Xiyin Wei

Reduced mitochondrial DNA copy number is correlated with tumor progression and prognosis in Chinese breast cancer patients.

Somatic mutations and large‐scale depletion in mitochondrial DNA (mtDNA) have been extensively detected in various human cancers. However, it still remains unclear whether the alterations in mtDNA content are related to the clinicopathological parameters and patient prognosis in breast cancer. In the present study, we analyzed the copy number of mtDNA in 59 cases of invasive breast tumors and paired nontumorous tissues using quantitative real‐time PCR. Our data showed that the level of mtDNA was significantly decreased in tumor tissues as compared to the adjacent nontumorous counterparts (P = 0.001). The reduced copy number in mtDNA was associated with an older onset age (≥50 years old, P = 0.035) as well as a higher histological grade (P = 0.012). Survival analysis measured by the Kaplan‐Meier curves and the log‐rank test indicated that patients with reduced mtDNA content had significantly poorer disease‐free survival (DFS, P = 0.0079) and overall survival (OS, P = 0.011) rate. In addition, tumors harboring mutations in displacement (D)‐loop region, particularly at the polycytidine stretch (T/N ratio = 64.3 ± 8.2%) or close to the replication origins of the heavy‐strand (T/N ratio = 68.7 ± 5.5%), had a significantly lower copy number of mtDNA than the ones without D‐loop alterations. Together, our results suggested that reduced copy number of mtDNA may be involved in breast neoplastic transformation or progression and mtDNA content might be potentially used as a tool to predict prognosis. Somatic mutation in the D‐loop region probably is one of key contributing factors leading to decreased mtDNA level in breast tumors. iubmb Life, 59: 450‐457, 2007

Anxa2 plays a critical role in enhanced invasiveness of the multidrug resistant human breast cancer cells

Multidrug resistance (MDR) is the major cause of failure in cancer chemotherapy. Recent reports even suggest that MDR is associated with elevated invasion and metastasis of tumor cells. In the current study, we used a proteomic approach to identify genes that play an important role in MDR induced cell migration. 2D-PAGE and MALDI-TOF/MS-based proteomics approach were used to separate and identify differentially expressed proteins between MCF-7 and MCF-7/ADR, a p-glycoprotein-overexpressing adriamycin-resistance breast cancer cell line. Annexin a2 (Anxa2) was identified as highly expressed in MCF-7/ADR cells, but not in MCF-7 cells. Small interference RNA-mediated gene suppression demonstrated that Anxa2 was required for enhanced cell proliferation and invasion of the MCF-7/ADR cells. Down-regulation of Anxa2 alone was not sufficient to revert the cell sensitivity to adriamycin, suggesting that Anxa2 was not required for MDR phenotype. Taken together, our results showed that expression of Anxa2 is enhanced when cancer cells, MCF-7, acquired drug resistance and it plays an essential role in MDR-induced tumor invasion.

Mitochondrial DNA depletion promotes impaired oxidative status and adaptive resistance to apoptosis in T47D breast cancer cells

The mutation and reduction of mitochondrial DNA (mtDNA) have been extensively detected in human cancers. The effects of mitochondrial dysfunction are particularly important in breast cancer, because estrogen-mediated metabolites generate large quantities of local reactive oxygen species in the breast, which directly bind to mtDNA and facilitate neoplastic transformation. To further elucidate the molecular roles of mtDNA in breast cancer, we determined the oxidative status of a breast tumor cell line lacking mtDNA (T47D ρ0) and analyzed its susceptibility after exposure to various anticancer drugs as well as different proapoptotic signals. Our data showed that T47D ρ0 cells generated significantly increased levels of lactate with concomitantly reduced oxygen consumption and ATP production compared with the wild-type (WT). The amount of reactive oxygen species generation in ρ0 cells was lowered to approximately 12% that of parental cells, as evidenced by the oxidation of redox-sensitive probes. Although mtDNA depletion did not affect the expression of superoxide dismutase or its activity, the activities of antioxidant enzymes, catalase and glutathione peroxidase, were significantly higher in ρ0 cells compared with WT cells. In addition, mtDNA-depleted cells displayed a decreased sensitivity and accumulation of chemotherapeutic drugs (doxorubicin, vincristine, and paclitaxel), potentially because of the upregulated expression of multidrug resistance 1 (MDR1) gene and its product P-glycoprotein. When compared with their WT counterparts, T47D ρ0 cells were also more resistant to apoptosis induced by varying concentrations of staurosporine and anti-Fas antibody. Altogether, our results indicate the importance of intact mtDNA for maintaining the proper intracellular oxidative status. These data provide evidence for a possible role of mtDNA content reduction in acquiring an apoptosis-resistant phenotype during breast tumor progression and might contribute to effective therapeutic strategies for this common malignancy.

