Elevated plasma neurofilament light chain in immune-mediated neurological disorders (IMND) detected by immunomagnetic reduction (IMR).

BACKGROUND: In cases of immune-mediated neurological disorders (IMND), different syndromes are associated with antibodies against neuronal surface antigens, intra-neuronal antigens, astrocytic aquaporin, and gangliosides. These autoantibodies can be pathogenic or connected to neuroinflammation and resulting neuronal injuries. This study aims to identify a blood biomarker that can detect neuronal damage in individuals with IMND. To this end, we use immunomagnetic reduction (IMR) nanobead technology to measure plasma neurofilament light chain (NfL). METHODS: The patients with IMND were enrolled in the Chang Gung Memorial Hospital at Keelung from 2018 to 2023. Seronegative patients were excluded based on the results of antibody tests. The healthy controls (HC) were community-dwelling adults from the Northeastern Taiwan Community Medicine Research Cohort (NTCMRC) conducted by the Community Medicine Research Center of the Keelung CGMH from 2020 to 2022. IMR technique detects magnetic susceptibility via measuring magnetic signal reduction caused by antigen-antibody immunocomplex formation on magnetic nanobeads. The plasma level of NfL was determined by the magnetic susceptibility changes in IMR. RESULTS: The study enrolled 57 IMND patients from the hospital and 73 HC participants from the communities. The plasma NfL was significantly higher in the IMND than in the HC (11.022 ± 2.637 vs. 9.664 ± 2.610 pg/mL, p = 0.004), regardless of age effects on plasma NfL in an analysis of covariance (ANCOVA) (F = 0.720, p = 0.950). In the receiver of operation curve analysis, the area under curve for plasma NfL to discriminate IMND and HC was 0.664 (95% CI = 0.549 to 0.739, p = 0.005). The subgroup analysis of plasma NfL in the IMND patients showed no difference between peripheral immune-mediated neuropathy (IMN) and central immune-mediated encephalomyelitis (IMEM) (11.331 ± 2.895 vs. 10.627 ± 2.260 pg/mL, p = 0.322), nor between tumor and non-tumor IMND (10.784 ± 3.446 vs. 11.093 ± 2.391 pg/mL, p = 0.714). Additionally, the antibody class of ganglioside antibodies in IMN did not have an impact on plasma NfL level (p = 0.857). CONCLUSION: Plasma NfL measurement is a reliable indicator of axonal injuries in patients with IMND. It is equally effective in detecting nerve injuries in inflammatory peripheral neuropathies and central neuroinflammation. The IMR nanobead technology offers a feasible method of detecting plasma NfL, which helps identify axonal injuries in IMND.

Cucurbitane-type triterpenoids from the vines of Momordica charantia and their anti-inflammatory, cytotoxic, and antidiabetic activity.

Phytochemical investigation of the ethanol extract from wild Momordica charantia vines has resulted in isolation of seven cucurbitane-type triterpenoids, including six undescribed compounds, kuguaovins H‒M, and the known compound, momordicoside K. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses, including 1D and 2D NMR, and MS experiments. The chemical structure of momordicoside K was determined for the first time by X-ray crystallographic analysis and its absolute configuration assigned. The cytotoxicity against four human tumor cell lines and anti-inflammatory activities on LPS-stimulated RAW264.7 macrophages were evaluated. Of the isolates, kaguaovin L exhibited potential cytotoxicity against MCF-7, HEp-2, Hep-G2, and WiDr cancer cell lines and showed moderate anti-NO production activity. In addition, kuguaovins H and J also showed the stimulatory effect of GLP-1 secretion on the murine intestinal secretin tumor cell line (STC-1).

Case Report: Plasma Biomarkers Reflect Immune Mechanisms of Guillain-Barré Syndrome.

