Exploring the association of metal mixture in blood to the kidney function and tumor necrosis factor alpha using machine learning methods.

This research aimed to approach relationships between metal mixture in blood and kidney function, tumor necrosis factor alpha (TNF-α) by machine learning. Metals levels were measured by Inductively Couple Plasma Mass Spectrometry in blood from 421 participants. We applied K Nearest Neighbor (KNN), Naive Bayes classifier (NB), Support Vector Machines (SVM), random forest (RF), Gradient Boosting Decision Tree (GBDT), Categorical boosting (CatBoost), eXtreme Gradient Boosting (XGBoost), Whale Optimization-based XGBoost (WXGBoost) to identify the effect of plasma metals, TNF-α, and estimated glomerular filtration rate (eGFR by CKD-EPI equation). We conducted not only toxic metals, lead (Pb), arsenic (As), cadmium (Cd) but also included trace essential metals, selenium (Se), copper (Cu), zinc (Zn), cobalt (Co), to predict the interaction of TNF-α, TNF-α/white blood count, and eGFR. The high average TNF-α level group was observed among subjects with higher Pb, As, Cd, Cu, and Zn levels in blood. No associations were shown between the low and high TNF-α level group in blood Se and Co levels. Those with lower eGFR group had high Pb, As, Cd, Co, Cu, and Zn levels. The crucial predictor of TNF-α level in metals was blood Pb, and then Cd, As, Cu, Se, Zn and Co. The machine learning revealed that As was the major role among predictors of eGFR after feature selection. The levels of kidney function and TNF-α were modified by co-exposure metals. We were able to acquire highest accuracy of over 85% in the multi-metals exposure model. The higher Pb and Zn levels had strongest interaction with declined eGFR. In addition, As and Cd had synergistic with prediction model of TNF-α. We explored the potential of machine learning approaches for predicting health outcomes with multi-metal exposure. XGBoost model added SHAP could give an explicit explanation of individualized and precision risk prediction and insight of the interaction of key features in the multi-metal exposure.

Efficacy of GMI, a fungal immunomodulatory protein, for head and neck cancer patients with chemotherapy-related oral mucositis: An open-labeled prospective single-arm study.

BACKGROUND: Cancer patients usually suffer from intensive chemotherapy-related oral mucositis (OM), yet limited effective treatment can rapidly alleviate OM severity. METHODS: This prospective study examined the efficacy of Reishimmune-S containing one fungal immunomodulatory protein, GMI on OM in patients with head and neck cancer. Patients with head and neck cancer and the diagnosis of chemotherapy-related OM were enrolled randomizedly to receive standard supportive care with/without Reishimmune-S 500 mg/day orally for consecutive 14 days. Due to intolerance to standard supportive care alone in the control arm, only the experimental arm with Reishimmune-S supplementation was analyzed in our trial. OM grading was evaluated as the primary outcome on day 1, 8, and 15. Secondary outcomes were absolute neutrophil counts and quality of life assessed by the EORTC-QLQ-H&N 35 questionnaire on day 1, 8, and 15. RESULTS: Reishimmune-S supplement significantly reduced OM grading both at day 8 and 15. Trouble with social contact and weight loss conditions were also improved by Reishimmune-S. Reishimmune-S did not significantly affect absolute neutrophil counts during the 15-day follow-up. CONCLUSION: Reishimmune-S supplement potentially alleviates the severity of chemotherapy-mediated OM.

Generation of Functional Cardiomyocytes from Human Gastric Fibroblast-Derived Induced Pluripotent Stem Cells.

Coronary artery diseases are major problems of the world. Coronary artery disease patients frequently suffer from peptic ulcers when they receive aspirin treatment. For diagnostic and therapeutic purposes, the implementation of panendoscopy (PES) with biopsy is necessary. Some biopsy samples are wasted after the assay is completed. In the present study, we established a protocol for human gastric fibroblast isolation and induced pluripotent stem cell (iPSC) generation from gastric fibroblasts via PES with biopsy. We showed that these iPSCs can be differentiated into functional cardiomyocytes in vitro. To our knowledge, this is the first study to generate iPSCs from gastric fibroblasts in vitro.

