Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
BMC Cancer
2017 Feb 01;171:93. doi: 10.1186/s12885-017-3085-z.
Show Gene links
Show Anatomy links
The marine triterpene glycoside frondoside A induces p53-independent apoptosis and inhibits autophagy in urothelial carcinoma cells.
Dyshlovoy SA
,
Madanchi R
,
Hauschild J
,
Otte K
,
Alsdorf WH
,
Schumacher U
,
Kalinin VI
,
Silchenko AS
,
Avilov SA
,
Honecker F
,
Stonik VA
,
Bokemeyer C
,
von Amsberg G
.
???displayArticle.abstract???
BACKGROUND: Advanced urothelial carcinomas represent a considerable clinical challenge as they are difficult to treat. Platinum-based combination regimens obtain response rates ranging from 40 to 70% in first-line therapy of advanced urothelial carcinoma. In the majority of cases, however, the duration of these responses is limited, and when progression occurs, the outcome is generally poor. Therefore, novel therapeutic strategies are urgently needed. The purpose of the current research is to investigate the anticancer effects and the mode of action of the marine triterpene glycoside frondoside A in p53-wild type and p53-deficient human urothelial carcinoma cells.
METHODS: Activity of frondoside A was examined in the human urothelial carcinoma cell lines RT112, RT4, HT-1197, TCC-SUP, T-24, and 486p. Effects of frondoside A on cell viability, either alone or in combination with standard cytotoxic agents were investigated, and synergistic effects were analyzed. Pro-apoptotic activity was assessed by Western blotting and FACS, alone and in combination with a caspases-inhibitor. The impact of functional p53 was investigated by siRNA gene silencing and the p53 inhibitor pifithrin-α. Effects on autophagy were studied using LC3B-I/II and SQSTM/p62 as markers. The unpaired Student''s t-test was used for comparison of the data sets.
RESULTS: Frondoside A shows high cytotoxicity in urothelial carcinoma cells with IC50s ranging from 0.55 to 2.33 μM while higher concentrations of cisplatin are required for comparable effects (IC50 = 2.03 ~ 5.88 μM). Induction of apoptosis by frondoside A was associated with the regulation of several pro-apoptotic factors, like caspase-3, -8, and -9, PARP, Bax, p21, DNA fragmentation, and externalization of phosphatidylserine. Remarkably, inhibition of p53 by gene silencing or pifithrin-α pretreatment, as well as caspase inhibition, did not suppress apoptotic activity of frondoside A, while cisplatin activity, in contrast, was significantly decreased. Frondoside A inhibited pro-survival autophagy, a known mechanism of drug resistance in urothelial carcinoma and showed synergistic activity with cisplatin and gemcitabine.
CONCLUSIONS: A unique combination of properties makes marine compound frondoside A a promising candidate for the treatment of human urothelial carcinomas.
Fig. 1. Structure and effect of FrA on the viability of human urothelial cancer cells. a Structure of frondoside A (FrA). b Cytotoxicity of FrA, determined with a trypan blue-based viability assay. Cells were treated with FrA or Cis for 48 h
Fig. 2. Induction of caspase-independent apoptosis in FrA-treated cells. a Western blotting analysis of protein extracts of RT112 cells treated with FrA for 48 h. b Cell cycle analysis of RT112 cells treated with FrA for 48 h. Apoptotic cells are detectable as a sub-G1 population. Cell cycle phase distribution was quantified using the Cell Quest Pro software. c, d Flow cytometry analysis of RT112 cells treated with FrA using an annexin-V-FITC/PI double staining (c) and quantification of positive cells (d). Cells were pretreated with 100 μM of the pan-caspase inhibitor zVAD for 1 h and then treated with indicated concentrations of FrA or with anisomycin (Aniso, positive control) for 48 h. Apoptotic cells appearing in the right lower and upper quadrants were quantified using the Cell Quest Pro software
Fig. 3. Effect of p53 silencing on cytotoxicity of FrA. a, b Western blotting analysis of p53 expression in RT112 cells treated with FrA for 48 h (a) or transfected with p53 siRNA versus cells transfected with scrambled siRNA (b). c Viability of transfected cells treated with FrA or Cis for 48 h. Cell viability was analyzed by flow cytometry analysis using annexin-V-FITC/PI double staining. Viable cells appearing in the lower left quadrant were quantified using the Cell Quest Pro software. d, e Effect of pifithrin-α (Pif-α)–an inhibitor of p53 activity–on the cytotoxic activity of FrA and Cis in nontransfected RT112 cells. Cells were pretreated with 40 μM of Pif-α for 30 min and then cotreated with FrA (d) or Cis (e) for 48 h. Cell viability was determined using MTT-assay
Fig. 4. Effect of FrA on mitogen-activated protein kinases (MAPK). a, b RT112 cells were treated with FrA for 1 h (a) or 48 h (b), and then protein extracts were analyzed by Western blotting. c Effect of SP600125 (a specific JNK1/2 inhibitor) on the survival of RT112 cells treated with FrA. Drugs were combined in the constant molar ratio C(FrA) : C(SP600125) = 1 : 15, and cells were cotreated with the individual drugs or their combination for 48 h. The data were generated using trypan blue-based viability assay. Cells were pretreated with SP600125 in 50 μL/well culture media for 1 h. The combinational index (CI) was calculated with the CompuSyn v.1.0. software
Fig. 5. Inhibition of autophagy in urothelial cancer cells under FrA-treatment. a, b Time- (a) and dose-dependent (b) effects of FrA, bafilomycin A1 (BafA1), and chloroquine (CQ) on levels of LC3B-I/II and SQSTM/p62 in RT112 cells. Cells were treated for 0.5–48 h (a) or for 48 h (b), followed by protein extraction and analysis by Western blotting. The established autophagy inhibitors BafA1 and CQ were used as positive controls. c, d Accumulation of autophagosomes/autolysosomes. Cells were treated with the indicated concentrations of FrA, BafA1, and CQ for 48 h, fixed, permeabilized, and incubated with the anti-LC3B-I/II antibody, followed by treatment with Alexa Fluor 488-conjugated secondary antibody. The pictures were made at × 400 (c) or × 1000 (d) magnification. LC3B-I/II-positive organelles (autophagosomes/autolysosomes) appearing as dots are indicated by arrows
Fig. 6. Effect of FrA in combination with cisplatin and gemcitabine. Cells were cotreated with different concentrations of the single substances or their combination for 48 h at the constant molar ratios C(FrA) : C(Cis) = 1 : 4 (a) and C(FrA) : C(Gem) = 1 : 0.25 (b). The combinational index (CI) values were calculated with CompuSyn software. The viability was examined using a trypan blue-based viability assay
Al Shemaili,
Pharmacokinetics in Mouse and Comparative Effects of Frondosides in Pancreatic Cancer.
