[Effect of Signal Transduction Pathway Gene Mutations on the One- course Induced Remission Rate and Analysis of Clinical Characteristics in Patients with CBF-AML].

OBJECTIVE: To investigate the effect of other gene mutations outside the fusion gene on the first complete remission (CR1) induced by one course of induction chemotherapy in patients with core binding factor-associated acute myeloid leukemia (CBF-AML). METHODS: DNA was extracted from bone marrow or peripheral blood samples of newly diagnosed CBF-AML patients admitted to the Hematology Department of the Second Hospital of Shanxi Medical University from January 2015 to January 2019. Next-generation sequencing was used for detection of 34 kinds of hematologic malignancy-related gene mutations in patients with CBF-AML, the effect of related gene mutations on the first complete remission (CR1) rate in one course of induction chemotherapy was analyzed by combineation with clinical characteristics. RESULTS: 34 kinds of genes in bone marrow or peripheral blood of 43 patients were detected by high throughput sequencing and the gene mutations were detected in 16 out of 34 genes. The mutation rate of KIT gene was the highest (48.8%), followed by NRAS (16.3%), ASXL1 (16.3%), TET2 (11.6%), CSF3R (9.3%), FLT3 (9.3%), KRAS (7.0%). The detection rates of mutations in different functional genes were as follows: genes related with signal transduction pathway (KIT, FLT3, CSF3R, KRAS, NRAS, JAK2, CALR, SH2B3, CBL) had the highest mutation frequency (72.1% (31/43); epigenetic modification gene mutation frequency was 30.2% (13/43), including ASXL1, TET2, BCOR); transcriptional regulation gene mutation frequency was 7.0% (3/43), including ETV6, RUNX1, GATA2). Splicing factor related gene mutation frequency was 2.3% (1/43), including ZRSR2). The CR1 rate was 74.4% after one course of induction chemotherapy. At first diagnosis, patients with low expression of WT1 (the median value of WT1 was 788.9) were more likely to get CR1 (P=0.032) and the RFS of patients who got CR1 after one course of induction chemotherapy was significantly longer than that of patients without CR1 [7.6 (2.2-44.1) versus 5.8 (1-19.4), (P=0.048)]. The rate of CR1 in the signal transduction pathway gene mutation group was significantly lower than that in non-mutation group (64.5% vs 100%) (P=0.045), while the level of serum hydroxybutyrate dehydrogenase (HBDH) was significantly higher than that in non-mutation group [(418 (154-2702) vs 246 (110-1068)] (P=0.032). There was no difference in CD56 expression between the two groups (P=0.053), which was limited to the difference between (≥20%) expression and non-expression. (P=0.048). CONCLUSION: CBF-AML patients with signal transduction pathway gene mutation are often accompanied by high HBDH level and CD56 expression, moreover, the remission rate induced by one course of treatment is low.

Correlation of Musashi-1, Lgr5, and pEGFR expressions in human small intestinal adenocarcinomas.

Recent studies have revealed that Musashi-1 and Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) were putative stem cell genes. The epidermal growth factor receptor (EGFR) has also been extensively studied; it was known as an oncogenic driver in cancers. Overexpressions of Musashi-1, EGFR, and Lgr5 have been reported in some tumor tissues and cell lines. In this study, we used immunohistochemical analysis to investigate the expression pattern of Musashi-1, Lgr5, and pEGFR in 38 small intestinal adenocarcinomas (SIAs) resection specimens, 20 matched normal specimens and tried to analyze the correlations among them. The positive rate of Musashi-1, Lgr5, and pEGFR in SIAs, respectively, was 71 % (27/38), 55 % (21/38), and 45 % (17/38). Compared with the adjacent normal small intestinal mucosa, Musashi-1, Lgr5, and pEGFR protein were overexpressed in SIAs (P< 0.05). Furthermore, Musashi-1 and Lgr5 expressions were significantly correlated with the depth of wall invasion (P = 0.0011, P = 0.0017, respectively). Musashi-1 expression was closely correlated with Lgr5 (P = 0.015, r = 0.392). However, pEGFR expression was not associated with age, gender, tumor size, differentiation, depth of invasion, lymphatic metastasis, TNM stage, and pEGFR expression was not correlated with Musashi-1 or Lgr5 (P > 0.05, r = 0.064; P > 0.05, r = 0.307, respectively). Thus, we suggest that Musashi-1, Lgr5, and pEGFR are overexpressed in human SIAs and may play roles in human SIA carcinogenesis and progression.

