Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer / 华中科技大学学报（医学）（英德文版）

The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy.

MiR-21 mediates the radiation resistance of glioblastoma cells by regulating PDCD4 and hMSH2 / 华中科技大学学报（医学）（英德文版）

The purpose of this study was to investigate the molecular mechanism by which miR-21 and its target genes mediate radiation resistance of glioblastoma cells. Real-time PCR was employed to detect miR-21 expression in normal brain tissues, glioblastoma tissues and glioblastoma cell lines (A172, T98G and U87MG). T98G cells were transfected with anti-miR-21 oligonucleotides, or plasmids containing PDCD4 or hMSH2 (PDCD4-pcDNA3 and hMSH2-pcDNA3). The survival curve was obtained to investigate the sensitivity of T98G cells to radiation. Cell apoptosis was measured by using the Caspase-3/7 kit and cell cycle by flow cytometry. Western blotting was performed to detect the expression of hMSH2 and PDCD4 in miR-21-inhibiting T98G cells. The results showed that miR-21 expression in glioblastoma cells and tissues was conversely associated with the radiation sensitivity. Over-expression of miR-21 resulted in radiation resistance, while knockdown of miR-21 led to higher sensitivity of glioblastma cells to radiation. After miR-21 knockdown, the apoptosis of T98G cells was significantly increased and the G(2) phase arrest was more significant. In addition, miR-21 knockdown increased the expression of endogenous PDCD4 and hMSH2, which contributed to the apoptosis and G(2) arrest of T98G cells. The findings suggested that miR-21 may mediate the resistance of glioblastoma cells against radiation via its target genes PDCD4 and hMSH2. MiR-21 and its target genes may be used as potential molecular targets for clinical radiotherapy sensitization in the future.

MiR-21 mediates the radiation resistance of glioblastoma cells by regulating PDCD4 and hMSH2 / 华中科技大学学报（医学）（英德文版）

The purpose of this study was to investigate the molecular mechanism by which miR-21 and its target genes mediate radiation resistance of glioblastoma cells. Real-time PCR was employed to detect miR-21 expression in normal brain tissues, glioblastoma tissues and glioblastoma cell lines (A172, T98G and U87MG). T98G cells were transfected with anti-miR-21 oligonucleotides, or plasmids containing PDCD4 or hMSH2 (PDCD4-pcDNA3 and hMSH2-pcDNA3). The survival curve was obtained to investigate the sensitivity of T98G cells to radiation. Cell apoptosis was measured by using the Caspase-3/7 kit and cell cycle by flow cytometry. Western blotting was performed to detect the expression of hMSH2 and PDCD4 in miR-21-inhibiting T98G cells. The results showed that miR-21 expression in glioblastoma cells and tissues was conversely associated with the radiation sensitivity. Over-expression of miR-21 resulted in radiation resistance, while knockdown of miR-21 led to higher sensitivity of glioblastma cells to radiation. After miR-21 knockdown, the apoptosis of T98G cells was significantly increased and the G2 phase arrest was more significant. In addition, miR-21 knockdown increased the expression of endogenous PDCD4 and hMSH2, which contributed to the apoptosis and G2 arrest of T98G cells. The findings suggested that miR-21 may mediate the resistance of glioblastoma cells against radiation via its target genes PDCD4 and hMSH2. MiR-21 and its target genes may be used as potential molecular targets for clinical radiotherapy sensitization in the future.

Effect of silencing Survivin gene expression by RNA interference on the radiosensitivity of cervical carcinoma cell line SiHa / 肿瘤