2016, Pubmed,
Echinobase
Al Shemaili,
Pharmacokinetics in Mouse and Comparative Effects of Frondosides in Pancreatic Cancer.
2016,
Pubmed
,
Echinobase
Aminin,
Anticancer activity of sea cucumber triterpene glycosides.
2015,
Pubmed
,
Echinobase
Bamford,
The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website.
2004,
Pubmed
Berggren,
p53 mutations in urinary bladder cancer.
2001,
Pubmed
Bröker,
Cell death independent of caspases: a review.
2005,
Pubmed
Chipuk,
Do inducers of apoptosis trigger caspase-independent cell death?
2005,
Pubmed
Dreicer,
Second-line chemotherapy for advanced urothelial cancer: because we should or because we can?
2009,
Pubmed
Dyshlovoy,
Proteomic profiling of germ cell cancer cells treated with aaptamine, a marine alkaloid with antiproliferative activity.
2012,
Pubmed
Dyshlovoy,
The marine triterpene glycoside frondoside A exhibits activity in vitro and in vivo in prostate cancer.
2016,
Pubmed
Dyshlovoy,
Marine alkaloid Monanchocidin a overcomes drug resistance by induction of autophagy and lysosomal membrane permeabilization.
2015,
Pubmed
Elliott,
In vitro cultivation of epithelial cells derived from tumors of the human urinary tract.
1976,
Pubmed
Elliott,
Properties of cell lines established from transitional cell cancers of the human urinary tract.
1977,
Pubmed
Erdal,
Induction of lysosomal membrane permeabilization by compounds that activate p53-independent apoptosis.
2005,
Pubmed
George,
p53 gene and protein status: the role of p53 alterations in predicting outcome in patients with bladder cancer.
2007,
Pubmed
Grieco,
Integrative modelling of the influence of MAPK network on cancer cell fate decision.
2013,
Pubmed
Janakiram,
Sea Cucumbers Metabolites as Potent Anti-Cancer Agents.
2015,
Pubmed
,
Echinobase
Jänicke,
The dark side of a tumor suppressor: anti-apoptotic p53.
2008,
Pubmed
Kim,
Recent advances in treatment of advanced urothelial carcinoma.
2012,
Pubmed
Klionsky,
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
2016,
Pubmed
Kroemer,
Caspase-independent cell death.
2005,
Pubmed
Lin,
Inhibition of High Basal Level of Autophagy Induces Apoptosis in Human Bladder Cancer Cells.
2016,
Pubmed
Liu,
Role of JNK activation in apoptosis: a double-edged sword.
2005,
Pubmed
Malats,
P53 as a prognostic marker for bladder cancer: a meta-analysis and review.
2005,
Pubmed
Massari,
Emerging concepts on drug resistance in bladder cancer: Implications for future strategies.
2015,
Pubmed
Mathew,
Role of autophagy in cancer.
2007,
Pubmed
Oing,
Second Line Chemotherapy for Advanced and Metastatic Urothelial Carcinoma: Vinflunine and Beyond-A Comprehensive Review of the Current Literature.
2016,
Pubmed
Ojha,
Inhibition of grade dependent autophagy in urothelial carcinoma increases cell death under nutritional limiting condition and potentiates the cytotoxicity of chemotherapeutic agent.
2014,
Pubmed
Otto,
Stress-activated kinase pathway alteration is a frequent event in bladder cancer.
2012,
Pubmed
Pelageev,
Quinone-carbohydrate nonglucoside conjugates as a new type of cytotoxic agents: synthesis and determination of in vitro activity.
2014,
Pubmed
Philchenkov,
Caspases and cancer: mechanisms of inactivation and new treatment modalities.
2004,
Pubmed
Rosenberg,
Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial.
2016,
Pubmed
Siddik,
Cisplatin: mode of cytotoxic action and molecular basis of resistance.
2003,
Pubmed
Silchenko,
Constituents of the sea cucumber Cucumaria okhotensis. Structures of okhotosides B1-B3 and cytotoxic activities of some glycosides from this species.
2008,
Pubmed
,
Echinobase
Sui,
Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment.
2013,
Pubmed
Tait,
Caspase-independent cell death: leaving the set without the final cut.
2008,
Pubmed
Yang,
The role of autophagy in cancer: therapeutic implications.
2011,
Pubmed
Yu,
Restoring p53-mediated apoptosis in cancer cells: new opportunities for cancer therapy.
2006,
Pubmed