This case series reported a group of patients with Guillain-Barré syndrome (GBS) and their plasma cytokine changes before and after immunotherapy. We aimed to understand GBS's pathogenesis and pathophysiology through observing the interval differences of the representative cytokines, which were the thymus and activation regulated chemokine (TARC) for T-cell chemotaxis, CD40 ligand (CD40L) for cosimulation of B and T cells, activated complement component C5/C5a, and brain-derived neurotrophic factor (BDNF) for survival and regenerative responses to nerve injuries. The fluorescence magnetic bead-based multiplexing immunoassay simultaneously quantified the five cytokines in a single sample. From June 2018 to December 2019, we enrolled five GBS patients who had completed before-after blood cytokine measurements. One patient was diagnosed with paraneoplastic GBS and excluded from the following cytokine analysis. The BDNF level decreased consistently in all the patients and made it a potential biomarker for the acute stage of GBS. Interval changes of the other four cytokines were relatively inconsistent and possibly related to interindividual differences in the immune response to GBS triggers, types of GBS variants, and classes of antiganglioside antibodies. In summary, utilizing the multiplexing immunoassay helps in understanding the complex immune mechanisms of GBS and the variation of immune responses in GBS subtypes; this method is feasible for identifying potential biomarkers of GBS.

Ellagic Acid Resensitizes Gemcitabine-Resistant Bladder Cancer Cells by Inhibiting Epithelial-Mesenchymal Transition and Gemcitabine Transporters.

Gemcitabine (GCB) resistance is a major issue in bladder cancer chemoresistance, but its underlying mechanism has not been determined. Epithelial-mesenchymal transition (EMT) has been shown to be comprehensively involved in GCB resistance in several other cancer types, but the direct connection between EMT and GCB remains unclear. This study was designed to elucidate the mechanism of EMT-related GCB resistance in bladder cancer and identify a potential phytochemical to modulate drug sensitivity. The biological effects of ellagic acid (EA) or its combined effects with GCB were compared in GCB-resistant cells and the GCB-sensitive line in terms of cell viability, apoptosis, motility, and in vivo tumorigenicity. The molecular regulation of EMT-related GCB resistance was evaluated at both the mRNA and protein expression levels. Our results indicated that TGF-ß/Smad induced the overactivation of EMT in GCB-resistant cells and reduced the expression of GCB influx transporters (hCNT1 and hENT1). Moreover, ellagic acid (EA) inhibited the TGF-ß signaling pathway both in vitro and in vivo by reducing Smad2, Smad3, and Smad4 expression and thereby resensitized GCB sensitivity. In conclusion, our results demonstrate that TGF-ß/Smad-induced EMT contributes to GCB resistance in bladder cancer by reducing GCB influx and also elucidate the novel mechanisms of EA-mediated inhibition of TGF-ß/Smad-induced EMT to overcome GCB resistance. Our study warrants further investigation of EA as an effective therapeutic adjuvant agent for overcoming GCB resistance in bladder cancer.

Decreased drug resistance of bladder cancer using phytochemicals treatment.

The aim of the study is to investigate the ability of phytochemicals to overcome the multiple drug resistance (MDR) of bladder cancer. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic sensitivity of T24-GCB cells, a GCB resistant cell line, to different phytochemicals, including capsaicin, quercetin, curcumin, and resveratrol, and their combination with gemcitabine. Western blot analysis was used to detect the expression of membranous ABCC2 and metabolic proteins, DCK, TK1, and TK2 in tumor cells. Animal models were used to confirm the treatment efficacy of phytochemicals in combination with gemcitabine to bladder cancer. The observed/expected ratio of cytotoxicity analysis revealed that capsaicin has synergistic effect with gemcitabine to T24-GCB cells in a dose-dependent pattern. Quercetin, curcumin, and resveratrol have additive effect with gemcitabine to T24-GCB cells. Capsaicin and quercetin alone and combination with gemcitabine decreased the expression of ABCC2 and DCK and TKs, in T24-GCB cells. On the contrary, resveratrol and curcumin alone and combination with gemcitabine increased the expression of ABCC2 but decreased cytoplasmic kinases simultaneously. In xenografted subcutaneous tumor model on nude mice, combination treatment of capsaicin and gemcitabine demonstrated the highest tumor suppression effect when compared to capsaicin or gemcitabine treatment alone. The MDR of bladder cancer is closely related to membranous ABCC2, cytoplasmic DCK, and TKs expression. Capsaicin owns the strongest synergistic cytotoxic effect of gemcitabine to T24-GCB cells. This combination regimen may provide as an adjunctive treatment for overcoming MDR in bladder cancer.