Circulating endothelial cells and tumor blood volume as predictors in lung cancer

The current criteria for evaluating antiangiogenic efficacy is insufficient as tumor shrinkage occurs after blood perfusion decreases. Tumor blood volume (BV) in computed tomography perfusion imaging and circulating endothelial cells (CEC) might predict the status of angiogenesis. The present study aimed to validate their representation as feasible predictors in non‐small‐cell lung carcinoma (NSCLC). A total of 74 patients was categorized randomly into two arms undergoing regimens of vinorelbine and cisplatin (Navelbine and platinum [NP]) with rh‐endostatin or single NP. The response rate, perfusion imaging indexes and activated CEC (aCEC) during treatment were recorded. Progression‐free survival (PFS) was determined through follow up. Correlations among the above indicators, response and PFS were analyzed: aCEC increased significantly in cases of progressive disease after single NP chemotherapy (P = 0.024). Tumor BV decreased significantly in cases with a clinical benefit in the combined arm (P = 0.026), whereas inverse correlations existed between ∆aCEC (post‐therapeutic value minus the pre‐therapeutic value) and PFS (P = 0.005) and between ∆BV and PFS (P = 0.044); a positive correlation existed between ∆aCEC and ∆BV. Therefore, both aCEC and tumor BV can serve as predictors, and detection of both indicators can help evaluate the chemo‐antiangiogenic efficacy in NSCLC more accurately.

C-FLIP(L) contributes to TRAIL resistance in HER2-positive breast cancer

Breast cancers with HER2 amplification have a poorer prognosis than the luminal phenotypes. TRAIL activates apoptosis upon binding its receptors in some but not all breast cancer cell lines. Herein, we investigated the expression pattern of c-FLIP(L) in a cohort of 251 invasive breast cancer tissues and explored its potential role in TRAIL resistance. C-FLIP(L) was relatively high-expressed in HER2-positive breast cancer in comparison with other molecular subtypes, co-expressed with TRAIL death receptors, and inversely correlated with the apoptosis index. Downregulation of c-FLIP(L) sensitized SKBR3 cells to TRAIL-induced apoptosis in a concentration- and time-dependent manner and enhanced the activities and cleavages of caspase-8 and caspase-3, without altering the surface expression of death receptors. Together, our results indicate that c-FLIP(L) promotes TRAIL resistance and inhibits caspase-3 and caspase-8 activation in HER2-positive breast cancer.

RNAi-mediated silencing of Anxa2 inhibits breast cancer cell proliferation by downregulating cyclin D1 in STAT3-dependent pathway

Although the upregulated expression of Anxa2 has been implicated in carcinogenesis, cancer progression, and poor prognosis of cancer patients, the detailed molecular mechanisms involved in these processes remain unclear. In this study, we investigated the effect of Anxa2 downregulation with small interference RNA on breast cancer proliferation. To explore molecular mechanisms underlying Anxa2-mediated cancer cell proliferation. We analyzed cell cycle distribution and signaling pathways using semi-quantitative real-time PCR and Western blotting. Anxa2 depletion in breast cancer cells significantly inhibited cell proliferation by decelerating cell cycle progression. The retarded G1-to-S phase transition in Anxa2-silenced cells was attributed to the decreased levels of cyclin D1, which is a crucial promoting factor for cell proliferation because it regulates G1-to-S phase transition during cell cycle progression. We provided evidence that Anxa2 regulates epidermal growth factor-induced phosphorylation of STAT3. The reduced expression of phosphorylated STAT3 is the main factor responsible for decreased cyclin D1 levels in Anxa2-silenced breast cancer cells. Our results revealed the direct relationship between Anxa2 and activation of STAT3, a key transcription factor that plays a pivotal role in regulating breast cancer proliferation and survival. This study provides novel insights into the functions of Anxa2 as a critical molecule in cellular signal transduction and significantly improves our understanding of the mechanism through which Anxa2 regulates cell cycle and cancer cell proliferation.

