Comprehensive optimization of urinary exfoliated tumor cells tests in bladder cancer with a promising microfluidic platform.

BACKGROUND: Enrichment of urinary exfoliated tumor cells (UETCs) is a noninvasive way of bladder cancer diagnosis, but the lack of specific capture and identification of tumor cells from the urine remains a limitation that impedes the development of liquid biopsy. METHODS: The CytoBot® 2000, a novel circulating cell isolation and enrichment platform, was used for UETCs isolation after comprehensive optimization. The commercial cell lines of bladder cancer were used in spiking assay for cell recovery test. The flow cytometry and immunofluorescent staining assays were performed for expression validation of capture target and identification markers. The performance of optimized platform was validated by 159 clinical samples and analyzed using receiver operator characteristic curve. RESULTS: The chip that had a pore diameter of 15*20 µm could reduce the background residues while maintaining a higher cell recovery rate. We found that the cell capture ability of chip significantly improved after anti-EpCam antibody encapsulation, but not with T4L6FM1. In identification system optimization, the spiking assay and validation of clinical sample showed that the performance of CK20 and DBC-1 were better that pan-CK in tumor cell identification, in addition, the staining quality is more legible with CK20. CONCLUSION: The optimized capture chip is more specific for UETCs isolation. CK20 and DBC-1 are both sensitive biomarkers of UETCs in bladder cancer diagnosis. The performance of this optimized platform is excellent in clinical test that improves the accuracy of urine cell testing and provides a new alternative for the clinical application of BLCA liquid biopsy assessment.

Ultrasensitive and High Reproducible Detection of Urinary Metabolites Using the Tip-Contact Extraction Method Coupled with Negative LDI-MS.

More and more evidence has proved that urinary metabolites can instantly reflect disease state. Therefore, ultra-sensitive and reproducible detection of urinary metabolites in a high-throughput way is urgently desirable for clinical diagnosis. Matrix-free laser desorption/ionization mass spectrometry (LDI-MS) is a high-throughput platform for metabolites detection, but it is encountered by severe interference from numerous salts in urine samples, because the crystallized urine salt on dried samples could result in poor reproducibility in LDI-MS detection. The present work proposed a tip-contact extraction (TCE) technique to eliminate interference from the urine salt. Vertical silicon nanowire arrays decorated with the fluorinated ethylene propylene film (FEP@VSiNWs) could effectively extract metabolites from the urine sample dropping on its surface. High salt tolerance was observed in the subsequent LDI-MS detection of the metabolites extracted on the tip of FEP@VSiNWs even in the presence of 1 M urea. Stable and reproducible mass spectra for non-target metabolic analysis were obtained in real urine samples with different dilution folds. Urinary metabolites collected from bladder cancer (BC) patients were reliably profiled by the TCE method coupled with negative LDI-MS. Based on this platform, potential metabolic biomarkers that can distinguish BC patients and normal controls were uncovered.

Meta-analysis of the effect of antithrombotic drugs on perioperative bleeding in BPH surgery.

Effects of antithrombotic agents on the bleeding risk after transurethral resection of the prostate (TURP) were assessed in patients with benign prostatic hyperplasia (BPH). Controlled clinical trials on the effects of perioperative anticoagulant therapy on postoperative bleeding in BPH patients published during January 1990 and February 2019 were searched in PubMed, Embase and the Cochrane Library. Two independent reviewers screened the studies according to the inclusion and exclusion criteria, extracted the data, evaluated the quality, and conducted a meta-analysis using the RevMan 5.3 software. A total of 20 studies were included. Analysis of these studies found that compared with interrupted use of antithrombotic agents, continuous use of antithrombotic drugs led to more frequent post-TURP bleeding (OR=4.34, 95% CI=2.29-8.23), and higher transfusion rate (2.96, 1.19-7.36). Compared with patients who never used antithrombotic agents, those who used antithrombotic agents continuously had higher bleeding risk (5.52, 1.64-18.66). Those who continued using antithrombotic agents during laser treatment had higher transfusion rate than those who stopped using them before the operation (5.39, 1.49-19.53), but it had no significant difference in clot retention, blood transfusion rate, intraoperative hemoglobin decrease and postoperative catheter-indwelling time compared with those who never used antithrombotic agents (P>0.05). Those who continued using antithrombotic agents during TURP showed less intraoperative hemoglobin decrease (-0.46, -0.58-0.35) than the patients who underwent low molecular weight heparin substitution. Interruption of antithrombotic agents during TURP can prevent the risk of postoperative bleeding; continuous use of antithrombotic agents is safe and feasible during laser treatment of BPH; whether low molecular weight heparin substitution is necessary during the discontinuation of antithrombotic agents is controversial.

lncRNA UCA1 Functions as a ceRNA to Promote Prostate Cancer Progression via Sponging miR143.

