TRPC6 Knockout Alleviates Renal Fibrosis through PI3K/AKT/GSK3B Pathway.

OBJECTIVE: Renal fibrosis is the ultimate pathway of various forms of acute and chronic kidney damage. Notably, the knockout of transient receptor potential channel 6 (TRPC6) has shown promise in alleviating renal fibrosis. However, the regulatory impact of TRPC6 on renal fibrosis remains unclear. METHODS: In vivo, TRPC6 knockout (TRPC6-/-) mice and age-matched 129 SvEv (WT) mice underwent unilateral renal ischemia-reperfusion (uIR) injury surgery on the left renal pedicle or sham operation. Kidneys and serum were collected on days 7, 14, 21, and 28 after euthanasia. In vitro, primary tubular epithelial cells (PTECs) were isolated from TRPC6-/- and WT mice, followed by treatment with transforming growth factor ß1 (TGFß1) for 72 h. The anti-fibrotic effect of TRPC6-/- and the underlying mechanisms were assessed through hematoxylin-eosin staining, Masson staining, immunostaining, qRT-PCR, and Western blotting. RESULTS: Increased TRPC6 expression was observed in uIR mice and PTECs treated with TGFß1. TRPC6-/- alleviated renal fibrosis by reducing the expression of fibrotic markers (Col-1, α-SMA, and vimentin), as well as decreasing the apoptosis and inflammation of PTECs during fibrotic progression both in vivo and in vitro. Additionally, we found that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase 3 beta (GSK3ß) signaling pathway, a pivotal player in renal fibrosis, was down-regulated following TRPC6 deletion. CONCLUSION: These results suggest that the ablation of TRPC6 may mitigate renal fibrosis by inhibiting the apoptosis and inflammation of PTECs through down-regulation of the PI3K/AKT/GSK3ß pathway. Targeting TRPC6 could be a novel therapeutic strategy for preventing chronic kidney disease.

[The CD133 polyclonal antibody generation and cancer stem cells identification].

OBJECTIVE: To generate the cancer stem cells (CSCs) specific protein CD133 polyclonal antibody for the study of the biological characteristics of CSCs in tumor tissues and CSCs screening for the mouse model. METHODS: The extracellular peptide of the human CD133 was injected into rabbits to generate polyclonal antibody which was used for glioblastoma(GBM) Western blot and immunohistochemistry. RESULTS: The CD133 antiserum we made could detect both overexpressed myc-CD133 and endogenous CD133 efficiently by Western blot. Immunohistochemistry indicated that the CD133 polyclonal antibody can label CSCs in GBM sections. CONCLUSION: High efficient and specific CD133 antibody was generated successfully and could be used to label CSCs in tumor sections and screen CSCs for the mouse model.

[Expression of DOG-1 in gastrointestinal stromal tumor and its diagnostic application].

OBJECTIVE: To investigate the expression of DOG-1 in gastrointestinal stromal tumors (GIST) and its diagnostic application. METHODS: Immunohistochemical EnVision technique was used to assess the expression of DOG-1 in 84 cases of GIST in comparison with CD117 and CD34. RESULTS: All 84 cases of GIST consisted of variable proportions of spindle and epithelioid tumor cells or just one type of the tumor cell. The expression rates of DOG-1, CD117 and CD34 were 91.3% (42/46), 95.7% (44/46) and 82.6% (38/46), in the group of very low and low risk GIST, and were 100% (38/38), 100% (38/38) and 78.9% (30/38), respectively, in the group of moderate and high risk GIST. True leiomyomas, schwannomas, fibromatosis and normal gastrointestinal mucoca did not express these markers. Moreover, the sensitivity and specificity of DOG-1 in the detection of GIST were similar to those of CD117, without statistical difference (P > 0.05) between the two markers. However, the sensitivity and specificity of DOG-1 detection of moderate and high risk GIST were significantly higher than those of CD34 (P < 0.01). CONCLUSIONS: DOG-1 is a novel marker of gastrointestinal stromal tumors. It has the sensitivity and specificity higher than CD34, especially in the detection of moderate and high risk GIST. Combined DOG-1 and CD117 immunohistochemistry will likely improve the diagnostic accuracy of GIST.

Impact of KRAS mutation and PTEN expression on cetuximab-treated colorectal cancer.