Molecular alterations of EGFR in small intestinal adenocarcinoma.

BACKGROUND AND AIMS: Molecular testing for epidermal growth factor receptor (EGFR) mutations has recently become a standard practice for the management of patients with non-squamous none small cell lung cancer. Primary small intestine adenocarcinoma (SIA) is an uncommon malignancy, and EGFR mutation in the cancer has not been well characterized due to its rarity. METHODS: A micro-tissue array with 53 SIAs and 24 surgically resected primary non-ampullary SIAs were studied. EGFR mutations were analyzed by DNA sequencing in 24 cases with formalin-fixed paraffin-embedded blocks. All 77 cases were examined by immunohistochemistry (IHC) using antibodies specific for the EGFR E746-A750 deletion in exon 19 (DEL), L858R point mutation in exon 21 (L858R), and total EGFR. EGFR amplifications were detected by fluorescence in situ hybridization. RESULTS: A positive reaction of DEL-specific, L858R-specific, and total EGFR antibodies was detected in seven (9.1%), 5 (6.5%) and 35 (45.5%) of 77 SIAs by IHC, respectively. Positive reaction of the three antibodies was not significantly correlated with patient's age, gender, differentiation, and stage. EGFR gene amplification was assayed in 77 SIAs in micro-tissue array. Of 24 SIA samples that had DNA sequencing, two (8.3%) harbored exon 19 deletion and one (4.2%) harbored L858R point mutation. Only one case with EGFR amplification and two cases with polysomy were shown. CONCLUSIONS: Our findings suggested that mutations and amplification in EGFR genes are minor events, and most of SIAs may be unsuitable to EGFR-TKIs treatment.

Expression of Cav-1 in tumour cells, rather than in stromal tissue, may promote cervical squamous cell carcinoma proliferation, and correlates with high-risk HPV infection.

Altered expression of caveolin-1 (Cav-1) is observed in various types of cancers. However, little research has been reported regarding the correlation between the expression of Cav-1 and cervical cancer. Here, we investigated the clinical significance of Cav-1 expression using quantum dot (QD)-based immunofluorescence staining in cervical cancer and its correlation with high-risk human papilloma virus (HPV) infection detected by chromogenic in situ hybridization. Our results showed that the positive rates of Cav-1 protein in normal cervical mucosa, CIN, cervical adenocarcinoma and SCC were: 0, 33, 19 and 55%, respectively. The differences in Cav-1 protein expression in cervical SCC compared to the other three groups were all statistically significant. Absence of stromal Cav-1 protein in 58 cases of cervical SCC was 67%. The positive rates of the Cav-1 protein in tumour and stromal cells of cervical SCC were not correlated with clinicopathological parameters. In the cervical SCC tissues, Cav-1 expression in tumour cells was not associated with stromal Cav-1 expression, but a positive correlation existed with the PCNA protein and high-risk of HPV infection. The results presented here suggest that expression of Cav-1 in the tumour cells, rather than in the stromal tissue surrounding the tumour, may promote cervical SCC cell proliferation, and correlates with high-risk HPV infection.

Differential expression and function of the caveolin-1 gene in non-small cell lung carcinoma.

