naphthoquinones and Urinary-Bladder-Neoplasms

Naphthoquinones are natural plants products or synthesized compounds. They have α, β-cyclic aromatic dienones structure with a naphthalene skeleton. Little is known about naphthoquinone and nothing about naphtho [2,3-b] thiophen-4,9-quinone effects on bladder cancer. In this study, a naphthoquinone containing a hetero sulfur atom was synthesized using classical synthetic method. The molecular structure was elucidated by NMR techniques and the antitumor effects were evaluated on bladder tumor cell lines with different

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Naphthoquinones; Reactive Oxygen Species; Thiophenes; Urinary Bladder; Urinary Bladder Neoplasms

Shikonin, a natural naphthoquinone compound, has a wide range of pharmacological effects, but its anti-tumor effect and underlying mechanisms in bladder cancer remain unclear.. We aimed to investigate the role of shikonin in bladder cancer in vitro and in vivo in order to broaden the scope of shikonin's clinical application.. We performed MTT and colony formation to detect the inhibiting effect of shikonin on bladder cancer cells. ROS staining and flow cytometry assays were performed to detect the accumulation of ROS. Western blotting, siRNA and immunoprecipitation were used to evaluate the effect of necroptosis in bladder cancer cells. Transmission electron microscopy and immunofluorescence were used to examine the effect of autophagy. Nucleoplasmic separation and other pharmacological experimental methods described were used to explore the Nrf2 signal pathway and the crosstalk with necroptosis and autophagy. We established a subcutaneously implanted tumor model and performed immunohistochemistry assays to study the effects and the underlying mechanisms of shikonin on bladder cancer cells in vivo.. The results showed that shikonin has a selective inhibitory effect on bladder cancer cells and has no toxicity on normal bladder epithelial cells. Mechanically, shikonin induced necroptosis and impaired autophagic flux via ROS generation. The accumulation of autophagic biomarker p62 elevated p62/Keap1 complex and activated the Nrf2 signaling pathway to fight against ROS. Furthermore, crosstalk between necroptosis and autophagy was present, we found that RIP3 may be involved in autophagosomes and be degraded by autolysosomes. We found for the first time that shikonin-induced activation of RIP3 may disturb the autophagic flux, and inhibiting RIP3 and necroptosis could accelerate the conversion of autophagosome to autolysosome and further activate autophagy. Therefore, on the basis of RIP3/p62/Keap1 complex regulatory system, we further combined shikonin with late autophagy inhibitor(chloroquine) to treat bladder cancer and achieved a better inhibitory effect.. In conclusion, shikonin could induce necroptosis and impaired autophagic flux through RIP3/p62/Keap1 complex regulatory system, necroptosis could inhibit the process of autophagy via RIP3. Combining shikonin with late autophagy inhibitor could further activate necroptosis via disturbing RIP3 degradation in bladder cancer in vitro and in vivo.

Topics: Autophagy; Cell Death; Humans; Kelch-Like ECH-Associated Protein 1; Naphthoquinones; Necroptosis; NF-E2-Related Factor 2; Reactive Oxygen Species; Urinary Bladder Neoplasms

Topics: Animals; Apoptosis; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthoquinones; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Fatty acid synthase (FASN) catalyzing the terminal steps in the de novo biogenesis of fatty acids is correlated with low survival and high disease recurrence in patients with bladder cancer. Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels and provides a growth advantage to tumors. However, it is unclear whether the change of PKM2 has an effect on FASN and what is the mechanisms underlying. Here we describe a novel function of PKM2 in control of lipid metabolism by mediating transcriptional activation of FASN, showing the reduced expression of sterol regulatory element binding protein 1c (SREBP-1c). We first discovered that PKM2 physically interacts with the SREBP-1c using biochemical approaches, and downregulation of PKM2 reduced the expression of SREBP-1c by inactivating the AKT/mTOR signaling pathway, which in turn directly suppressed the transcription of major lipogenic genes FASN to reduce tumor growths. Furthermore, either PKM2 inhibitor-Shikonin or FASN inhibitor-TVB-3166 alone induced a strong antiproliferative and anticolony forming effect in bladder cancer cell line. The combination of both inhibitors exhibits a super synergistic effect on blocking the bladder cancer cells growth. It provides a new target and scientific basis for the treatment of bladder cancer.