AIM: To investigate the prognostic value of KRAS mutation, and phosphatase and tensin (PTEN) expression in Chinese metastatic colorectal cancer metastatic colorectal cancer (mCRC) patients treated with cetuximab. METHODS: Ninety Chinese mCRC patients treated with cetuximab were evaluated for KRAS mutation and PTEN protein expression by DNA sequencing of codons 12 and 13 and immunohistochemistry, respectively. We then selected 61 patients treated with cetuximab, either in combination with chemotherapy, or alone as a second-line or third-line regimen to assess whether KRAS mutation or PTEN protein expression is associated with the response and the survival time of mCRC patients treated with cetuximab. RESULTS: KRAS mutation was found in 30 (33.3%) tumor samples from the 90 patients, and positive PTEN expression was detected in 58 (64.4%) of the 90 patients. Among the 61 patients who were treated with cetuximab as a second-line or third-line regimen, the resistance to cetuximab was found in 22 patients with KRAS mutation and in 39 patients without KRAS mutation, with a response rate of 4.5% and 46.1% respectively (P = 0.001), a shorter median progression-free survival (PFS) time of 14 ± 1.3 wk and 32 ± 2.5 wk respectively (P < 0.001), a median overall survival (OS) time of 11 ± 1.2 mo and 19 ± 1.8 mo respectively (P < 0.001), as well as in 24 patients with negative PTEN expression and in 37 patients with positive PTEN expression respectively (P < 0.001), with a responsive rate of 4.2% and 48.6% respectively, a shorter median PFS survival time of 17 ± 2.0 wk and 28 ± 1.9 wk respectively (P = 0.07), and a median OS time of 11 ± 1.3 mo and 18 ± 1.9 mo respectively (P = 0.004). Combined KRAS mutation and PTEN expression analysis showed that the PFS and OS time of patients with two favorable prognostic factors were longer than those of patients with one favorable prognostic factor or no favorable prognostic factor (P < 0.001). CONCLUSION: KRAS mutation and PTEN protein expression are significantly correlated with the response rate and survival time of Chinese mCRC patients treated with cetuximab.

Cetuximab enhances the effect of oxaliplatin on hypoxic gastric cancer cell lines.

Hypoxia is recognized as an important factor contributing to cancer development and drug resistance. Cetuximab, a chimeric monoclonal antibody to EGFR, is known to inhibit HIF-1 alpha expression levels and to enhance the cytotoxicity of chemotherapeutic agents. We demonstrated that hypoxia induced drug resistance in gastric cancer cells. Cetuximab enhanced oxaliplatin-induced cytotoxicity and apoptosis in normoxia and caused a reversal of drug resistance in hypoxia. Normoxic and hypoxic gastric cancer cells were treated with cetuximab, oxaliplatin or the combination and assessed for cell growth, proliferation, and apoptosis. Combination treatment resulted in a marked inhibition of HIF-1 alpha expression levels in hypoxic cells and caused a significant reduction in the expression of activated phosphorylated AKT, ERK1/2, p-BAD and VEGF in both normoxia and hypoxia with greater levels of inhibition in hypoxia. In summary, cetuximab inhibits HIF-1 alpha expression via the MAPK/ERK and PI3K/AKT signaling pathways and functions to overcome drug resistance induced by hypoxia. Cetuximab-oxaliplatin combination therapy may therefore emerge as an attractive treatment strategy for advanced gastric cancer.

Phase II trial of XELOX as first-line treatment for patients with advanced gastric cancer.

BACKGROUND: The prognosis of patients with advanced gastric cancer (AGC) remains poor, and no single chemotherapy regimen is recognized as a global standard. A phase II trial was conducted to determine the efficacy and tolerability of capecitabine and oxaliplatin (XELOX) given every 3 weeks in combination in patients with AGC. METHODS: Patients with previously untreated AGC received intravenous oxaliplatin 130 mg/m(2) over 2 h on day 1 plus oral capecitabine 1,000 mg/m(2) twice daily on days 1-14, every 3 weeks. Treatment was continued for 8 cycles or until disease progression or intolerable toxicity. RESULTS: Fifty patients were enrolled. In total, 210 cycles of XELOX were delivered. The OVERALL response rate was 42% (95% CI 28.6-56.7), with 2 complete and 19 partial responses. At 15.2 months of median follow-up, median time to progression and overall survival were 5.8 (95% CI 3.4-8.2) and 11.1 (95% CI 5.6-16.5) months, respectively. The most common hematological adverse event was neutropenia (56% of patients); grade 3-4 neutropenia was observed in 6 patients, with neutropenic fever in only 2 patients. The most common non-hematological toxicities were vomiting (34%), hand-foot syndrome (26%), diarrhea (24%) and neurosensory toxicity (22%). There were no treatment-related deaths. CONCLUSIONS: XELOX is active for the first-line treatment of AGC with a manageable tolerability profile.

[Prognosis of 84 intrahepatic cholangiocarcinoma patients].

