Therapeutic Potential of Regorafenib in Cisplatin-Resistant Bladder Cancer with High Epithelial-Mesenchymal Transition and Stemness Properties.

Bladder cancer is becoming one of the most common malignancies across the world. Although treatment strategy has been continuously improved, which has led to cisplatin-based chemotherapy becoming the standard medication, cancer recurrence and metastasis still occur in a high proportion of patients because of drug resistance. The high efficacy of regorafenib, a broad-spectrum kinase inhibitor, has been evidenced in treating a variety of advanced cancers. Hence, this study investigated whether regorafenib could also effectively antagonize the survival of cisplatin-resistant bladder cancer and elucidate the underlying mechanism. Two types of cisplatin-resistant bladder cancer cells, T24R1 and T24R2, were isolated from T24 cisplatin-sensitive bladder cancer cells. These cells were characterized, and T24R1- and T24R2-xenografted tumor mice were created to examine the therapeutic efficacy of regorafenib. T24R1 and T24R2 cells exhibited higher expression levels of epithelial-mesenchymal transition (EMT) and stemness markers compared to the T24 cells, and regorafenib could simultaneously inhibit the viability and the expression of EMT/stemness markers of both T24R1 and T24R2 cells. Moreover, regorafenib could efficiently arrest the cell cycle, promote apoptosis, and block the transmigration/migration capabilities of both types of cells. Finally, regorafenib could significantly antagonize the growth of T24R1- and T24R2-xenografted tumors in mice. These results demonstrated the therapeutic efficacy of regorafenib in cisplatin-resistant bladder cancers. This study, thus, provides more insights into the mechanism of action of regorafenib and demonstrates its great potential in the future treatment of cisplatin-resistant advanced bladder cancer patients.

Modulation of mitochondrial apoptosis by ß2-adrenergic receptor blockage in colorectal cancer after radiotherapy: an in-vivo and in-vitro study.

Colorectal cancer (CRC) is one of the leading causes of malignancy-related deaths worldwide. Radiotherapy is often combined with surgery to treat patients with more advanced CRC. Despite impressive initial clinical responses, radiotherapy resistance is the main reason for most treatment failures in colorectal cancer. The G protein-coupled adrenergic receptor (AR) has shown to involve in the development and radiotherapy resistance of CRC. The ß2-AR blockage (ICI-118,551) can use to inhibit the progression of CRC through downregulating EGFR-Akt-ERK1/2 signaling. Since catecholamines-activated the G protein-coupled AR activation has been shown to result in radioresistant, co-treatment with both ß2-AR blockage and radiation may be improved the clinical outcome of CRC. We demonstrated that selective ß2-AR blockage, but not selective ß1-AR blockage, significantly enhanced radiation-induced apoptosis in CRC cells with wild-type p53 in vitro. The molecular mechanism of the apoptotic pathway was possibly triggered by a change in the mitochondrial membrane permeability and release of cytosolic cytochrome C through phospho-P53 mitochondrial translocation. We also found that a P53 knockout in the HCT116 cells was correlated with reversing ß2-AR blockage-mediated apoptosis induction after radiation treatment. Furthermore, the ß2-AR blockage significantly inhibited CRC cell-xenograft growth in vivo. Our study suggests that ß2-AR blockage may be used as adjunct agent for improving the clinical outcomes of CRC following radiotherapy by inducing apoptosis in CRC cells.

The cancer stemness inhibitor napabucasin suppresses small cell lung cancer growth through SOX2 expression.

Small cell lung cancer (SCLC) is a high-grade malignancy of neuroendocrine origin characterized by aggressive cell growth and a poor survival rate of patients. Currently, the treatment options for SCLC remain limited despite platinum-based chemotherapy. Systemic chemotherapy is effective for SCLC, but most patients eventually acquire drug resistance, which leads to treatment failure. Stemness-high cancer cells show characteristics of advanced tumorigenesis and metastasis and have high potential in promoting treatment resistance and disease relapse. Napabucasin (BBI608), a novel small-molecule drug targeting on signal transducer and activator of transcription 3 (STAT3), was shown to suppress the progression and metastasis of stemness-high cancer stem cells in various cancers. Here, we demonstrated that napabucasin significantly decreased viability and colony formation and induced the arrest of S-phase cell cycle and apoptosis in cisplatin-resistant SCLC cells. Findings from mechanistic studies further indicated that napabucasin directly downregulated the expression of SOX2 in cisplatin-resistant SCLC cells; however, dysfunctional SOX2 expression in SCLC cells was associated with interference in the napabucasin-mediated reduction of cell viability. In contrast, napabucasin-induced viability reduction was restored in these cells when SOX2 expression was upregulated. Furthermore, napabucasin significantly inhibited cisplatin-resistant SCLC cell xenograft growth in vivo by downregulating SOX2 and inducing apoptosis. These data demonstrate that napabucasin may be a novel drug for the clinical treatment of cisplatin-resistant SCLC.

