Human Bladder Cancer T24 Cells Research Articles

Transcriptome Analysis Reveals Anti-Cancer Effects of Isorhapontigenin (ISO) on Highly Invasive Human T24 Bladder Cancer Cells.

Bladder cancer, the most common malignancy of the urinary tract, has a poor overall survival rate when the tumor becomes muscle invasive. The discovery and evaluation of new alternative medications targeting high-grade muscle invasive bladder cancer (MIBC) are of tremendous importance in reducing bladder cancer mortality. Isorhapontigenin (ISO), a stilbene derivative from the Chinese herb Gnetum cleistostachyum, exhibits a strong anti-cancer effect on MIBCs. Here, we report the whole transcriptome profiling of ISO-treated human bladder cancer T24 cells. A total of 1047 differentially expressed genes (DEGs) were identified, including 596 downregulated and 451 upregulated genes. Functional annotation and pathway analysis revealed that ISO treatment induced massive changes in gene expression associated with cell movement, migration, invasion, metabolism, proliferation, and angiogenesis. Additionally, ISO treatment-activated genes involved in the inflammatory response but repressed genes involved in hypoxia signaling, glycolysis, the actin cytoskeleton, and the tumor microenvironment. In summary, our whole transcriptome analysis demonstrated a shift in metabolism and altered actin cytoskeleton in ISO-treated T24 cells, which subsequently contribute to tumor microenvironment remodeling that suppresses tumor growth and progression.

Structural features of Prussian Blue-related iron complex - FeT of activity to peroxidate unsaturated fatty acids.

Aim: FeT is a complex of Fe3+, ferricyanide and tartrate, similar in structure to Prussian Blue. Its synthesis was planned to produce a potential antiproliferative drug. Methods: Dynamic light scattering was applied to study nanostructures formed by FeT complexes, while their biological activity was tested following changes in cell proliferation using cultured T24 human bladder cancer cells. Results: The antiproliferative activity of FeT derived from its ability to peroxidate unsaturated fatty acids, which can cause cell death through oxidative stress and/or ferroptosis. FeT molecules associate into drop-like nanostructures in water solutions, between 10-130 nm, which can bind albumin.Conclusion: Fatty acid peroxidation is significantly activated by light. The characteristics and reactivity of FeT represent a prospective application in medicine.

Molecular mechanism of anticancer effect of heat shock protein 90 inhibitor BIIB021 in human bladder cancer cell line

Bladder cancer is a type of urologic malignancy that exhibits significant morbidity, mortality, and treatment costs. Inhibition of heat shock protein 90 (HSP90) activity has been a promising pharmacological strategy for blocking of bladder cancer pathogenesis. BIIB021 is a next-generation HSP90 inhibitor which interrupts ATP hydrolysis process of HSP90 and inhibits the stabilization and correct folding of client proteins. In current study, we aimed to investigate the molecular mechanism of the anticancer activity of BIIB021 in human bladder cancer T24 cells. Our results revealed that nanomolar concentration of BIIB021 decreased viability of T24 cell. BIIB021 downregulated HSP90 expression in T24 cells and inhibited the refolding activity of luciferase in the presence of T24 cell lysate. PCR array data indicated a significant alteration in transcript levels of cancer-related genes involved in metastases, apoptotic cell death, cell cycle, cellular senescence, DNA damage and repair mechanisms, epithelial-to-mesenchymal transition, hypoxia, telomeres and telomerase, and cancer metabolism pathways in T24 cells. All findings hypothesize that BIIB021 could exhibit as effective HSP90 inhibitor in the future for treatment of bladder cancer patients.

SIRT1 Promotes Cisplatin Resistance in Bladder Cancer via Beclin1 Deacetylation-Mediated Autophagy.

