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The current study investigated the mechanisms by which licochalcone C induces apoptosis of T24 human malignant bladder cancer cells. Cell viability was evaluated using an MTT assay. Apoptosis was investigated using a morphological assay, flow cytometry and a caspase-3 activity assay. Alterations in the gene expression levels of Bcl-2 family members were measured by semi-quantitative reverse transcription-polymerase chain reaction assays. The protein levels of pro-caspase-3 and cleaved poly(ADP ribose) polymerase were measured using western blotting. The results indicated that licochalcone C induced T24 cell apoptosis in a concentration-dependent manner. Licochalcone C treatment reduced the levels of the anti-apoptotic mRNAs (Bcl-2, Bcl-w and Bcl-XL) and increased expression of the pro-apoptotic mRNAs (Bax and Bim). The Bcl-2 family inhibitor (ABT-737) reduced apoptosis induced by licochalcone C in T24 cells. The current study demonstrated that licochalcone C may be a potential adjuvant therapeutic agent for bladder cancer. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to investigate the expression of EGFR and the autophagy-related markers Beclin1 and LC3 in cervical cancer. Methods: Beclin1, LC3, and EGFR expression were analyzed in 80 samples of cervical squamous cell carcinoma (SCC), 40 samples of high-grade cervical intraepithelial neoplasia (CIN), and 40 samples of normal cervical tissues by immunohistochemistry. The protein expression rates were analyzed with χ2 and Fisher's exact tests. Differences in overall survival (OS) were determined using the Kaplan-Meier method and log-rank tests. Results: Cervical cancer, high-grade CIN, and normal cervical epithelial cells expressed Beclin1 in 26.2%, 77.5%, and 82.5% of patients, respectively, and expressed LC3 in 28.8%, 70.0%, and 75.0% of patients, respectively. There was a significant difference between cervical SCC and high-grade CIN or normal cervical epithelial cells (P=0.000). Cervical cancer cells, high-grade CIN cells, and normal cervical epithelial cells expressed EGFR in 68.8%, 62.5%, and 12.5% of patients, respectively. There was a significant difference between cervical SCC or high-grade CIN and normal cervical epithelial cells (P=0.000). No significant association between Beclin1 or LC3 or EGFR expression and various clinicopathological parameters was observed in cervical SCC. There was no significant correlation between Beclin1, LC3, EGFR expression, and 5-year OS rates of cervical SCC patients. Beclin1- or LC3-negativity with EGFR-positivity in cervical SCC was associated with a higher Federation International of Gynecology and Obstetrics (FIGO) stage (P=0.011 and P=0.013, respectively) and pelvic lymph node metastasis (P=0.036 and P=0.092, respectively). The 5-year OS rates did not significantly differ between Beclin1- or LC3-positive and -negative patients with positive EGFR. Conclusion: Autophagy was downregulated and EGFR was upregulated in cervical SCC. Autophagy downregulation combined with EGFR upregulation promotes the progression of cervical SCC. [ABSTRACT FROM AUTHOR]

Multidrug resistance (MDR) is a major obstacle to the chemotherapeutic treatment of breast cancer. Germacrone, the main component of Rhizoma Curcuma, has been shown to possess antitumor, anti-inflammatory and immunomodulatory properties. The aim of the present study was to investigate the effect of germacrone on MCF-7/Adriamycin (ADR) multidrug-resistant human breast cancer cells. The treatment of MCF-7/ADR cells with a combination of germacrone and ADR resulted in an increase in cytotoxicity compared with that of ADR alone, as determined using an MTT assay. Flow cytometric analysis revealed that germacrone promoted cell apoptosis in a dose-dependent manner, whilst treatment with germacrone plus ADR enhanced the apoptotic effect synergistically. Furthermore, the results from the western blot analysis demonstrated that augmenting ADR treatment with germacrone resulted in a reduction of anti-apoptotic protein expression levels (bcl-2) and enhancement of pro-apoptotic protein expression levels (p53 and bax) in MCF-7/ADR cells compared with the levels achieved by treatment with ADR alone. In addition, germacrone significantly reduced the expression of P-glycoprotein via the inhibition of the multidrug resistance 1 (MDR1) gene promoter. These findings demonstrate that germacrone has a critical role against MDR and may be a novel MDR reversal agent for breast cancer chemotherapy. [ABSTRACT FROM AUTHOR]