Adjuvant therapy by high-speed burr may cause intraoperative bone tumor seeding: an animal study.

BACKGROUND: Bone tumors are often treated with intralesional curettage. High-speed burring, an adjuvant therapy, was performed to maximize the tumor cell killing; however, tumor recurrence might still occur, which may be caused by residual tumor or local tumor spread during surgery. METHODS: A porcine cadaver (femur) was utilized to determine whether the use of a high-speed burr causes bone cement spray. To mimic residual tumor after curettage, luminescent cement was smeared on two locations of the bone cavity, the wall and the bottom. The cavity in the femoral bone was then placed in the middle of a sheet of drawing paper featuring 10 cm, 20 cm, and 30 cm concentric circles. The luminescent cement was then burred totally with a high-speed burr. RESULTS: The intensity of the area in the wall in circle I was 72.6% ± 5.8%; within circle II, it was 22.1% ± 4.2%; and within circle III, it was 5.4% ± 1.5%. The intensity of the area within the bottom of the femoral bone within circle I was 66.5% ± 6.1%, within circle II was 28.1 ± 4.8%, and within circle III, it was 5.4% ± 1.4%. The amount of luminescent cement seeding decreased with distance, but there was no difference while burring at different locations of the bone cavity. Under the handpiece cover, a greater amount of cement spray was retained in circle I during burring of the cement in the bottom of the cavity and less was sprayed out in circle III. CONCLUSIONS: High-speed burring may cause explosive bone cement spray, which could extend to 20 cm. The intensities of spray did not decrease, even when the handpiece cover was used. The wide range of bone cement spray caused by high-speed burr was inspected in this pilot study, which may lead to tumor seeding. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

Cytokine changes during treatment of anti-Caspr2 encephalitis: a case report.

BACKGROUND: Cytokines are effective molecules of immune reactions. They work in inflammatory sites as well as circulate in the blood. Cytokines in the cerebrospinal fluid have been suggested to be markers of autoimmune encephalitis and reflect disease progression. However, studies on blood cytokines in autoimmune encephalitis are scarce. We report a case presenting with serial changes in blood cytokine levels in a male patient with anti-contactin-associated protein 2 (Caspr2) encephalitis. CASE PRESENTATION: A 61-year-old man without systemic disease presented with ataxia and speech disturbance 1 week. After admission, he further developed visual hallucinations, psychosis, and consciousness deterioration. Brain magnetic resonance imaging and infection and tumor surveillances were negative. 18F-fluorodeoxyglucose positron emission tomography of brain revealed frontal and occipital hypometabolism and anterior cingulate gyrus and mesial temporal hypermetabolism. Autoimmune studies confirmed Caspr2 antibodies in his blood. After receiving a diagnosis of anti-Caspr2 encephalitis, the patient received steroids, plasmapheresis, and zonisamide. He recovered well and was totally independent 6 months after disease onset. A cytokine profiler array kit was used to investigate neuroimmune mechanisms during the disease course. Several cytokines showed significant changes in plasma levels, such as B cell activating factor for B cell proliferation; thymus and activation-regulated chemokine for T cell chemoattraction; soluble CD40 ligand for Th2 cell mediation; C5/C5a for complement activation; brain-derived neurotrophic factor for neuronal survival response; and dipeptidyl peptidase 4, retinol binding protein, dickkopf-related protein, and epidermal growth factor for response to environmental provocation. The concentration of cytokines was verified using Luminex multiplexing assay. CONCLUSIONS: Due to their easy accessibility, blood cytokines are potential biomarkers of autoimmune encephalitis. Based on the investigating platform of this single case study, future larger scale studies are warranted.

Cucurbitane-Type Triterpenoids from the Vines of Momordica charantia and Their Anti-inflammatory Activities.

