Hypoxia-inducible factor-2a is associated with ABCG2 expression, histology-grade and Ki67 expression in breast invasive ductal carcinoma.

BACKGROUND: Breast cancer is the most common cancer and the leading cause of cancer mortality in women worldwide. Hypoxia is an important factor involved in the progression of solid tumors and has been associated with various indicators of tumor metabolism, angiogenesis and metastasis. But little is known about the contribution of Hypoxia-Inducible Factor-2a (HIF-2a) to the drug resistance and the clinicopathological characteristics in breast cancer. METHODS: Immunohistochemistry was employed on the tissue microarray paraffin sections of surgically removed samples from 196 invasive breast cancer patients with clinicopathological data. The correlations between the expression of HIF-2a and ABCG2 as well as other patients' clinicopathological data were investigated. RESULTS: The results showed that HIF-2a was expressed in different intensities and distributions in the tumor cells of the breast invasive ductal carcinoma. A positive staining for HIF-2a was defined as a brown staining observed mainly in the nucleus. A statistically significant correlation was demonstrated between HIF-2a expression and ABCG2 expression (p = 0.001), histology-grade (p = 0.029), and Ki67 (p = 0. 043) respectively. CONCLUSION: HIF-2a was correlated with ABCG2 expression, histology-grade and Ki67 expression in breast invasive ductal carcinoma. HIF-2a could regulate ABCG2 in breast cancer cells, and could be a novel potential bio-marker to predict chemotherapy effectiveness. The hypoxia/HIF-2a/ABCG2 pathway could be a new mechanism of breast cancer multidrug-resistance. VIRTUAL SLIDES: http://www.diagnosticpathology.diagnomx.eu/vs/2965948166714795.

[Effects of artificial microRNA targeting glucosylceramide synthase on drug sensitivity of breast cancer cells].

OBJECTIVE: To explore the inhibitory effects on glucosylceramide synthase (GCS) expression and drug sensitivity in breast cancer cells by transfecting artificial microRNA targeting GCS. METHODS: Two microRNA expression vectors targeting GCS were constructed and transfected into MCF-7/ADR cells via Lipofectamine 2000. The levels of GCS mRNA and protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. Methyl thiazolyl tetrazolium (MTT) assay was used to assess the chemosensitivity of MCF-7/ADR cells to adriamycin (ADM) and vincristine. RESULTS: After transfection of two microRNA expression vectors, the expression of GCSmRNA in MCF-7/ADR cells was 0.098 ± 0.005 and 0.143 ± 0.007 respectively. Compared with the control cells (0.875 ± 0.008), the difference was significant (P < 0.01). The expression of GCS protein (0.127 ± 0.004, 0.165 ± 0.008) in MCF-7/ADR cells was lower than that in the control cells (0.765 ± 0.007; P < 0.01). Furthermore, in comparison with the control cells, the resistance factor to adriamycin significantly dropped to 4.06 and 6.06 while the drug resistance to vincristine decreased to 8.30 and 12.67 respectively (P < 0.01). CONCLUSION: Artificial microRNA targeting GCS inhibits the GCS expression and restores significantly the sensitivity of breast cancer cells to anticancer drugs. These findings may provide a novel strategy of enhancing the chemotherapy sensitivity of breast cancer.

[Significance of MUM-1/IRF4 protein expression in follicular lymphoma].

OBJECTIVE: To study the expression of MUM-1/IRF4 and its significance in follicular lymphoma. METHODS: Ninety-eight cases of follicular lymphoma were enrolled into the study. They were graded according to the 2008 WHO criteria. The expression of MUM-1/IRF4 protein and other markers (CD10, bcl-6, bcl-2 and Ki-67) was studied using tissue microarray and immunohistochemistry. RESULTS: Amongst the 98 cases studied, there were 24 grade 1 cases, 30 grade 2 cases, 26 grade 3A cases and 18 were grade 3B cases. The rates of expression of MUM-1/IRF4, CD10, bcl-6, bcl-2 and Ki-67 (≥ 25%) were 39.8% (39/98), 62.2% (61/98), 80.6% (79/98), 87.8% (86/98) and 50.0% (49/98), respectively. MUM-1/IRF4 predominantly expressed in high-grade follicular lymphoma and showed a significantly positive correlation with lymphoma grade (r = 0.628, P = 0.000) and Ki-67 index (r = 0.473, P = 0.000). MUM-1/IRF4 expression had a significantly negative correlation with CD10 expression (r = -0.597, P = 0.000), but no correlation with bcl-6 and bcl-2 expression. CONCLUSIONS: MUM-1/IRF4 expression is significantly higher in high-grade follicular lymphoma, indicating that these cases have a high proliferative activity, more aggressive behavior and poorer prognosis. MUM-1/IRF4, when strongly expressed, is another helpful marker for the diagnosis of high-grade follicular lymphoma.