Cilostazol Induces eNOS and TM Expression via Activation with Sirtuin 1/Krüppel-like Factor 2 Pathway in Endothelial Cells.

Cilostazol was suggested to be beneficial to retard in-stent atherosclerosis and prevent stent thrombosis. However, the mechanisms responsible for the beneficial effects of cilostazol are not fully understood. In this study, we attempted to verify the mechanism of the antithrombotic effect of cilostazol. Human umbilical vein endothelial cells (HUVECs) were cultured with various concentrations of cilostazol to verify its impact on endothelial cells. KLF2, silent information regulator transcript-1 (SIRT1), endothelial nitric oxide synthase (eNOS), and endothelial thrombomodulin (TM) expression levels were examined. We found cilostazol significantly activated KLF2 expression and KLF2-related endothelial function, including eNOS activation, Nitric oxide (NO) production, and TM secretion. The activation was regulated by SIRT1, which was also stimulated by cilostazol. These findings suggest that cilostazol may be capable of an antithrombotic and vasculoprotective effect in endothelial cells.

In silico immune infiltration profiling combined with functional enrichment analysis reveals a potential role for naïve B cells as a trigger for severe immune responses in the lungs of COVID-19 patients.

COVID-19, caused by SARS-CoV-2, has rapidly spread to more than 160 countries worldwide since 2020. Despite tremendous efforts and resources spent worldwide trying to explore antiviral drugs, there is still no effective clinical treatment for COVID-19 to date. Approximately 15% of COVID-19 cases progress to pneumonia, and patients with severe pneumonia may die from acute respiratory distress syndrome (ARDS). It is believed that pulmonary fibrosis from SARS-CoV-2 infection further leads to ARDS, often resulting in irreversible impairment of lung function. If the mechanisms by which SARS-CoV-2 infection primarily causes an immune response or immune cell infiltration can be identified, it may be possible to mitigate excessive immune responses by modulating the infiltration and activation of specific targets, thereby reducing or preventing severe lung damage. However, the extent to which immune cell subsets are significantly altered in the lung tissues of COVID-19 patients remains to be elucidated. This study applied the CIBERSORT-X method to comprehensively evaluate the transcriptional estimated immune infiltration landscape in the lung tissues of COVID-19 patients and further compare it with the lung tissues of patients with idiopathic pulmonary fibrosis (IPF). We found a variety of immune cell subtypes in the COVID-19 group, especially naïve B cells were highly infiltrated. Comparison of functional transcriptomic analyses revealed that non-differentiated naïve B cells may be the main cause of the over-active humoral immune response. Using several publicly available single-cell RNA sequencing data to validate the genetic differences in B-cell populations, it was found that the B-cells collected from COVID-19 patients were inclined towards naïve B-cells, whereas those collected from IPF patients were inclined towards memory B-cells. Further differentiation of B cells between COVID-19 mild and severe patients showed that B cells from severe patients tended to be antibody-secreting cells, and gene expression showed that B cells from severe patients were similar to DN2 B cells that trigger extrafollicular response. Moreover, a higher percentage of B-cell infiltration seems associated with poorer clinical outcome. Finally, a comparison of several specific COVID-19 cases treated with targeted B-cell therapy suggests that appropriate suppression of naïve B cells might potentially be a novel strategy to alleviate the severe symptoms of COVID-19.

A Double Triage and Telemedicine Protocol to Optimize Infection Control in an Emergency Department in Taiwan During the COVID-19 Pandemic: Retrospective Feasibility Study.

