Validation of hyperthermia as an enhancer of chemotherapeutic efficacy: insights from a bladder cancer organoid model.

OBJECTIVE: This study aimed to evaluate the combined efficacy of hyperthermia and chemotherapy using a bladder cancer organoid model and to explore hyperthermia-related molecular pathways. METHOD: Tumor organoids were generated by embedding RT4 bladder cancer cells into Matrigel. The resulting organoids were treated with pirarubicin or gemcitabine at 37 °C or 42 °C. Proliferation was determined by Ki67 immunofluorescence staining, and apoptosis was assessed using a TdT-mediated dUTP nick end labeling (TUNEL) assay. RNA sequencing was used to identify the differentially expressed genes. RESULTS: Bladder cancer organoids were successfully established and exhibited robust proliferative abilities. Treatment with gemcitabine or pirarubicin under hyperthermic conditions caused pronounced structural damage to the organoids and increased cell death compared to that in the normothermically treated group. Furthermore, Ki67 labeling and TUNEL assays showed that the hyperthermia chemotherapy group showed a significantly reduced proliferation rate and high level of apoptosis. Finally, RNA sequencing revealed the IFN-γ signaling pathway to be associated with hyperthermia. CONCLUSION: Overall, hyperthermia combined with chemotherapy exerted better therapeutic effects than those of normothermic chemotherapy in grade 1-2 non-muscle-invasive bladder cancer, potentially through activation of the IFN-γ-JAK-STAT pathway.

Study on the Anti-Ulcerative Colitis Effect of Pseudo-Ginsenoside RT4 Based on Gut Microbiota, Pharmacokinetics, and Tissue Distribution.

The purpose of this study was to explore the therapeutic effect of the oral administration of pseudo-ginsenoside RT4 (RT4) on ulcerative colitis (UC), and to determine the rate of absorption and distribution of RT4 in mice with UC. Balb/c mice were induced using dextran sulfate sodium salts (DSS) to establish the UC model, and 10, 20, or 40 mg/kg of RT4 was subsequently administered via gavage. The clinical symptoms, inflammatory response, intestinal barrier, content of total short-chain fatty acids (SCFAs), and gut microbiota were investigated. Caco-2 cells were induced to establish the epithelial barrier damage model using LPS, and an intervention was performed using 4, 8, and 16 µg/mL of RT4. The inflammatory factors, transient electrical resistance (TEER), and tight-junction protein expression were determined. Finally, pharmacokinetic and tissue distribution studies following the intragastric administration of RT4 in UC mice were performed. According to the results in mice, RT4 decreased the disease activity index (DAI) score, restored the colon length, reduced the levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß), and boosted the levels of immunosuppressive cytokine IL-10, increased the content of SCFAs, improved the colonic histopathology, maintained the ultrastructure of colonic mucosal epithelial cells, and corrected disturbances in the intestinal microbiota. Based on the results in caco-2 cells, RT4 reduced the levels of TNF-α, IL-6, and IL-1ß; protected integrity of monolayers; and increased tight-junction protein expression. Additionally, the main pharmacokinetic parameters (Cmax, Tmax, t1/2, Vd, CL, AUC) were obtained, the absolute bioavailability was calculated as 18.90% ± 2.70%, and the main distribution tissues were the small intestine and colon. In conclusion, RT4, with the features of slow elimination and directional distribution, could alleviate UC by inhibiting inflammatory factors, repairing the intestinal mucosal barrier, boosting the dominant intestinal microflora, and modulating the expression of SCFAs.

Prenatal evaluation of fetal atrioventricular valves by real-time 4D volume imaging with electronic matrix probe.