Topics: Azetidines; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Fatty Acid Synthase, Type I; Gene Expression Regulation, Neoplastic; Humans; Lipogenesis; Membrane Proteins; Naphthoquinones; Nitriles; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Pyrazoles; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Thyroid Hormone-Binding Proteins; Thyroid Hormones; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms

Bladder cancer is a "Warburg-like" tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment.. HTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hexosaminidase assay. Western blot and Blue-native analysis was performed for protein expression of PKM2. Shikonin, an herb that is known to bind and inhibit PKM2, was utilized to determine if PKM2 has a role in glucose usage and cellular proliferation in bladder cancer cells by caspase activity assay. Institutional review board approval was obtained to collect healthy control and bladder cancer patient urine samples. The ScheBo M2-PK EDTA Plasma Test was performed on urine samples to assess urine Tumor M2-PK values.. The three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL). Similarly, low doses of glucose (25mg/dL) result in reduced proliferation. Increased cell growth in higher glucose concentration correlated with up-regulation of PKM2 protein expression. Shikonin, a PKM2 inhibitor, reduced cell proliferation and switched PKM2 isoforms from the dimer to tetramer. Lastly, dimer PKM2 (Tumor-M2PK) levels were assessed in the urine samples from bladder cancer (Bca) patients and healthy controls. Tumor M2-PK significantly correlated with the presence of BCa in our subjects.. Our studies demonstrate the potential of PKM2, specifically the dimer (Tumor-M2PK) as a target of drug therapy and as a urinary marker for bladder cancer.

Topics: Adult; Aged; Biomarkers, Tumor; Carrier Proteins; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Drugs, Chinese Herbal; Female; Glucose; Glycolysis; Humans; Male; Membrane Proteins; Middle Aged; Naphthoquinones; Protein Structure, Quaternary; Pyruvate Kinase; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Urinary Bladder Neoplasms

Cisplatin-based chemotherapy often results in the development of chemo-resistance when used to treat bladder cancer (BC), which is difficult to overcome. Recent data indicate that pyruvate kinase M2 (PKM2), a glycolytic enzyme for Warburg effect, is strongly upregulated in BC, and contributes to the cisplatin resistance in BC. However, the underlying mechanisms remain unclear. In this study, we also found that the expression level of PKM2 is also higher in cisplatin resistant BC cells and tumors. Down-regulation of PKM2 by siRNA or inhibition of PKM2 by shikonin re-sensitized the cisplatin resistant T24 cells. Shikonin and cisplatin together exhibit significantly greater killing effects than when used alone. Interestingly, we found shikonin kills the T24 cisplatin resistant cells by inducing necroptosis, as the cell death could not inhibited by apoptosis inhibitor, z-VAD, but compromised by RIP3 inhibitor, GSK872, or RIP3 siRNA. In contrast, shikonin induced apoptosis in T24 parental cells. We further investigate the underlying mechanism, and found that the dysregulation of Bcl-2 family proteins, including Bcl-2, PUMA, Bax, play an important role in deciding that shikonin kills the BC cells by necroptosis or apoptosis. Collectively, our results suggested that inducing necroptosis is an alternative way to overcome the apoptosis resistant in BC therapy, and orchestrating the regulation of Bcl-2, PUMA, and Bax in BC cisplatin resistant cells may improve the therapy effect of cisplatin in BC tumor.