BACKGROUND: Frontline health care workers, including physicians, are at high risk of contracting coronavirus disease (COVID-19) owing to their exposure to patients suspected of having COVID-19. OBJECTIVE: The aim of this study was to evaluate the benefits and feasibility of a double triage and telemedicine protocol in improving infection control in the emergency department (ED). METHODS: In this retrospective study, we recruited patients aged ≥20 years referred to the ED of the National Taiwan University Hospital between March 1 and April 30, 2020. A double triage and telemedicine protocol was developed to triage suggested COVID-19 cases and minimize health workers' exposure to this disease. We categorized patients attending video interviews into a telemedicine group and patients experiencing face-to-face interviews into a conventional group. A questionnaire was used to assess how patients perceived the quality of the interviews and their communication with physicians as well as perceptions of stress, discrimination, and privacy. Each question was evaluated using a 5-point Likert scale. Physicians' total exposure time and total evaluation time were treated as primary outcomes, and the mean scores of the questions were treated as secondary outcomes. RESULTS: The final sample included 198 patients, including 93 cases (47.0%) in the telemedicine group and 105 cases (53.0%) in the conventional group. The total exposure time in the telemedicine group was significantly shorter than that in the conventional group (4.7 minutes vs 8.9 minutes, P<.001), whereas the total evaluation time in the telemedicine group was significantly longer than that in the conventional group (12.2 minutes vs 8.9 minutes, P<.001). After controlling for potential confounders, the total exposure time in the telemedicine group was 4.6 minutes shorter than that in the conventional group (95% CI -5.7 to -3.5, P<.001), whereas the total evaluation time in the telemedicine group was 2.8 minutes longer than that in the conventional group (95% CI -1.6 to -4.0, P<.001). The mean scores of the patient questionnaire were high in both groups (4.5/5 to 4.7/5 points). CONCLUSIONS: The implementation of the double triage and telemedicine protocol in the ED during the COVID-19 pandemic has high potential to improve infection control.

Ginkgo biloba Induces Thrombomodulin Expression and Tissue-Type Plasminogen Activator Secretion via the Activation of Krüppel-Like Factor 2 within Endothelial Cells.

The effects of thrombo-prevention, such as antiplatelet and anticoagulant activity, have been reported with the usage of Ginkgo biloba extract (GbE); however, the detailed mechanism has not yet been fully investigated, especially the role of Krüppel-like factor 2 (KLF2). This study aimed to investigate whether GbE can activate KLF2 and then induce thrombomodulin (TM) and tissue-type plasminogen activator (t-PA) secretion to enhance the effects of thrombo-prevention. Different concentrations of GbE were incubated with human umbilical vein endothelial cells (HUVECs) to evaluate its effect on endothelial cells. We found that KLF2 expression is correlated to the risk of atherosclerosis and venous thromboembolism in clinical practice. In the HUVEC cell model, GbE stimulated the expression of KLF2 in a dose-dependent manner. Moreover, TM and t-PA secretion increased when the cells were cultured with GbE. Both the expressions and activities of TM and t-PA in the GbE-treated cells declined after KLF2 was blocked by shKLF2. In sum, with GbE treatment, KLF2 expression in human endothelial cells was significantly activated, which in turn induced an increase in the protein expression and activity of TM and t-PA. After shRNA inhibited the KLF2 expression, GbE stopped inducing the expression and activity of TM and t-PA. These findings suggest that GbE exerts an antithrombotic effect on endothelial cells by increasing the TM expression and t-PA secretion; further, KLF2 is a key factor in this mechanism.

Early high-frequency spinal cord stimulation treatment inhibited the activation of spinal mitogen-activated protein kinases and ameliorated spared nerve injury-induced neuropathic pain in rats.

BACKGROUND: Neuromodulation therapies offer a treatment option that has minimal side effects and is relatively safe and potentially reversible. Spinal cord stimulation (SCS) has been used to treat various pain conditions for many decades. High-frequency SCS (HFSCS) involves the application of a single waveform at 10,000 Hz at a subthreshold level, therefore providing pain relief without any paresthesia. METHODS: We tested whether early HFSCS treatment attenuated spared nerve injury (SNI)-induced neuropathic pain. The phosphorylation profile of mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38, was evaluated to elucidate the potential underlying mechanism. RESULTS: SNI of rat unilateral sciatic nerves induced mechanical hyperalgesia in the ipsilateral hind paws. Rats were assigned to SCS sessions with HFSCS (frequency 10 kHz; pulse width 30 µs; pulse shape of charge-balanced, current controlled; delivered continuously for 72 h), or sham stimulation immediately after SNI. Tissue samples were examined at 1, 3, 7, and 14 days after SNI. Behavioral studies showed that HFSCS applied to the T10/T11 spinal cord significantly attenuated SNI-induced mechanical hyperalgesia compared with the sham stimulation group. Moreover, western blotting revealed a significant attenuation of the activation of ERK1, ERK2, JNK1, and p38 in the dorsal root ganglia and the spinal dorsal horn. CONCLUSION: Application of HFSCS provides an effective treatment for SNI-induced persistent mechanical hyperalgesia by attenuating ERK, JNK, and p38 activation in the dorsal root ganglia and the spinal dorsal horn.