A new type of signaling network element, called cancer signaling bridges (CSB), has been shown to have the potential for systematic and fast-tracked drug repositioning. On the basis of CSBs, we developed a computational model to derive specific downstream signaling pathways that reveal previously unknown target-disease connections and new mechanisms for specific cancer subtypes. The model enables us to reposition drugs based on available patient gene expression data. We applied this model to repurpose known or shelved drugs for brain, lung, and bone metastases of breast cancer with the hypothesis that cancer subtypes have their own specific signaling mechanisms. To test the hypothesis, we addressed specific CSBs for each metastasis that satisfy (i) CSB proteins are activated by the maximal number of enriched signaling pathways specific to a given metastasis, and (ii) CSB proteins are involved in the most differential expressed coding genes specific to each breast cancer metastasis. The identified signaling networks for the three types of breast cancer metastases contain 31, 15, and 18 proteins and are used to reposition 15, 9, and 2 drug candidates for the brain, lung, and bone metastases. We conducted both in vitro and in vivo preclinical experiments as well as analysis on patient tumor specimens to evaluate the targets and repositioned drugs. Of special note, we found that the Food and Drug Administration-approved drugs, sunitinib and dasatinib, prohibit brain metastases derived from breast cancer, addressing one particularly challenging aspect of this disease. [ABSTRACT FROM AUTHOR]

microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play important roles in tumor initiation and progression. Recently, we examined the global miRNA expression profile of esophageal squamous cell carcinoma (ESCC) and demonstrated that miR-92a was highly expressed in tumor tissues. In this study, we found that the upregulation of miR-92a was significantly correlated with the status of lymph node metastasis and TNM stage in 107 ESCC patients. Moreover, the upregulation of miR-92a was associated with poor survival of ESCC patients and might be used as an independent prognostic factor. Next, we investigated the role and mechanism of miR-92a in ESCC cells, and found that miR-92a modulated the migration and invasion but not apoptosis and proliferation of ESCC cells in vitro. We further demonstrated that miR-92a directly targeted the CDH1 3′- UTR and repressed the expression of CDH1, a tumor metastasis suppressor. In addition, restoring of miR-92a-resistant CDH1 expression in miR-92a-overexpression cells recovered the pro-metastasis activity of miR-92a. Taken together, we demonstrated that miR-92a promotes ESCC cell migration and invasion at least partially via suppression of CDH1 expression, and patients with up-regulated miR-92a are prone to lymph node metastasis and thus have poor prognosis. [ABSTRACT FROM AUTHOR]

OBJECTIVE To investigate the association between DNA polymorphisms, including single-nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms, in exon 1 and promoter of the CDH1 gene, and the risk of transitional cell carcinoma (TCC) of the urinary bladder (TCCB). PATIENTS, SUBJECTS AND METHODS This was a hospital-based case-control study of 180 patients with TCCB and 110 normal controls. Genomic DNA was extracted from blood samples of all participants and genotypes determined using polymerase chain reaction and DNA sequencing techniques. Haplotypes were analysed using appropriate software. RESULTS SNPs were detected at −160A/C, −73A/C and 178C/T; an inserted oligonucleotide of 5′-CCGTGCCCCAGCC-3′ was identified at 234 bp. The −160A allele frequency in the case group was 0.67, statistically higher than in the control group (0.42; P < 0.001), and higher in invasive carcinoma (0.77) than in superficial carcinoma (0.60). For −73C/A and 178C/T SNPs there was no difference among genotypes. The 234 repeat oligonucleotide insertion (2I) frequencies in cases was 0.27, statistically higher than in the control group (0.17; P = 0.01). The most common haplotype in controls was C-A-T-I (28%), the frequency of which was higher than in the TCCB group (6%). The A-A-T-2I was the only variation distribution carrying the −160A allele and was at a statistically higher frequency in the TCCB group (37%, the most common haplotype in cases) than in the control group. CONCLUSION The −160A, 234 2I allele and haplotype A-A-T-2I were risk factors of TCCB. Haplotype C-A-T-I might act as a protective factor for TCCB. [ABSTRACT FROM AUTHOR]