Topics: Aged; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; Carrier Proteins; Cell Line, Tumor; Cisplatin; Female; Humans; In Vitro Techniques; Male; Membrane Proteins; Middle Aged; Naphthoquinones; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Urinary Bladder Neoplasms

Chemoresistance to cisplatin is a principal cause of treatment failure and mortality of advanced bladder cancer (BC). The underlying mechanisms remain unclear, which hinders the development of preventive strategies. Recent data indicate that pyruvate kinase M2 (PKM2), a glycolytic enzyme for Warburg effect, is strongly upregulated in BC. This study explores the role of PKM2 in chemoresistance and whether inhibiting PKM2 augments the chemosensitivity to cisplatin and reduces BC growth and progression. We found that Shikonin binds PKM2 and inhibits BC cell survival in a dose-dependent but pyruvate kinase activity-independent manner. Down-regulation of PKM2 by shRNA blunts cellular responses to shikonin but enhances the responses to cisplatin. Shikonin and cisplatin together exhibit significantly greater inhibition of proliferation and apoptosis than when used alone. Induced cisplatin-resistance is strongly associated with PKM2 overexpression, and cisplatin-resistant cells respond sensitively to shikonin. In syngeneic mice, shikonin and cisplatin together, but not as single-agents, markedly reduces BC growth and metastasis. Based on these data, we conclude that PKM2 overexpression is a key mechanism of chemoresistance of advanced BC to cisplatin. Inhibition of PKM2 via RNAi or chemical inhibitors may be a highly effective approach to overcome chemoresistance and improve the outcome of advanced BC.

Topics: Actins; Aged, 80 and over; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Naphthoquinones; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Polymerization; Protein Kinase Inhibitors; Pyruvate Kinase; RNA, Small Interfering; Up-Regulation; Urinary Bladder Neoplasms

Survivin is overexpressed in transitional cell carcinoma (TCC), the most common type of bladder cancer. Previous reports demonstrated that knockdown of survivin by siRNA induced apoptosis of TCC cells. The present study evaluated the therapeutic effects of sepantronium bromide (YM155), a novel small molecule survivin inhibitor under clinical trials, on TCC cells in vitro. BFTC905, a grade III TCC cell line derived from a patient of blackfoot disease in Taiwan, was the most gemcitabine-resistant cell line when compared to BFTC909, TSGH8301 and T24 in cytotoxicity assay, resulting from upregulation of securin and bcl-2 after gemcitabine treatment. YM155 caused potent concentration‑dependent cytotoxicity in 4 TCC cell lines (IC50s ≤20 nM), but exhibited no cytotoxicity in survivin-null primary human urothelial cells. For BFTC905 cells, addition of gemcitabine and/or cisplatin, the standard TCC chemotherapy regimen, to YM155 did not exert additive cytotoxic effects. Molecular analyses indicated that YM155 inhibited the proliferation of BFTC905 cells by increasing p27kip1, suppressing Ki-67, and inducing quiescence. In addition, YM155 elicited apoptosis manifested with DNA fragmentation through suppressing the expression of survivin, securin and bcl-2. Furthermore, YM155 induced autophagy in BFTC905 cells as autophagic inhibitor, 3-methyladenine, attenuated YM155-induced LC3B-II levels and reversed the cytotoxicity of YM155. mTOR inhibitors sirolimus and everolimus did not increase YM155-induced expression of LC3B-II nor augment YM155-induced cytotoxicity. These results indicate that YM155 exerts its lethal effect on BFTC905 cells via apoptotic and autophagic death pathways and suggest that YM155 may be a potential drug for the therapy of gemcitabine-resistant bladder cancer.

Topics: Adenine; Antimetabolites, Antineoplastic; Apoptosis; Autophagy; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Deoxycytidine; DNA Fragmentation; Drug Resistance, Neoplasm; Everolimus; Gemcitabine; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Ki-67 Antigen; Microtubule-Associated Proteins; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Securin; Sirolimus; Survivin; TOR Serine-Threonine Kinases; Urinary Bladder Neoplasms; Urothelium