Effects of ABT-737 combined with irradiation treatment on uterine cervical cancer cells.

The aim of the present study was to examine the role of ABT-737, an inhibitor of B-cell lymphoma 2 (Bcl-2), in enhancing the effect of irradiation on uterine cervical cancer. Based on The Cancer Genomic Atlas (TCGA), Bcl-2 mRNA expression was associated with the Tumor-Node-Metastasis stage of cervical cancer. Therefore, it was hypothesized that Bcl-2 inhibition may decrease the progression of cervical cancer. ABT-737 was added to irradiation treatment to evaluate its effectiveness in inhibiting cancer cell progression. SiHa and CaSki cervical cancer cells were selected for in vitro assays. Patients with advanced stage III uterine cancer had slightly increased mRNA expression levels of Bcl-2 compared with patients with stage I cancer, although the difference was not significant. ABT-737 and radiation administration induced a synergistic cytotoxic effect based on the MTT assay and flow cytometry results, where an increase in apoptosis was observed. The apoptotic percentages were significantly increased in the cells treated with a combination of ABT-737 and irradiation. Loss of mitochondrial membrane potential and gain of reactive oxygen species (ROS) were detected by flow cytometry in CaSki and SiHa cells treated with ABT-737 and radiation. Additionally, the protein expression levels of the cleaved forms of poly ADP ribose polymerase and caspase-7 were increased following the combined treatment. In conclusion, ABT-737 and irradiation may induce apoptosis via loss of mitochondrial membrane potential and a ROS-dependent apoptotic pathway in CaSki and SiHa cells. The present study indicates that ABT-737 may be a potential irradiation adjuvant when treating cervical cancer.

Ni-induced TGF-ß signaling promotes VEGF-a secretion via integrin ß3 upregulation.

Nickel compounds are associated with lung and skin cancer incidence increase and accumulation of nickel in the body contributes to carcinogenesis. Upregulation of certain integrins in the primary tumor is associated with cancer metastasis and poor prognosis. However, the molecular mechanisms of nickel-induced cancer metastasis are still unclear. The purpose of the present study was to investigate the effects of nickel chloride (NiCl2 ) on the progression of cancer during metastasis. The results of showed that NiCl2 induces the expression of integrin ß3 mRNA and protein in a dose- and time-dependent manner. Inhibition of integrin αvß3 activation by ITGB3 ligand mimetics and GR144053, as well as downregulation of ITGB3 by lentiviral shRNA gene silencing, diminished NiCl2 -induced secretion of vascular endothelial growth factor-a (VEGF-a). Furthermore, pretreatment with type I TGF-ß receptor inhibitor, SB525334, suppressed the expression of ITGB3 at cell surface and secretion of VEGF-a in NiCl2 -treated cells. In conclusion, NiCl2 induces the expression of ITGB3 through TGF-ß signaling activation, followed by increasing VEGF-a secretion, revealing a novel role for ITGB3 in nickel compound-induced cancer metastasis and tumor angiogenesis.

Hypericin inhibits hepatitis C virus replication via deacetylation and down-regulation of heme oxygenase-1.

