Eight undescribed alkaloids named corydalisine D-K (1-7), including one isoquinoline benzopyranone alkaloid (1), one benzocyclopentanone alkaloid (2), four benzofuranone alkaloids (3, 4, and 5a/5b) and two protoberberine alkaloids (6 and 7), along with fourteen known ones, were isolated from the Corydalis saxicola. Their structures, including absolute configurations, were unambiguously identified using spectroscopic techniques, single-crystal X-ray diffraction and electron circular dichroism calculation. Compounds 2, 14 and 21 exhibit antiproliferative activity against five cancer cell lines. The aporphine alkaloid demethylsonodione (compound 14), which exhibited the best activity (IC50 = 3.68 ± 0.25 μM), was subjected to further investigation to determine its mechanism of action against the T24 cell line. The molecular mechanism was related to the arrest of cell cycle S-phase, inhibition of CDK2 expression, accumulation of reactive oxygen species (ROS), induction of cell apoptosis, inhibition of cell migration, and activation of p38 MAPK signaling pathway. The results indicated that 14 could be used as a potential candidate agent for further development of anti-bladder transitional cell carcinoma.

Quinones are chemical compounds produced from the oxidation of phenols. Among the quinones, naphthoquinones stand out as potential antitumor agents. Bladder tumour is the tenth most diagnosed in the world. Based on this, using a urothelial carcinoma cell line (T24), two naphthoquinones had their cytotoxicity tested by the MTT colorimetric method and were submitted to assays of clonogenic survival, morphology, cell cycle, cell migration and species reactive oxygen. The results showed 8-methoxy-α-lapachone and lausone presented selectivity indexes (19.5 and 28.0, respectively) for T24 cells. Moreover, the two naphthoquinones reduced the cell viability, interfered with the process of cell migration, changed the cell cycle kinectics and induced the production of species reactive oxygen (ROS). Additionaly, 8-methoxy-α-lapachone altered the morphology of the cells. In conclusion, the studied naphthoquinones showed potential antiproliferative effects in bladder cancer cells, interfering in cellular processes, possibly through oxidative stress.

This study investigated whether melatonin (Mel) would promote cisplatin to suppress the proliferation and growth of bladder cancer (BC) cells by inhibiting cellular prion protein (PrPC)-mediated cell stress and cell proliferation signaling. An immunohistochemical staining of tissue arrays from BC patients demonstrated that the PrPC expression was significantly upregulated from stage I to III BC (p < 0.0001). The BC cellline of T24 was categorized into G1 (T24), G2 (T24 + Mel/100 μM), G3 (T24+cisplatin/6 μM), G4 (PrPC overexpression in T24 (i.e., PrPC-OE-T24)), G5 (PrPC-OE-T24+Mel), and G6 (PrPC-OE-T24+cisplatin). When compared with a human uroepithelial cell line (SV-HUC-1), the cellular viability/wound healing ability/migration rate were significantly increased in T24 cells (G1) and further significantly increased in PrPC-OE-T24 cells (G4); and they were suppressed in Mel (G2/G5) or cisplatin (G3/G6) treatment (all p < 0.0001). Additionally, the protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrPC), cell cycle/mitochondrial functional integrity (cyclin-D1/clyclin-E1/ckd2/ckd4/mitochondrial-cytochrome-C/PINK1), and cell stress (RAS/c-RAF/p-MEK1/2, p-ERK1/2) markers showed a similar pattern of cell viability among the groups (all p < 0.001). After the BC cell line of UMUC3 was implanted into nude mouse backs, by day 28 mthe BC weight/volume and the cellular levels of PrPC/MMP-2/MMP-9 were significantly, gradually reduced from groups one to four (all p < 0.0001). The protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrPC), cell cycle/mitophagy (cyclin-D1/clyclin-E1/ckd2/ckd4/PINK1), and cell stress (RAS/c-RAF/p-MEK1,2/p-ERK1,2) signaling were significantly, progressively reduced from groups one to four, whereas the protein expressions of apoptotic (Mit-Bax/cleaved-caspase-3/cleaved-PARP) and oxidative stress/mitochondrial damaged (NOX-1/NOX-2/cytosolic-cytochrome-C/p-DRP1) markers expressed an opposite pattern of cell proliferation signaling among the groups (all p < 0.0001). Mel-cisplatin suppressed BC cell growth/proliferation via inhibiting the PrPC in upregulating the cell proliferation/cell stress/cell cycle signaling.

