Qici Sanling Decoction Suppresses Glutamine Consumption and Bladder Cancer Cell Growth through Inhibiting c-Myc Expression

Traditional Chinese medicine (TCM) is widely used as an alternative therapy for cancer treatment in China. Glutamine catabolism plays an important role in cancer development. Qici Sanling decoction (QCSL) suppresses bladder cancer growth. However, the association between QCSL and glutamine catabolism remains unknown. In this study, different doses of QCSL were applied to T24 cells, followed by the measurements of cell viability and apoptosis using CCK-8 and Annexin V/PI assay, respectively. Furthermore, glutamine consumption was detected using the glutamine assay kit. QCSL was observed to inhibit cell growth and induced cell apoptosis in a dose-dependent manner. Analysis of glutamine consumption revealed that QCSL suppressed glutamine consumption in T24 cells. Furthermore, QCSL decreased the mRNA and protein levels of c-Myc, GLS1, and SLC1A5. All these effects induced by QCSL could be alleviated by c-Myc overexpression, indicating c-Myc was involved in the protective role of QCSL in bladder cancer. In addition, QCSL was found to inhibit tumor growth in the xenograft tumor model. The similar results were obtained in tumor samples that protein levels of c-Myc, GLS1, and SLC1A5 were decreased upon treatment with QCSL. In conclusion, QCSL suppresses glutamine consumption and bladder cancer cell growth through inhibiting c-Myc expression.

Exosome-Transmitted miR-128 Targets CCL18 to Inhibit the Proliferation and Metastasis of Urothelial Carcinoma

Objective: To investigate the regulatory function of exosome-transmitted miR-128 and chemokine (C-C motif) ligand 18 (CCL18) on urothelial carcinomas (UCs).Methods: Tumor tissues, paracancerous tissues, and serum were collected from 20 patients with UCs (diagnosed at Beijing Friendship Hospital, Capital Medical University). CCL18 was detected by immunohistochemistry and ELISA. PCR was used to measure the expression levels of CCL18 and mir-183, miR-128, mir-33a in UCs. We acquired exosomes from mesenchymal stem cells and synthesized exosomes overexpressing miR-128 (HMSC-128-EV). The effects of miR-128 on the migration and invasion abilities, apoptosis and epithelial-mesenchymal transition of BUC T24 cells were investigated by co-culturing HMSC-128-EV. The therapeutic potential of miR-128 on disease models was explored by injecting HMSC-128-EV into nude mice.Results: The expression of CCL18 in UCs was significantly higher than that in normal tissues (p < 0.05), and the serum level of CCL18 in patients with UC was significantly increased compared with those in healthy controls (p < 0.05). CCL18 overexpression or downregulation enhanced or suppressed the proliferation, migration and invasion of BUC T24 cells, resectively (p < 0.05). The exosome-transmitted miR-128 can inhibit cell proliferation (p < 0.05), invasion (p < 0.05), and migration (p < 0.05) in UCs, and these effects can be reversed by CCL18. In terms of apoptosis, miR-128 was able to promote the occurrence of BUC T24 apoptosis (p < 0.05), which can also be reversed by CCL18. In addition, miR-128 can inhibit the proliferation (p < 0.05) and metastasis (p < 0.05) of UCs in nude mice.Conclusion: The miR-128 inhibits the proliferation, invasion, migration of UCs, and promotes its apoptosis by regulating CCL18 secretion.

Indoleamine 2,3-Dioxygenase-1 Expression is Changed During Bladder Cancer Cell Invasion

The severity of the bladder carcinoma (BC) is directly linked to cell invasion and metastasis. Indoleamine 2,3-dioxygenase-1 (IDO-1) is an INF-γ-induced immunomodulating enzyme that has been linked to the cancer cell invasiveness. Because IDO1 is variable among the tumors, we analyzed its expression in the BC invasion using BC mice models and cell culture. MB49 cells were orthotopically or ectopically inoculated in C57Bl6 mice to evaluate IDO1 by immunohistochemistry. For in vitro experiments, expression of IDO1 and INF-γ was evaluated in grade-1 (RT4) and in grade-3 (T24) BC cell lines. Invading and non-invading T24 cells were separated using the Matrigel/Transwell system, of which total RNA was extracted immediately or after 2 weeks of subculture. Finally, IDO1 was silenced in T24 cells to verify its role on cell invasiveness. In both animal models, IDO1 was differentially expressed between non-invading and invading cells. In cell culture, T24 cells expressed more IDO1 than RT4 cells, independently of the INF-γ expression. IDO1 was differentially expressed between non-invading and invading T24 cells, a difference that was lost by long-time subculture. IDO1 silencing resulted in diminished cell invasiveness. In conclusion, IDO1 expression is changed during bladder carcinoma invasion, playing an important role in this process.