Objective: To explore the influence of silencing Survivin expression by RNA interference on the radiosensitivity of cervical carcinoma SiHa cells. Methods: The recombinant eukaryotic expression plasmid pSurvivin-shRNA was constructed and transfected into SiHa cells. The mRNA and protein expressions of Survivin were determined by RT-PCR and Western blotting, respectively. The activity of caspase-3 was measured by kinase activity determination method. The apoptotic rate was determined by flow cytometry. The changes of radiosensitivity were explored by colony formation test. Results: Compared with SiHa cells transfected with pNeg-shRNA (SiHa/ pNeg-shRNA cells) and untransfected SiHa cells, the expressions of survivin in the cells transfected with pSurvivin-shRNA (Si-Ha/ pSurvivin-shRNA cells) were inhibited significantly at both mRNA and protein levels. The activity of caspase-3 in SiHa/pSurvivin-shRNA cells was significantly enhanced, and the D405 was 1.34 ± 0.05 (P < 0.05). The apoptotic rate of SiHa/pSurvivin-shRNA cells was (5.13 ± 0.81)% and (11.27 ± 1.89)% after 24 hand 48 h X-ray irradiation at 6 MV and 8 Gy, respectively, which was significantly higher than SiHa/pNeg-shRNA cells and untransfected cells (P < 0.05). The colony formation ability of SiHa/pSurvivin-shRNA cells was markedly decreased after irradiation (P < 0.05), which indicated that the sensitivity of SiHa/pSurvivin-shRNA cells was enhanced. Conclusion: The inhibition of Survivin by RNAi could enhance the radiosensitivity of SiHa cells by increasing caspase-3 activity and inducing apoptosis.

Expression of thymidine phosphorylase in cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the thymidine phosphorylase (TP) expression in different types of cancer and its correlation with tumor microvessel density (MVD).</p><p><b>METHODS</b>The expression of TP and MVD was detected by immunohistochemistry method. In a series of 251 cancer patients there were 48 patients with gastric cancer, 53 with colorectal cancer, 47 with breast cancer, 56 with cervical cancer, 47 with lung cancer. Normal gastric (n = 25), colorectal (n = 25), cervical (n = 17) and lung (n = 25) tissues around the cancer were also examined.</p><p><b>RESULTS</b>The TP expression rate was 64.6% in gastric cancer, 67.9% in colorectal cancer, 80.9% in breast cancer, 82.1% in cervical cancer, and 63.8% in lung cancer, which was significantly higher than that in normal tissues (P = 0.0000). TP expression was positively correlated with MVD in gastric, colorectal, breast, and cervical cancers. The correlation was not statistically significant in lung cancer.</p><p><b>CONCLUSION</b>This study indicates that TP overexpression in cancer may be associated with tumor angiogenesis.</p>

First-line Xeloda (Capecitabine) treatment for advanced and recurrent colorectal cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of capecitabine as first-line therapy in patients with advanced and recurrent colorectal cancer.</p><p><b>METHODS</b>From December 2000 to November 2001, sixty patients with advanced and recurrent colorectal cancer received first-line capecitabine treatment given at a dose of 1250 mg/m(2) twice daily, on days 1 - 14 every 21 days. At least 2 cycles were administered.</p><p><b>RESULTS</b>The overall response rate was 23.3% with 14 PR, 24 SD (40.0%) and 15 PD. The median survival time was 14.7 months. The survival rate was 63.9% at 12-months and 33.4% at 24-months. Grade III-IV adverse effects were diarrhea in 4 patients (6.6%), anemia in 2 (3.3%) and hand-foot syndrome (HFS) in 1 (1.7%); Grade I-II adverse effects were hyperpigmentation in 20 (33.3%), HFS in 18 (30.0%) and diarrhea in 10 (16.7%).</p><p><b>CONCLUSION</b>Capecitabine is an efficacious and better-tolerated alternative treatment for the patients with advanced and recurrent colorectal cancer.</p>

Result of phase II clinical trial of herceptin in advanced Chinese breast cancer patients / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To observe the clinical efficacy and adverse effects of herceptin for advanced Chinese breast cancer patients.</p><p><b>METHODS</b>Thirty-one pathologically proved advanced breast cancer women were treated by herceptin. In the first week, a loading dose 4 mg/kg was administered by intravenous infusion and from the second week, a routine dose of 2 mg/kg was given every week for at least 3 months.</p><p><b>RESULTS</b>There were 2 CR, 6 PR, 7 SD, and 16 PD among 31 patients after treatment by herceptin, the response rate being 25.8%. In factors influencing the prognosis, age and general condition were factors favoring the results, and pathological type, site of metastasis, grade of her-2 over expression and prior treatment were irrelevant to the results. The adverse effects were mild but different from those of the common anticancer drugs.</p><p><b>CONCLUSION</b>Herceptin is effective and well tolerated by the Chinese breast cancer patients.</p>