Autophagy-dependent cisplatin resistance poses a challenge in bladder cancer treatment. SIRT1, a protein deacetylase, is involved in autophagy regulation. However, the precise mechanism through which SIRT1 mediates cisplatin resistance in bladder cancer via autophagy remains unclear. In this study, we developed a cisplatin-resistant T24/DDP cell line to investigate this mechanism. The apoptosis rate and cell viability were assessed using flow cytometry and the CCK8 method. The expression levels of the relevant RNA and protein were determined using RT-qPCR and a Western blot analysis, respectively. Immunoprecipitation was utilized to validate the interaction between SIRT1 and Beclin1, as well as to determine the acetylation level of Beclin1. The findings indicated the successful construction of the T24/DDP cell line, which exhibited autophagy-dependent cisplatin resistance. Inhibiting autophagy significantly reduced the drug resistance index of these cells. The T24/DDP cell line showed a high SIRT1 expression level. The overexpression of SIRT1 activated autophagy, thereby further promoting cisplatin resistance in the T24/DDP cell line. Conversely, inhibiting autophagy counteracted the cisplatin-resistance-promoting effects of SIRT1. Silencing SIRT1 led to increased acetylation of Beclin1, the inhibition of autophagy, and a reduction in the cisplatin resistance of the T24/DDP cell line. Introducing a double mutation (lysine 430 and 437 to arginine, 2KR) in Beclin-1 inhibited acetylation and activated autophagy, effectively reversing the decreased cisplatin resistance resulting from SIRT1 silencing. In summary, our study elucidated that SIRT1 promotes cisplatin resistance in human bladder cancer T24 cells through Beclin1-deacetylation-mediated autophagy activation. These findings suggest a potential new strategy for reversing cisplatin resistance in bladder cancer.

Brazilin inhibits bladder cancer by promoting cell necroptosis.

Brazilin possesses anticancer effects, but the mechanisms are poorly understood. This study investigated the mechanisms of brazilin-induced cell death in the T24 human bladder cancer cell line. Low serum cell culture and the lactate dehydrogenase assay were used to confirm the antitumor effect of brazilin. Annexin V and propidium iodide double staining, transmission electron microscopy, fluo-3-AM assay for Ca2+ mobilization and caspase activity assay were performed to identify the type of cell death after brazilin treatment. Mitochondria membrane potentials were measured using JC-1. Quantitative real-time polymerase chain reaction and western blot analyses were performed to verify the expression of the necroptosis-related genes and proteins receptor interacting protein 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL). The results showed that brazilin induced necrosis in T24 cells and upregulated the mRNA and protein levels of RIP1, RIP3 and MLKL and Ca2+ influx. The necroptosis-mediated cell death was rescued by the necroptosis inhibitor necrostatin-1 (Nec-1), but not by the apoptosis inhibitor z-VAD-fmk. Brazilin repressed caspase 8 expression and decreased the mitochondrial membrane potentials; both effects were partially reversed by Nec-1. Brazilin induced physiological and morphological changes in T24 cells and RIP1/RIP3/MLKL-mediated necroptosis might be involved. In conclusion, the results confirm the involvement of necroptosis in brazilin-induced cell death and suggest that brazilin could be explored as an anticancer agent against bladder cancer.

Methylseleninic acid induces apoptosis of human bladder cancer cells through the ROS-mediated mitochondrial pathway.

As the most common selenium derivative, methylseleninic acid (MSA) has attracted wide attention. Its apoptotic induction ability and the possible molecular mechanism in human bladder cancer (BC) J82 and T24 cells were investigated in the present study. We found that the survival of J82 and T24 cells were inhibited in a dose-dependent manner after MSA treatment. Propidium iodide (PI) staining and Annexin V-fluorescein isothiocyanate/PI double staining clarified that MSA stocked cells at G2 /M phase and caused apoptosis in J82 and T24 cells. Further, typical morphological features of apoptotic cells were also observed. Accumulation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential were also detected by dichlorodihydrofluorescein diacetate and Rhodamin123 staining. Meanwhile, pretreatment with N-acetylcysteine, an ROS scavenging agent, found that the apoptosis of BC cells induced by MSA was related to the production of ROS. Western blot analysis results showed that MSA interrupted Bax/Bcl-2 balance, stimulated cytochrome c release into the cytoplasm, activated caspase-9 and caspase-3, and finally induced the apoptosis of the BC cells. These findings demonstrated that MSA was able to induce apoptosis in J82 and T24 cells through ROS-mediated mitochondrial apoptosis.