Forced Overexpression of Signal Transducer and Activator of Transcription 3 (STAT3) Activates Yes-Associated Protein (YAP) Expression and Increases the Invasion and Proliferation Abilities of Small Cell Lung Cancer (SCLC) Cells.

Background: We sought to investigate the interaction between signal transducer and activator of transcription 3 (STAT3) and the Yes-associated protein (YAP) signaling pathway in human small cell lung cancer (SCLC) cells. Methods: The STAT3-overexpressing SCLC cell lines H146 and H446 were established by plasmid DNA transfection for in vitro and in vivo experiments. Results: Overexpression of STAT3 increased YAP protein expression in H146 and H446 cells. STAT3 overexpression significantly increased YAP mRNA expression and the mRNA expression of the YAP signaling downstream genes CTGF and CYR61 in H146 and H446 cells (p < 0.05). We showed that STAT3 overexpression promoted EMT (epithelial−mesenchymal transition) with increased matrix metalloproteinase (MMP)-2 and MMP9 expression. Transwell assays showed that STAT3 overexpression increased the invasion ability of H146 and H446 cells. In addition, STAT3-overexpressing H146 cells grew significantly more rapidly than control H146 cells in the xenograft mouse model (p < 0.05). Immunohistochemistry (IHC) staining and Western blotting (WB) showed that STAT3-overexpressing H146 tumors had increased p-STAT3 and YAP staining and protein expression compared with control tumors. Increased EMT was also observed in STAT3-overexpressed xenograft tumors. Conclusions: The results of our study suggest that the overexpression of STAT3 promotes SCLC EMT, invasion, and proliferation through the activation of the YAP signaling pathway.

Nuclear p120 catenin is a component of the perichromosomal layer and coordinates sister chromatid segregation during mitosis in lung cancer cells.

Abnormal expression of p120 catenin is associated with the malignant phenotype in human lung cancer. Numerous studies have focused on the function of p120 catenin in the juxta-membrane compartment. However, the role of nuclear p120 catenin remains unclear. In this study, the dynamic changes in nuclear p120 catenin localization during cell cycle progression were investigated. Immunofluorescent staining, FACS analysis, and western blotting revealed that nuclear p120 catenin is a major architectural constituent of the chromosome periphery during mitosis. During mitosis, granule-like p120 catenin dispersed into a cloudy-like structure and formed cordon-like structures surrounding the condensed chromosomes to create the peri-chromosomal layer. Interestingly, lumican and p120 catenin colocalized at the spindle fiber where the perichromosomal layer connects to the condensed chromosomes during mitosis. Furthermore, downregulation of p120 catenin using a specific siRNA induced cell cycle stalling in the G2/M phase and promoted aneuploidy. This study validates the role of nuclear p120 catenin in the formation of the chromosome periphery and reveals the p120 catenin-lumican interaction may couple orientation of cell division with the segregation of sister chromatids during mitosis. Our data suggest the protective role of p120 catenin in maintaining the integrity of chromosomes, and also warrants further studies to evaluate the contribution of the loss of p120 catenin to the creation of gene rearrangement in cancer evolution and tumor progression.

Intracavernous Injection of Autologous Platelet-Rich Plasma Ameliorates Hyperlipidemia-Associated Erectile Dysfunction in a Rat Model.