Detection of hemorrhagic hypointense foci in radiation injury region using susceptibility-weighted imaging.

BACKGROUND: Susceptibility-weighted imaging (SWI), a novel, highly sensitive 3D gradient echo MR imaging technique, is used to detect hemorrhage. PURPOSE: To evaluate SWI at 3.0T for detection and visualization of hemorrhage at radiation injury region after radiotherapy for brain glioma. MATERIAL AND METHODS: In 16 patients who had radiation injury in the vicinity of the previously resected and irradiated high-grade brain glioma, SWI examinations were performed on a 3T MR scanner. The presence of intralesional hypointense foci on SWI was evaluated by two neuroradiologists. Frequency of these foci on SWI was assessed and the number of these foci was counted. Diagnosis of radiation injury was assigned by means of histopathology or follow-up MR image. RESULTS: In all 16 cases with cerebral radiation injury, nine were verified by means of histopathologic examination, seven by follow-up image. While in one patient quality of SWI was poor, in all remaining patients diagnostic-quality SWI was obtained. The intralesional hypointense foci were detected in 12 of 15 patients. These hypointense foci were nodular, angular, or tubular regions of low signal intensity on SWI. The distribution of these foci was diffusive (n=5) or scattered (n=7). Number of these foci per cm(2) on SWI was 7.25 ± 3.67. CONCLUSION: SWI is a novel and promising technique for evaluation of hemorrhage at radiation injury regions in the vicinity of the previously treated gliomas.

Screening of deoxyribozyme with high reversal efficiency against multidrug resistance in breast carcinoma cells.

Specific inhibition of P-glycoprotein (Pgp) expression, which is encoded by multidrug resistance gene-1 (MDR1), is considered a well-respected strategy to overcome multidrug resistance (MDR). Deoxyribozymes (DRz) are catalytic nucleic acids that could cleave a target RNA in sequence-specific manner. However, it is difficult to select an effective target site for DRz in living cells. In this study, target sites of DRz were screened according to MDR1 mRNA secondary structure by RNA structure analysis software. Twelve target sites on the surface of MDR1 mRNA were selected. Accordingly, 12 DRzs were synthesized and their suppression effect on the MDR phenotype in breast cancer cells was confirmed. The results showed that 4 (DRz 2, 3, 4, 9) of the 12 DRzs could, in a dose-dependent response, significantly suppress MDR1 mRNA expression and restore chemosensitivity in breast cancer cells with MDR phenotype. This was especially true of DRz 3, which targets the 141 site purine-pyrimidine dinucleotide. Compared with antisense oligonucleotide or anti-miR-27a inhibitor, DRz 3 was more efficient in suppressing MDR1 mRNA and Pgp protein expression or inhibiting Pgp function. The chemosensitivity assay also proved DRz 3 to be the best one to reverse the MDR phenotype. The present study suggests that screening targets of DRzs according to MDR1 mRNA secondary structure could be a useful method to obtain workable ones. We provide evidence that DRzs (DRz 2, 3, 4, 9) are highly efficient at reversing the MDR phenotype in breast carcinoma cells and restoring chemosensitivity.

[Study on the sensitizing potential of shuanghuanglian injection using popliteal lymph node assay in C57BL/6J mice].

