplasm and its morphologic change were observed. After the transplanted neoplasms were uniform with the ameliorated method in another 10 BALB/c nude mice, they were divided into 2 groups and injected ASODN and PBS into the neoplasm respectively. Seven days later, the tumor were measured, its morphology were observed, and the apoptotic cells were detected with a TUNEL kit. RESULTS: After 72 h treatment there were DNA ladders and early apoptosis peak in hTERT ASODN treated HL-60 cells but was none in SODN treated and blank control cells. In tumor formation experiment, neoplasms were formed in ASODN treated group at 16-17 d and untreated group at 12-13 d. Neoplasm was formed in 2 of 5 ASODN treated mice and 4 of 5 untreated mice respectively. In untreated mice tumor tissues were rich in blood vasa and stromal tissue compared with that in ASODN treated mice. In tumor therapy experiment, before treatment, there was no difference in the average neoplasm physical volume between ASODN treated group [(100.9 +/- 24.6) mm3] and PBS treated group [(98.4 +/- 23.1) mm3] (P > 0.05). After treatment, the neoplasm volume in ASODN treated group [(422.7 +/- 326.4) mm3] was smaller than that in PBS treated group [(786.4 +/- 357.6) mm3] (P < 0.05). Histologically, there were many apoptosis cells in ASODN treated group, but was seldom seen in PBS treated group. The TUNEL positive cells in ASODN treated group were much more than that in PBS treated group (P < 0.05). CONCLUSION: The hTERT ASODN induces apoptosis of HL-60 cells in vitro, reduces the tumor formation in BALB/c nude mice and inhibits the growth of the transplanted neoplasm.
Subject(s)
Apoptosis/drug effects , Oligodeoxyribonucleotides, Antisense/pharmacology , Telomerase/genetics , Transfection , Animals , HL-60 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: To screen effective sequences of small interfering RNA targeting MDR1 gene in human gastric cancer SGC7901/VCR cells. METHODS: Four siRNAs (MDR1si326, MDR1si1513, MDR1si2631 and MDR1si3071) targeting MDR1 gene were designed and synthesized by in vitro transcription. The siRNA duplexes were used to transfect into the human gastric cancer SGC7901/VCR cells. The expression level of MDR1 mRNA and P-gp were detected by RT-PCR and Western blotting, respectively. The accumulation of intracellular adriamycin (ADR) was examined by flow cytometry and the cell sensitivity to ADR was demonstrated by MTT. RESULTS: The SGC7901/VCR cells treated with 4 siRNAs led to reversal effect on multidrug resistance to different extents. Among the SGC7901/VCR cells treated by siRNAs for 48 h, the expression level of MDR1 mRNA in cells of MDR1si326 or MDR1si2631 group (0.42 +/- 0.07 or 0.49 +/- 0.02) was more decreased than that in cells of MDR1si1513 or MDR1si3071 group (P < 0.05). The accumulation of ADR in cells of MDR1si326 group was the most; in cells of MDR1si2631 group, more; in cells of MDR1si3071 group, lower and in cells of MDR1si1513 group, the lowest (P < 0.05). The relative reversal efficiency of cells of MDR1si2631 group to ADR was the highest and in cells of MDR1si326 group, higher (P < 0.05). There was no significant difference in the relative reversal efficiency between the cells of MDR1si1513 and MDR1si3071 groups (P > 0.05). The expression level of P-gp in cells of MDR1si326 group was the lowest among the SGC7901/VCR cells treated by siRNAs for 72 h. CONCLUSION: The MDR1si326 with most, MDR1si2631 with more, MDR1si3071 with less and MDR1si1513 with least reversal effects on MDR1 gene mediated multidrug resistance were found in the human gastric cancer SGC7901/VCR cells.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Drug Resistance, Multiple/drug effects , RNA, Small Interfering/pharmacology , Stomach Neoplasms/metabolism , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antibiotics, Antineoplastic/metabolism , Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cell Line, Tumor , Doxorubicin/metabolism , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/drug effects , Genes, MDR , Humans , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Stomach Neoplasms/pathology , Transfection , Vincristine/pharmacologyABSTRACT