Seven new cucurbitane-type triterpenoids, kuguaovins A-G (1-7), and five known ones were isolated from the rattans of wild Momordica charantia. Their structures were established by spectroscopic data analyses, including 1D and 2D NMR, IR, and MS techniques. The absolute configurations of the cucurbitanes were determined from NOESY data and partially by X-ray crystallographic analysis. In pharmacological studies, compounds 1-7 and 9-12 exhibited weak anti-inflammatory effects (IC50 = 15-35 µM), based on an anti-NO production assay.

Concomitant Guillain-Barré Syndrome and Acute Transverse Myelitis in an Older Adult-A Case Report.

PURPOSE: Guillain-Barré syndrome concomitant with spinal cord involvement, which is defined as Guillain-Barré syndrome and acute transverse myelitis overlap syndrome, is rarely seen in the elders. Here we present a 68-year-old female patient who developed Guillain-Barré syndrome, as well as acute transverse myelitis at the same episode. CASE REPORT: This patient developed acute weakness of lower limbs, which then rapidly became tetraplegia and hyporeflexia within 5 days. She also had impaired pinprick and vibration sensations below T4, as well as urinary and defecation incontinence. The nerve conduction studies revealed a motorsensory axonal neuropathy. Cerebrospinal fluid analysis showed albuminocytological dissociation and elevated IgG index. The spinal magnetic resonance imaging study revealed heterogeneously contrastenhanced, long-segmental intramedullary lesion from C2 to T3. Other laboratory findings, including blood anti-aquaporin 4 antibody, were not remarkable. The patient's tetraplegia was gradually improved by plasmapheresis and methylprednisolone pulse therapy. CONCLUSION: Although Guillain-Barré syndrome and acute transverse myelitis overlap syndrome is occasionally seen in young adults, it could still occur in the elderly patients. Plasmapheresis and steroid pulse therapy could be beneficial to improve functional outcome of patients with this immunemediated neurological disease.

Different FDG-PET metabolic patterns of anti-AMPAR and anti-NMDAR encephalitis: Case report and literature review.

INTRODUCTION: 18 F-fluorodeoxyglucose (FDG)-PET metabolic patterns of brain differ among autoimmune encephalitis with different neuronal surface antigens. In this case report, we compared the topographical relationship of cerebral glucose metabolism and antigen distribution in the patients with anti-NMDAR and anti-AMPAR encephalitis. Literature review summarized the common features of brain metabolism of autoimmune encephalitis. METHODS: The cerebral glucose metabolism was evaluated by FDG-PET/CT during acute-to-subacute stage of autoimmune encephalitis and after treatment. The stereo and quantitative analysis of cerebral metabolism used standardized z-score and visualized on three-dimensional stereotactic surface projection. To map NMDAR and AMPAR in human brain, we adopted genetic atlases from the Allen Institute and protein atlases from Zilles's receptor densities. RESULTS: The three-dimensional stereotactic surface projection displayed frontal-dominant hypometabolism in a 66-year-old female patient with anti-AMPAR encephalitis and occipital-dominant hypometabolism in a 29-year-old female patient with anti-NMDAR encephalitis. Receptor density maps revealed opposite frontal-occipital gradients of AMPAR and NMDAR, which reflect reduced metabolism in the correspondent encephalitis. FDG-PET hypometabolic areas possibly represent receptor hypofunction with spatial correspondence to receptor distributions of the autoimmune encephalitis. The reversibility of hypometabolism was in line with patients' cognitive improvement. The literature review summarized six features of metabolic anomalies of autoimmune encephalitis: (a) temporal hypermetabolism, (b) frontal hypermetabolism and (c) occipital hypometabolism in anti-NMDAR encephalitis, (d) hypometabolism in association cortices, (e) sparing of unimodal primary motor cortex, and (e) reversibility in recovery. CONCLUSIONS: The distinct cerebral hypometabolic patterns of autoimmune encephalitis were representative for receptor hypofunction and topographical distribution of antigenic receptors. The reversibility of hypometabolism marked the clinical recovery of autoimmune encephalitis and made FDG-PET of brain a valuable diagnostic tool.