MP14-13 SYNERGISTIC ANTITUMOR ACTIVITY OF PI3K INHIBITION AND IMMUNE CHECKPOINT BLOCKADE IN BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023MP14-13 SYNERGISTIC ANTITUMOR ACTIVITY OF PI3K INHIBITION AND IMMUNE CHECKPOINT BLOCKADE IN BLADDER CANCER Vincent D'Andrea, Chong-Xian Pan, and Juan D. Garisto Vincent D'AndreaVincent D'Andrea More articles by this author , Chong-Xian PanChong-Xian Pan More articles by this author , and Juan D. GaristoJuan D. Garisto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Immune checkpoint blockade (ICB) produces a durable response in only 20% of patients with bladder cancer and adjuncts are needed to render this treatment more robust. We sought to determine the synergistic effects of PI3K inhibition and ICB when utilized in combination for the treatment of bladder cancer using a mouse model. METHODS: Human and mouse bladder cancer cell lines were utilized for in vitro tests of a previously-established PI3K inhibitor, copanlisib. These cells lines were subsequently injected subcutaneously into laboratory mice to determine in vivo activity and the anti-tumor mechanisms of action of PI3K inhibition, ICB (achieved with anti-PD-1 therapy), and synergistic therapy between the two. RESULTS: PI3K inhibition with copanlisib showed a dose-dependent suppression of cell growth in T24 (human bladder cancer cell line) and UPPL (mouse bladder cancer cell line) (Figure 1).Mouse bladder cancer models were generated by subcutaneously implanting UPPL cells in transgenic mice. Compared to the treatments with copanlisib and anti-PD-1 therapy alone, which only slightly delayed tumor growth, the combination treatment significantly delayed tumor growth in these models (Figure 2a). For injected mice, the median survivals of the of the control, copanlisib, and combination groups were 48, 37, and 65 days, respectively (Figure 2b). Tumor volume in the combination group was the smallest when compared with the control group (p<0.001), copanlisib (p=0.001), and anti-PD-1 (p<0.001) monotherapy (Figure 2c). CONCLUSIONS: Combination PI3K inhibition and ICB functions synergistically to promote a pro-inflammatory tumor reaction and has significant anti-tumor effects in bladder cancer. This regimen serves as an enticing possibility to render immunotherapy treatment in bladder cancer more robust, and warrants further investigation for safety and efficacy in human clinical trials. Source of Funding: U54 Grant, R01 Grant, VA Merit Review Grant © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e187 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Vincent D'Andrea More articles by this author Chong-Xian Pan More articles by this author Juan D. Garisto More articles by this author Expand All Advertisement PDF downloadLoading ...

Selective Cytotoxicity and Changes in Protein Expression of T24 Bladder Carcinoma Permanent Cell Line after Treatment with Hemocyanins.

Some molluscan hemocyanins (Hcs) have significant immunological and antitumor potential, enabling their application in oncology. The antitumor activity of Hcs from marine snails Rapana venosa (RvH), giant keyhole limpet Megathura crenulata (KLH) and garden snails Helix lucorum (HlH), as well as their different derivatives, were studied in vitro on a permanent T24 cell line of bladder cancer and normal urothelial cell line HL 10/29 compared to doxorubicin. The antiproliferative activity of the tested Hcs was determined using the WST-1 assay and BrdU ELISA assay. Morphological changes in both urothelial cell lines were confirmed by fluorescence microscopy. The proteomic analysis of a bladder cancer cell line before and after treatment with functional unit (FU) βc-HlH-h using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry revealed differences in the expression of some proteins. Studies prove that the T24 tumor cell line is dose- and time-dependent, sensitive to the action of the tested isoforms, and it glycosylated FU of these hemocyanins. Selective inhibition of T24 cell growth was observed after incubation with structural subunits (βc-HlH, RvHI and RvHII) and FUs (βc-HlH-h and RvHII-e). Additionally, fluorescent microphotographs did not show apoptotic or necrotic alterations in the normal urothelial cell line HL 10/29. The FU βc-HlH-h demonstrated the highest antiproliferative effect (similarly to doxorubicin), in which predominantly apoptotic and less late apoptotic or necrotic changes in the tumor cells were observed. Several downand up-regulated proteins identified by proteome analysis may be associated with the apoptosis pathway. The present study illustrated the selectivity of the cytotoxic effect of Hcs against the Т24 cancer cell line. This is the first report of protein expression in T24 human bladder cancer cells under the influence of FU βc-HlH-h. That is probably due to the specific oligosaccharide structures rich in methylated hexoses exposed on the surface of βc-HlH-h.