BACKGROUND: The purpose of this study is to evaluate the feasibility using real-time four-dimensional (RT 4D) volume imaging with electronic matrix probe to observe the morphology of atrioventricular valves in normal and abnormal fetuses, measure the area and circumference of atrioventricular valves in normal fetuses and analyze the correlation with gestational age. METHODS: RT 4D volume imaging with electronic matrix probe was used to collect cardiac volume data of 162 normal fetuses with the gestational age from 22 to 32 weeks and 19 fetuses with atrioventricular valves abnormalities were also enrolled. All the volume data were analyzed and processed in real-time. The morphology of mitral and tricuspid valves was observed in surface mode. The area and circumference of valves were measured in a 4D render view at the end of diastole and analyzed the correlation with gestational age. RESULTS: In 148 of 162 fetuses (91%), the 4D rendered image could be successfully obtained, which clearly showed the morphology of the atrioventricular valves. The area and circumference of mitral and tricuspid valves were positively correlated with gestational age (P < 0.01). Furthermore, 4D rendered images were successfully obtained in 17 of 19 fetuses (89%) with atrioventricular valves abnormalities. CONCLUSIONS: The reference range of the area and circumference of atrioventricular valves in normal fetuses at different gestational weeks could be determined by using the RT 4D volume imaging with electronic matrix probe, which can provide certain diagnostic information for the clinic. The RT 4D images could display the valves morphology vividly in both normal and abnormal fetuses, including some subtle lesions which are not identified by traditional two-dimensional (2D) echocardiography. It is feasible to use the RT 4D volume imaging with electronic matrix probe to perform the prenatal evaluation in the fetal atrioventricular valves.

ALDOLASE A regulates invasion of bladder cancer cells via E-cadherin-EGFR signaling.

Glycolysis and glycogenesis are known to be tightly associated with cancer cell migration. However, their roles in bladder cancer have not been reported. In this study, ALDOLASE A (ALDOA) was identified in a coexpression network generated using glycolysis- and glycogenesis-related genes in Kyoto Encyclopedia of Genes and Genomes. ALDOA was located in the central region in the network, and the cancer genome atlas (TCGA) data suggest that ALDOA expression levels are associated with viability in patients with cancer at the middle and late stages. Bladder cancer cell lines, T24 and RT4, were used to knockdown (sh) or overexpress (OE) ALODA to analyze its role. The sh-ALDOA reduced cell viability, colony formation rate, and invasion cell number; while OE had an opposite effect compared with sh-ALDOA. Further, the sh-ALDOA expression induced E-cadherin level while reduced N-cadherin and vimentin levels. The OE cells reduced E-cadherin and induced N-cadherin and vimentin levels. In addition, epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and AKT serine/threonine kinase (AKT) phosphorylation levels are all reduced in sh-ALODA while activated in OE cells compared with the control group. But either sh-ALODA or OE did not change total protein levels of EGFR, MAPK, and AKT. To further analyze E-cadherin function in ALDOA regulation on bladder cancer cells, sh-ALDOA and sh-E-cadherin were cotransfected in T24 and RT4 cells. The results indicated that sh-ALDOA and sh-E-cadherin expressions eliminated sh-ALDOA function, resulting similar cell viability, colony formation rate, and invasion cell number with control group. Also, sh-ALDOA and shE-cadherin expressions increased EGFR, MAPK, and AKT phosphorylation levels; and the levels were similar to the control group. But, sh-ALDOA and sh-E-cadherin expressions did not change N-cadherin and vimentin levels, which maintain similar levels with sh-ALDOA-expressing cells. Taken together, these results suggest that ALDOA might play an important function in bladder cancer and its action may be though E-cadherin-EGFR signaling.

Upregulation of microRNA 344a-3p is involved in curcumin induced apoptosis in RT4 schwannoma cells.

BACKGROUND: Schwannoma arising from peripheral nervous sheaths is a benign tumor. METHODS: To evaluate cell cytotoxicity, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction and terminal deoxynucleotidyltransferase UTP nick-end labeling (TUNEL) assays were used. A microRNA (miRNA) array was used to identify the miRNAs involved in curcumin-induced apoptosis. To examine miRNA expression, quantitative RT-PCR was used. RESULTS: In this study, curcumin exerted cellular cytotoxicity against RT4 schwannoma cells, with an increase in TUNEL-positive cells. Curcumin also activated the expression of apoptotic proteins, such as polyADP ribose polymerase, caspase-3, and caspase-9. The miRNA array revealed that seven miRNAs (miRNA 350, miRNA 17-2-3p, let 7e-3p, miRNA1224, miRNA 466b-1-3p, miRNA 18a-5p, and miRNA 322-5p) were downregulated following treatment with both 10 and 20 µM curcumin in RT4 cells, while four miRNAs (miRNA122-5p, miRNA 3473, miRNA182, and miRNA344a-3p) were upregulated. Interestingly, transfection with a miRNA 344a-3p mimic downregulated the mRNA expression of Bcl2 and upregulated that of Bax, Curcumin treatment in RT 4 cells also reduced the mRNA expression of Bcl2 and enhanced expression of Bax, Overexpression of miRNA344a-3p mimic combined with curcumin treatment activated the expression of apoptotic proteins, including procaspase-9 and cleaved caspase-3 while inhibition of miRNA 344a-3p using miR344a-3p inhibitor repressed cleaved caspase-3 and -9 in curcumin treated RT-4 cells compared to control. CONCLUSIONS: Our findings demonstrate that curcumin induces apoptosis in schwannoma cells via miRNA 344a-3p. Thus, curcumin may serve as a potent therapeutic agent for the treatment of schwannoma.