The modulation study of multiple drug resistance in bladder cancer by curcumin and resveratrol.

Gemcitabine (GCB), which functions via the inhibition of DNA synthesis, is commonly used in the treatment of bladder cancer; however, its response rate is not satisfactory due to the development of drug resistance. The potential for phytochemicals to reverse drug resistance in bladder cancer tumor cells was evaluated. A human bladder cancer cell line, T24, was cultured, and GCB-resistant cells (T24-GCB) were also established. The acquired resistance of T24-GCB to GCB was measured using an MTT assay. The gene expression of ATP-binding cassette (ABC) transporter protein family members was analyzed using reverse transcription-quantitative PCR analysis, and western blotting was performed to verify ABC family protein, cytoplasmic thymidine kinase (TK) and poly (ADP-ribose) polymerase (PARP) expression on whole cell lysates. Subsequently, resveratrol and curcumin were used to evaluate their modulation potential in decreasing the drug resistance of T24-GCB cells to GCB using MTT and migration assays. T24-GCB cells have increased drug resistance ability, with an 18.75-fold higher ID50 value compared with native T24 cells (105 vs. 5.6 nM). T24-GCB cells also exhibit increased cross resistance to mitomycin C and paclitaxel. The mRNA expression of ABCC2 in T24-GCB cells increased compared with that in native T24 cells. Via western blot analysis, it was determined that the expression of ABCC2 protein was also increased in T24-GCB cells. Conversely, the expression of ABCB1, ABCG2, deoxycytidine kinase (DCK), TK1 and TK2 decreased. Following curcumin and resveratrol treatment alone or combined with GCB, additive cytotoxic enhancement was observed, and the migratory abilities of T24-GCB cells were significantly decreased. Western blot analysis revealed that ABCC2 protein expression increased, and DCK, TK1 and TK2 expression decreased following co-treatment of T24-GCB cells with GCB + curcumin or resveratrol compared with GCB alone. Of note, there was a marked increase in cleaved-PARP expression in T24-GCB cells treated with a combination of GCB + curcumin or resveratrol. Both curcumin and resveratrol could reverse the drug resistance of T24-GCB cells in an additive pattern though PARP enhancement without changes in ABCC2 and DCK, TK1 and TK2 expression.

Stroke Rate Increases Around the Time of Cancer Diagnosis.

Objective: To test whether strokes increase around the time of cancer diagnosis, we comprehensively examined the correlations of cancer and stroke by employing a population-based cohort study design. Methods: One million people insured under the Taiwan's National Health Insurance program in 2005 were randomly sampled to create the study's dataset. According to the presence of cancer and/or stroke, patients were separated into cancer and stroke, cancer-only, and stroke-only groups. Diagnoses of cancer, stroke, and comorbidities were defined according to ICD9-CM codes. Cancer and non-cancer populations were matched by age at cancer diagnosis, gender, and stroke risk factors, and each patient with cancer was matched with two non-cancer controls nested in the same year of cancer diagnosis. The hazards of stroke and cumulative incidences within a year after cancer diagnosis were evaluated using Fine and Gray's subdistributional hazard model. Results: The temporal distribution of first-ever stroke in patients with both cancer and stroke was a sharpened bell shape that peaked between 0.5 years before and after cancer diagnosis. Frequencies of stroke were further adjusted by number of cancer survivors. The monthly event rate of stroke remained nested around the time of cancer diagnosis in all strokes. Brain malignancies, lung cancer, gastric cancer, prostate cancer, and leukemia patients obtained higher ratio of stroke, while breast cancer and thyroid cancer patients had low percentage of combining stroke. When compared to non-cancer matched control, the hazard of stroke within one year after cancer diagnosis was increased by cancer at a subdistributional hazard ratio of 1.72 (95% confident interval 1.48 to 2.01; p < 0.0001). Conclusions: Cancer increased the risk of stroke and stroke events were nested around the time of cancer diagnosis, occurring 0.5 years prior to cancer on average regardless of stroke type.

Association between gender and stoke recurrence in ischemic stroke patients with high-grade carotid artery stenosis.