(-)-Epigallocatechin-3-Gallate Prevents IL-1β-Induced uPAR Expression and Invasiveness via the Suppression of NF-κB and AP-1 in Human Bladder Cancer Cells.

(-)-Epigallocatechin-3-O-gallate (EGCG), a primary green tea polyphenol, has powerful iron scavengers, belongs to the family of flavonoids with antioxidant properties, and can be used to prevent cancer. Urokinase-type plasminogen activator receptors (uPARs) are glycosylphosphatidylinositol (GPI)-anchored cell membrane receptors that have crucial roles in cell invasion and metastasis of several cancers including bladder cancer. The mechanism of action of EGCG on uPAR expression has not been reported clearly yet. In this study, we investigated the effect of EGCG on interleukin (IL)-1β-induced cell invasion and uPAR activity in T24 human bladder cancer cells. Interestingly, nuclear factor (NF)-κB and activator protein (AP)-1 transcription factors were critically required for IL-1β-induced high uPAR expression, and EGCG suppressed the transcriptional activity of both the ERK1/2 and JNK signaling pathways with the AP-1 subunit c-Jun. EGCG blocked the IL-1β-stimulated reactive oxygen species (ROS) production, in turn suppressing NF-κB signaling and anti-invasion effects by inhibiting uPAR expression. These results suggest that EGCG may exert at least part of its anticancer effect by controlling uPAR expression through the suppression of ERK1/2, JNK, AP-1, and NF-κB.

Iron oxide-alginate-berbamine nanocomposites trigger reactive oxygen species-induced apoptosis through down-regulation of PI3K/AKT signaling in human bladder cancer cells

Globally, bladder cancers are the most common cancers with 81,180 new cases in 2022. Being one of the most important urological cancers it has 17,100 estimated deaths in 2022. Unequivocally the best-known treatment so far is radical cystectomy followed by chemotherapy. Despite the treatment strategies including surgical intervention and adjuvant therapy recurrent rate and tumor development prevention remains as a potential problem. Hence targeted treatment strategies gained more interests and the current exploration examines the potential of iron oxide-alginate-berbamine nanocomposites on human bladder cancer T24 cells. The synthesis of iron oxide-alginate-berbamine nanocomposites was with iron (III) nitrate hexahydrate, sodium alginate, and the phytocomponent berbamine. The iron oxide-alginate-berbamine nanocomposites were then treated with human bladder cancer T24 cells. Interestingly, there was a significant dose-dependent reduction in the cell survival rate (2.5–15 µg/ml). Furthermore, the nanocomposites activated caspase-3,8 and -9 almost two-fold. These caspases are the key players in extrinsic and intrinsic apoptosis pathways. The nanocomposites also elevated the ROS status, thereby inactivating the PI3K/Akt signaling pathway. Thus, the current outcomes indicate that iron oxide-alginate-berbamine nanocomposites could effectively inhibit proliferation and induce apoptosis and block the PI3K/Akt pathway in T24 cells.

Cytotoxic Homo- and Hetero-Dimers of o-toluidine, o-anisidine, and Aniline Formed by In Vitro Metabolism.