Bupivacaine modulates the apoptosis and ferroptosis in bladder cancer via PI3K/AKT pathway

Background The study aimed to explore the effects of local anesthetic bupivacaine on bladder cancer cells in vivo and in vitro. Methods After T24 cells and 5637 cells were treated with different doses of bupivacaine (0-16 mM) for 24 h, MTT assay was used to detect the cytotoxicity, and bupivacaine (0.25, 0.5, 1 mM) was selected for subsequent experiments. Apoptosis was detected by Hoechst 33342 staining and TUNEL. The contents of Fe2+, MDA, GSH and ROS were detected by the corresponding kit. Mitochondrial membrane potential was detected by JC-1 kit. HE staining, TUNEL and immunohistochemistry were used to detect the xenografted tumors. Protein expression was detected by western blot. Results Bupivacaine significantly inhibited the activity of T24 cells and 5637 cells at 0.25-16 mM. Bupivacaine significantly promoted cell apoptosis with increasd concentration. bupivacaine inhibited the expression of Bcl-2 and increased the expression of Bax and cytochrome C. Moreover, bupivacaine promoted the increase of Fe2+ and ROS, and inhibited the expression of xCT and GPX4. Further results showed that bupivacaine decreased mitochondrial membrane potential, reduced GSH, and increased MDA levels. Besides, bupivacaine attenuated the phosphorylation of PI3K, Akt, and mTOR, which might be involved in the regulation of bupivacaine-induced apoptosis and ferroptosis. In addition, bupivacaine suppressed the growth of xenografted tumors, induced apoptosis and ferroptosis, and inhibited the activity of PI3K/AKT signaling pathway in xenografted tumors. Conclusion Bupivacaine could induce apoptosis and ferroptosis by inhibiting PI3K / Akt signaling pathway in bladder cancer cells.

The effect of hypoxia on PD-L1 expression in bladder cancer

Introduction Recent data has demonstrated that hypoxia drives an immunosuppressive tumour microenvironment (TME) via various mechanisms including hypoxia inducible factor (HIF)-dependent upregulation of programmed death ligand 1 (PD-L1). Both hypoxia and an immunosuppressive TME are targetable independent negative prognostic factors for bladder cancer. Therefore we sought to investigate whether hypoxia is associated with upregulation of PD-L1 in the disease. Materials and methods Three human muscle-invasive bladder cancer cell lines (T24, J82, UMUC3) were cultured in normoxia (20% oxygen) or hypoxia (1 and 0.1% oxygen) for 24 h. Differences in PD-L1 expression were measured using Western blotting, quantitative polymerase chain reaction (qPCR) and flow cytometry (≥3 independent experiments). Statistical tests performed were unpaired t tests and ANOVA. For in silico work an hypoxia signature was used to apply hypoxia scores to muscle-invasive bladder cancers from a clinical trial (BCON; n = 142) and TCGA (n = 404). Analyses were carried out using R and RStudio and statistical tests performed were linear models and one-way ANOVA. Results When T24 cells were seeded at < 70% confluence, there was decreased PD-L1 protein (p = 0.009) and mRNA (p < 0.001) expression after culture in 0.1% oxygen. PD-L1 protein expression decreased in both 0.1% oxygen and 1% oxygen in a panel of muscle-invasive bladder cancer cells: T24 (p = 0.009 and 0.001), J82 (p = 0.008 and 0.013) and UMUC3 (p = 0.003 and 0.289). Increasing seeding density decreased PD-L1 protein (p < 0.001) and mRNA (p = 0.001) expression in T24 cells grown in both 20 and 1% oxygen. Only when cells were 100% confluent, were PD-L1 protein and mRNA levels higher in 1% versus 20% oxygen (p = 0.056 and p = 0.037). In silico analyses showed a positive correlation between hypoxia signature scores and PD-L1 expression in both BCON (p = 0.003) and TCGA (p < 0.001) cohorts, and between hypoxia and IFNγ signature scores (p < 0.001 for both). Conclusion Tumour hypoxia correlates with increased PD-L1 expression in patient derived bladder cancer tumours. In vitro PD-L1 expression was affected by cell density and decreased PD-L1 expression was observed after culture in hypoxia in muscle-invasive bladder cancer cell lines. As cell density has such an important effect on PD-L1 expression, it should be considered when investigating PD-L1 expression in vitro.

Novel Dihydroartemisinin Derivative Mito-DHA5 Induces Apoptosis Associated with Mitochondrial Pathway in Bladder Cancer Cells

Background: Bladder cancer is the second most common genitourinary malignancy and the eleventh most common cancer worldwide. Dihydroartemisinin (DHA), a first-line antimalarial drug, has been found to have potent antitumor activity. In our previous study, a novel dihydroartemisinin derivative Mito-DHA5 synthesized in our laboratory has a stronger anti-tumor activity than DHA. In this study, we investigated the apoptotic effect of Mito-DHA5 on bladder cancer T24 cells and molecular mechanisms underlying. Methods: Antitumor activity in vitro was evaluated by MTT and cloning formation assays. Mitochondrial membrane potential (MMP) was detected by JC-1 probe and ROS levels were measured by specific kit. The expression of caspase-3, Bcl-2 and Bax in T24 cells was evaluated by Western blotting. Results: The results showed that Mito-DHA5 reduced cell viability with an IC50 value of 3.2 μM in a dose-dependent manner, induced T24 cell apoptosis at both early and late stages, increased the production of ROS and decreased MMP. Mito-DHA5 could down-regulate the expression of Bcl-2 and Caspase-3, and up-regulate the expression of Bax and cleaved Caspase-3. Conclusion: These data suggested that Mito-DHA5 had a potent inhibitory effect on T24 bladder cancer cell growth and induced these cells apoptosis associated with mitochondrial pathway.