The pursuit of novel therapeutic agents in cancer relies on the identification and validation of molecular targets. Hallmarks of cancer include self-sufficiency in growth signals and evasion from apoptosis; genes that regulate these processes may be optimal for therapeutic attack. Here we describe a loss-of-function screen for genes required for the proliferation and survival of cancer cells using an RNA interference library. We used a doxycycline-inducible retroviral vector for the expression of small hairpin RNAs (shRNAs) to construct a library targeting 2,500 human genes. We used retroviral pools from this library to infect cell lines representing two distinct molecular subgroups of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like DLBCL and germinal centre B-cell-like DLBCL. Each vector was engineered to contain a unique 60-base-pair ‘bar code’, allowing the abundance of an individual shRNA vector within a population of transduced cells to be measured using microarrays of the bar-code sequences. We observed that a subset of shRNA vectors was depleted from the transduced cells after three weeks in culture only if shRNA expression was induced. In activated B-cell-like DLBCL cells, but not germinal centre B-cell-like DLBCL cells, shRNAs targeting the NF-κB pathway were depleted, in keeping with the essential role of this pathway in the survival of activated B-cell-like DLBCL. This screen uncovered CARD11 as a key upstream signalling component responsible for the constitutive IκB kinase activity in activated B-cell-like DLBCL. The methodology that we describe can be used to establish a functional taxonomy of cancer and help reveal new classes of therapeutic targets distinct from known oncogenes. [ABSTRACT FROM AUTHOR]

Pharmacological inhibitors of JNK (SP600 125) and p38 (PD169316) sensitize tumor cells to Fas-mediated apoptosis. PD169316 is less potent than SP600125 and diminishes its effect when present together. Because the p38 isoforms that promote (p38α) or inhibit (p38β) apoptosis are both suppressed by PD169316, we investigated their regulatory involvement in Fas-signaling. We report here, that p38α, but not p38β, exerts its proapoptotic effect by inhibiting the phosphorylation and presence of c-FLIPS, but not c-FLIPL, in the DISC to promote caspase-8 activation and type I signaling in Fas-activated Jurkat cells. Its effect was enhanced by enforced expression of Flag- tagged p38α and was attenuated by its inactive mutant (p38α-AGF) or by translational silencing. By contrast, type II signaling was facilitated by p38α-dependent mitochondrial presence of tBid and inhibition of Bcl-2 (Ser70) phosphorylation as well as by p38α-dependent mitochondrial localization of Bax and inhibition of phosphorylation of Bad (Ser112/Ser155). Potentiation of Fas-mediated apoptosis by the inhibition of JNK½ correlated with the loss of Bad (Ser136) phosphorylation and was dependent on the stimulatory effect of p38α on DISC and the downstream effects of both p38α and p38β. These data underscore the need to reassess the findings obtained with pan-p38 inhibitors and suggest that activation of p38α coupled with targeted inhibition of p38β and JNK½ should optimally sensitize tumor cells to Fas-mediated apoptosis. [ABSTRACT FROM AUTHOR]

• 19 isoquinoline alkaloids, including two new ones (1 and 14), were targeted obtained from Corydalis saxicola by SMART. • Structures were determined by NMR, HR-ESI-MS, X-ray, and calculate ECD. • The cytotoxicity of all isolates was evaluated against six human cancer cell lines. In the present study, HSQC-based Small Molecule Accurate Recognition Technology (SMART) strategy discovery of diverse cytotoxic isoquinolines. Two new isoquinoline alkaloids, corydalisines A and B (1 and 14), along with 17 known isoquinoline alkaloids (2 - 13, 15 - 19) were obtained from Corydalis saxicola. The structures were characterized using spectroscopic data analysis (NMR, HR-ESI-MS, IR, UV and X-ray diffraction), and the absolute configurations of new compounds were established by ECD data analysis. Biologically, Compounds 3, 7 , and 17 - 19 exhibited moderate cytotoxicity against six human cancer cell lines, with IC 50 values of 4.16-48.66 μM. [Display omitted] [ABSTRACT FROM AUTHOR]