BACKGROUND: Aloperine (ALO) is an important active component of quinolizidine alkaloids in Sophora flavescens A and Sophora alopecuroides L, and has effective anticancer activity against multiple cancers. However, the influence and mechanism of ALO on migration, invasion, and adhesion in bladder cancer cells remain unclear.
OBJECTIVE: The aim of this study is to determine the anticancer effect of ALO on migration, invasion, and adhesion in bladder cancer cells and to investigate its potential TIMP-4-related mechanism.
METHODS: Cell viability, cytotoxicity, wound healing, Transwell invasion, cell adhesion, real-time qPCR, western blot, and ELISA assays were performed to analyze the effect of ALO on migration, invasion, and adhesion in bladder cancer 5637 and UM-UC-3 cells. Furthermore, the anti-TIMP-4 antibody was used to explore the potential effect on ALO-inhibited bladder cancer cells.
RESULTS: We have found that ALO significantly suppressed migration, invasion, and adhesion in bladder cancer cells. Furthermore, ALO could downregulate the expression of MMP-2 and MMP-9 mRNAs and proteins, and increase the expression of TIMP-4 mRNA and protein. Moreover, the anti- TIMP-4 antibody reversed the prevention of migration, invasion, and adhesion in ALO-treated bladder cancer cells.
CONCLUSIONS: The data in this study suggest that ALO suppressed migration, invasion, and adhesion in bladder cancer cells by upregulating the expression of TIMP-4.

The edible Rosa hybrida (RH) petal is utilized in functional foods and cosmetics. Although the biological function of RH petal extract is known, mechanism of action studies involving tumor-associated angiogenesis have not yet been reported. Herein, we investigated the regulatory effect of the ethanol extract of RH petal (EERH) on tumor growth and tumor angiogenesis against bladder cancer. EERH treatment inhibited the bladder carcinoma T24 cell and 5637 cell proliferation because of G1-phase cell cycle arrest by inducing p21WAF1 expression and reducing cyclins/CDKs level. EERH regulated signaling pathways differently in both cells. EERH-stimulated suppression of T24 and 5637 cell migration and invasion was associated with the decline in transcription factor-mediated MMP-9 expression. EERH oral administration to xenograft mice reduced tumor growth. Furthermore, no obvious toxicity was observed in acute toxicity test. Decreased CD31 levels in EERH-treated tumor tissues led to examine the angiogenic response. EERH alleviated VEGF-stimulated tube formation and proliferation by downregulating the VEGFR2/eNOS/AKT/ERK1/2 cascade in HUVECs. EERH impeded migration and invasion of VEGF-induced HUVECs, which is attributed to the repressed MMP-2 expression. Suppression of neo-microvessel sprouting, induced by VEGF, was verified by treatment with EERH using the ex vivo aortic ring assay. Finally, kaempferol was identified as the main active compound of EERH. The present study demonstrated that EERH may aid the development of antitumor agents against bladder cancer.

Photodynamic therapy (PDT) is a potential non-invasive approach for application in oncological diseases, based on the activation of a photosensitizer (PS) by light at a specific wavelength in the presence of molecular oxygen to produce reactive oxygen species (ROS) that trigger the death tumor cells. In this context, porphyrins are interesting PS because they are robust, have high chemical, photo, thermal, and oxidative stability, and can generate singlet oxygen (1O2). However, porphyrins exhibit low solubility and a strong tendency to aggregate in a biological environment which limits their clinical application. To overcome these challenges, we developed hybrid nanostructures to immobilize 5,10,15,20-tetrakis[(4-carboxyphenyl) thio-2,3,5,6-tetrafluorophenyl] (P), a new third-generation PS. The biological effect of this system was evaluated against bladder cancer (BC) cells with or without light exposition. The nanostructure composed of lipid carriers coated by porphyrin-chitosan (P-HNP), presented a size of ca. 130 nm and low polydispersity (ca. 0.25). The presence of the porphyrin-chitosan (P-chitosan) on lipid nanoparticle surfaces increased the nanoparticle size, changed the zeta potential to positive, decreased the recrystallization index, and increased the thermal stability of nanoparticles. Furthermore, P-chitosan incorporation on nanoparticles increased the stability and enhanced the self-organization of the system and the formation of spherical structures, as observed by small-angle X-ray scattering (SAXS) analysis. Furthermore, the immobilization process maintained the P photoactivity and improved the photophysical properties of PS, minimizing its aggregation in the cell culture medium. In the photoinduction assays, the P-HNP displayed high phototoxicity with IC50 3.2-folds lower than free porphyrin. This higher cytotoxic effect can be correlated to the high cellular uptake of porphyrin immobilized, as observed by confocal images. Moreover, the coated nanoparticles showed mucoadhesive properties interesting to its application in vivo. Therefore, the physical and chemical properties of nanoparticles may be relevant to improve the porphyrin photodynamic activity in BC cells.

Hybrid nanoparticles composed of different biopolymers for delivery of enzyme/prodrug systems are of interest for cancer therapy. Hyaluronic acid-coated chitosan nanoparticles (CS/HA NP) were prepared to encapsulate individually an enzyme/pro-drug complex based on horseradish peroxidase (HRP) and indole-3-acetic acid (IAA). CS/HA NP showed size around 158 nm and increase to 170 and 200 nm after IAA and HRP encapsulation, respectively. Nanoparticles showed positive zeta potential values (between +20.36 mV and +24.40 mV) and higher encapsulation efficiencies for both nanoparticles (up to 90 %) were obtained. Electron microscopy indicated the formation of spherical particles with smooth surface characteristic. Physicochemical and thermal characterizations suggest the encapsulation of HRP and IAA. Kinetic parameters for encapsulated HRP were similar to those of the free enzyme. IAA-CS/HA NP showed a bimodal release profile of IAA with a high initial release (72 %) followed by a slow-release pattern. The combination of HRP-CS/HA NP and IAA- CS/HA NP reduced by 88 % the cell viability of human bladder carcinoma cell line (T24) in the concentrations 0.5 mM of pro-drug and 1.2 μg/mL of the enzyme after 24 h.