This study was purposed to investigate the inhibition of hTERT antisense oligodeoxynucleotide (ASODN) on the proliferation and telomerase activity in HL-60 cells and to explore the relativity between the telomerase activity and the expression of hTERT gene in HL-60 cells. After treated by hTERT ASODN the expression of hTERT was detected by RT-PCR, the morphological changes of HL-60 cells was observed with inverted microscopy, the cell proliferation was measured by MTT method, and the telomerase activity was determined with TRAP-ELISA and TRAP-PAGE. The results showed that after sealing hTERT gene with ASODN for 72 hours, the expression of hTERT gene was significantly inhibited, the cell growth was repressed and the ability of proliferation decreased, and the effect was specific in sequence and dependent in dose and time. OD(450-690) values were 2.648 +/- 0.42, 1.504 +/- 0.47, 1.223 +/- 0.39, 0.944 +/- 0.16 respectively, as the cells were treated with 0, 10, 20, 30 micromol/L ASODN for 72 hours. The difference was significant as compared 10, 20, 30 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 20 micromol/L SODN group (2.376 +/- 0.65) with untreated group (2.648 +/- 0.42) (P > 0.05). TRAP-PAGE detection revealed that comparing ASODN groups with SODN groups the telomerase image bands were decreased and least was found in groups of 30 +/- mol/L. It is concluded that the hTERT ASODN may inhibit the proliferation and down-regulate the telomerase activity in HL-60 cells by sealing the expression of hTERT gene.
Subject(s)
Cell Proliferation/drug effects , Oligonucleotides, Antisense/genetics , Telomerase/metabolism , HL-60 Cells , Humans , Oligonucleotides, Antisense/biosynthesis , Telomerase/biosynthesis , Telomerase/genetics , Telomerase/pharmacology , TransfectionABSTRACT
To investigate the effects of c-myc antisense oligodeoxynucleotide (ASODN) on the telomerase activity and the induction of apoptosis in HL-60 cells, and to explore the relationship between the telomerase activity and the expression of c-myc gene in HL-60 cells, after treatment by c-myc ASODN, the expression of c-myc was detected by RT-PCR, the apoptosis, cell cycle were detected with agarose gel electrophoresis and flow cytomety, and the telomerase activity was determined with TRAP-ELISA. The results showed that after blocking c-myc gene with ASODN for 72 hours, it is obvious that the expression of c-myc gene was inhibited. The percentage of S phase HL-60 cells decreased from 55.6% to 30%, the early apoptosis peak appeared (the percentage of apoptosis cells were 25.2%) and the DNA ladders were shown. OD(450 - 690) were 2.648 +/- 0.42, 2.324 +/- 0.36, 2.162 +/- 0.38, 1.952 +/- 0.14, 1.805 +/- 0.40, 1.616 +/- 0.41 and 2.466 +/- 0.29, respectively, as the cells were treated with 0, 1, 2, 3, 4, 5 micromol/L ASODN and 5 micromol/L SODN for 72 hours. The difference was significant when compared 3, 4, 5 micromol/L groups with 0 micromol/L ASODN group respectively (P < 0.05), but the difference was no significant when compared 1, 2 micromol/L ASODN and 5 micromol/L SODN groups with 0 micromol/L ASODN group (P > 0.05). It is concluded that the c-myc gene ASODN may induce the apoptosis of cells, inhibit cells from G(1) phase into S phase and regulate the telomerase activity down in HL-60 cells by blocking the expression of c-myc gene.