This study was designed to clarify the function of caveolin-1 (Cav-1) in the development of lung cancer by investigating the mutation and protein expression of the Cav-1 gene in non-small cell lung carcinoma (NSCLC). Quantum dot immunofluorescence histochemistry was used to evaluate Cav-1 protein expression and subcellular localization in the lung cancer tissue microarray including 140 cases of lung cancer and 20 cases of non-cancerous lung tissue. Mutation of the Cav-1 gene in exon 1 and exon 3 was detected by polymerase chain reaction-single strand conformation polymorphism and sequencing. The positive rates of Cav-1 expression were 49.3% (69/140) in NSCLC group, significantly lower than the 100% (20/20) rate in the control group. Adenocarcinomas (16.7%), adenosquamous carcinomas (38.4%), squamous cell carcinomas (67.1%) and large cell lung cancers (66.7%) displayed Cav-1 positive staining, suggesting a gradient of Cav-1 expression according to tumor histotype-related aggressiveness. High-expression of Cav-1 protein was statistically correlated with pathologic TNM stage and lymph node metastasis. No mutation could be detected in exon 1 and exon 3 from all Cav-1 protein negative expression of NSCLC samples. Cav-1 immunoreactivity in lung cancer is histotype-dependent, increased Cav-1 expression indicates the malignant progression and high invasion features of NSCLCs. Deregulation of Cav-1 expression in NSCLCs may not correlate with mutation.

Expression of putative stem cell genes Musashi-1 and beta1-integrin in human colorectal adenomas and adenocarcinomas.

BACKGROUND AND AIMS: Recent studies revealed that Musashi-1and beta1-integrin were putative stem cell genes. Overexpressions of Musashi-1 and beta1-integrin have been reported in some tumor tissues and cell lines. This study was to detect expressions of the two genes in colorectal adenomas and carcinomas and to analyze the correlation between Musashi-1 and beta1-integrin. METHODS: Musashi-1 and beta1-integrin immunoreactivity was studied immunohistochemically in tissue microarray-based samples containing 69 colorectal adenocarcinomas, eight normal mucosa, and eight adenomas, and their messenger RNA (mRNA) expression level was detected by RT-PCR in resected specimens including the three types of tissue. RESULTS: A percentage of 66.7% (46/69) and 59.2% (41/69) of colorectal adenocarcinomas were immunoreactive with Musashi-1 and beta1-integrin, respectively. The expressions of Musashi-1 and beta1-integrin protein were significantly higher in tissue samples of stage III than those of stage I-II (P = 0.0252; P = 0.0018, respectively). beta1-integrin expression was higher in group of adenocarcinomas than that of adenomas (P = 0.0276). Musashi-1 expression was closely correlated with beta1-integrin (rs = 0.631, P = 0.0001). Significant differences of Musashi-1 and beta1-integrin mRNA expression levels were found between the normal colorectal mucosa, adenoma, and adenocarcinoma tissues (P = 0.01; P = 0.03, respectively). CONCLUSIONS: Musashi-1 and beta1-integrin may be involved in human colorectal tumor carcinogenesis and progression. Our observations also indicate the need for further investigations to test in vivo whether cells with these markers have stem cell properties.

Studies of chitosan/Kollicoat SR 30D film-coated tablets for colonic drug delivery.

The aim of the study was to define in vitro and in vivo characteristics of chitosan/Kollicoat SR30D film-coated tablets of theophylline for colonic delivery. The tablet cores were coated to different film thicknesses with blends of Kollicoat SR30D and chitosan (2.5:1, 3.5:1, and 5:1, w/w). Swelling and drug release studies were carried out in simulated gastric fluid, simulated intestinal fluid and simulated colonic fluid, respectively. The mechanism of drug release was determined using the Korsmeyer-Peppas model. The in vivo degradation of the tablets was also studied in rats. The swelling behavior and drug release depended on the composition of the coating, as well as the ratio of Kollicoat SR30D to chitosan. The coating was susceptible to enzymatic action, and more accessible to bacterial enzymes than beta-glucosidase enzyme. The extent of swelling and digestion correlated with the amount of chitosan within the coating. The drug release data fit well into the Korsmeyer-Peppas equation, indicating that the drug release was controlled by polymer relaxation. The in vivo pharmacokinetic studies of the coated tablets showed delayed T(max), decreased C(max) and prolonged MRT. Chitosan/Kollicoat SR30D coated tablets could deliver the drug to the targeted site for local action.

Design and in vitro/in vivo evaluation of multi-layer film coated pellets for omeprazole.