The progression from a lower to a higher invasive stage of bladder cancer is associated with severe alterations in glucose and pyruvate metabolism.

Cancer cells present a particular metabolic behavior. We hypothesized that the progression of bladder cancer could be accompanied by changes in cells glycolytic profile. We studied two human bladder cancer cells, RT4 and TCCSUP, in which the latter represents a more invasive stage. The levels of glucose, pyruvate, alanine and lactate in the extracellular media were measured by Proton Nuclear Magnetic Resonance. The protein expression levels of glucose transporters 1 (GLUT1) and 3 (GLUT3), monocarboxylate transporter 4 (MCT4), phosphofructokinase-1 (PFK1), glutamic-pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) were determined. Our data showed that glucose consumption and GLUT3 levels were similar in both cell lines, but TCCSUP cells displayed lower levels of GLUT1 and PFK expression. An increase in pyruvate consumption, concordant with the higher levels of lactate and alanine production, was also detected in TCCSUP cells. Moreover, TCCSUP cells presented lower protein expression levels of GPT and LDH. These results illustrate that bladder cancer progression is associated with alterations in cells glycolytic profile, namely the switch from glucose to pyruvate consumption in the more aggressive stage. This may be useful to develop new therapies and to identify biomarkers for cancer progression.

Novel ginsenoside monomer RT4 promotes colitis repair in mice by regulating miR-144-3p/SLC7A11 signaling pathway.

BACKGROUND: Ginsenoside RT4 (RT4) is a new biologically active compound extracted from ginseng that possesses numerous medicinal and pharmacological properties. However, its potential therapeutic effect of ginsenoside RT4 on ulcerative colitis remains unknown. METHODS AND RESULTS: In this study, we investigated the anti-inflammatory effects of ginsenoside RT4 and its underlying molecular mechanism in the treatment of ulcerative colitis mice induced with dextran sulfate sodium (DSS). Our results demonstrate that ginsenoside RT4 effectively reduced weight, shortening of colonic tract length, colonic bowel damage, and disease activity index (DAI) scores in DSS-induced colitis mice. Additionally, ginsenoside RT4 regulates miR-144-3p expression in DSS-induced colitis mice, and we further confirmed that the solute carrier family 7 member 11 (SLC7A11) was the target gene of miR-144-3p by database analysis. Finally, ginsenoside RT4 inhibits the activation of the miR-144-3p/SLC7A11 signaling pathway, which alleviates colitis. Ginsenoside RT4 significantly decreased the expression of pro-inflammatory cytokines TNF-α and IL-1ß and increased the anti-inflammatory cytokine IL-10. CONCLUSIONS: These findings suggest that ginsenoside RT4 may have therapeutic potential for treating ulcerative colitis by downregulating levels of miR-144-3p/SLC7A11 signaling pathway, which are expected to be useful in treating clinical ulcerative colitis.

The protective role of mesencephalic astrocyte-derived neurotrophic factor in endoplasmic reticulum stress in RT4-D6P2T schwannoma sells with the S63del MPZ mutation.