Several aromatic amine compounds are urinary bladder carcinogens. Activated metabolites and DNA adducts of polycyclic aromatic amines, such as 4-aminobiphenyl, have been identified, whereas those of monocyclic aromatic amines, such as o-toluidine (o-Tol), o-anisidine (o-Ans), and aniline (Ani), have not been completely determined. We have recently reported that o-Tol and o-Ans are metabolically converted in vitro and in vivo to cytotoxic and mutagenic p-semidine-type dimers, namely 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD) and 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), respectively, suggesting their roles in urinary bladder carcinogenesis. In this study, we found that when o-Tol and o-Ans were incubated with S9 mix, MMBD and MxMxBD as well as two isomeric heterodimers, MMxBD and MxMBD, were formed. Therefore, any two of o-Tol, o-Ans, and Ani (10 mM each) were incubated with the S9 mix for up to 24 h and then subjected to LC-MS to investigate their metabolic kinetics. Metabolic conversions to all nine kinds of p-semidine-type homo- and hetero-dimers were observed, peaking at 6 h of incubation with the S9 mix; MxMxBD reached the peak at 6.1 ± 1.4 μM. Homo- and hetero-dimers containing the o-Ans moiety in the diamine structure showed a faster dimerization ratio, whereas levels of these dimers, such as MxMxBD, markedly declined with further incubation. Dimers containing o-Tol and Ani were relatively stable, even after incubation for 24 h. The electron-donating group of the o-Ans moiety may be involved in rapid metabolic conversion. In the cytotoxic assay, dimers with an o-Ans moiety in the diamine structure and MMBD showed approximately two- to four-fold higher cytotoxicity than other dimers in human bladder cancer T24 cells. These chemical and biological properties of homo- and hetero-dimers of monocyclic aromatic amines may be important when considering the combined exposure risk for bladder carcinogenesis.

Magnetic multi-enzyme cascade combined with liquid chromatography tandem mass spectrometry for fast DNA digestion and quantitative analysis of 5-hydroxymethylcytosine in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone

Long-term exposure to halobenzoquinones (HBQs) can induce genomic damages and abnormal epigenetic modifications. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has shown unique advantages in identification and sensitive analysis of these structurally modified DNA lesions. Prior to MS analysis, genomic DNA needs to be fully digested into mono-nucleosides. Here, we prepared Supernuclease (SN)-, snake venom phosphodiesterase (SVP)- and calf intestinal alkaline phosphatase (CIP)- individually immobilized magnetic nanoparticles (MNPs), and combined them according to certain formula to construct a recyclable SN-SVP-CIP magnetic nanoparticles (SNSC-MNPs) cascade for rapid and efficient DNA digestion. The SNSC-MNPs cascade can fully digest genomic DNA into mono-nucleosides within 30 min. The SNSC-MNPs cascade coupled with HPLC-MS/MS method can accurately and sensitively detect 5-hydroxymethylcytosine (5hmC) changes in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone. The immobilization of enzymes on MNPs can enhance the stability and enzymatic activity of the three enzymes, which guarantees the reusability and longtime preservation of the cascades. The relative digestive efficiencies are among 86% -106% up to ten times of reuse. The newly synthesized SNSC-MNPs cascade coupled with HPLC-MS/MS method is promising for fast identification and analysis of structural modifications in genomic DNA.

High P2X6 receptor expression in human bladder cancer predicts good survival prognosis

As alterations in purinergic signaling have been observed in bladder diseases, we aimed to assess the potential prognostic role of purinergic receptors in bladder cancer in a translational approach based on clinical databases and in vitro data. The prognostic role of purinergic receptors in the survival of patients with bladder cancer and the expression profile of the altered P2 receptors in normal and in tumor samples were determined using The Cancer Genome Atlas databank. In T24 and RT4 human bladder cancer cell lines, the P2 purinergic receptors were characterized by RT-PCR and RT-qPCR analysis including radiotherapy exposure as treatment. The cell number and the cumulative population doubling were also assessed. The expression profile of P2X6 receptor in the cancer pathological stage and in the nodal metastasis status was in agreement with Kaplan-Meier analysis, indicating that high expression of this receptor was related to an increased survival rate in patients with bladder cancer. Of all the P2 receptors expressed on T24 cell line, P2X6 presented high expression after radiotherapy, while it was not altered in RT4 cells. In addition, irradiation promoted a decrease of T24 cell number, but did not change the cell number of RT4 after the same time and radiation dose. Along 7days after irradiation exposure, both cells regrew. However, while P2X6 receptor was downregulated in T24 cells, it was upregulated in RT4 cells. Our findings indicated that high P2X6 receptor expression induced by radiation in T24 cell line may predict a good survival prognostic factor.