The purpose of the study is to perform the in vitro and in vivo evaluation of multi-layer film coatings for omeprazole. The system consists of drug-layered or drug-containing core pellets coated with salt (sodium chloride and disodium hydrogen phosphate), hydroxypropyl methyl cellulose (HPMC), and enteric film-coating layer, respectively. The drug-layered core pellets were prepared by a coating layer of omeprazole on inert pellet cores in fluidized bed coater. An in vitro/in vivo gastro-resistance study was conducted, and a dissolution study was performed in pH 7.4 phosphate buffer for omeprazole release. The multi-layer coated pellets were stable in gastric pH conditions and upper gastrointestinal (GI) tract in rats. Salt layer improved the drug stability, and its coating levels had little influence on the dissolution profiles of omeprazole. The rate of drug release was significantly delayed by HPMC layer. The salt layer could function as a separated layer, and substitute part of the HPMC layer and decrease the coating levels of HPMC. The bioavailability (AUC) of the multi-layer coated drug-layered and drug-containing pellets was 3.48+/-0.86 and 2.97+/-0.57 microg*h/ml, respectively. The drug-layered pellets with multi-layer film coatings not only provided delayed and rapid release of omeprazole, but also could provide a good stable property for omeprazole. It was confirmed that rapid in vitro drug release rate resulted in better absorption.

Role of Hypoxia-inducible factor-1 alpha and Survivin in colorectal carcinoma progression.

BACKGROUND AND AIMS: Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is the main active subunit of HIF-1 that promoted tumor cells survival and critical steps in tumor progression and aggressiveness. The authors aimed to investigate the role of HIF-1 alpha and Survivin in colorectal cancer (CRC) progression. MATERIALS AND METHODS: Plasmid expressing small interfering RNA (siRNA) against HIF-1 alpha was constructed and transfected into LS174T cells with Lipofectamine. The LS174T cells were incubated for 24 h under hypoxic condition. The inhibitory effects of siRNA on HIF-1 alpha gene was determined by semiquantitative reverse transcriptase polymerase chain reaction and Western blot. Expression of HIF-1 alpha and Survivin was investigated by immunohistochemistry in colorectal adenocarcinomas tissue microarrays. RESULTS: HIF-1 alpha and Survivin expressions were markedly downregulated after the siRNA expression vector against HIF-1 alpha was transfected into the LS174T cells. Of the eight adenoma lesions, one case (12.25%) and four cases (50%) were positive for HIF-1 alpha and Survivin, respectively. Of the 69 cases of CRCs, 46 cases (66.7%) and 39 cases (56.5%) were positive for HIF-1 alpha and Survivin, respectively. The positive rate of HIF-1 alpha protein in CRCs was significantly higher than that in colorectal adenoma lesions (P < 0.05). HIF-1 alpha protein expression was significantly higher in patients with stage III than in patients with stage I-II CRCs (P < 0.01). In addition, overexpression of HIF-1 alpha in higher stages of CRCs was found to correlate positively with Survivin levels (P < 0.001). CONCLUSIONS: Our data demonstrate that HIF-1 alpha and Survivin are mostly expressed in invasive CRCs. Inhibition of HIF-1 alpha may lead to exploration of its potential as a diagnostic tool and possibly a target for gene therapy for colorectal carcinoma.

Biphasic drug release: permeability and swelling of pectin/ethylcellulose films, and in vitro and in vivo correlation of film-coated pellets in dogs.

The major objectives of this study were i) to evaluate the permeability and swelling characteristics of isolated films prepared by mixing of pectin with ethylcellulose; and ii) to assess the absorption and in vitro/in vivo correlation (IVIVC) of 5-FU film-coated colon-targeted pellets in dogs. Free films were prepared by casting and solvent evaporation method. These free films were evaluated by swelling experiment and permeability to 5-FU in different media. Pectin/ethylcellulose films had suitable characteristics for colonic delivery; and when the addition of pectin was up to the ratio of 30%, the swelling and permeability of the mixed films was significantly increased in the simulated colonic fluid (SCF). Pharmacokinetic study in dogs gave Tmax/Cmax of 14 h/1.6 microg/ml and 16 h/1.7 microg/ml for total weight gain (TWG)-22% and 18% coated pellets, respectively. The plasma 5-FU levels of the TWG-22% and 18% coated pellets were maintained at a much lower level with a mean residence time (MRT) of 18-20 h, longer than 2.1 h for 5-FU uncoated pellets, confirming delayed absorption. There was no statistically significant difference in the area under the plasma concentration vs. times curve (AUC) values between the uncoated pellets and the coated pellets. Moreover, a good linear regression relationship was observed between the percent in vitro dissolution in SCF and the percent absorption or percent AUC. It was concluded that i) pectin within the mixed films were susceptible to colonic enzymes, and the film-coated pellets are potentially useful for colonic drug delivery; and ii) in vitro dissolution testing in SCF could be used to establish certain IVIVC for the colon-specific drug delivery systems activated by microflora.