UCA1 (urothelial carcinoma associated 1) is a long non-coding RNA (lncRNA) that was found overexpressed in various human cancers including prostate cancer (PCa). However, the aspect of UCA1-miRNA-mRNA interaction in PCa remains unclear. In this study, we confirmed the role of UCA1 in PCa and found that UCA1 downregulation inhibited cell proliferation of PCa cells. Then we demonstrated that repressed UCA1 promoted the microRNA-143 (miR-143) expression and miR-143 could bind to the predicted binding site of UCA1. We then proved the anti-tumor role of miR-143 in PCa. Furthermore, we found that miR-143 displays its role in PCa via modulating the MYO6 expression. In summary, our study demonstrated that UCA1 exerts oncogenes activity in PCa, acting mechanistically by upregulating MYO6 expression through "sponging" miR-143.

Combinatorial Inhibition of mTORC2 and Hsp90 Leads to a Distinctly Effective Therapeutic Strategy in Malignant Pheochromocytoma.

BACKGROUND: Malignant pheochromocytoma (mPCC) is an uncommon tumor with poor prognosis, and no effective therapeutic strategy exists as yet. Discovering new and effective therapeutic strategies to improve prognosis is an urgent need. OBJECTIVE: To investigate whether a combinatorial inhibition of both mTORC2 and Hsp90 in PC12 cells could lead to a distinct anti-tumor effect in vitro and in vivo that was greater than the inhibition of mTORC2 or Hsp90 alone. METHODS: Targeting mTORC2 was assessed by knockdown of Rictor using shRNA, and 17-AAG was used to inhibit Hsp90 function. RESULTS: Combinatorial inhibition of both mTORC2 and Hsp90 could lead to a distinct anti-tumor effect in vitro that was greater than the inhibition of mTORC2 or Hsp90 alone. Inhibiting Hsp90 specifically could inhibit tumor growth of sh-Rictor cells in vivo, suggesting that the combinatorial inhibition of both mTORC2 and Hsp90 could lead to a distinct anti-tumor effect in vivo. Western blotting has shown that both p-Akt Ser473 and p-Akt Thr450 showed significantly decreased expression after targeting mTORC2, while p-Akt Thr308 did not. However, all three different p-AKTs, including p-Akt Ser473, p-Akt Thr450 and p-Akt Thr308, showed a significantly decreased expression in combinatorial inhibition of both mTORC2 and Hsp90. Collectively, it revealed that combinatorial inhibition of mTORC2 and Hsp90 could destabilize the Akt signaling. CONCLUSION: Our results demonstrated that combinatorial inhibition of mTORC2 and Hsp90 could increase their anti-tumor effect and destabilize the Akt signaling in PC12 cells, suggesting a combinatorial inhibition of both mTORC2 and Hsp90 which might be an effective therapeutic strategy for mPCC.

Pathologically examining a minimum of three lymph nodes could better determine node negativity in patients with non-metastatic chromophobe renal cell carcinoma.

OBJECTIVE: To evaluate the prognostic value of lymph node (LN) involvement for patients with chromophobe renal cell carcinoma (chRCC) and ascertain the minimum number of LNs that need to be pathologically examined to reliably diagnose a patient with node negative chRCC. METHODS: From 2004 to 2014, non-metastatic chRCC patients receiving radical nephrectomy together with lymphadenectomy were identified from the Surveillance, Epidemiology and End Results (SEER) database. The primary outcome was overall survival (OS). RESULTS: Two hundred and forty-six patients received lymph node dissection during the surgery. Of the patients, 24 (10%) had pathologically confirmed positive LN. Multivariate Cox regression model showed that positive LN was an independent unfavorable predictor for OS (HR = 2.83, 95%CI = 1.14-6.98, P = 0.024). More importantly, LN(-) patients with at least three LNs dissected had significantly better OS compared with when 1-2 LNs were examined (P = 0.048). Multivariate analysis confirmed that in LN(-) patients, the examination of three or more LNs could independently predict better OS compared with patients with only 1-2 LNs dissected (HR≥3LNs = 0.362, 95% CI = 0.135-0.972, P = 0.044). Additionally, the likelihood of finding at least one positive LN was significantly higher on dissection of ≥3 LNs compared with examination of 1-2 LNs (15% vs 5%, P = 0.018). Decision curve analysis found a better clinical validity of the '3 LNs examined'-based classification compared with the traditional LN(-)/LN(+) classification. CONCLUSION: The proportion of positive LNs in chRCC was far from neglectable and LN metastasis could independently predict unfavorable OS. We recommended a minimum of three LNs should be pathologically examined in order to reliably determine node negative.

