Dyshlovoy SA et al. (2017), The marine triterpene glycoside frondoside A in...

ECB-ART-45163

BMC Cancer 2017 Feb 01;171:93. doi: 10.1186/s12885-017-3085-z.

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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 .

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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.

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Genes referenced: bax gemin5 impact LOC100893907 LOC115919910 LOC584760 LOC592256 osbpl9

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	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

References [+] :

Al Shemaili, Pharmacokinetics in Mouse and Comparative Effects of Frondosides in Pancreatic Cancer. 2016, Pubmed, Echinobase