Clerodane diterpene induces apoptosis/anoikis and suppresses migration and invasion of human bladder cancer cells through the histone deacetylases, integrin–focal adhesion kinase, and matrix metalloproteinase 9 signalling pathways

Clerodane diterpene, a class of bicyclic diterpenoids, is found in hundreds of plant species. 16-hydroxycleroda-3,13-dien-15,16-olide (CD) can be isolated from the plant Polyalthia longifolia and has been applied against oral cancer and glioma by xenograft model. In this study, we aim to explore its antitumour action by examining its histone deacetylase (HDAC) activity and integrin-associated intracellular signalling pathway on T24 human bladder cancer (BC) cells. Our results revealed that CD-inhibited colony formation, HDAC activity, HDAC (1, 2 and 3) mRNA and cell spreading on fibronectin-coated surfaces in a concentration-dependent manner. Furthermore, decreased cFLIP and increased caspase-8 cleavage accompanied CD-induced cell death. At non-toxic concentrations, CD blocked the migration and invasion of T24 cells. CD hindered migration and invasion by the downregulation of fibronectin, integrin α5β1, β-catenin, FAK, vinculin and Rho A, as well as by reduction of phosphorylated glycogen synthase kinase 3β (pGSK3β), pSrc, pstat3 and pNFκB. We observed that the MMP9 gene was closely linked with prognosis of patients with bladder cancer. MMP9 protein levels and activity were largely attenuated by CD in a concentration-dependent manner. In conclusion, CD-induced caspase-8-dependent apoptosis and suppressed migration and invasion by blocking several intracellular signalling pathways, including downregulation of HDAC activity and integrin-FAK and MMP9 pathways.

Potentiation of Anticancer Effect by Combination of Mushroom Extract and Grapeseed Extract on Human Bladder Cancer Cells

Background: Although bladder cancer is the second-leading urologic cancer next to prostate cancer in the United States, few effective and satisfactory therapeutic options have yet been established. As we have been searching for certain natural remedies with anticancer activity, we came across the two natural products, namely mushroom extract (PL) and grapeseed extract (GSP). Accordingly, we investigated if the combination of these two extracts could further potentiate anticancer effect on bladder cancer cells. Materials and Methods: Human bladder cancer T24 cells were separately treated with PL, GSP, or their combination, and cell viability was determined by MTT assay. To explore the anticancer mechanism, severity of oxidative stress was assessed by lipid peroxidation (LPO) assay, activities of epigenetic regulators were determined by enzymatic assays, and apoptotic regulators were examined by Western blot analysis. Results: PL and GSP alone led to a ~20% and ~25% cell viability reduction at 20 μg/ml and 25 μg/ml, respectively. When the same concentrations of PL and GSP were then combined, cell viability was drastically reduced to merely ~15% ~85% reduction) in a synergistic manner. Concomitantly, the PL/GSP combination led to a ~3-fold increase in oxidative stress, assessed by LPO assay. Activities of DNA methyltransferase and histone deacetylase were significantly lost by ~50% and 55% with the PL/GSP combination, respectively. Moreover, Western blot analysis revealed that bcl-2 (anti-apoptotic regulator) was down-regulated while Bax (pro-apoptotic regulator) was up-regulated with the combination, implying induction of apoptosis. Conclusions: This study shows that the combination of PL and GSP can synergistically induce the significant reduction (~85%) in T24 cell viability, demonstrating an extensive potentiation of anticancer effect. Such a profound effect is accompanied by increased oxidative stress, chromatin modifications, and ultimate apoptosis. Therefore, the PL/GSP combination may offer a more effective and safer regimen for bladder cancer.