Subject(s)
Apoptosis/drug effects , Oligonucleotides, Antisense/pharmacology , Proto-Oncogene Proteins c-myc/biosynthesis , Telomerase/metabolism , Dose-Response Relationship, Drug , Flow Cytometry , HL-60 Cells , Humans , Oligonucleotides, Antisense/genetics , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
OBJECTIVE: To detect methylation in promoter region of hMSH2 gene in esophageal cancer. METHODS: Specimens of cancer and normal tissues freshly removed from 32 cases of esophageal cancer patients without previous radiotherapy, chemotherapy or other treatment were preserved at -80 degrees C within 30 min. Methylation specific PCR (MSP) was used to detect methylation of mismatch repair gene (MMR) hMSH2 in promoter region in esophageal cancer and normal esophageal tissues. RESULTS: The frequencies of methylation of hMSH2 gene in promoter region of cancer and normal esophageal tissues were 32.4% (11/32) and 0/30 (0%), respectively, and significant difference was found between the two groups (P < 0.01). The frequency of methylation in elder patients (> or = 70 years old) was significantly higher than that in younger patients (< 70 years old) (P < 0.05). Methylation was less frequently found in grade I-II (18.2%) than in grade III-IV (70.0%) (P < 0.05). CONCLUSION: Methylation of hMSH2 gene in promoter region is related to patients' age and histopathological grade of the esophageal cancer.
Subject(s)
DNA Methylation , Esophageal Neoplasms/genetics , MutS Homolog 2 Protein/genetics , Promoter Regions, Genetic , Aged , Base Pair Mismatch , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Esophageal Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Male , TransfectionABSTRACT
AIM: To study the alteration of nuclear matrix proteins (NMPs) in gastric cancer. METHODS: The NMPs extracted from 22 cases of gastric cancer and normal gastric tissues were investigated by SDS-PAGE technique and the data were analyzed using Genetools analysis software. RESULTS: Compared with normal gastric tissue, the expression of 30 ku and 28 ku NMPs in gastric cancer decreased significantly (P=0.002, P=0.001, P<0.05). No significant difference was found in the expression of the two NMPs between the various differentiated grades (P=0.947, P=0.356) and clinical stages of gastric cancer (P=0.920, P=0.243, P>0.05). CONCLUSION: The results suggested that the alteration of NMPs in gastric cancer occurred at the early stage of gastric cancer development.
Subject(s)
Gastric Mucosa/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Stomach Neoplasms/metabolism , Cell Nucleus/metabolism , Electrophoresis, Polyacrylamide Gel , HumansABSTRACT
OBJECTIVE: To observe the expression of DNA mismatch repair gene hMSH2 mRNA in esophageal cancer tissues. METHODS: This study included 32 esophageal cancer patients who received no previous radiotherapy, chemotherapy or other treatments. Within 30 min following surgical removal of the tumor tissues, specimens of the tumor, the tissue adjacent to the tumor and normal tissue at the esophageal stump (1 cmx1 cmx1 cm in size for each specimen) were obtained for examining hMSH2 expression with hMSH2 ISH detection kit. RESULTS: The positivity rate of hMSH2 was 46.88% in the esophageal cancer tissues, 53.12% in the adjacent tissues, and 84.38% in normal tissues at the esophageal stump, showing significant difference of the former two tissues from the normal tissue (P<0.05). No significant correlation was noted between the positivity rate of hMSH2 and such factors as the patients' age, sex, tumor size, tumor location, pathological type, histological grade, lymphatic metastasis or degree of tumor invasion (P>0.05). CONCLUSION: The deletion of hMSH2 is an early event in the development of esophageal cancer.