BACKGROUND: Hepatitis C virus (HCV) is a globally prevalent pathogen and a leading cause of death and morbidity. Traditional therapy with pegylated interferon- and ribavirin has had only limited success, with some adverse effects. Direct-acting antivirals (DAAs) are effective in suppressing HCV replication, but are expensive. PURPOSE: Hypericin has been reported to be a good antiviral agent for inhibiting HCV replication, however, little is known about its mechanisms of action. The aim of this study is to elucidate the mode of action of hypericin in Ava5 human hepatoma cell line (Huh7 derivative) harboring HCV subgenomic replicon RNA. METHODS: To determine the non-structure protein 5A (NS5A) mRNA and NS3 protein expression levels, real-time PCR and Western blot analysis were performed, respectively. To investigate how hypericin inhibits HCV replication, 5-aza-2'-deoxycytidine (5-Aza-dC) and chidamide were used for determining histone modification. Furthermore, shRNA was applied to confirm the role of heme oxygenase (HO-1) in HCV repression. RESULTS: Hypericin in experiment were tested and showed no cytotoxicity. Hypericin reduced HO-1 and NS5A in a time- and dose- dependent manner. Chidamide, but not 5-Aza-dc, restored hypericin-induced reduction in HCV NS3 expression and reversed HO-1 expression in Ava5 cells. LY294002 inhibited HCV replication via HO-1 down-regulation. Constitutive expressed p-AKT was not involved in hypericin-induced reduction in HCV replication. In addition, shHO-1 inhibited HCV replication. CONCLUSION: In conclusion, hypericin inhibits HCV replication via down-regulation of HO-1 expression and deacetylation.

Semiparametric Estimation of Longitudinal Medical Cost Trajectory.

Estimating the average monthly medical costs from disease diagnosis to a terminal event such as death for an incident cohort of patients is a topic of immense interest to researchers in health policy and health economics because patterns of average monthly costs over time reveal how medical costs vary across phases of care. The statistical challenges to estimating monthly medical costs longitudinally are multifold; the longitudinal cost trajectory (formed by plotting the average monthly costs from diagnosis to the terminal event) is likely to be nonlinear, with its shape depending on the time of the terminal event, which can be subject to right censoring. The goal of this paper is to tackle this statistically challenging topic by estimating the conditional mean cost at any month t given the time of the terminal event s. The longitudinal cost trajectories with different terminal event times form a bivariate surface of t and s, under the constraint t ≤ s. We propose to estimate this surface using bivariate penalized splines in an Expectation-Maximization algorithm that treats the censored terminal event times as missing data. We evaluate the proposed model and estimation method in simulations and apply the method to the medical cost data of an incident cohort of stage IV breast cancer patients from the Surveillance, Epidemiology and End Results-Medicare Linked Database.

Metformin alleviates nickel-induced autophagy and apoptosis via inhibition of hexokinase-2, activating lipocalin-2, in human bronchial epithelial cells.

Autophagy is an intracellular recycling and degradation process for regulating tumor progression, survival and drug resistance. Nickel compounds have been identified as human carcinogens. However, the role of nickel-induced autophagy in lung carcinogenesis has not yet been fully elucidated. In this study, we determined that hexokinase 2 (HK2), which phosphorylates glucose and regulates autophagy, is the key mediator in nickel-induced autophagy in lung bronchial epithelial cells. We attempted to investigate the effects of the antidiabetic drug metformin on HK2 expression and lung cancer chemoprevention. Our results showed that metformin decreases nickel-induced autophagy and activation of apoptosis through inhibition of HK2 gene, protein and activity. Furthermore, we demonstrated that lipocalin 2 (LCN2), which is released by neutrophils at sites of infection and inflammation is involved in HK2-driven autophagy pathway. Knockdown of endogenous HK2 and LCN2 by shRNA reduced nickel-elicited autophagy and apoptosis, illustrating that metabolic alteration and inflammatory action are important in nickel-elicited carcinogenesis. We also determined the association between nickel-induced autophagy and apoptosis. Inhibition of nickel-induced autophagy abolished apoptotic cell death in chloroquine-treated, shLC3 Beas-2B cells and Atg5-/- MFFs. From TGCA database and immunohistochemistry analysis, HK2 and LCN2 expression increased in lung squamous cell carcinoma and their related adjacent normal tissues. Taken together, our results demonstrated that metformin alleviates NiCl2-induced autophagy and apoptosis via HK2-driven LCN2 activation in human bronchial epithelial cells. This novel mechanism provides a strategy for targeting nickel-elicited lung cancer progression, as well as for preventing HK2 cumulative damage triggered by environmental carcinogens.