Perfluorinated iodine alkanes (PFIs) can serve as an important raw materials for the synthesis of various perfluorinated chemical products through telomerization reaction. The estrogenic effects of PFIs have been reported previously by some in vitro and in vivo screening assays. To explore the potential epigenetic toxicity of PFIs, activation of lncRNAs was screened, and the cell motility changes induced by perfluorooctyl iodide (PFOI) were analyzed in this study. High metastatic bladder cell line (T24) was used to investigate the cellular migration function affected by PFOI. PFOI exposure significantly induced the upregulation of lncRNA anril, thorlnc, hotairm1, meg3, and malat1. The migration and invasion of T24 cells were also enhanced upon PFOI exposure. The transcription level of matrix metalloenzyme genes, epidermal growth factors, cytoskeleton genes, and the upstream factors involved in cell motility pathways were examined to illustrate possible mechanisms. Additionally, the basic role of malat1 in cellular motility was investigated by lncRNA knockdown and migration assays. The knockdown of malat1 inhibited the cellular motility induced by PFOI. The levels of MMP-2/-9 genes were also down-regulated by the treatment of si-malat1. Overall, the perturbation of cytoskeleton genes (E-cadherin/N-cadherin) may account for the impact on the motility of T24 cells. Our studies indicate that perfluorinated chemicals might regulate the lncRNAs, thus promoting the metastasis of the tumor cells.

The present study aimed to investigate the roles of Notch1 in the biological processes of bladder cancer cells (BCCs) in vitro. Short hairpin (sh)RNA targeting Notch1 was designed and constructed, and the T24 and 5637 BCCs were selected for transfection. The cells were classified into two groups: shRNA negative control (NC) and Notch1 shRNA. MTT and Transwell assays, and flow cytometry were performed to examine the changes in cell proliferation, invasiveness, and apoptosis, respectively. In addition, reverse transcription-quantitative PCR and western blot analysis was used to detect the mRNA and protein expression levels of apoptosis-related proteins (Bax, Bid and Bcl2) and epithelial-mesenchymal transition factors (vimentin and E- and N-cadherin). Compared with that in the shRNA NC group, the Notch1 shRNA group showed significantly decreased cell proliferation rate and invasiveness; increased apoptotic rate; elevated mRNA expression levels of Bad, Bid and E-cadherin; and reduced mRNA expression levels of Bcl2, N-cadherin and vimentin. The trends for protein expression levels were the same as those for mRNA levels. Notch1 silencing inhibited invasion and promoted apoptosis of BCCs.

Chemoresistance represents one of the main obstacles in treating several types of cancer, including bladder and ovarian cancers, and it is characterized by an increase of cellular antioxidant potential. Nrf2 and YAP proteins play an important role in increasing chemoresistance and in inducing antioxidant enzymes. It has been reported that Ailanthone (Aila), a compound extracted from the Ailanthus Altissima, has an anticancer activity toward several cancer cell lines, including chemoresistant cell lines. We have examined the effect of Aila on proliferation, migration and expression of Nrf2 and YAP proteins in A2780 (CDDP-sensitive) and A2780/CP70 (CDDP-resistant) ovarian cancer cells. Furthermore, to clarify the mechanism of Aila action we extended our studies to sensitive and CDDP-resistant 253J-BV bladder cancer cells, which have been used in a previous study on the effect of Aila. Results demonstrated that Aila exerted an inhibitory effect on growth and colony formation of sensitive and CDDP-resistant ovarian cancer cells and reduced oriented cell migration with higher effectiveness in CDDP resistant cells. Moreover, Aila strongly reduced Nrf2 and YAP protein expression and reduced the expression of the Nrf2 target GSTA4, and the YAP/TEAD target survivin. In CDDP-resistant ovarian and bladder cancer cells the intracellular oxidative stress level was lower with respect to the sensitive cells. Moreover, Aila treatment further reduced the superoxide anion content of CDDP-resistant cells in correlation with the reduction of Nrf2 and YAP proteins. However, Aila treatment increased Nrf2 and YAP mRNA expression in all cancer cell lines. The inhibition of proteolysis by MG132, a proteasoma inhibitor, restored Nrf2 and YAP protein expressions, suggesting that the Aila effect was at post-translational level. In accordance with this observation, we found an increase of the Nrf2 inhibitor Keap1, a reduction of p62/SQSTM1, a Nrf2 target which leads Keap1 protein to the autophagic degradation, and a reduction of P-YAP. Moreover, UCHL1 deubiquitinase expression, which was increased in bladder and ovarian resistant cells, was down-regulated by Aila treatment. In conclusion we demonstrated that Aila can reduce proliferation and migration of cancer cells through a mechanism involving a post translational reduction of Nrf2 and YAP proteins which, in turn, entailed an increase of oxidative stress particularly in the chemoresistant lines.