Li-cadherin is inversely correlated with galectin-3 expression in gastric cancer.

The aims of this study were to examine the expressions of Li-cadherin and Galectin-3 in gastric cancer, and the correlation between Li-cadherin and Galectin-3 in gastric cancer was also analyzed. The present study investigated the expression level of Li-cadherin and Galectin-3 by immunohistochemistry and semiquantitative polymerase chain reaction (PCR), and correlated this with clinicopathologic parameters in 91 cases of gastric cancer. The correlation between expression levels of Li-cadherin and Galectin-3 was analyzed by Spearman correlation analysis. The expression level of Li-cadherin mRNA was correlated to differentiation and lymph node metastasis, and the expression level of Galectin-3 was related to TNM staging, differentiation and lymph node metastasis. On Spearman correlation analysis, a definitive negative correlation was found between the expression levels of Li-cadherin and Galectin-3 in gastric cancerous tissues. We postulate that interaction between Li-cadherin and Galectin-3 may play an important role in the development of gastric cancer.

Study on colon-specific pectin/ethylcellulose film-coated 5-fluorouracil pellets in rats.

The purpose of the present study is to assess the biodistribution and pharmacokinetics of pectin/ethylcellulose film-coated and uncoated pellets containing 5-fluorouracil (5-FU) in rats. Both coated and uncoated pellets were orally administered to the rats at a dosage equivalent to 15mg/kg. 5-FU concentrations in different parts of the gastrointestinal (GI) tract and plasma were quantitatively analyzed using a high-performance liquid chromatography (HPLC) assay. 5-FU released from uncoated pellets mainly distributes in the upper GI tract, however, 5-FU released from coated pellets mainly distributes in the cecum and colon. In plasma, the observed mean C(max) from the coated pellets group (3.65+/-2.3microg/mL) was lower than that of the uncoated pellets group (23.54+/-2.9microg/mL). The AUC values obtained from the uncoated pellets and the coated pellets were 49.08+/-3.1 and 9.06+/-1.2microgh/mL, respectively. The relatively high local drug concentration with prolonged exposure time provides a potential to enhance anti-tumor efficacy with low systemic toxicity for the treatment of colon cancer.

Selective drug delivery to the colon using pectin-coated pellets.

A combination of ethylcellulose and pectin, when applied as a film coat, has a potential value as a colon-specific delivery system. Dispersions of pectin in ethylcellulose were used as the film former for coating of 5-aminosalicylic acid (5-ASA) pellet cores. Drug release behavior was assessed, in vitro, under simulating conditions in term of pH and time in vivo during transit to the colon. Negligible drug release occurred during first 5 h, when the coated pellets were in the simulated gastric and small intestinal conditions. After that, rat cecal contents were added into the pH 6.8 medium to simulate the in vivo condition where there is the digestion of bacteria in the colon. Drug release depended on the composition of the mixed film, as well as the ratio of ethylcellulose to pectin. Drug release profiles seemed to conform to the mechanism involving the osmotically driven release and formation of channels in the film caused by dissolution of pectin. Channel formation was, in most cases, activated by the presence of rat cecal contents, showing that the pectin in the mixed film was subjected to enzymic breakdown. In conclusion, pectin could be used as an additive in ethylcellulose films to control the release of colonic delivery system. In addition, the mechanism of the hydrophilic drug release from pellets coated with ethylcellulose aqueous dispersions containing an aqueous gel-forming polymer (pectin) is also discussed.