CIP2A depletion potentiates the chemosensitivity of cisplatin by inducing increased apoptosis in bladder cancer cells.

Poor response and chemotherapy resistance to cisplatin (DDP)­based therapy frequently lead to treatment failure in advanced bladder cancer; however the underlying mechanism is extremely complex and unclear. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A), a recently identified human oncoprotein, has been shown to play important regulatory roles in cancer cell survival. The present study aimed to investigate the correlation of CIP2A with sensitivity to DDP in bladder cancer cells. In the present study, knockdown of CIP2A was performed using short hairpin­RNA. IC50 determination was used to estimate the chemosensitivity of cells to DDP. Apoptosis and DNA damage indicators were tested in vitro and in vivo to clarify the role of CIP2A in enhancing DDP sensitivity. We observed that CIP2A knockdown enhanced DDP sensitivity. CIP2A depletion accelerated the process of DNA damage caused by DDP treatment. Furthermore, DDP triggered inhibition of CIP2A by preventing AKT Ser473 phosphorylation. In vivo, CIP2A suppression increased the cytotoxicity of DDP, which resulted in a decrease in the subcutaneous tumor growth in a xenograft mouse model. Our findings revealed that the mechanism underlying the involvement of CIP2A in DDP sensitivity enhancement is that CIP2A mediates DDP­induced cell apoptosis and DNA damage. CIP2A is a potential target to improve the response to DDP­based therapy in bladder cancer patients.

Nomograms to Predict Individual Prognosis of Patients with Primary Small Cell Carcinoma of the Bladder.

Objectives: To develop reliable nomograms to estimate individualized overall survival (OS) and cancer specific survival (CSS) for patients with primary small cell carcinoma of the bladder (SCCB) and compare the predictive value with the AJCC stages. Patients and Methods: 582 eligible SCCB patients identified in the Surveillance, Epidemiology, and End Results (SEER) dataset were randomly divided into training (n=482) and validation (n=100) cohorts. Akaike information criterion was used to select the clinically important variables in multivariate Cox models when establishing nomograms. The performance of nomograms was bootstrapped validated internally and externally using the concordance index (C-index) with 95% confidence interval (95% CI) and calibration curves and was compared with that of the AJCC stages using C-index, Kaplan-Meier curves and decision curve analysis (DCA). Results: Two nomograms shared common indicators including age, tumor size, T stage, lymph node ratio, metastases, chemotherapy, radiation and radical cystectomy, while marriage and gender were only incorporated in the OS nomogram. The C-indices of nomograms for OS and CSS were 0.736 (95%CI 0.711-0.761) and 0.731(95%CI 0.704-0.758), respectively, indicating considerable predictive accuracy. Calibration curves showed consistency between the nomograms and the actual observation. The results remained reproducible when nomograms were applied to the validation cohort. Additionally, comparisons between C-indices, Kaplan-Meier curves and DCA proved that the nomograms obtained obvious superiority over the AJCC stages with wide practical threshold probabilities. Conclusions: We proposed the first two nomograms for individualized prediction of OS and CSS in SCCB patients with satisfactory predictive accuracy, good robustness and wide applicability.

Effectiveness of urine fibronectin as a non-invasive diagnostic biomarker in bladder cancer patients: a systematic review and meta-analysis.