BACKGROUND: Endoplasmic reticulum stress (ERS) occurred in S63del mutant CMT1B mice model, and few drugs has been studied. Mesencephalic astrocyte-derived neurotrophic factor (MANF) can inhibit ERS. This study aimed at investigating the effect of MANF on ERS of RT4-D6P2T schwannoma cells with S63del MPZ Mutation. METHODS: Experimental grouping: blank control group, blank control + MANF group, lentivirus group, lentivirus + MANF group, S63del MPZ group, S63del MPZ + MANF group. CCK8 and Annexin-FITC/PI were used to detect cell proliferation and apoptosis. JC-1 was used to detect ΔΨm. MANF, GRP78 and CHOP mRNA and protein were detected by using RT-qPCR, western blotting and immunofluorescence. ER-Tracker and mito-tracker were used to observe the morphology of endoplasmic reticulum (ER) and mitochondria. RESULTS: Cell proliferation decreased (p < 0.001) and apoptosis increased (p < 0.001) in S63del MPZ group; cell proliferation increased (p = 0.005) and apoptosis decreased (p < 0.001) in S63del MPZ + MANF group. ΔΨm decreased (p < 0.001), MANF, GRP78, CHOP, ATF6, P-PERK/PERK, P-IRE1/IRE1, Bax and Caspase3 increased (p < 0.001) and Bcl2 decreased (p < 0.001) in S63del MPZ group. MANF, GRP78, CHOP, ATF6, P-PERK/PERK, P-IRE1/IRE1, Bax and Caspase3 decreased (p < 0.001) and Bcl2 increased (p < 0.001) in S63del MPZ group. CONCLUSIONS: ERS occurred in RT4-D6P2T cells with S63del MPZ mutation, and MANF exerted protective effect in RT4-D6P2T cells with S63del MPZ mutation.

Hypoxia-Induced Suppression of Antiapoptotic Bcl-2 Expression in Human Bladder Tumor Cells Is Regulated by Caveolin-1-Dependent Adenosine Monophosphate-Activated Protein Kinase Activity.

PURPOSE: Adenosine monophosphate-activated protein kinase (AMPK) is thought to inhibit cell proliferation or promote cell death, but the details remain unclear. In this study, we propose that AMPK inhibits the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) by relying on the hypoxia-inducible factor 1 alpha (HIF-1α)-induced caveolin-1 (Cav-1) expression pathway in noninvasive human bladder tumor (RT4) cells. METHODS: In cells exposed to a hypoxic environment (0.5% oxygen), the levels of expression and phospho-activity of the relevant signaling enzymes were examined via Western blots and reverse transcription-polymerase chain reaction. Cell proliferation was assessed using a Cell Counting Kit-8 assay. RESULTS: The level of expression of Cav-1 was very low or undetectable in RT4 cells. Hypoxia was associated with significantly decreased cell growth, along with marked induction of HIF-1α and Cav-1 expression; additionally, it suppressed the expression of the antiapoptotic marker Bcl-2 while leaving AMPK activity unchanged. Under hypoxic conditions, HIF-1α acts as a transcription factor for Cav-1 mRNA gene expression. The cell growth and Bcl-2 expression suppressed under hypoxia were reversed along with decreases in the induced HIF-1α and Cav-1 levels by AMPK activation with metformin (1mM) or phenformin (0.1mM). In addition, pretreatment with AMPK small interfering RNA not only increased the hypoxia-induced expression of HIF-1α and Cav-1, but also reversed the suppression of Bcl-2 expression. These results suggest that HIF-1α and Cav-1 expression in hypoxic environments is regulated by basal AMPK activity; therefore, the inhibition of Bcl-2 expression cannot be expected when AMPK activity is suppressed, even if Cav-1 expression is elevated. CONCLUSION: For the first time, we find that AMPK activation can regulate HIF-1α induction as well as HIF-1α-induced Cav1 expression, and the hypoxia-induced inhibitory effect on the antiapoptotic pathway in RT4 cells is due to Cav-1-dependent AMPK activity.

Mitochondrial Damage-Associated Molecular Patterns of Injured Axons Induce Outgrowth of Schwann Cell Processes.