Expression levels and significance of hypoxia inducible factor-1 alpha and vascular endothelial growth factor in human colorectal adenocarcinoma.

BACKGROUND: Hypoxia-inducible factor 1 (HIF-1), a transcription factor, is overexpressed in common human cancers and their metastases. This study aimed at determining the expression levels of HIF-1alpha and vascular endothelial growth factor (VEGF) in SW480 cells and in colorectal adenocarcinoma tissue and ascertaining whether HIF-1alpha and VEGF play important roles in tumor angiogenesis. METHODS: HIF-1alpha mRNA expression was analyzed using in situ hybridization and RT-PCR. HIF-1alpha and VEGF protein were detected in SW480 cells and colorectal adenomas and adenocarcinomas by immunohistochemistry using streptavidin/peroxidase (SP). Western blot was used to detect HIF-1alpha protein extracted from SW480 cells. Microvessel density (MVD) in colorectal carcinomas was determined by anti-CD34 immunostaining in colorectal carcinomas. RESULTS: Optical density values representing HIF-1alpha mRNA expression levels were found to be significantly higher in SW480 cells in hypoxic conditions than in cells under normoxic conditions (P < 0.05) or in hypoxic conditions but treated with genistein (P < 0.05). The levels of HIF-1alpha and VEGF protein expression in SW480 cells were significantly higher in the hypoxia group than in the normoxia group (P < 0.01, P < 0.05, respectively) and hypoxia/genistein group (P < 0.01, P < 0.05, respectively). The positive expression rates of HIF-1alpha mRNA changed dramatically when comparing colorectal adenomas with adenocarcinomas of different Dukes' stages (P < 0.05). HIF-1alpha mRNA was also expressed at higher levels in adenocarcinomas than that in adenomas (P < 0.01). HIF-1alpha protein expression correlated significantly with VEGF protein expression and MVD. CONCLUSIONS: Hypoxia induces the expression of HIF-1alpha and VEGF in colorectal adenocarcinomas. HIF-1alpha may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinomas.

[Expression and pathobiological implication of hypoxia-inducible factor-1alpha in human colorectal carcinoma].

OBJECTIVE: To investigate the transcription level and protein expression of HIF-1alpha and VEGF in SW480 cell line and colorectal adenocarcinoma, and to determine whether HIF-1alpha plays a role in angiogenesis through its regulation of VEGF. METHODS: HIF-1alpha mRNA expression was analyzed by in situ hybridization. HIF-1alpha and VEGF protein expressions were determined by immunochemical streptavidin/peroxidase (SP) in SW480 cells and colorectal carcinoma tissue samples and Western blot, using proteins extracted from SW480 cells. Tumor tissue microvessel density (MVD) was determined by CD34 immunostaining of colorectal carcinomas. RESULTS: The levels of HIF-1alpha mRNA changed significantly in response to different oxygen concentrations and an addition of genistein in SW480 cells. Immunocytochemistry revealed that the levels of HIF-1alpha, VEGF protein expression in SW480 cells were significantly higher under hypoxia than those in nomoxia (P < 0.01, P < 0.05 respectively). However, addition of genistein, an inhibitor of HIF-1alpha, suppressed such responses to hypoxia. Western blot analysis showed that SW480 cells exposed to hypoxia expressed a high level of HIF-1alpha protein, compared to a weak expression in nomoxia. The addition of genistein in hypoxia suppressed the over-expression of HIF-1alpha. The positive rates of HIF-1alpha mRNA by in situ hybridization in colorectal adenomas and adenocarcinomas were 38.9% (7/18) and 67.7% (42/62), respectively. The percentage of HIF-1alpha mRNA positive cells varied significantly from colorectal adenomas to adenocarcinomas at different Duke stages (P < 0.05), and HIF-1alpha mRNA was higher in adenocarcinomas than in adenomas (P < 0.01). The positive rates of HIF-1alpha and VEGF protein expression in adenocarcinomas were 43.5% (27/62) and 37.1% (23/62), respectively. The expression of VEGF elevated as the Duke tumor staging increased. The conformation rate of HIF-1alpha and VEGF was 74.2% (46/62). MVD was significantly higher in HIF-1alpha and/or VEGF positive tumors than those without (P < 0.01 and P < 0.05 respectively). Among the four groups, i.e. HIF-1alpha+/VEGF+, HIF-1alpha+/VEGF-, HIF-1alpha+/VEGF- and HIF-1alpha-/VEGF-, the difference of MVD was highly significant (P < 0.01). HIF-1alpha expression was correlated significantly with VEGF expression and microvessel density. CONCLUSIONS: These findings suggest hypoxia induces the expression of HIF-1alpha and VEGF in colorectal adenocarcinoma. HIF-1alpha may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinoma.