BACKGROUND: Previous researches pointed out that the measurement of urine fibronectin (Fn) could be a potential diagnostic test for bladder cancer (BCa). We conducted this meta-analysis to fully assess the diagnostic value of urine Fn for BCa detection. METHODS: A systematic literature search in PubMed, ISI Web of Science, EMBASE, Cochrane library, and CBM was carried out to identify eligible studies evaluating the urine Fn in diagnosing BCa. Pooled sensitivity, specificity, and diagnostic odds ratio (DOR) with their 95% confidence intervals (CIs) were calculated, and summary receiver operating characteristic (SROC) curves were established. We applied the STATA 13.0, Meta-Disc 1.4, and RevMan 5.3 software to the meta-analysis. RESULTS: Eight separate studies with 744 bladder cancer patients were enrolled in this meta-analysis. The pooled sensitivity, specificity, and DOR were 0.80 (95%CI = 0.77-0.83), 0.79 (95%CI = 0.73-0.84), and 15.18 (95%CI = 10.07-22.87), respectively, and the area under the curve (AUC) of SROC was 0.83 (95%CI = 0.79-0.86). The diagnostic power of a combined method (urine Fn combined with urine cytology) was also evaluated, and its sensitivity and AUC were significantly higher (0.86 (95%CI = 0.82-0.90) and 0.89 (95%CI = 0.86-0.92), respectively). Meta-regression along with subgroup analysis based on various covariates revealed the potential sources of the heterogeneity and the detailed diagnostic value of each subgroup. Sensitivity analysis supported that the result was robust. No threshold effect and publication bias were found in this meta-analysis. CONCLUSIONS: Urine Fn may become a promising non-invasive biomarker for bladder cancer with a relatively satisfactory diagnostic power. And the combination of urine Fn with cytology could be an alternative option for detecting BCa in clinical practice. The potential value of urine Fn still needs to be validated in large, multi-center, and prospective studies.

Prognostic value of site-specific metastases and therapeutic roles of surgery for patients with metastatic bladder cancer: a population-based study.

BACKGROUND: We aimed to evaluate the prognostic value of site-specific metastases in patients with metastatic bladder cancer and analyze the roles that surgeries play in the treatment of this malignancy. MATERIALS AND METHODS: A population-based retrospective study using Surveillance, Epidemiology and End Results dataset was performed and metastatic bladder cancer patients were classified according to the sites of metastases (bone, brain, liver, lung and distant lymph nodes). Kaplan-Meier analysis with log-rank test was used for survival comparisons. Multivariate Cox regression model was employed to analyze the effect of distant metastatic sites on overall survival (OS) and cancer-specific survival (CSS). RESULTS: A total of 1862 patients with metastatic bladder cancer from 2010 to 2014 were identified. Bone, lung and distant lymph nodes were the most common metastatic sites. Patients with bone, brain, liver and lung involvement had worse OS and CSS compared to patients without the corresponding sites of metastases. Multivariate analysis showed that bone, brain, liver and lung metastases were independent prognostic factors for both OS and CSS, while distant node metastasis was not. Moreover, patients with a single metastatic site had more favorable OS (p<0.001) and CSS (p<0.001) than patients with multisite metastases. Among single-site metastatic patients, distant nodes and liver metastases represented the best and the worst prognosis, respectively. Moreover, radical cystectomy was an independent predictor for better OS and CSS, while in patients with liver metastasis and multiple metastatic sites, RC did not bring benefits. Besides, in patients with a single metastatic site, metastasectomy seemed to be associated with favorable OS (p=0.042), especially for patients with age <65 years (p=0.006) and for muscle-invasive bladder cancer patients (p=0.031). CONCLUSION: Distant metastatic sites have differential impact on survival outcomes in patients with metastatic bladder cancer. Surgeries, including radical cystectomy and metastasectomy, might still lead to survival benefits for highly selected patients.

The Triglycerides and Glucose Index rather than HOMA-IR is more associated with Hypogonadism in Chinese men.

Previous studies have reported that insulin resistant and low testosterone are related. The triglyceride and glucose index (TyG) well mirrors insulin sensitivity. No study investigated the application of TyG in male hypogonadism. We aimed to explore whether TyG was associated with hypogonadism, and also evaluate the ability of TyG compared to HOMA-IR as a possible hypogonadism predictor. A total of 4299 male subjects were enrolled from 22 sites in East China. Hypogonadism was defined as total testosterone <11.3 nmol/L. 695 (16.2%) hypogonadal men had significantly higher TyG index. The prevalence of hypogonadism stepwise increased across increasing TyG quartiles (P < 0.01). TyG was negatively associated with sex hormones and hypogonadism after adjustment for age, current smoking status, hypertension and overweight/obesity (all P for trend <0.01). The full-adjusted odds ratio was 6.1 for the highest quartile compared with the lowest quartile of TyG (95% CI 4.51, 8.25, P < 0.001). On ROC curve analysis, a larger area under the curve was found for TyG (0.71, 95% CI 0.69,0.73) than for HOMA-IR (0.68, 95% CI 0.66,0.70). Thus, the TyG was significantly associated with a higher prevalence of hypogonadism in Chinese men. TyG had a better predictive power for hypogonadism than HOMA-IR.