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.

PTCSC1 Promotes Bladder Cancer Progression by AKT Pathway

In bladder cancer the expression of PTCSC1is elevated, but how PTCSC1influencetumor progression are still unclear. We found that in human bladder cancer PTCSC1 is upregulated. The study showedthat the expression level of PTCSC1 is higher in bladder cancer tissuesthan that of PTCSC1 in para-carcinoma tissue. Furthermore, PTCSC1promoted bladder cancer cell migration and invasion, and decreased expression of PTCSC1 inhibited the expression of matrix metalloprotease MMP2and MMP9, and increased the expression level of E-cadherin. PTCSC1 promoted tumor growth in a mouse model of human bladder cancer. Additionally,PTCSC1 shRNA caused a significant decrease in p-AKTexpression in T24 cells and BIU-87 cells, but the overexpression ofPTCSC1got an opposite result.Allthe results showed that PTCSC1 can influencebladder cancer cellmigration and invasion ability by the AKT pathways. PTCSC1 may be an effective therapeutic target in bladder cancer.

Oridonin Promotes Apoptosis and Restrains the Viability and Migration of Bladder Cancer by Impeding TRPM7 Expression via the ERK and AKT Signaling Pathways

Background. Oridonin is a powerful anticancer compound found in Rabdosia rubescens. However, its potential impact on bladder cancer remains uninvestigated. In this work, we aimed to detect the anticancer effect of oridonin on bladder cancer and explore the molecular mechanisms involved. Methods. The anticancer activity of oridonin was assessed in vitro with a CCK8 assay, an annexin V-FITC apoptosis analysis, and colony formation and Transwell migration assays which were performed with the human bladder cancer cell line T24. Levels of apoptosis-related proteins, melastatin transient receptor potential channel 7 (TRPM7), and signaling molecules were examined in oridonin-treated T24 cells by western blotting or RT-PCR. Oridonin anticancer efficacy was further validated in vivo with a T24 xenograft mouse model. Results. Oridonin repressed the proliferative, colony-forming, and migratory capacities of T24 cells, triggered extensive apoptosis in vitro, and retarded tumor growth in vivo. Moreover, oridonin treatment significantly increased expression levels of p53 and cleaved caspase-3 and reduced expression of TRPM7, p-AKT, and p-ERK. Conclusion. Oridonin exhibited outstanding antiproliferative and antimigratory effects on bladder cancer, and these effects were at least partially associated with targeting of TRPM7 through inactivation of the ERK and AKT signaling pathways. These findings provide insight for the clinical application of oridonin in bladder cancer prevention.

The Targeting Mechanism of Long Intergenic Non-Coding RNA 01503 to miR-338-3p and Its Regulation on Cell Behaviors of Bladder Cancer T24 Cells

This study aimed to investigate the effects of long intergenic non-coding RNA 01503 (LINC01503) on cell behaviors of T24 bladder cancer cells, including proliferation and apoptosis, as well as its mechanism for targeting miR-338-3p. First, bladder cancer tissues and the corresponding adjacent tissues were obtained and subjected to RT-qPCR to analyze their LINC01503 and miR-338-3p expressions. Afterward, si-LINC01503, miR-338-3p mimics were transfected into T24 cells and then CCK-8 assay and flow cytometry were applied to analyze the cell behaviors of the T24 cells, including proliferation and apoptosis. In additionally, Western blot was carried out to analyze the protein expressions of Ki-67, Bcl-2, and Bax. Finally, dual luciferase reporter and RT-qPCR assay were used to confirm the targeted function of LINC01503 to miR-338-3p. Our data clearly demonstrates a highly expressed LINC01503 and a lowly expressed miR-338-3p in bladder cancer. Interfering LINC01503 was found to obviously influence some protein expressions, such that it down-regulated Ki-67 and Bcl-2 expressions, but upregulated Bax expression, thus resulting in reduced proliferation and enhanced apoptosis. Meanwhile, miR-338-3p overexpression was found to influence these protein expressions, which was similar to functions of the interfering LINC01503. Furthermore, it was found that LINC01503 directly binds to miR-338-3p and that LINC01503 overexpression could obviously down-regulate miR-338-3p expression, thus weakening the influence of interfering LINC01503 on behaviors of the T24 cells. Our results demonstrate that interfering LINC01503 could affect cell behaviors of T24 bladder cancer cells, inhibit proliferation, and induce apoptosis, which was mediated by the targeted binding of interfering LINC01503to miR-338-3p.