Effect of antisense oligodeoxynucleotide of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer.

AIM: To explore the effect of antisense oligodeoxynucleotide (As-ODN) of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer. METHODS: As-ODN was transfected into SW480 cells by liposomal transfection reagent. Telomerase activity of SW480 cells was examined by telomeric repeat amplification protocol (TRAP) and enzyme-linked immunosorbent assay (ELISA). Apoptosis was analyzed by morphology and flow cytometry. RESULTS: The telomerase activity in SW480 cells transfected with 1.0 micromol/L of As-ODN for 2-5 days, was significantly decreased in a time-dependent manner, and the cells underwent apoptosis. The missense ODN (Ms-ODN) and the control group transfected with SW480 cells did not show these changes. CONCLUSION: As-ODN can specifically inhibit the telomerase activity of SW480 cells and induce apoptosis.

[Relationship of MAGE-A1 expression with Ki-67 and tumor-infiltrating lymphocyte response in non-small cell lung carcinoma].

BACKGROUND & OBJECTIVE: Recent studies revealed a possible close association between the expression of some members of tumor-specific antigen MAGE (melanoma antigen) family and actively proliferated infantile cells. But the correlation of MAGE-A1 expression with proliferation of tumor cells and immune response at host local site has not been reported to date. Our study was to investigate the expression of MAGE-A1 in non-small cell lung carcinoma (NSCLC), and its relationship with Ki-67 expression, tumor-infiltrating lymphocyte (TIL) response, histologic grade, and pathological type. METHODS: Thirty NSCLC samples in formalin-fixed, paraffin-embedded sections were examined for MAGE-A1, Ki-67 and TIL response using SP immunohistochemical technique. RESULTS: The positive expression rate of MAGE-A1 was 80.00%(24/30) with high expression rate of 58.33%(14/24) and low expression rate of 41.67%(10/24). The positive expression rate of Ki-67 was 93.33%(28/30) with high expression rate of 57.14%(16/28) and low expression rate of 42.86% (12/28). TIL response was observed in 22 patients. There was a significant relationship between MAGE-A1 positive expression and Ki-67 positive expression (rs=0.578, P< 0.005), as well as between MAGE-A1 positive expression and TIL response (rs=0.505, P< 0.005). However, MAGE-A1 expression was not significantly correlated with histologic grade and pathological type (P >0.05). CONCLUSION: The NSCLC cells with MAGE-A1 positive expression possess high proliferation activity; meanwhile, the up-regulation of MAGE-A1 indicates the increase of antigen in tumor cells and the increase of local TIL response, indicating that MAGE-A1 may have potential to be used as a target for immunotherapy in NSCLC patient.

Telomerase activity and cell apoptosis in colon cancer cell by human telomerase reverse transcriptase gene antisense oligodeoxynucleotide.

AIM: To evaluate the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynucleotide (As-ODN) on telomerase activity and cell apoptosis in colon cancer cell line SW480. METHODS: As-ODN was transfected into cells SW480 by liposomal transfection. Cultured cells were divided into three groups: ASODN (5'GGAGCGCGCGGCATCGCGGG-3'), sense oligodeoxynucleotide (5'-CCCGCGATGCCGCGCGCTCC-3', S-ODN) and control. The concentration of oligodeoxynucleotide and liposome was 10 micromol/L and 16 mg/L, respectively. The activity of telomerase was examined by telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (ELISA), and cell apoptosis was observed by morphology and flow cytometry in each group. RESULTS: Telomerase activity began to be down-regulated or inhibited when cells SW480 were treated with As-ODN for 72 h, and cell apoptosis was induced. CONCLUSION: It is suggested that hTERT As-ODN might specially inhibit the activity of telomerase in colon cancer cells and it is further proved that the hTERT gene has a significant correlation with telomerase activity. Further evidence is needed to prove whether hTERT As-ODN is a potential tool for the treatment of colon cancer.