Targeting of CCN2 suppresses tumor progression and improves chemo-sensitivity in urothelial bladder cancer.

Urothelial bladder cancer (UBC) is the most common urinary neoplasm in China. CCN family protein 2 (CCN2), a cysteine-rich matricellular protein, is abnormally expressed in several cancer types and involved in tumor progression or chemo-resistance. However, detailed expression patterns and effects of CCN2 in UBC still remain unknown. We found that down-regulation of CCN2 suppressed proliferation, migration and invasion of UBC cells in vitro and targeting of CCN2 decelerated xenograft growth in vivo. When treated with mitomycin C (MMC), CCN2-scilencing UBC cells showed lower survival and higher apoptotic rates and these effects were probably mediated via inactivation of Akt and Erk pathways. We also demonstrated the clinical significance of CCN2 expression, which was higher in UBC tissues and associated with advanced tumor stage and high pathologic grade. Taken together, our data suggest that CCN2 is an oncogene in UBC and might serve as a matricellular target for improving chemotherapeutic efficacy.

Serum Uric Acid Is Associated with Erectile Dysfunction: A Population-Based Cross-Sectional Study in Chinese Men.

The role that serum uric acid (UA) plays in the pathophysiological development of erectile dysfunction (ED) is controversial. We aimed to screen the factors related with ED, and to examine the association between serum UA and ED. Our data were derived from a cross-sectional Survey on Prevalence in East China for Metabolic Diseases and Risk Factors study in 2014-2015. Questionnaire of International Index of Erectile Dysfunction-5 was used for assessment of ED. Data were collected in three general communities respectively. A total of 1365 men were enrolled with an overall mean age 55.5 ± 10.8 years (range: 20-83 years). The prevalence of ED was 62.4% (51.4% standardized) in the population. Males with ED were older, and more prone to have a higher follicle-stimulating hormone, luteinizing hormone, sex hormone-binding globulin, glycated hemoglobin, fasting plasma glucose levels and lower free androgen index (FAI), UA levels, and more likely to have diabetes and elevated blood pressure compared with those without ED. Age and UA were independent influencing factors for ED. Besides, UA was positively correlated with FAI after adjustment for age. In conclusion, our study demonstrated the protective role that UA might play in development of ED.

CIP2A mediates fibronectin-induced bladder cancer cell proliferation by stabilizing ß-catenin.

BACKGROUND: Fibronectin (FN) is associated with tumorigenesis and progression in bladder cancer, however, the underlying mechanisms causing this remain largely unknown. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A) has been shown to play important regulatory roles in cancer proliferation. Here, we investigated whether FN regulates CIP2A expression to promote bladder cancer cell proliferation. METHODS: The correlations of stromal FN with CIP2A and proliferating cell nuclear antigen (PCNA) expression were analyzed in a cohort bladder cancer patients. The roles of FN and CIP2A in regulating bladder cancer cell proliferation were evaluated in cell and animal models. Cycloheximide treatment was used to determine the effects of CIP2A on ß-catenin stabilization. The CIP2A-ß-catenin interaction was confirmed by immunofluorescence staining and co-immunoprcipitation. RESULTS: In this study, we found that stromal FN expression correlated positively with the levels of CIP2A and PCNA in bladder cancer tissues. Meanwhile, in human bladder cancer cell lines (T24 and J82), exogenous FN significantly promoted cell proliferation, however, CIP2A depletion inhibited this process. Furthermore, the interaction between CIP2A and ß-catenin enhanced the stabilization of ß-catenin, which was involved in FN-induced cell proliferation. In vivo, CIP2A depletion repressed FN-accelerated subcutaneous xenograft growth rates. CONCLUSIONS: These data reveal that CIP2A is a crucial mediator of FN-induced bladder cancer cell proliferation via enhancing the stabilization of ß-catenin. Promisingly, FN and CIP2A could serve as potential therapeutic targets for bladder cancer treatment.

How do organ-specific metastases affect prognosis and surgical treatment for patients with metastatic upper tract urothelial carcinoma: first evidence from population based data.