INTRODUCTION: Hyperlipidemia is associated with an increased risk of erectile dysfunction (ED) mediated by endothelial damage. Platelet-rich plasma (PRP) contains numerous angiogenic growth factors. Currently, evidence supporting the use of PRP for ED treatment is limited. AIM: We investigated PRP in a rat model of hyperlipidemia-associated ED. METHODS: Thirty 2-month-old male Sprague-Dawley rats were randomly divided into 3 groups. 20 rats were fed a high-fat diet for 5 months and were randomly divided into 2 groups: (i) rats in the H group received supernatant injection into the corpus cavernosum weekly for 4 weeks; (ii) rats in the H + PRP group received PRP injection into the corpus cavernosum weekly for 4 weeks. 10 rats were fed a standard diet for 5 months and received supernatant injection into the corpus cavernosum weekly for 4 weeks (N group). 7 days after the 4th injection, all rats underwent erectile function testing and then euthanasia. MAIN OUTCOME MEASURES: Erectile function was evaluated by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP). Serum and penile tissue were collected for metabolic variable assessment and histochemical examination, respectively. RESULTS: Intracavernous pressure/MAP and area under the curve/MAP ratios were significantly higher in the N and H + PRP groups than in the H group. Insulin-like growth factor-1, brain-derived neurotrophic factor, and vascular endothelial growth factor levels were significantly higher in the H + PRP group than in the N and H groups. Corporal neuronal nitric oxide synthase, endothelial nitric oxide synthase, and endothelial cells were weakly expressed in the H group compared with the N and H + PRP groups. Intracorporal oxidative stress and apoptotic index were significantly higher in the H group than in the N and H + PRP groups. CONCLUSIONS: This preclinical evidence suggests that clinical trials of PRP in men with ED should be considered. PRP may play a role in ED management. Huang YC, Wu CT, Chen MF, et al. Intracavernous Injection of Autologous Platelet-Rich Plasma Ameliorates Hyperlipidemia-Associated Erectile Dysfunction in a Rat Model. Sex Med 2021;9:100317.

The influence of smoking exposure and cessation on penile hemodynamics and corporal tissue in a rat model.

BACKGROUND: While epidemiological studies have clearly documented that smoking cessation significantly enhances sexual health, the underlying mechanism remains largely unknown. Thus, we wished to explore possible mechanisms by using a rat model of smoking-associated erectile dysfunction (ED). METHODS: Forty 8-week old male Sprague-Dawley rats were divided into 4 groups. Ten rats were exposed only to room air (N group). The remaining 30 rats were passively exposed to cigarette smoke over a 12-week period. At the end of 12 weeks, the smoking (S, n=10) group underwent immediate erectile function testing and were sacrificed. The remaining 20 rats were exposed to room air only for 4 (Q4W, n=10) or 8 (Q8W, n=10) weeks and then underwent erectile function testing and sacrifice. Erectile function was evaluated by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP). After blood collection for serum testosterone determination, rats were sacrificed to obtain corporal tissue for immunohistochemistry. RESULTS: Mean ICP/MAP ratio was significantly lower in the S group compared to the N and Q8W groups (0.52±0.11, 0.94±0.05, and 0.94±0.12, respectively, P=0.0189). Smooth muscle/collagen ratio was also significantly lower in the S group compared to the N and Q8W groups (11.8±0.94, 17.5±1.82, and 16.4±0.60, respectively, P=0.0008). Oxidative stress and apoptotic indices were significantly higher in the S group compared to the N and Q8W groups. Neuronal and endothelial nitric oxide synthases were significantly less expressed in the S group compared to the N and Q8W groups. CONCLUSIONS: Smoking cessation is associated with partial recovery of penile hemodynamics in a rat model of smoking associated ED.

Flavopereirine Suppresses the Growth of Colorectal Cancer Cells through P53 Signaling Dependence.

Colorectal cancer (CRC) is a significant cause of morbidity and mortality worldwide. The outcome of CRC patients remains poor. Thus, a new strategy for CRC treatment is urgently needed. Flavopereirine is a ß-carboline alkaloid extracted from Geissospermum vellosii, which can reduce the viability of various cancer cells through an unknown mode of action. The aim of the present study was to investigate the functional mechanism and therapeutic potential of flavopereirine on CRC cells in vitro and in vivo. Our data showed that flavopereirine significantly lowered cellular viability, caused intrinsic and extrinsic apoptosis, and induced G2/M-phase cell cycle arrest in CRC cells. Flavopereirine downregulated Janus kinases-signal transducers and activators of transcription (JAKs-STATs) and cellular myelocytomatosis (c-Myc) signaling in CRC cells. In contrast, the enforced expressions of constitutive active STAT3 and c-Myc could not restore flavopereirine-induced viability reduction. Moreover, flavopereirine enhanced P53 expression and phosphorylation in CRC cells. CRC cells with P53 knockout or loss-of-function mutation significantly diminished flavopereirine-mediated viability reduction, indicating that P53 activity plays a major role in flavopereirine-mediated CRC cell growth suppression. Flavopereirine also significantly repressed CRC cell xenograft growth in vivo by upregulating P53 and P21 and inducing apoptosis. In conclusion, flavopereirine-mediated growth suppression in CRC cells depended on the P53-P21, but not the JAKs-STATs-c-Myc signaling pathway. The present study suggests that flavopereirine may be efficacious in the clinical treatment of CRC harboring functional P53 signaling.