Upregulation of microRNA-4417 and Its Target Genes Contribute to Nickel Chloride-promoted Lung Epithelial Cell Fibrogenesis and Tumorigenesis.

Nickel compounds have been classified as carcinogens and shown to be associated with induction of epithelial-mesenchymal transition (EMT) in fibrogenesis and tumorigenesis, as well as the crucial role of microRNAs (miRNAs) and their related genes in controlling EMT and cancer metastasis. Thus, the mechanisms involved in the regulation of EMT in nickel-treated cells are of potential interest in understanding lung fibrosis and tumor progression. We investigated the miRNA-dependent mechanisms involved in nickel-induced EMT in lung epithelial cells. Nickel increased miR-4417 expression and decreased its target gene TAB2 expression. Treatment of cells with TGF-ß inhibitor SB525334 significantly blocked NiCl2 and TGF-ß-induced EMT. The expression of miR-4417 was abolished by SB525334 in TGF-ß-treated cells, but not in nickel-treated cells. Both overexpression of miR-4417 and silencing of TAB2 induced fibronectin expression, but did not reduce E-cadherin expression. Moreover, oral administration of nickel promoted lung tumor growth in nude mice that had received BEAS-2B transformed cells by intravenous injection. The induction of EMT by nickel is mediated through multiple pathways. Induction of abundant miR-4417 and reduction of TAB2 expression following nickel exposure and may be involved in nickel-induced fibronectin. These findings provide novel insight into the roles of nickel in fibrogenesis and tumor progression.

Pulsed Radiofrequency Attenuates Complete Freund's Adjuvant-Induced Epigenetic Suppression of Potassium Chloride Cotransporter 2 Expression.

Background: Pulsed radiofrequency (PRF) treatment offers pain relief for patients suffering from chronic pain who do not respond well to conventional treatments. We tested whether PRF treatment attenuated complete Freund's adjuvant (CFA)-induced inflammatory pain. Epigenetic modification of potassium-chloride cotransporter 2 (KCC2) gene expression was examined to elucidate the potential contributing mechanism. Methods: Male Sprague-Dawley rats were injected with CFA into the plantar surface of the left hind paw to induce inflammation. PRF (20 minutes of 500-kHz RF pulses, delivered at a rate of 2 Hz, maximum temperature 42ºC) was delivered to the L5 and L6 anterior primary ramus just distal to the intervertebral foramen of adult CFA or saline rats. The hind paw withdrawal threshold to von Frey filament stimuli and withdrawal latency to radiant heat were determined before and after CFA. Acetyl-histone H3 and H4 was determined by chromatin immunoprecipitation in spinal dorsal horn. KCC2 expression was determined by Western blot. Inhibitory synaptic function was evaluated by patch clamp in lamina II neurons. Results: KCC2 gene expression was suppressed through histone hypoacetylation, resulting in decreased efficacy of GABAergic signaling in CFA rats. PRF increased histone acetylation and KCC2 expression, partially restored the GABA synaptic function, and relieved sensitized pain behavior. Conclusion: These findings suggest that PRF might be an alternative therapy for inflammatory pain. One of the underlying mechanisms is through modification of KCC2, which is an important determinant for the efficacy of inhibitory neurotransmission in the spinal cord, and its expression levels are regulated by histone acetylation epigenetically following inflammation.

Pulsed radiofrequency attenuates diabetic neuropathic pain and suppresses formalin-evoked spinal glutamate release in rats.