Expression level of beta protein 1 mRNA in Chinese breast cancer patients: A potential molecular marker for poor prognosis

Recent studies revealed high ectopic beta protein 1 (BP1) expression in breast cancer. Remarkably, up to 100% (18/18) of estrogen receptor (ER)‐negative tumors and 89% (25/28) of tumors from African American women were BP1‐positive. However, the role of BP1 in breast cancer development and its clinical significance still has not been well defined. In the present study, we analyzed the quantitative level of BP1 mRNA in breast carcinomas using real‐time polymerase chain reaction and aimed to elucidate its association with tumor characteristics and patient prognosis. Our data showed that BP1 mRNA was expressed at significantly higher levels in tumors with lymph node metastasis, with a high histological grade, and in those that were of ER‐negative status. Furthermore, overexpression of BP1 was significantly associated with poor outcome of patients harboring tumors with a high histological grade and negative ER. Using both in vitro and in vivo systems, we also showed that the transcript level of BP1 was positively correlated to the growth rate of breast tumor cells. Taken together, our results support the notion that BP1 might contribute to breast neoplastic transformation or tumor progression and suggest for the first time that BP1 mRNA level has potential as a prognostic predictor for breast cancer. (Cancer Sci 2008; 99: 173–178)

Critical role for c-FLIPL on Fas resistance in colon carcinoma cell line HT-29

The cellular Fas‐associated death domain‐like interleukin‐1β‐converting enzyme inhibitory protein‐long form (c‐FLIPL) is a key regulator of Fas signaling, although owing a dominant‐negative homologue of caspase‐8, the role of c‐FLIPL remains controversial. In the present study, two pairs of small interfering RNA (siRNA) directed against c‐FLIPL were used to assess the effect of c‐FLIPL on Fas‐mediated apoptosis of colon carcinoma in vitro. HT‐29 cell line was selected for overexpression of c‐FLIPL and Fas with RT—PCR and flow cytometry analyses. After electroporation, the mRNA level of c‐FLIPL was significantly decreased (control siRNA versus c‐FLIPL siRNA, 77.97 ± 5.61% versus 26.22 ± 3.79%) and the maximum interfering efficiency was around 66.49% using semi‐quantitative RT—PCR analysis. Knockdown of c‐FLIPL with the specific siRNA sensitized colon carcinoma cells to Fas‐mediated apoptosis (control siRNA versus c‐FLIPL siRNA, 5.68 ± 2.11% versus 29.50 ± 2.27%) using DNA content analysis and Annexin V—FITC analysis. In conclusion, our study indicated that c‐FLIPL might be a suppressor of Fas‐mediated apoptosis in Fas antigen expressing colon carcinoma and therefore a potential target for novel anticancer therapies.

Evaluation of primary thyroid lymphoma by ultrasonography combined with contrast-enhanced ultrasonography: A pilot study.

OBJECTIVE To evaluate the features of primary thyroid lymphoma (PTL) by ultrasonography (US) combined with contrast-enhanced ultrasonography (CEUS). MATERIALS AND METHODS In this retrospective study, 20 patients (8 male and 12 female) with PTL were evaluated by conventional US and among them, 10 patients underwent CEUS examination. The appearance of US features was classified into three types: Diffusive mass type, multiple nodular type and mixed type. The CEUS patterns included diffusive homogeneous enhancement and diffusive heterogeneous enhancement pattern. Parameters of CEUS time-intensive curve were analyzed in primary tumor and involved lymph nodes compared to ipsilateral common carotid artery. RESULTS Of 20 patients with PTL, 18 presented an enlarging neck mass that grew rapidly with an average duration of 3.2 months, and 17 were associated with Hashimotos thyroiditis. In conventional US, all patients had marked hypoechoic masses. Among them, 12 patients were diffusive mass type, 6 were multiple nodular type and 2 were mixed type. For CEUS patterns, 8 were diffusive homogeneous enhancement and 2 were diffusive heterogeneous enhancement. Necrosis areas were showed in diffuse heterogeneous pattern which were hardly seen in conventional US. In the quantitative analysis of CEUS parameters, the time to peak of time-intensive curve in the primary tumors or involved lymph nodes was longer than that of the ipsilateral common carotid artery (P = 0.004). CONCLUSION PTL mainly demonstrated as a diffusive mass type with marked hypoechogenecity on conventional US and diffusive homogeneous enhancement pattern on CEUS. And the heterogeneous enhancement pattern is also helpful for detecting necrosis areas of PTL.