OBJECTIVE: To investigate the sensitizing potential of Shuanghuanglian Injection (SHL) by comparing the popliteal lymph node (PLN) response in mice induced by SHL and chemicals. METHODS: Sixty female C57BL/6J mice were equally and randomly divided into six groups, i.e. the blank control group (A) and five treated groups treated respectively with phenobarbital 1 mg/mouse (B), mercuric chloride ( HgCl2) 50 microg/mouse (C), D-penicillamine 2 mg/mouse (D), and SHL in low (1 mg/mouse) and high (5 mg/mouse) dosages (E and F) via subcutaneous injection into left pad of hind foot. Animals were sacrificed on the 8th day after injection, their bilateral PLNs were isolated and weighed respectively to calculate the PLN mass index (MI). Then the PLNs get from four mice in each group were fixed with 4% paraformaldehyde solution for histopathologic examination; the other six PLNs were prepared into single-cell suspensions to calculate cell index (CI) for comparing the changes of PLN in various groups. RESULTS: MI and CI in Group F reached to > or = 2 and > or = 5 (average) respectively, which was higher than those in Group A (P<0.05). Pathological examination showed that the left PLN in Group F enlarged, with remarkable germinal center and increased high endothelial venules proliferation. CONCLUSION: SHL could induce significant PLN response in C57BL/6J mice, suggesting it has certain sensitizing potential.

Lymphangiogenesis in gastric carcinoma correlates with prognosis.

Lymphatic metastasis is an important way that gastric carcinomas can spread. However, little is known about the mechanisms of lymphangiogenesis and its clinical significance in gastric carcinomas. In the present study, lymphatic vessel density (LVD), VEGF-C expression, and proliferative activity of lymphatic endothelium were determined in human gastric carcinomas and xenografts of gastric cancer cells in nude mice. The development of lymphangiogenesis and its correlation with patient prognosis were investigated. The results showed that lymphatic vessels were observed mainly in peripheral tumour tissue with significantly (p < 0.05) higher P-LVD (peri-tumoural-LVD) than I-LVD (intra-tumoural-LVD). The expression of VEGF-C was heterogeneous within tumours, with a significantly higher expression (immunostaining score) at the margin than at the tumour centre (p < 0.05). A significant correlation was found between VEGF-C expression at the margin (but not at the centre) and P-LVD (r = 0.72, p < 0.01). High proliferative activity of lymphatic endothelium was also observed in the peripheral tissues, with a significant correlation between proliferative activity of lymphatic endothelium and VEGF-C expression (p < 0.05). These data imply that the increased lymphatics may have been newly formed following stimulation by VEGF-C. High VEGF-C expression at the margin of gastric carcinomas could induce lymphangiogenesis in the peri-tumoural stroma and contribute to the increased P-LVD. The data from mice tumour xenografts also suggested that VEGF-C produced from the transplanted gastric carcinoma cells could induce lymphangiogenesis around them. In patients, VEGF-C expression at tumour margins was associated with nodal metastasis, lymphatic vessel invasion, poor recurrence-free survival, and poor overall survival, and could serve as an independent predictor for patients with gastric carcinoma.

Clinicopathological significance of peritumoral lymphatic vessel density in gastric carcinoma.

Lymphangiogenesis has recently been considered important for spread of malignant tumors. In the present study, lymphatic vessel density (LVD) including peritumoral LVD (P-LVD) and intratumoral LVD (I-LVD) was determined, respectively, by immunohistochemical staining with the antibody to LYVE-1 in 63 cases of early gastric carcinoma and 105 cases of advanced gastric carcinoma. The aim of the study is to investigate whether or not increased LVD could be a risk factor for nodal metastasis and survival. We conclude that increased P-LVD, but not I-LVD, could serve as an independent risk factor for nodal metastasis, recurrence and overall survival in gastric carcinoma.

Distributions of angiogenesis and lymphangiogenesis in gastrointestinal intramucosal tumors.