BACKGROUND AND OBJECTIVE: The incidence of intrahepatic cholangiocarcinoma (ICC) is low. Current treatment for ICC is unsatisfied. This study was to investigate the prognosis of patients with resectable or unresectable ICC. METHODS: Clinical data of 84 patients with pathologically confirmed ICC treated at Cancer Center, Sun Yat-sen University from January 1997 to December 2007 were reviewed. Survival and prognosis were analyzed by Kaplan-Meier method and Cox regression model. RESULTS: Of the 84 patients, 56 (66.7%) had resectable ICC, and 28 (33.3%) had unresectable ICC. Among the 56 patients with resectable ICC, 27 (48.2%) underwent radical resection, and 29 (51.8%) underwent palliative resection. The 2-year overall survival rate was 3.1% in unresectable ICC group; it was significantly higher in radical resection group than in palliative resection group (P<0.01). For the patients with resectable ICC, univariate analysis revealed that operation pattern, histological type, tumor size and number, lymph node metastasis, intrahepatic metastasis, portal vein thrombus, postoperative serum level of albumin, preoperative serum levels of CEA, CA199, TBIL, ALT and AST were related to the prognosis; multivariate analysis found that operation pattern, histological type, tumor number, preoperative serum levels of CEA, CA199 and TBIL were independent prognostic factors. For the patients with unresectable ICC, univariate analysis found that histological type and preoperative serum level of CA199 were related to the prognosis; whereas multivariate analysis found that histological type was the only independent prognostic factor. Chemotherapy showed no survival benefit in both resectable and unresectable ICC groups (P=0.30, P=0.78). CONCLUSIONS: Radical resection is the main effective treatment for ICC patients to achieve long-term survival. Preoperative serum levels of CEA, CA199 and TBIL are significant prognostic factor for patients with resectable ICC.

[Preparation and identification of the Syntenin1 polyclonal antibody, a cancer metastasis related gene].

OBJECTIVE: To express the GST fusion protein, GST-Syntenin1 in E. coli, and to prepare the polyclonal antibody of Syntenin1. METHODS: CDS fragment of Syntenin1 was obtained by RT-PCR from normal mouse brain and subcloned into pGEX-4T-2 to generate pGEX-4T-2-Syntenin1 recombinant. The confirmed recombinant was transformed into the BL21 competent cells and induced with IPTG. The recombinant fusion protein was purified with immobilized Glutathione Sepharose and confirmed by SDS-PAGE. The purified fusion protein was mixed with the Freund's adjuvant, and then injected into New Zealand white rabbits by hypodermic injection. The polyclonal antibody titer and specification were identified by Western blot. RESULTS: Syntenin1 polyclonal antibody bind Sytenin1 protein specifically and the antiserum tiger reached to 1 : 20 000. CONCLUSION: The Syntenin1 polyclonal antibody with high titer and high specificity was prepared successfully. This will be very helpful for the further study on Syntenin1 function and molecule mechanism of cancer metastasis.

[Responses of 109 adult soft tissue sarcoma patients to chemotherapy].

BACKGROUND & OBJECTIVE: The response of adult soft tissue sarcoma (STS) to chemotherapy is uncertain. This study was to evaluate the role of chemotherapy in treating adult soft tissue sarcoma. METHODS: Clinical data of 109 adult soft tissue sarcoma patients, treated with chemotherapy at Cancer Center of Sun Yat-sen University from Jan. 2000 to Dec. 2005, were analyzed. RESULTS: Of the 109 patients, 66 received palliative chemotherapy, 40 received adjuvant chemotherapy, and 3 received neoadjuvant chemotherapy. The overall response rate for first line chemotherapy was 22.7%. The median survival was 16.9 months. The 1-and 2-year survival rates were 63.6% and 33.3%. The patients with lung metastasis had a significantly longer median survival than those with liver metastasis did (25.1 months vs. 11.8 months, P<0.05). MAID and CYVADIC were the most commonly used first-line chemotherapy regimens; the response rates were 28.0% and 22.2%, respectively. When anthracycline and/or standard dose ifosfamide failed, the patients could still benefit from high dose ifosfamide (14.0 g/m(2)). The median survival was significantly shorter in the patients who got metastasis within 6 months after diagnosis than in those that got metastasis more than 6 months after diagnosis (11.8 months vs. 42.9 months, P=0.04). Of the 40 patients who received adjuvant chemotherapy, 16 developed progression during follow-up: 10 had relapse and 6 had distant metastasis. CONCLUSIONS: MAID and CYVADIC are two effective chemotherapy regimens for adult soft tissue sarcoma. We recommend to take a high dose ifosfamide when anthracycline and/or standard dose ifosfamide failed. The patients with liver metastasis are more resistant to chemotherapy than those with lung metastasis. Developing metastasis within 6 months after diagnosis is a poor prognostic factor.

Restoring atomic configuration at interfaces by image deconvolution.

The image deconvolution technique in combination with dynamical scattering effect correction developed previously for crystal defect investigation has been modified to meet the needs of interface studies and applied to a [111] twin model of Si. Elliptical windows are utilized as a new means for Fourier filtering and correcting the amplitudes of reflections. Images were simulated with a 200 kV field-emission high-resolution electron microscope. After image restoration, four images simulated with different defocus values were transformed into structure images with atomic columns revealed individually at correct positions. The effectiveness of the technique is discussed.