The association between gender and stroke outcome in patients with high-grade internal carotid artery (ICA) stenosis remains unclear. We investigate gender differences in clinical characteristics and outcomes in ischemic stroke patients with high-grade ICA stenosis. Three-hundred and seventy-two acute ischemic stroke patients with high-grade ICA stenosis were enrolled and followed up for 5 years. Demographic features, vascular risk factors, co-morbidities, and outcomes were compared between male and female genders. Two-hundred and seventy-three (73.4%) patients were males and 99 (26.6%) patients were females. The prevalence of diabetes mellitus and atrial fibrillation was higher in females (P = 0.031 and P = 0.043), whereas the prevalence of smoking was higher in males (P < 0.001). The 5-year mortality rate was not different between males and females (P = 0.437), whereas the 5-year recurrent stroke rate was significantly higher in males (OR, 2.14; 95% CI, 1.22-3.75; P = 0.004). After adjusting for the established clinical predictors of adverse outcomes, the multivariate Cox regression revealed that male gender is a significant predictor of recurrent ischemic stroke (HR, 1.95; 95% CI, 1.19-3.20; P = 0.008). In conclusion, male gender is associated with increased risk of recurrent ischemic stroke in patients with high-grade ICA stenosis during 5-year follow-up. Further prospective trial to assess whether male gender may benefit from more aggressive vascular risk factors control and treatment strategies for stroke prevention is warranted.

A new niclosamide derivatives-B17 can inhibit urological cancers growth through apoptosis-related pathway.

The incidence and mortality rate of urological cancers is increasing yearly. Niclosamide has been repurposed as an anti-cancer drug in recent years. Synthesized derivative of niclosamide was testified for its anti-cancer activity in urological cancers. MTT assay was used to measure the cytotoxicity effect of niclosamide and its derivatives in urological cancer cell lines. Migratory ability was monitored by scratch migration assay. Apoptosis and cell cycle changes were analyzed by annexin V and PI staining. The apoptosis-related signal proteins were evaluated by western blotting. T24 had the best drug sensitivity with the lowest IC50 in niclosamide and B17 treatment than DU145 and Caki-1 cells. After niclosamide and B17 treatment, the mitotic cells were decreased, but apoptotic bodies and morphology changes were not prominent in T24, Caki-1, and DU145 cells. The migratory ability was inhibited in niclosamide treatment than control group on Caki-1 cells and niclosamide and B17 treatment than control group on DU145 cells. Early apoptosis cells were increased after niclosamide and B17 treatment than control group without cell cycle changes in T24, Caki-1, and DU145 cells. Programmed cell death was activated majorly through PAPR and bcl-2 in T24 and caspase-3 in Caki-1 cells, respectively. Niclosamide and B17 derivative had good ability in inhibition proliferation and migratory ability in T24, Caki-1, and DU145 cells without prominent morphology and apoptotic body changes. UCC cells are more sensitive to niclosamide and B17 treatment. Early apoptosis was induced after niclosamide and B17 treatment through different mechanisms in T24, Caki-1, and DU145 cells.

miR-429 expression in bladder cancer and its correlation with tumor behavior and clinical outcome.

We previously showed that microRNA-429 (miR-429) played an important role in epithelial-mesenchymal transition (EMT) of urothelial cell carcinoma of the bladder. We herein evaluated the expression of miR-429 in bladder cancer and its potential relevance to clinicopathological characteristics and patient survival. Relative expression levels of miR-429 in surgical bladder cancer tissue specimens obtained from 76 patients with bladder cancer were measured by chromogenic in situ hybridization. miR-429 expression was significantly higher in specimens from alive patients than expired patients in both of 5-year overall survival (OS) (0.59 ± 0.09 vs. 0.27 ± 0.12; p < 0.05) and 5-year recurrence-free survival (RFS) (0.63 ± 0.10 vs. 0.33 ± 0.10; p < 0.05). The univariate Cox proportional hazards analysis revealed that tumor grade, stage, and miR-429 expression were significantly associated with patient survival. In multivariate analysis, tumor stage and miR-429 expression were significantly associated with 5-year OS (hazard ratio [HR] 4.70, p < 0.001) and 5-year-RFS (HR 2.20, p < 0.05). The Kaplan-Meier analysis showed that patients with miR-429 expression had significantly better 5-year OS and 5-year RFS rates than those without miR-429 expression (84.4% vs. 61.4%, p < 0.05 and 71.9% vs. 45.5%, p < 0.05, respectively). miR-429 may be considered as an adjunctive prognostic marker in addition to tumor grade and stage in bladder cancer.