BACKGROUND: Although angiogenesis and lymphangiogenesis in gastrointestinal cancers has been investigated in many studies, their distribution characteristics in gastrointestinal intramucosal tumors have not been well addressed. METHODS: We evaluated the blood microvessel density (BMVD) and lymphatic microvessel density (LMVD) by immunostaining with monoclonal antibodies of CD34 and D2-40 in 37 patients with stomach intramucosal carcinoma and 28 patients with colorectal intramucosal neoplasia. Microvessels with endothelial cells labeled by CD34 but not by D2-40 were recognized as blood microvessels; and microvessels with endothelial cells labeled by both CD34 and D2-40 were recognized as lymphatic vessels. Furthermore, the relationships between expression of vascular endothelial growth factor (VEGF), VEGF-C, and BMVD, LMVD were investigated as well. RESULTS: The LMVD was significantly higher in peritumoral tissues than in corresponding normal tissues in gastrointestinal intramucosal tumors (20.87 versus 14.56, P = 0.003). However, there was no significant difference in the BMVD between peritumoral tissues and corresponding normal tissues (P = 0.166). The BMVD in peritumoral tissues was higher in patients with lymph node metastases than in patients without lymph nodes metastases (P = 0.047). Our results did not show significant association between VEGF, VEGF-C and BMVD, LMVD. CONCLUSIONS: Our results suggested that the increase of lymphangiogenesis seems superior to the increase of angiogenesis in gastrointestinal intramucosal tumors.

Reversal of drug resistance in breast carcinoma cells by anti-mdr1 ribozyme regulated by the tumor-specific MUC-1 promoter.

Multidrug resistance (MDR) is a serious obstacle for cancer chemotherapy. The aim of this study was to reverse MDR of breast carcinoma cells specifically by degrading mdr1 mRNA with anti-mdr1 ribozyme. Our strategy was to limit the expression of ribozyme to only breast-derived cells, but not other type of cells. The results showed the recombinant ribozyme pEGFP-RZmuc was expressed in two kinds of breast carcinoma cells, but not in non-breast-derived cancer cells. Evaluation of chemosensitivity showed that a 15-fold reduction in drug resistance for Adriamycin and a 32-fold reduction in drug resistance for Vinblastine were observed in the transfected cells. Our results demonstrate the efficacy and selectivity of pEGFP-RZmuc to reverse MDR in drug resistant breast carcinoma cells in vitro.

Reversal MDR in breast carcinoma cells by transfection of ribozyme designed according the secondary structure of mdr1 mRNA.

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The present study aims to investigate whether the ribozyme could reverse MDR in breast carcinoma cells. In this study, two GUC sites (GUC106 and GUC135) on the surface of mdr1 mRNA were selected according to the secondary structure of the 5'-region of mdrl mRNA. The ribozyme gene RZ106 and RZ135 complementary to two sides bases of the target GUC were synthesized and cloned into the plasmid pEGFP -C1 which has EGFP (Enhanced Green Fluorescence Protein) as report gene and Kan/Neo as selection gene. After transfection with the recombinant plasmid and selected by G418, the stable cell clones were produced and used for detection. The alteration of mdr1 mRNA and P-gp in the treated cells was detected by RT-PCR, flow cytometry and Rh123 retention. The reversal efficiency of the drug resistance for adriamycin was determined by MTT assay. The results showed that after transfection with RZ106 and RZ135, the amount of the mdr1 mRNA and P-gp decreased significantly and the efflux function of P-gp was inhibited accordingly. Nine-fold and 16-fold reduction of resistance for adriamycin was observed in the two groups of treated cells. These results suggested that both ribozymes can reverse the MDR phenotype by inhibiting the expression of mdr1 mRNA and P-gp, and the RZ135 showed the better cleavage efficiency. The ribozyme strategy designed according the secondary structure of the target RNA could be a useful therapy for reversal of MDR.

[Reversion the multidrug resistance of human breast carcinoma cells by RNA interference targeting HIF-1 alpha gene].