Cyclooxygenase-2 Activity Regulates Recruitment of VEGF-Secreting Ly6Chigh Monocytes in Ventilator-Induced Lung Injury.

Mechanical ventilation is usually required for saving lives in critically ill patients; however, it can cause ventilator-induced lung injury (VILI). As VEGF-secreting Ly6Chigh monocytes are involved in VILI pathogenesis, we investigated whether cyclooxygenase-2 (COX-2) activity regulates the recruitment of VEGF-secreting Ly6Chigh monocytes during VILI. The clinically relevant two-hit mouse model of VILI, which involves the intravenous injection of lipopolysaccharide prior to high tidal volume (HTV)-mechanical ventilation, was used in this study. To investigate the role of COX-2 in the recruitment of VEGF-secreting Ly6Chigh monocytes during VILI, celecoxib, which is a clinical COX-2 inhibitor, was administered 1 h prior to HTV-mechanical ventilation. Pulmonary vascular permeability and leakage, inflammatory leukocyte infiltration, and lung oxygenation levels were measured to assess the severity of VILI. HTV-mechanical ventilation significantly increased the recruitment of COX-2-expressing Ly6Chigh, but not Ly6Clow, monocytes. Celecoxib significantly diminished the recruitment of Ly6Chigh monocytes, attenuated the levels of VEGF and total protein in bronchoalveolar lavage fluid, and restored pulmonary oxygenation during VILI. Our findings demonstrate that COX-2 activity is important in the recruitment of VEGF-secreting Ly6Chigh monocytes, which are involved in VILI pathogenesis, and indicate that the suppression of COX-2 activity might be a useful strategy in mitigating VILI.

Stomatin-like protein 2 regulates survivin expression in non-small cell lung cancer cells through ß-catenin signaling pathway.

The overexpression of stomatin-like protein-2 (SLP-2) is commonly observed in non-small cell lung cancer (NSCLC) cells. In the present study, we transfected a number of NSCLC cells with an SLP-2 shRNA-expressing vector (AdSLP2i) and examined its possible effects on cell growth and apoptosis. We found that suppression of SLP-2 expression inhibited cell growth, and that the apoptosis induced by SLP-2 suppression was correlated with decreased survivin protein expression. Moreover, the reduced survivin expression was found to be associated with reduced ß-catenin nuclear localization and appeared not to be modulated through the AKT signaling pathway. By using immunoprecipitation and proteomics to analyze protein-protein interactions in A549 cells with SLP-2 overexpression, we found that annexin A2 interacted with SLP-2 and ß-catenin directly. Our data further suggested that the knockdown of SLP-2 gene affected the SLP-2/Annexin A2/ß-catenin cascade formation, reduced the translocation of cytoplasmic ß-catenin into nucleus, and downregulated downstream target genes. The results presented in this study, together with our previous findings, suggest that SLP-2 promotes NSCLC cell proliferation by enhancing survivin expression mediated via ß-catenin pathway.

Downregulation of lumican accelerates lung cancer cell invasion through p120 catenin.

The overexpression of lumican has been found in lung cancer cells; however, the functional role of lumican in lung cancer cells remains unclear. In this study, we found lumican functioned as a tubulin-binding protein and the depletion of lumican by transfection with its specific shRNA increased lung cancer cell invasion. Such alterations led to morphological changes and actin cytoskeleton remodeling, including the induction of membrane ruffling or protrusion and stress fiber formation, correlated with the increased activities of Rac and Rho. The downregulation of lumican was also implicated in macrophage-conditioned media (maCM)-induced cell invasion. Immunofluorescence images and immunoprecipitation assays revealed the co-localization of p120-catenin (p120ctn) and lumican. Reduction in the levels of p120ctn induced membrane ruffling and the activation of the Rho family, which accelerated cell invasion. Our data indicated that lumican is associated with microtubule-modulated p120ctn signaling, providing important insights into lung cancer progression.

Evodiamine Induces Cell Growth Arrest, Apoptosis and Suppresses Tumorigenesis in Human Urothelial Cell Carcinoma Cells.