BACKGROUND: Pulsed radiofrequency (PRF) has been used to treat chronic pain for years, but its effectiveness and mechanism in treating diabetic neuropathic pain are still unexplored. The aim of this study was to elucidate the modulation of diabetic neuropathic pain induced by streptozotocin and the release of spinal excitatory amino acids by PRF. METHODS: Diabetes was induced by intraperitoneal administration of streptozotocin. Pulsed radiofrequency was applied to L5 and L6 dorsal roots at 42 °C for 2 min. The responses of all of the groups to thermal, mechanical and cold stimuli were measured for a period of 6 d after this process. Seven days after PRF treatment, intrathecal microdialysis was used to examine the effect of pulsed radiofrequency on the formalin-evoked spinal release of excitatory amino acids and concurrent behaviour responses from diabetic rats. RESULTS: Three weeks after intraperitoneal streptozotocin treatment and before PRF application, mechanical, thermal and cold hypersensitivity occurred. Application of PRF significantly alleviated hyperglycaemia-induced mechanical, thermal and cold hypersensitivity and also attenuated the increase in formalin-evoked CSF glutamate concentration, compared with sham treated diabetic rats. CONCLUSION: It may be concluded that PRF has an analgesic effect on neuropathic pain by suppressing the nociception-induced release of excitatory neurotransmitters. PRF may provide a novel promising therapeutic approach for managing diabetic neuropathic pain.

Immunomodulatory proteins FIP-gts and chloroquine induce caspase-independent cell death via autophagy for resensitizing cisplatin-resistant urothelial cancer cells.

BACKGROUND: Chloroquine, a lysosomal inhibitor, is used for malaria, rheumatoid arthritis, and lupus erythematosus therapy. In our previous study, FIP-gts, an immunomodulatory protein from Ganoderma tsugae, inhibited cell viability in lung cancer cells and urothelial cancer cells. Urothelial carcinoma is the most common type of bladder cancer. Cisplatin resistance is an important issue in urothelial carcinoma therapy. PURPOSE: The aim of this study is to investigate the effect of combination treatment with FIP-gts and chloroquine on cytotoxicity to resensitize the cisplatin-resistant cells. METHODS: FIP-gts and chloroquine cytotoxicity were determined by evaluating CCK-8 assay. Cell death pathways, ROS and cell cycle arrested were analysed through flow cytometry and Western blot. ShRNA targeting to autophagy-related genes were tested to evaluate their autophagic cell death for resistant urothelial cells. RESULTS: Using CCK-8 assay, chloroquine increased FIP-gts-induced cytotoxicity in parental and cisplatin-resistant urothelial cancer cell lines. On flow cytometry, chloroquine enhanced FIP-gts-mediated sub-G1 accumulation, annexin V positive signal and mitochondrial membrane potential loss. Caspase-3/PARP cascade and z-VAD-fmk were performed to prove that FIP-gts and chloroquine induced caspase-independent cell death. Using H2DCFDA staining and flow cytometry, FIP-gts and chloroquine did not induce ROS production. N-acetyl cysteine, a ROS scavenger, inhibited the cytotoxicity and LC3-II accumulation in FIP-gts and chloroquine-treated N/P cells. To elucidate the role of autophagy in caspase-independent cell death by FIP-gts and chloroquine, LC3 shRNA were used to inhibit autophagy in N/P cells. The capabilities of FIP-gts and chloroquine to induce cytotoxicity and sub-G1 phase accumulation were abolished in autophagy-defective cells. This is the first study to reveal the novel function of FIP-gts in triggering caspase-independent cell death in cisplatin-resistant urothelial cancer cells. CONCLUSION: Chloroquine enhanced FIP-gts-induced autophagy dependent caspase-independent cell death via abundant autophagosome accumulation. Combination treatment with FIP-gts and chloroquine may provide a new strategy for urothelial cancer therapy.

Relationship of Genetic Polymorphisms of Aldosterone Synthase Gene Cytochrome P450 11B2 and Mineralocorticoid Receptors with Coronary Artery Disease in Taiwan.