The activation of the epithelial-to-mesenchymal transition (EMT) program is a critical step in cancer progression and metastasis, but visualization of this process at the single-cell level, especially in vivo, remains challenging. We established an in vivo approach to track the fate of tumor cells based on a novel EMTdriven fluorescent color switching breast cancer mouse model and intravital two-photon laser scanning microscopy. Specifically, the MMTV-PyMT, Rosa26-RFP-GFP, and Fsp1-Cre triple transgenic mouse model was used to monitor the conversion of RFP-positive epithelial cells to GFP-positive mesenchymal cells in mammary tumors under the control of the Fsp1 (ATL1) promoter, a gatekeeper of EMTinitiation. RFP-positive cells were isolated from the tumors, sorted, and transplanted into mammary fat pads of SCID mice to monitor EMT during breast tumor formation. We found that the conversion from RFP- to GFP-positive and spindleshaped cells was a gradual process, and that GFP-positive cells preferentially localized close to blood vessels, independent of tumor size. Furthermore, cells undergoing EMT expressed high levels of the HGF receptor, c-Met, and treatment of RFP-positive cells with the c-Met inhibitor, cabozantinib, suppressed the RFP-to-GFP conversion in vitro. Moreover, administration of cabozantinib to mice with palpable RFP-positive tumors resulted in a silent EMT phenotype whereby GFP-positive cells exhibited reduced motility, leading to suppressed tumor growth. In conclusion, our imaging technique provides a novel opportunity for visualizing tumor EMT at the single-cell level and may help to reveal the intricacies underlying tumor dynamics and treatment responses. [ABSTRACT FROM AUTHOR]

Continued reliance on the androgen receptor (AR) is now understood as a core mechanism in castrationresistant prostate cancer (CRPC), the most advanced form of this disease. While established and novel AR pathway-targeting agents display clinical efficacy in metastatic CRPC, dose-limiting side effects remain problematic for all current agents. In this study, we report the discovery and development of ARN-509, a competitive AR inhibitor that is fully antagonistic to AR overexpression, a common and important feature of CRPC. ARN-509 was optimized for inhibition of AR transcriptional activity and prostate cancer cell proliferation, pharmacokinetics, and in vivo efficacy. In contrast to bicalutamide, ARN-509 lacked significant agonist activity in preclinical models of CRPC. Moreover, ARN-509 lacked inducing activity for AR nuclear localization or DNA binding. In a clinically valid murine xenograft model of human CRPC, ARN-509 showed greater efficacy than MDV3100. Maximal therapeutic response in this model was achieved at 30 mg/kg/d of ARN-509, whereas the same response required 100 mg/kg/d of MDV3100 and higher steady-state plasma concentrations. Thus, ARN-509 exhibits characteristics predicting a higher therapeutic index with a greater potential to reach maximally efficacious doses in man than current AR antagonists. Our findings offer preclinical proof of principle for ARN-509 as a promising therapeutic in both castration-sensitive and castration-resistant forms of prostate cancer. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: The expression of fibulin-5 was investigated in urothelial carcinomas of bladder and in normal bladder samples. The effects of fibulin-5 on cell proliferation, motility, and invasion were further explored in bladder cancer cell line 5637. Materials and methods: The expression of fibulin-5 in 20 bladder carcinoma samples and 7 normal bladder samples were detected by Western blot. Fibuin-5 cDNA was cloned into p-EGFP-N1 vector and transfected into bladder cancer cell line 5637. Growth curves were estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay and cell number count. The migration and invasion of the transfected cells and the control cells were measured by Boyden chamber assay. Results: Compared with 1.16 ± 0.28 in the normal control, the mean ± SD expression of fibulin-5 in low grade tumors and high grade tumors were 0.57 ± 0.32 and 0.44 ± 0.42 (P = 0.002 and P = 0.018, respectively), while the expression in Ta-T1 tumors and T2+ tumors were 0.55 ± 0.31 and 0.49 ± 0.43 (P = 0.002 and P = 0.015, respectively). The differences between low grade and high grade tumors or Ta-T1 and T2+ tumors were not statistically significant (P = 0.362 and P = 0.589, respectively). The growth rate of the fibulin-5 transfected GFP-F5 cells was remarkably reduced than that of the untransfected cells or the empty vector transfected GFP-t cells. Besides, compared with control cells, the GFP-F5 cells showed significant inhibition of cell invasion with slight inhibition of cell migration. Conclusions: Fibulin-5 is down-regulated in urothelial carcinoma of bladder and acts as a tumor suppressor gene by inhibiting proliferation and invasion of bladder cancer cells. [Copyright &y& Elsevier]