Expression and significance of PTEN, hypoxia-inducible factor-1 alpha in colorectal adenoma and adenocarcinoma.

AIM: To investigate the expression and significance of PTEN, hypoxia-inducible factor-1 alpha (HIF-1alpha), and targeting gene VEGF during colorectal carciogenesis. METHODS: Total 71 cases colorectal neoplasms (9 cases of colorectal adenoma and 62 colorectal adenocarcinoma) were formalin fixed and paraffin-embedded, and all specimens were evaluated for PTEN mRNA, HIF-1alpha mRNA and VEGF protein expression. PTEN mRNA, HIF-1alpha mRNA were detected by in situ hybridization. VEGF protein was identified by citrate-microwave SP immunohistochemical method. RESULTS: There were significant differences in PTEN, HIF-1alpha and VEGF expression between colorectal adenomas and colorectal adenocarcinoma (P<0.05). The level of PTEN expression decreased as the pathologic stage increased. Conversely, HIF-1alpha and VEGF expression increased with the Dukes stage as follows: stage A (0.1029+/-0.0457: 0.1207+/-0.0436), stage B (0.1656+/-0.0329: 0.1572+/-0.0514), and stage C+D (0.2335+/-0.0748: 0.2219+/-0.0803). For PTEN expression, there was a significant difference among Dukes stage A, B, and C+D, and the level of PTEN expression was found to be significant higher in Dukes stage A or B than that of Dukes stage C or D. For HIF-1alpha expression, there was a significant difference between Dukes stage A and B, and the level of HIF-1alpha expression was found to be significantly higher in Dukes stage C+D than that of Dukes stage A or B. The VEGF expression had similar results as HIF-1alpha expression. In colorectal adenocarcinoma, decreased levels of PTEN were significantly associated with increased expression of HIF-1alpha mRNA (r=-0.36, P<0.05) and VEGF protein (r=-0.48, P<0.05) respectively. The levels of HIF-1 were positively correlated with VEGF expression (r=0.71, P<0.01). CONCLUSION: Loss of PTEN expression and increased levels of HIF-1alpha and VEGF may play an important role in carcinogenesis and progression of colorectal adenocarcinoma.

[Expression of HIF-1 alpha and its relationship to apoptosis and proliferation in lung cancer].

BACKGROUND & OBJECTIVES: Hipoxia-inducible factor-1 is a transcriptive factor that regulates genes involved in metabolism, angiogenesis, proliferation, and apoptosis. This study was designed to investigate the expression of hypoxia inducible facter-1 alpha(HIF-1 alpha) and its relationship to bcl-2, Bax, PCNA in lung cancer. METHOD: Immunohistochemical streptavidin/peroxidase(SP) was used to examine the expression of HIF-1a, bcl-2, Bax, and PCNA in 60 cases of lung cancer. RESULTS: In 60 cases of lung cancer, positive rate for HIF-1a was 28.3% (17/60), specially the positive rate of small cell lung cancer(66.7%) was significantly higher than non-small cell lung cancer (21.6%). HIF-1a expression increased as clinical stage and metastasis increased(P < 0.01). The positive rate of bcl-2, Bax, and PCNA were 31.7% (19/60), 40.0% (24/60), 76.7% (46/60), respectively. Inverse relationship was found between the expression of HIF-1 alpha and bcl-2; while the correlation of HIF-1 alpha and Bax was positive(P < 0.01). The relationship between HIF-1 alpha and Bax was positive(P < 0.01). The relationship between HIF-1 alpha and PCNA was not observed(P > 0.05). CONCLUSION: HIF-1 alpha is correlated with apopotosis, but has no relationship with proliferation.