To evaluate the prognostic roles of organ-specific metastases and analyze the impact of organ-specific metastases on surgical resection of the primary tumor for metastatic upper tract urothelial carcinoma (UTUC) patients. A population-based study using Surveillance, Epidemiology, and End Results database was carried out. Kaplan-Meier analysis were used for survival comparisons. Multivariate Cox regression model was employed to analyze the effect of distant metastatic organs on overall survival (OS) and cancer specific survival (CSS). 337 patients from 2010 to 2014 were included. Patients with brain metastasis had significantly worse OS (p = 0.012) and CSS (p = 0.004). Liver metastasis could only independently predict unfavorable OS rather than CSS. Multivariate analysis showed that patients with bone, lung or distant lymph node metastasis was not independent prognostic factor for patients' survival. Surgical resection of the primary tumor was an independent favorable predictor for both OS (p = 0.004) and CSS advantages (p = 0.006). In subgroup analysis, patients with multiple organs of metastasis or distant lymph node involvement could benefit from surgical resection of the primary tumor. However, the presence of liver or lung metastasis could make such surgery become meaningless from the point of survival benefits. Our study showed that brain metastasis independently predicted both unfavorable OS and CSS for metastatic UTUC patients while liver metastasis was only associated with worse OS. More importantly, surgical resection of the primary tumor might still lead to survival benefits for highly selected patients.

Comparison of soil respiration in typical conventional and new alternative cereal cropping systems on the North China plain.

We monitored soil respiration (Rs), soil temperature (T) and volumetric water content (VWC%) over four years in one typical conventional and four alternative cropping systems to understand Rs in different cropping systems with their respective management practices and environmental conditions. The control was conventional double-cropping system (winter wheat and summer maize in one year--Con.W/M). Four alternative cropping systems were designed with optimum water and N management, i.e. optimized winter wheat and summer maize (Opt.W/M), three harvests every two years (first year, winter wheat and summer maize or soybean; second year, fallow then spring maize--W/M-M and W/S-M), and single spring maize per year (M). Our results show that Rs responded mainly to the seasonal variation in T but was also greatly affected by straw return, root growth and soil moisture changes under different cropping systems. The mean seasonal CO2 emissions in Con.W/M were 16.8 and 15.1 Mg CO2 ha(-1) for summer maize and winter wheat, respectively, without straw return. They increased significantly by 26 and 35% in Opt.W/M, respectively, with straw return. Under the new alternative cropping systems with straw return, W/M-M showed similar Rs to Opt.W/M, but total CO2 emissions of W/S-M decreased sharply relative to Opt.W/M when soybean was planted to replace summer maize. Total CO2 emissions expressed as the complete rotation cycles of W/S-M, Con.W/M and M treatments were not significantly different. Seasonal CO2 emissions were significantly correlated with the sum of carbon inputs of straw return from the previous season and the aboveground biomass in the current season, which explained 60% of seasonal CO2 emissions. T and VWC% explained up to 65% of Rs using the exponential-power and double exponential models, and the impacts of tillage and straw return must therefore be considered for accurate modeling of Rs in this geographical region.

Induction of DNA damage and ATF3 by retigeric acid B, a novel topoisomerase II inhibitor, promotes apoptosis in prostate cancer cells.

Retigeric acid B (RB) has been reported to exhibit its anti-tumor activity in vitro and in vivo. Here, we found that RB significantly inhibited activity of topoisomerase IIα (Topo IIα), leading to remarkable DNA damage in prostate cancer (PCa) cells as evidenced by a strong induction of γH2AX and DNA fragmentation. Activation of ATM and ATR sequentially led to induction of phospho-Chk1/2 and phospho-Cdc25 in response to RB. Blockade of ATM/ATR signaling resulted in the attenuation of RB-induced γH2AX, and partially rescued RB-mediated cell death. RB treatment also resulted in inactivation of DNA repair proteins such as phospho-BRCA1, impairment of HR, and NHEJ repair as indicated by DNA end-joining assays. Meanwhile, a stress-responsive gene activating transcription factor 3 (ATF3) was noted for its predominant expression in response to RB-induced DNA damage. Knockdown of ATF3 inhibited the RB-induced expression changes of cell cycle- and apoptosis-related genes such as DR5, DDIT4, CDC25A, GADD45A, and partially blocked RB-mediated inhibition on cell proliferation and induction of apoptosis, suggesting the crucial involvement of ATF3 in this event. Microarray data displayed that RB caused changes of genes required for damaged-DNA binding and repair, as well as ATF3 and its target genes. Our data firstly demonstrated that RB was a novel DNA Topo II inhibitor and triggered cell death by inducing DNA damage and stress-response, suggesting a promising anticancer agent.