Expression of p-FOXO3/FOXO3 in bladder cancer and its correlation with clinicopathology and tumor recurrence.

BACKGROUND: Survey for more accurate biomarkers for predicting and preventing the future recurrence in high risk patients is urgently needed. The transcription factor forkhead box-O3 (FOXO3) is a well-established tumor suppressor. Its phosphorylation (p-FOXO3) as well as deregulation is involved in cancer initiation, progression and drug resistance. Therefore, we proposed that p-FOXO3/FOXO3 ratio change may play important role in the bladder cancer recurrence. METHODS: Surgical specimens of cancer tissue were obtained from 75 patients with bladder cancer (30 of non-recurrent and 45 of recurrent). The relative expression levels of p-FOXO3/FOXO3 in cancer tissue were measured by immunohistochemistry (IHC) stain and graded according to stain intensity. The correlation p-FOXO3/FOXO3 with clinicopathological parameters and tumor recurrence was analyzed. RESULTS: For bladder cancer patients with tumor recurrence, higher tumor grade (82% vs 70%, P=0.04) and stage (≥II, 49% vs 33%, P=0.02) in these patients was seen. In IHC study of paired tumor tissues, 39 out of 75 (52%) patients have increased p-FOXO3/FOXO3 ratio and they are closely related to tumor grade (low grade vs high grade =29.4% vs 58.6%, P=0.01) but not related to stage (low stage vs high stage =46.5% vs 59.3%, P=0.26). Regarding to tumor recurrence, the p-FOXO3/FOXO3 ratio is significant higher in recurrent group than non-recurrent group patients (0.78±0.15 vs 1.25±0.11, P=0.03). As comparing the first recurrence and subsequent recurrence group patients, there is no difference in the level of p-FOXO3/FOXO3 ratio (1.25±0.11 vs 1.10±0.09, P=0.25). Interestingly, recurrent tumors in low grade bladder cancer patients have marked increased p-FOXO3/FOXO3 ratio than non-recurrent tumors (0.90±0.22 vs 0.15±0.12, P=0.02). CONCLUSION: Increased p-FOXO3/FOXO3 ratio has been observed in bladder cancer patients with tumor recurrence and it is closely related to higher tumor grade. Low grade bladder cancer is high risk in recurrence when p-FOXO3/FOXO3 ratio increased. These results implicated that p-FOXO3/FOXO3 ratio can be applied as a useful marker for further treatment decision making and prognostic of tumor recurrence in bladder cancer patients.

MiR-429 reverses epithelial-mesenchymal transition by restoring E-cadherin expression in bladder cancer.

Epithelial-mesenchymal transition (EMT) accompanying loss of E-cadherin is important for invasiveness and metastasis of bladder cancer. MicroRNAs (miRs) had been associated with cancer progression and differentiation in several cancers. Our goal is to find out the specific miR which modulates EMT in bladder cancer. Real-time quantitative polymerase chain reaction was used to measure the miRs expression in urothelial cell carcinoma (UCC) cell lines. MiR or siRNA mimics was used to regulate miR and mRNA level respectively. Migration and scratch assays were used to determine the migratory ability. Zymography assay was used to confirm the metalloproteinase activity. Western blotting was used to elucidate the mechanism which regulated by specific miR. MiR-429 was highly expressed in low grade UCC cell lines. Exogenous mimic of miR-429 treatment dramatically inhibited the migratory ability of T24 cells. MiR-429 downstream target ZEB1 was decreased, E-cadherin was restored, and ß-catenin was contrarily decreased by exogenous mimic of miR-429 treatment in T24 cells. Cell invasive ability was also inhibited by exogenous mimic of miR-429 treatment through inactivating the MMP-2 activity in T24 cells. E-cadherin protein expression level was inhibited by E-cadherin siRNA accompanied with increasing cell migratory ability when compared with control group in low grade TSGH8301 cells. MiR-429 decreased the cell migratory and invasive abilities through reducing ZEB1 and ß-catenin, restoring the E-cadherin expression and inactivation of MMP-2 of UCC cells. MiR-429 might be used as a progression marker of bladder cancer.