OBJECTIVE: To reverse the multidrug resistant (MDR) phenotype of human breast carcinoma cells by small hairpin RNA (shRNA) technique targeting hypoxia-inducible factor (HIF)-1alpha gene. METHODS: Small hairpin RNA (shRNA) eukaryotic expression vector targeting HIF-1alpha gene, named pSilencer-HIF, was constructed and transfected into MCF-7/ADR human breast cancer cells by liposome technique. Tumor cell livability (TCL) and Rhodamine 123 efflux assay were used to monitor the biological changes of the transfected cells. The mRNA and protein expression of HIF-1alpha and mdr-1 were investigated by RT-PCR and Western blot. RESULTS: The successful construction of pSilencer-HIF plasmid was confirmed by DNA sequencing. HIF-1alpha mRNA and protein levels were significantly decreased in MCF-7/ADR cells after the transfection and there was a direct correlation between HIF-1alpha and mdr-1 expression. By comparing the cells transfected with control vector and the MCF-7/ADR cells transfected with pSilencer-HIF, a reduced TCL from 76% to 43%, and an increased Rhodamine 123 fluorescence intensity from 22.0% to 86.6% were observed. CONCLUSIONS: pSilencer-HIF-1alpha has been successfully constructed. The inhibition of HIF-1alpha expression through shRNA technique can significantly reverse the multidrug resistance phenotype of MCF-7/ADR cells.

Lymphatic vessel density as a prognostic indicator for patients with stage I cervical carcinoma.

Lymphatic vessel density (LVD) was recently considered important for spread of several malignant tumors. However, there are no reports describing the situation in cervical carcinoma. The purpose of this study was to investigate whether LVD could serve as a risk factor for nodal metastasis and recurrence of cervical carcinoma in 147 cases of stage I patients. Other questions were if depth of invasion, proliferation rate, and tumor size could be used as predictive markers for Chinese patients with cervical carcinoma. The lymphatics were determined by immunohistochemistry with the antibody to LYVE-1, a specific lymphatic endothelium marker, and average LVD was calculated. Double immunohistochemistry and double immunofluorescence staining for LYVE-1/CD34 were used to distinguish between lymphatic and blood vessels. The results showed that average LVD in cervical carcinoma was statistically associated with inflammatory cell infiltration of carcinoma tissues, but not associated with other pathological parameters. Average LVD of the cases with nodal metastasis or recurrence was significantly higher than the cases without metastasis and recurrence in both stage IA and stage IB cervical carcinomas. The correlation between both depth of invasion and tumor size with nodal metastasis and recurrence of cervical carcinoma was also statistically significant. Ki-67 labeling index was found to be correlated with the recurrence of disease, but not to be correlated with nodal metastasis. We concluded that for the patients with stage I cervical carcinoma, increased LVD could serve as a high-risk factor for nodal metastasis and recurrence. Depth of invasion and tumor size could also be useful indicators.

[The relationship of abnormal expression of cell glucoprotein with recurrence of pleomorphic adenoma in salivary gland].

OBJECTIVE: To study the relationship of expression of mucin 1 and E-cadherin with recurrence of pleomorphic adenoma in salivary gland, and to investigate the signal to predict the recurrence potential of the tumor. METHODS: ; The capsule of tumor was observed by microscope. The expression of mucin 1 and E-cadherin in 33 cases of primary adenoma, 12 cases of recurrent pleomorphic adenomas and 7 cases of malignant pleomorphic adenomas were detected by immunohistochemistry. RESULTS: There was no significant difference about the status of capsule and the positive rate of mucin 1 expression between primary and recurrent pleomorphic adenoma (P > 0.05). The abnormal distribution of mucin 1 expression was observed in recurrent pleomorphic adenoma (6/8), which was characterized by the positive staining of the whole cytomembrane. On the other hand, positive staining of the primary pleomorphic adenoma was observed on the top of the membrane (19/21). The difference was statistically significant (P < 0.05). The staining pattern in malignant pleomorphic adenoma was similar with the recurrent ones except higher ratio of positive expression. No significant different was observed among the three kind of tumors on the expression rate of E-cadherin (P > 0.05). CONCLUSION: The status of capsule didn't have much actual usage in predicting the recurrence of pleomorphic adenoma. There was no significant relationship between the expression of E-cd and the recurrence of the tumor. The abnormal distribution of mucin 1 expression contributes to the invasiveness of the tumor and can be used as the predictive signal for recurrence of pleomorphic adenoma.

[Construction of glucosylceramide synthase-specific siRNA expression vector and its efficiency in reversal of drug resistance in breast carcinoma cells].