BACKGROUND/AIM: Evodiamine, an indole alkaloid derived from Evodia rutaecarpa, exhibits pharmacological activities including vasodilatation, analgesia, anti-cardiovascular disease, anti-Alzheimer's disease, anti-inflammation, and anti-tumor activity. MATERIALS AND METHODS: This study analyzes the anti-tumor effects of evodiamine on cellular growth, tumorigenesis, cell cycle and apoptosis induction of human urothelial cell carcinoma (UCC) cells. RESULTS: The present study showed that evodiamine significantly inhibited the proliferation of UCC cells in a dose- and time-dependent manner. Also, evodiamine suppressed the tumorigenesis of UCC cells in vitro. Moreover, evodiamine caused G2/M cell-cycle arrest and induced caspase-dependent apoptosis in UCC cells. Finally, we demonstrated that evodiamine exhibits better cytotoxic than 5-fluorouracil, a clinical chemotherapeutic drug, for UCC cells. CONCLUSION: Evodiamine induces growth inhibition, tumorigenesis suppression, cell-cycle arrest, and apoptosis induction in human UCC cells. Therefore, this agent displays a therapeutic potential for treating human UCC cells and is worthy for further investigation.

VEGF Production by Ly6C+high Monocytes Contributes to Ventilator-Induced Lung Injury.

BACKGROUND: Mechanical ventilation is a life-saving procedure for patients with acute respiratory failure, although it may cause pulmonary vascular inflammation and leakage, leading to ventilator-induced lung injury (VILI). Ly6C+high monocytes are involved in the pathogenesis of VILI. In this study, we investigated whether pulmonary infiltrated Ly6C+high monocytes produce vascular endothelial growth factor (VEGF) and contribute to VILI. METHODS: A clinically relevant two-hit mouse model of VILI, with intravenous lipopolysaccharide (LPS, 20 ng/mouse) immediately before high tidal volume (HTV, 20 mL/kg) ventilation (LPS+HTV), was established. Blood gas and respiratory mechanics were measured to ensure the development of VILI. Flow cytometry and histopathological analyses revealed pulmonary infiltration of leukocytes subsets. Clodronate liposomes were intravenously injected to deplete pulmonary monocytes. In vitro endothelial cell permeability assay with sorted Ly6C+high monocytes condition media assessed the role of Ly6C+high monocytes in vascular permeability. RESULTS: LPS+HTV significantly increased total proteins, TNF-α, IL-6, vascular endothelial growth factor (VEGF) and mononuclear cells in the bronchoalveolar lavage fluid (BALF). Pulmonary Ly6C+high monocytes (SSClowCD11b+F4/80+Ly6C+high), but not Ly6C+low monocytes (SSClowCD11b+F4/80+Ly6C+low), were significantly elevated starting at 4 hr. Clodronate liposomes were able to significantly reduce pulmonary Ly6C+high monocytes, and VEGF and total protein in BALF, and restore PaO2/FiO2. There was a strong correlation between pulmonary Ly6C+high monocytes and BALF VEGF (R2 = 0.8791, p<0.001). Moreover, sorted Ly6C+high monocytes were able to produce VEGF, resulting in an increased permeability of endothelial cell monolayer in an in vitro endothelial cell permeability assay. CONCLUSION: VEGF produced by pulmonary infiltrated Ly6C+high monocytes regulates vasculature permeability in a two-hit model of HTV-induced lung injury. Ly6C+high monocytes play an important role in the pathogenesis of VILI.

Selective ß2-AR Blockage Suppresses Colorectal Cancer Growth Through Regulation of EGFR-Akt/ERK1/2 Signaling, G1-Phase Arrest, and Apoptosis.

The stress-upregulated catecholamines-activated ß1- and ß2-adrenergic receptors (ß1/2-ARs) have been shown to accelerate the progression of cancers such as colorectal cancer (CRC). We investigated the underlying mechanism of the inhibition of ß1/2-ARs signaling for the treatment of CRC and elucidated the significance of ß2-AR expression in CRC in vitro and in clinical samples. The impacts of ß1/2-AR antagonists in CRC in vitro and CRC-xenograft in vivo were examined. We found that repression of ß2-AR but not ß1-AR signaling selectively suppressed cell viability, induced G1-phase cell cycle arrest, caused both intrinsic and extrinsic pathways-mediated apoptosis of specific CRC cells and inhibited CRC-xenograft growth in vivo. Moreover, the expression of ß2-AR was not consistent with the progression of CRC in vitro or in clinical samples. Our data evidence that the expression profiles, signaling, and blockage of ß2-AR have a unique pattern in CRC comparing to other cancers. ß2-AR antagonism selectively suppresses the growth of CRC accompanying active ß2-AR signaling, which potentially carries wild-type KRAS, in vitro and in vivo via the inhibition of ß2-AR transactivated EFGR-Akt/ERK1/2 signaling pathway. Thus, ß2-AR blockage might be a potential therapeutic strategy for combating the progressions of ß2-AR-dependent CRC.