Activated Schwann cells put out cytoplasmic processes that play a significant role in cell migration and axon regeneration. Following nerve injury, axonal mitochondria release mitochondrial damage-associated molecular patterns (mtDAMPs), including formylated peptides and mitochondrial DNA (mtDNA). We hypothesize that mtDAMPs released from disintegrated axonal mitochondria may stimulate Schwann cells to put out cytoplasmic processes. We investigated RT4-D6P2T schwannoma cells (RT4) in vitro treated with N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP) or cytosine-phospho-guanine oligodeoxynucleotide (CpG ODN) for 1, 6 and 24 h. We also used immunohistochemical detection to monitor the expression of formylpeptide receptor 2 (FPR2) and toll-like receptor 9 (TLR9), the canonical receptors for formylated peptides and mtDNA, in RT4 cells and Schwann cells distal to nerve injury. RT4 cells treated with fMLP put out a significantly higher number of cytoplasmic processes compared to control cells. Preincubation with PBP10, a selective inhibitor of FPR2 resulted in a significant reduction of cytoplasmic process outgrowth. A significantly higher number of cytoplasmic processes was also found after treatment with CpG ODN compared to control cells. Pretreatment with inhibitory ODN (INH ODN) resulted in a reduced number of cytoplasmic processes after subsequent treatment with CpG ODN only at 6 h, but 1 and 24 h treatment with CpG ODN demonstrated an additive effect of INH ODN on the development of cytoplasmic processes. Immunohistochemistry and western blot detected increased levels of tyrosine-phosphorylated paxillin in RT4 cells associated with cytoplasmic process outgrowth after fMLP or CpG ODN treatment. We found increased immunofluorescence of FPR2 and TLR9 in RT4 cells treated with fMLP or CpG ODN as well as in activated Schwann cells distal to the nerve injury. In addition, activated Schwann cells displayed FPR2 and TLR9 immunostaining close to GAP43-immunopositive regenerated axons and their growth cones after nerve crush. Increased FPR2 and TLR9 immunoreaction was associated with activation of p38 and NFkB, respectively. Surprisingly, the growth cones displayed also FPR2 and TLR9 immunostaining. These results present the first evidence that potential mtDAMPs may play a key role in the induction of Schwann cell processes. This reaction of Schwann cells can be mediated via FPR2 and TLR9 that are canonical receptors for formylated peptides and mtDNA. The possible role for FPR2 and TLR9 in growth cones is also discussed.

Chitosan hydrochloride has no detrimental effect on bladder urothelial cancer cells.

Bladder cancer is among the most common and aggressive human malignant carcinomas, thus targeting and removal of bladder cancer cells is still a challenge. Although it is well known that chitosan hydrochloride (CH-HCl) causes desquamation of normal urothelial cells, its effect on cancer urothelial cells has not been recognized yet. In this in vitro study, we analyzed the cytotoxicity of 0.05% CH-HCl on three urothelial models: two cancer urothelial models, i.e. invasive and papillary urothelial neoplasms, and a normal urothelial model. The cytotoxicity of CH-HCl was evaluated with viability tests, transepithelial resistance (TER) measurements, and electron microscopy. TER measurements showed that 15-minute treatment with CH-HCl caused no reduction in TER of the cancer models, whereas the TER of the normal urothelial model significantly decreased. Furthermore, after CH-HCl treatment, the viability of cancer cells was reduced by only 5%, whereas the viability of normal cells was reduced by 30%. Ultrastructural analysis revealed necrotic cell death in all cases. We have demonstrated that although CH-HCl increases the mortality of cancer urothelial cells, it increases the mortality of normal urothelial cells even more so. However, shorter 2-minute CH-HCl treatment only temporarily increases the permeability of normal urothelial model, i.e. disrupts tight junctions and reduces TER without comprising cell viability, and enables the complete recovery of the permeability barrier after 24h. Overall, our results suggest that CH-HCl cannot be used as a self-sufficient anticancer agent for urothelial bladder cancer treatment; nevertheless a possibility of its use as an enhancer of cytostatic treatment is discussed.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening.

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 µm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 µg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

Effect of Varying Magnetic Fields on Targeted Gene Delivery of Nucleic Acid-Based Molecules.

Several physical methods have been developed to introduce nucleic acid expression vectors into mammalian cells. Magnetic transfection (magnetofection) is one such transfection method, and it involves binding of nucleic acids such as DNA, RNA or siRNA to magnetic nanoparticles followed by subsequent exposure to external magnetic fields. However, the challenge between high efficiency of nucleic acid uptake by cells and toxicity was not totally resolved. Delivery of nucleic acids and their transport to the target cells require carefully designed and controlled systems. In this study, we introduced a novel magnetic system design providing varying magnet turn speeds and magnetic field directions. The system was tested in the magnetofection of human breast (MCF-7), prostate (DU-145, PC-3) and bladder (RT-4) cancer cell lines using green fluorescent protein DNA as a reporter. Polyethylenimine coated superparamagnetic iron oxide nanoparticles (SPIONs) were used as nucleic acid carriers. Adsorption of PEI on SPION improved the cytocompatibility dramatically. Application of external magnetic field increased intracellular uptake of nanoparticles and transfection efficiency without any additional cytotoxicity. We introduce our novel magnetism-based method as a promising tool for enhanced nucleic acid delivery into mammalian cells.