The aldosterone synthase gene, cytochrome P450 11B2 (CYP11B2), and mineralocorticoid receptor (MR) genes have been reported to be associated with coronary artery disease (CAD). In this study, we investigated the association of single nucleotide polymorphisms (SNPs) of CYP11B2 (CYP11B2 T-344C) and MR (MR C3514G and MR C4582A) with CAD in Taiwanese. Six hundred and nine unrelated male and female subjects who received elective coronary angiography were recruited from Chung Shan Medical University Hospital. The enrolled subjects were those who had a positive noninvasive test. CYP11B2 T-344C, MR C3514G and MR C4582A were determined by polymerase chain reaction-restriction fragment length polymorphism. We found that women with CYP11B2 C/C had a higher risk of developing CAD. However, there were no significant differences in the genotype distributions of MR C3514G and MR C4582A between the women with and without CAD. In multivariate analysis, CYP11B2 T-344C was most significantly associated with CAD in Taiwanese women. In conclusions, CYP11B2 C/C was more significantly associated with the development of CAD than diabetes mellitus or hypertension. This implies that CYP11B2 C/C plays a more important role than some conventional risk factors in the development of CAD in Taiwanese women.

Reduction of spinal glycine receptor-mediated miniature inhibitory postsynaptic currents in streptozotocin-induced diabetic neuropathic pain.

Diabetic neuropathic pain (DNP) is a common clinical problem, and the mechanisms underlying the onset and progression of this complication are poorly understood. The present study examined the glycine receptors (GlyR) in the control of synaptic input to dorsal horn neurons in diabetes. Male Sprague-Dawley rats with or without streptozotocin (STZ) intraperitoneal injections were used. Tactile sensitivities were assessed by measuring paw withdrawal thresholds to von Frey filaments for four weeks. The extent of GlyR-mediated inhibition controlling primary afferent-evoked excitation in dorsal horn neurons was examined by using the whole cell patch clamp recording technique in isolated adult rat spinal cord slices. The content of the spinal dorsal horn glycine levels was measured by microdialysis. An intrathecal glycine agonist injection was used to test whether mimicking endogenous glycine-receptor-mediated inhibition reduces DNP. We found that persistent hyperglycemia induced by the administration of STZ caused a decrease in the paw withdrawal latency to mechanical stimuli. The miniature inhibitory post-synaptic current (mIPSC) rise, decay kinetics and mean GlyR-mediated mIPSC amplitude were not affected in DNP. The mean frequency of GlyR-mediated mIPSC of lamina I neurons from DNP rats was, however, significantly reduced when compared with neurons from control rats. Principal passive and active membrane properties and the firing patterns of spinal lamina I neurons were not changed in DNP rats. Spinal microdialysis rats had a significantly decreased glycine level following its initial elevation. The intrathecal administration of glycine diminished tactile pain hypersensitivity in DNP rats. In conclusion, these results indicate that long-lasting hyperglycemia induced by STZ injections leads to a reduced glycinergic inhibitory control of spinal lamina I neurons through a presynaptic mechanism.

Clinical implication of voltage-dependent anion channel 1 in uterine cervical cancer and its action on cervical cancer cells.

Two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry were performed to investigate the influence of human nonmetastatic clone 23 type 1 (nm23-H1), a metastasis-associated gene on proteomic alterations in cancer cells of the uterine cervix. It was validated by RT-PCR and Western blot analysis. The expression of voltage-dependent anion channel 1 (VDAC1) was increased in nm23-H1 gene silenced SiHa or CaSki cervical cancer cells. The clinical implication was shown that cervical cancer tissues with positive VDAC1 immunoreactivity exhibited deep stromal invasion (>10 mm in depth) and large tumor size (> 4 cm in diameter). Cervical cancer patients with positive VDAC1 immunoreactivity displayed higher recurrence and poorer overall survival than those with negative VDAC1. Silencing of VDAC1 reduced cell proliferation and migratory ability. Mitochondrial membrane potential was decreased and reactive oxygen species generation was increased in the VDAC1 gene-silenced cervical cancer cells. Cell cycle progression and autophagy were not changed in VDAC1 silencing cells. The cytotoxicity of cisplatin was significantly enhanced by knockdown of cellular VDAC1 and the compounds that interfere with hexokinase binding to VDAC. Therapeutic strategies may be offered using VDAC1 as a target to reduce cell growth and migration, enhance the synergistic therapeutic efficacy of cisplatin and reduce cisplatin dose-limiting toxicity.