MicroRNA-145 regulates oncolytic herpes simplex virus-1 for selective killing of human non-small cell lung cancer cells.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, and novel treatment modalities to improve the prognosis of patients with advanced disease are highly desirable. Oncolytic virotherapy is a promising approach for the treatment of advanced NSCLC. MicroRNAs (miRNAs) may be a factor in the regulation of tumor-specific viral replication. The purpose of this study was to investigate whether miRNA-145 regulated oncolytic herpes simplex virus-1 (HSV-1) can selectively kill NSCLC cells with reduced collateral damage to normal cells. METHODS: We incorporated 4 copies of miRNA-145 target sequences into the 3'-untranslated region of an HSV-1 essential viral gene, ICP27, to create AP27i145 amplicon viruses and tested their target specificity and toxicity on normal cells and lung cancer cells in vitro. RESULTS: miRNA-145 expression in normal cells was higher than that in NSCLC cells. AP27i145 replication was inversely correlated with the expression of miRNA-145 in infected cells. This oncolytic HSV-1 selectively reduced cell proliferation and prevented the colony formation of NSCLC cells. The combination of radiotherapy and AP27i145 infection was significantly more potent in killing cancer cells than each therapy alone. CONCLUSIONS: miRNA-145-regulated oncolytic HSV-1 is a promising agent for the treatment of NSCLC.

CK2 inhibitors enhance the radiosensitivity of human non-small cell lung cancer cells through inhibition of stat3 activation.

CK2 interacts and phosphorylates >300 proteins, including Stat3, and is linked to a number of human cancers. Constitutively activated Stat3 has been reported in 50% of human lung cancers. Inhibition of CK2 activity can induce apoptosis and suppression of Stat3 activation in cancer cells. This study examined the effects of CK2 inhibitors on growth inhibition of lung cancer cells and the therapeutic potential on lung cancer. The CK2 inhibitor and radiation both suppressed cancer cell growth in a dose-dependent manner. Besides, the cytotoxic effect of irradiation could be augmented by CK2 inhibitors (p<0.05, two-way analysis of variance and Tukey's Honestly Significant Difference). Moreover, the growth inhibition of CK2 inhibitor and irradiation was both associated with suppression of Stat3 activation. Taken together, inhibition of CK2 activity appears to be a promising treatment strategy for non-small cell lung cancer and CK2 inhibition results in reduced Stat3 activation. Our data warrant further effort to develop CK2-targeted radiosensitizer for lung cancer treatment.

WIF-1 promoter region hypermethylation as an adjuvant diagnostic marker for non-small cell lung cancer-related malignant pleural effusions.

PURPOSE: Malignant pleural effusion is an important staging criterion in non-small cell lung cancer (NSCLC). Although cytologic examination remains the major diagnostic tool for NSCLC-related malignant pleural effusion, sometimes other invasive methods maybe required. Aberrant activation of Wnt signaling pathway due to Wnt inhibitory factor-1 (WIF-1) promoter region hypermethylation is common in NSCLC, and can be specifically detected by methylation-specific polymerase chain reaction (MSP). We hypothesized that WIF-1 promoter region MSP can be used to improve the diagnostic yield of NSCLC-related malignant pleural effusion. METHODS: We performed WIF-1 promoter region MSP in 36 definite malignant pleural effusions from consecutive NSCLC patients and 35 pleural effusion specimens of benign origin. Pleural effusion cells were collected for DNA extraction. After bisulfite treatment, DNA was amplified by methylation-specific and unmethylation-specific primers, respectively, to identify the methylation status of WIF-1 promoter region. RESULTS: The results of WIF-1 promoter region MSP were positive in 25 (69.4%) of 36 NSCLC patients with malignant pleural effusion. In addition, the results of WIF-1 promoter region MSP were negative in all 35 patients with pleural effusion of benign origin. The age, gender, and smoking status of patients were not correlated with the methylation status of WIF-1 promoter region in NSCLC-related malignant pleural effusion. CONCLUSIONS: WIF-1 promoter region MSP might be used as an adjuvant tool to complement cytologic examination for the diagnosis of NSCLC-related malignant pleural effusion.