Graphene Oxide and Graphene Quantum Dots as Delivery Systems of Cationic Porphyrins: Photo-Antiproliferative Activity Evaluation towards T24 Human Bladder Cancer Cells.

The development of new photodynamic therapy (PDT) agents designed for bladder cancer (BC) treatments is of utmost importance to prevent its recurrence and progression towards more invasive forms. Here, three different porphyrinic photosensitizers (PS) (TMPyP, Zn-TMPyP, and P1-C5) were non-covalently loaded onto graphene oxide (GO) or graphene quantum dots (GQDs) in a one-step process. The cytotoxic effects of the free PS and of the corresponding hybrids were compared upon blue (BL) and red-light (RL) exposure on T24 human BC cells. In addition, intracellular reactive oxygen species (ROS) and singlet oxygen generation were measured. TMPyP and Zn-TMPyP showed higher efficiency under BL (IC50: 0.42 and 0.22 μm, respectively), while P1-C5 was more active under RL (IC50: 0.14 μm). In general, these PS could induce apoptotic cell death through lysosomes damage. The in vitro photosensitizing activity of the PS was not compromised after their immobilization onto graphene-based nanomaterials, with Zn-TMPyP@GQDs being the most promising hybrid system under RL (IC50: 0.37 μg/mL). Overall, our data confirm that GO and GQDs may represent valid platforms for PS delivery, without altering their performance for PDT on BC cells.

Betulinic acid induces autophagy-dependent apoptosis via Bmi-1/ROS/AMPK-mTOR-ULK1 axis in human bladder cancer cells.

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark, exhibits antitumor effects against solid malignancies and triggers autophagy and/or apoptosis in human cancer cells. Nonetheless, the relationship between autophagy and apoptosis and the potential modulatory actions of BA on autophagy-dependent bladder cancer cell death remain unclear. The present study showed that BA exposure significantly suppressed viability, proliferation, and migration of EJ and T24 human bladder cancer cells. These effects reflected caspase 3-mediated apoptosis and could be attenuated or abolished by inhibiting ROS production with N-acetyl-L-cysteine, inhibiting autophagy with chloroquine, or silencing ATG7 with targeted siRNA. BA-induced autophagy was evidenced by epifluorescence imaging of lentivirus-induced expression of mCherry-GFP-LC3B and increased expression of two autophagy-related proteins, LC3B-II and TEM. Moreover, enhanced AMPK phosphorylation and decreased mTOR and ULK-1 phosphorylation suggested BA activates autophagy via the AMPK/mTOR/ULK1 pathway. Accordingly, exposure to dorsomorphin (Compound C), an AMPK inhibitor, and AICAR, an AMPK activator, respectively inhibited and stimulated BA-induced autophagy in EJ and T24 cells. The effects of Bmi-1 overexpression in vitro and decreased Bmi-1 expression in BA-treated T24 cell xenografts in nude mice suggested that downregulation of Bmi-1 is the underlying mechanism in BA-mediated, autophagy-dependent apoptosis.

The Inhibitory Effect of Sulforaphane on Bladder Cancer Cell Depends on GSH Depletion-Induced by Nrf2 Translocation

Sulforaphane (SFN), an isothiocyanate (ITCs) derived from glucosinolate that is found in cruciferous vegetables, has been reported to exert a promising anticancer effect in a substantial amount of scientific research. However, epidemical studies showed inconsistencies between cruciferous vegetable intake and bladder cancer risk. In this study, human bladder cancer T24 cells were used as in vitro model for revealing the inhibitory effect and its potential mechanism of SFN on cell growth. Here, a low dose of SFN (2.5 µM) was shown to promote cell proliferation (5.18–11.84%) and migration in T24 cells, whilst high doses of SFN (>10 µM) inhibited cell growth significantly. The induction effect of SFN on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression at both low (2.5 µM) and high dose (10 µM) was characterized by a bell-shaped curve. Nrf2 and glutathione (GSH) might be the underlying mechanism in the effect of SFN on T24 cell growth since Nrf2 siRNA and GSH-depleting agent L-Buthionine-sulfoximine abolished the effect of SFN on cell proliferation. In summary, the inhibitory effect of SFN on bladder cancer cell growth and migration is highly dependent on Nrf2-mediated GSH depletion and following production. These findings suggested that a higher dose of SFN is required for the prevention and treatment of bladder cancer.