Subject(s)
Base Pair Mismatch , DNA Repair , DNA-Binding Proteins/genetics , Esophageal Neoplasms/genetics , Proto-Oncogene Proteins/genetics , Aged , Female , Humans , Male , Middle Aged , MutS Homolog 2 Protein , RNA, Messenger/analysisABSTRACT
AIM: To study methylation, frequencies of homozygous deletion and mutation of p16 gene in gastric carcinoma. METHODS: The methylation pattern in exon 1 and exon 2 of p16 gene was studied with polymerase chain reaction (PCR), using methylation sensitive restriction endonuclease HpaII and methylation insensitive restriction endonuclease MspI. PCR technique was used to detect homozygous deletions of exon 1 and exon 2 of p16 gene and single strand conformation polymorphism (SSCP) technique was used to detect the mutation of the gene. RESULTS: Hypermethylation changes in exon 1 and exon 2 of p16 gene were observed in 25 % and 45 % of 20 gastric cancer tissues, respectively, while no methylation abnormality was found in normal tissues. The homozygous deletion frequency of exon 1 and exon 2 of p16 gene in 20 gastric cancer tissues was 20 % and 10 %, respectively. No mutation was found in exon 1 of p16 gene, while abnormal single strands were found in 2 (10 %) cases in exon 2 as detected by SSCP. CONCLUSION: The results suggest that hypermethylation and abnormality of p16 gene may play a key role in the progress of gastric cancer. Hypermethylation of exon 2 of p16 gene may have effects on the carcinogenesis of gastric mucosa and may be a later event.
Subject(s)
DNA Methylation , Genes, p16 , Mutation , Stomach Neoplasms/genetics , DNA Mutational Analysis , Gene Deletion , HumansABSTRACT
AIM: To detect the loss of heterozygosity (LOH) frequency of microsatellite sites D9s171, D9s1604 of p16 gene and expression of hMSH2 mRNA in various differentiated types of gastric cancer, adjacent cancer tissues and normal gastric mucosa. METHODS: LOH was detected by polymerase chain reaction (PCR)-denaturing polyacrylamide gel electrophoresis-silver staining. The expression of hMSH2 mRNA was examined with in situ hybridization. RESULTS: The frequency rate of LOH was significantly higher in gastric cancers than that in adjacent cancer tissues (P=0.032). No significant difference was noted among various differentiated types and various clinical stages of gastric cancers. The significantly reduced expression of hMSH2 mRNA positive signal cells exhibited in gastric cancers, in comparison with that in the adjacent cancer tissues and normal gastric mucosa, respectively (P=0.001). No significant difference was noted among various clinical stages of gastric cancers (P>0.05). The difference of positive signal cells in poorly differentiated cancers and those in well and moderately differentiated cancers were significant (P<0.001). CONCLUSION: The frequencies of LOH in two microsatellite sites, D9s171 and D9s1604, in p16 genome were associated with development of gastric cancer and no significant correlation was demonstrated between the LOH frequency and the cell differentiated types of tumor cells or clinical stages. There was a positive relationship between the expression of hMSH2 mRNA and the differentiated types of gastric cancer.
Subject(s)
DNA-Binding Proteins , Genes, p16 , Microsatellite Repeats/genetics , Proto-Oncogene Proteins/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Humans , Loss of Heterozygosity , MutS Homolog 2 Protein , Proto-Oncogene Proteins/genetics , RNA, Messenger/metabolismABSTRACT
AIM:To investigate the expression of somatostatin mRNA in various differentiated types of gastric carcinoma.METHODS: By using in situ hybridization and immunohistochemical techniques, the expression of somatostatin mRNA and somatostatin immunoreactivity in the normal gastric mucosa, the poorly, moderately and well-differentiated gastric carcinomas, and various clinical stages of carcinoma were observed.RESULTS: In comparison with the normal gastric mucosa, the significantly increased expression of somatostatin mRNA positive cells was displayed in gastric carcinoma (t = 2.681, P < 0.01). The positive signal cells were distributed in a scattered form or aggregated as a mass or a cord, and the positive cells were more significantly enhanced in poorly differentiated carcinomas than those in well and moderately differentiated carcinomas (t = 2.962, P < 0.01). The somatostatin mRNA hybridization signals in stages III and IV of gastric carcinoma were significantly higher than those in stages I and II. The results of somatostatin immunoreactivity were consistent with those of in situhybridization.CONCLUSION: The alteration of the expression of somatostatin mRNA was associated with the development of gastric carcinoma and may play an important role in theprocess of tumor differentiation.