Evasion of Cell Senescence Leads to Medulloblastoma Progression.

How brain tumors progress from precancerous lesions to advanced cancers is not well understood. Using Ptch1(+/-) mice to study medulloblastoma progression, we found that Ptch1 loss of heterozygosity (LOH) is an early event that is associated with high levels of cell senescence in preneoplasia. In contrast, advanced tumors have evaded senescence. Remarkably, we discovered that the majority of advanced medulloblastomas display either spontaneous, somatic p53 mutations or Cdkn2a locus inactivation. Consistent with senescence evasion, these p53 mutations are always subsequent to Ptch1 LOH. Introduction of a p53 mutation prevents senescence, accelerates tumor formation, and increases medulloblastoma incidence. Altogether, our results show that evasion of senescence associated with Ptch1 LOH allows progression to advanced tumors.

Bacille Calmette-Guerin can induce cellular apoptosis of urothelial cancer directly through toll-like receptor 7 activation.

Immunotherapy using bacille Calmette-Guerin (BCG) instillation is the mainstay treatment modality for superficial urothelial cancer (UC) through toll-like receptor (TLR) activation of cognitive immune response. We investigated the roles of TLR7 in the activation of apoptosis in UC cells after BCG treatment. The in vitro cytotoxicity effect of BCG on UC cells was measured by a modified 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium assay. Expressions of TLR7 mRNA and protein in native UC cells prior to and after BCG treatment were analyzed using real-time quantitative polymerase chain reaction and western blot methods. Phagocytotic processes after BCG treatment in UC cells were observed microscopically using a specific immunostain, subsequent cellular apoptosis-related signals induced by TLR7 were analyzed by western blot. Low-grade UC cells, TSGH8301, showed significant cellular death (4.23-fold higher than the high-grade UC cells T24 and J82) when treated with BCG and the BCG cytotoxicity was displayed in a dose-time-dependent manner. TSGH8301 cells had the highest content of TLR7 mRNA, 7.2- and 4.5-fold higher than that of T24 and J82 cells, respectively. TLR7 protein expression was also significantly increased in TSGH8301 cells. Phagocytosis-related markers, including beclin 1, ATG2, and LC3, were increased when TSGH8301 cells were treated by BCG. Interleukin-1 receptor-associated kinases 2 and 4 were also increased markedly in TSGH8301 cells. On the contrary, cellular apoptosis of TSGH8301 cells decreased by 34% when TLR7 activation was suppressed by the TLR antagonist IRS661 after BCG treatment. Our findings suggest that well differentiated TCC cells have higher expression of TLR7 and BCG can drive cellular death of TCC cells directly via TLR7 activation and related apoptotic pathway.

Structure-based hybridization, synthesis and biological evaluation of novel tetracyclic heterocyclic azathioxanthone analogues as potential antitumor agents.

A series of tetracyclic heterocyclic azathioxanthones were synthesized and evaluated for cell proliferations, topoisomerase inhibitions, and NCI-60 cell panel assay, respectively. Compounds 5, 7, 8, 16, and 19 were selected for topoisomerase assay after MTT assay. 7 not only showed cytotoxic effect (IC50 = 2.84 ± 0.64 µM) in PC-3 cells but also revealed topoisomerases inhibition with IC50 (10-25 µM) and increased apoptotic cleavage of PARP and caspase 3 activity. The overall of novel azathioxanthones with different cytostatic and cytotoxic activities should be further developed as new potential candidates for anticancer drugs.