OBJECTIVE: To construct a glucosylceramide synthase (GCS)-specific small interfering RNA (siRNA) expression vector and to investigate the inhibitory effect of this siRNA on GCS expression and drug resistance in breast carcinoma cells. METHODS: Two GCS gene-specific siRNAs were designed and cloned into the expression vector pSUPER to generate the plasmids pSUPER-GCS1 and pSUPER-GCS2. Human adriamycin (ADM)-resistant breast carcinoma cells of the line MCF-7/ADR and human adriamycin-sensitive breast carcinoma cells of the line MCF-7 were cultured and transfected with pSUPER-GCS1, pSUPER-GCS2, and blank vector pSUPER as controls. The expression of GCS mRNA was assayed by RT-PCR and the expression of GCS protein was observed by flow cytometry. The 50% inhibition concentration of ADM on MCF-7/ADR cells was evaluated by MTT method. Flow cytometry was performed to determine the ratio of apoptosis. RESULTS: Double enzyme digestion analysis and DNA sequencing confirmed that pSUPER-GCS1 and pSUPER-GCS2 were successfully constructed. The GCS protein positive rate of the MCF-7/ADR cells 48 hours after transfection with pSUPER-GCS1 and pSUPER-GCS2 were 8.3% +/- 1.0% and 9.2% +/- 0.8% respectively, significantly lower than that before transfection (68.3% +/- 0.6%), with a inhibition rate of 89.4% and 88.5% respectively (both P < 0.01). Forty-eight hours after transfection with pSUPER-GCS1 and pSUPER-GCS2, the relative reversal rates of sensitivity to ADM of the MCF-7/ADR cells were 93.7% and 91.6%. Flow cytometry showed that the apoptotic rate of the MCF-7/ADR cells was 0.80 +/- 0.06 before transfection, 15.38 +/- 1.16 after transfection with pSUPER-GCS1 and 13.92 +/- 1.73 after transfection with pSUPER-GCS2 (both P < 0.05), and was 0.87 +/- 0.12 in the cells transfected with blank vector (P > 0.05). CONCLUSION: A GCS-specific small interfering RNA expression vector has been constructed successfully that suppresses the GCS expression and reverses the multidrug resistance in breast carcinoma cells by increasing the ratio of apoptosis in drug-resistant cells.

[Expression of mucin 1 and tumor invasiveness in breast carcinoma].

OBJECTIVE: To study the relationship of abnormal expression of mucin 1 with the invasiveness of breast carcinoma cells. METHODS: Immunohistochemistry was used to detect the protein expression of mucin 1 in 5 specimens of juxta-cancerous normal tissues, 20 specimens of benign breast tumors, 35 specimens of early breast carcinoma, 22 specimens of infiltrating cancerous tissues, and 20 specimens of lymph node foci with metastatic breast carcinoma. Human breast cancer cells of the line MCF-7 were cultured and transfected with antisense oligodeoxynucleotide (ASODN) of mucin 1. The mucin 1 mRNA expression in the cells was detected by RT-PCR and the protein expression of mucin 1 in the cells was detected by flow cytometry. The cell invasiveness was detected by Matrigel invasion assays. RESULTS: Top membrane positive expression of mucin 1 was observed in the normal breast tissues and breast benign tumors and whole membrane positive expression of mucin 1 was observed in the 30 of the 35 specimens of early breast carcinoma, 18 of the 22 specimens of breast infiltrating carcinoma, and 17 of the 20 specimens of lymph node metastatic tissues. The mRNA and protein expressions of mucin 1 in the breast carcinoma cells treated with ASODN of mucin 1 were significantly decreased (both P < 0.05). The number of invasive cells decreased significantly in the cell treated with ASODN of mucin 1 in comparison with those treated with sense nucleotide (P < 0.05). CONCLUSION: The abnormal distribution of mucin 1 contributes to the invasiveness of carcinoma cells and may not make difference in the lymphogenous metastasis of the carcinoma. The invasiveness of breast carcinoma cells can be inhibited by the ASODN complementary to the start site of mucin1 mRNA.