SHOX2 promotes migration, invasion and stemness of bladder cancer cells in vitro

To explore the role of human short stature homeobox 2 (SHOX2) in regulating the migration, invasion and stemness of human bladder cancer cells. We analyzed SHOX2 gene expression in bladder cancer and adjacent tissues based on TCGA database. Univariate survival analysis of SHOX2 gene expression in TCGA-BLCA data was performed using GEPIA. The probable function of SHOX2 was predicted using GSEA. Human bladder cancer T24 cell models of SHOX2 knockdown or overexpression were assessed for changes in migration and invasion abilities using wound healing assay and Transwell assay, and their cancer stem cell-like characteristics were evaluated using tumorsphere formation assay and colony formation assay. Western blotting was used to detect the expressions of epithelial mesenchymal transition (EMT) markers Ecadherin and vimentin and the TGF-β signaling network component TβR-I in the cells. SHOX2 expression was significantly higher in bladder cancer tissues than in the adjacent tissues (P < 0.05), especially in paired tissue specimens (P < 0.01), and was negatively correlated with the overall survival of the patients (P < 0.05). SHOX2 gene expression was correlated positively with EMT-related (P < 0.05) and stemness-related gene signatures (P < 0.01). In T24 cells, SHOX2 knockdown significantly suppressed cell migration and invasion, which was significantly enhanced by SHOX2 overexpression (P < 0.01). The cancer stem cell-like characteristics of T24 cells was repressed by SHOX2 knockdown but significantly enhanced by SHOX2 overexpression (P < 0.01). SHOX2 knockdown induced morphological changes of the cells into epithelioid cells, whereas SHOX2 overexpression induced a mesenchymal morphology of the cells. SHOX2 knockdown increased E-cadherin expression and decreased vimentin and TβR-I expression, while SHOX2 overexpression increased the expressions of vimentin and TβR-I in the cells. SHOX2 promotes the migration, invasion and stemness of human bladder cancer cells possibly by regulating EMT via the TGF-β signaling pathway.

Extraction, Purification, Characterization, and Bioactive Properties of Polysaccharides from Sphallerocarpus gracilis

AbstractFour new polysaccharides, SGP‐1‐4, are isolated and purified from Sphallerocarpus gracilis (S. gracilis) . The monosaccharide analysis revealed that the isolated polysaccharides are composed of different molar ratios of glucose (Glu), galactose (Gal), rhamnose (Rha), mannose (Man), and arabinose (Ara). Congo red test showed that no triple helical structure is detected in SGP‐1‐4. The Zeta potentials of SGP‐1‐4 are negative. The antioxidant activities are evaluated using DPPH and Superoxide radical assays. In vitro antitumor activities are investigated by MTT assay and Calcein‐AM/PI staining confocal imaging. The hypolipidemic activities are measured by the bile‐salt binding capacity in vitro. The results indicated that SGP‐1‐4 possessed significant antioxidant and hypolipidemic abilities. At the concentration of 0.2 mg/mL, the purified polysaccharides possess no significant effects against MGC‐803 (Human gastric cancer cells), A2780 (Human ovarian cancer cells), T24 (Human bladder cancer cells), CNE‐2 (Human nasopharyngeal cancer cells), and 4T1 (Mouse breast cancer cells) tumor cells in vitro. However, the SGP‐2 shows potential as a DOX‐chemotherapy against 4T1 cells, which indicated that SGP‐2 could exert antitumor effects. These findings provide a scientific basis for the further use of S. gracilis polysaccharides as a natural antioxidant, antitumor, and hypolipidemic agent in industry.