Quinone-containing molecules have been developed against cancer mainly for their redox cycling ability leading to reactive oxygen species (ROS) formation. We have previously shown that donor-acceptor phenylaminonaphthoquinones are biologically active against a panel of cancer cells. In this report, we explored the mechanisms involved in cancer cell growth inhibition caused by two phenylaminonaphthoquinones, namely Q7 and Q9, with or without ascorbate (ASC). The results show that Q7 and Q9 are both redox cyclers able to form ROS, which strongly inhibit the proliferation of T24 cells. Q9 was a better redox cycler than Q7 because of marked stabilization of the semiquinone radical species arising from its reduction by ascorbate. Indeed, ASC dramatically enhances the inhibitory effect of Q9 on cell proliferation. Q9 plus ASC impairs the cell cycle, causing a decrease in the number of cells in the G2/M phase without involving other cell cycle regulating key proteins. Moreover, Q9 plus ASC influences the MAPK signaling pathways, provoking the appearance of a senescent cancer cell phenotype and ultimately leading to necrotic-like cell death. Because cellular senescence limits the replicative capacity of cells, our results suggest that induction of senescence may be exploited as a basis for new approaches to cancer therapy.

Topics: Aminophenols; Aniline Compounds; Antineoplastic Agents; Ascorbic Acid; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cellular Senescence; Drug Synergism; Humans; Imidazoles; MAP Kinase Signaling System; Naphthoquinones; Necrosis; Oxidation-Reduction; Phenotype; Pyridines; Reactive Oxygen Species; Urinary Bladder Neoplasms

To study in vitro the molecular mechanism of apoptosis caused by beta-lapachone, a quinone obtained from the bark of the lapacho tree (Tabebuia avellanedae).. The study was carried out on human bladder carcinoma T24 cell line. Determination of cell viability was done using trypan blue exclusion method, apoptosis quantitative estimation - by DAPI staining and agarose gel electrophoresis for DNA fragmentation. Flow cytometry analysis, RT-PCR and Western blot analysis, colorimetric assay of caspase activity were applied as well.. It was found that in micromolar range of concentrations beta-lapachone inhibited the viability of T24 cells by inducing apoptosis, which could be proved by formation of apoptotic bodies and DNA fragmentation. Treatment of T24 cells with beta-lapachone resulted in a down-regulation of Bcl-2 expression and up-regulation of Bax expression. beta-lapachone-induced apoptosis was also associated with activation of caspase-3 and caspase-9, inhibition of IAP expression, and degradation of poly (ADP-ribose) polymerase, phospholipase C-gamma1 and beta-catenin proteins. At the same time Fas and FasL levels were inhibited upon treatment with beta-lapachone in a concentration-dependent manner.. beta-lapachone-induced apoptosis in T24 cells is mediated, at least in part, by the mitochondrial-signaling pathway.

Topics: Apoptosis; Caspases; Cell Division; Cell Line, Tumor; Cell Survival; Enzyme Activation; Fas Ligand Protein; fas Receptor; Humans; Membrane Glycoproteins; Naphthoquinones; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Inhibitors; RNA, Messenger; Tumor Necrosis Factors; Urinary Bladder Neoplasms

Topics: Animals; Animals, Newborn; Carcinogens; Carcinoma, Hepatocellular; Dogs; Female; Fibroma; Fibrosarcoma; Granuloma; Hydroxylation; Liver Neoplasms; Lymphoma, Non-Hodgkin; Male; Mice; Naphthalenes; Naphthoquinones; Neoplasms, Experimental; Nitroso Compounds; Urinary Bladder Neoplasms; Urine

Topics: Adult; Esophageal Neoplasms; Female; Humans; Lung Neoplasms; Male; Middle Aged; Naphthoquinones; Peritoneal Neoplasms; Radiation-Sensitizing Agents; Radiotherapy Dosage; Rectal Neoplasms; Urinary Bladder Neoplasms

Topics: Agar; Aniline Compounds; Animals; Carcinogens; Carcinosarcoma; Escherichia coli; Genetics, Microbial; Injections, Intraperitoneal; Leukemia, Experimental; Male; Mutagens; Mutation; Naphthalenes; Naphthoquinones; Neoplasms, Experimental; Peritoneal Neoplasms; Peritoneum; Rats; Sarcoma, Experimental; Urinary Bladder Neoplasms