Revisiting old drugs as novel agents for retinoblastoma: in vitro and in vivo antitumor activity of cardenolides

Abstract

Purpose: Intra-arterial delivery of chemotherapeutic agents offers a new and exciting opportunity for the treatment of advanced intraocular retinoblastoma. It allows local delivery of relatively high doses of chemotherapy agents while bypassing general blood circulation. For this reason, this study was undertaken to revisit some of the FDA-approved drugs for the treatment of retinoblastoma.

Methods: High-throughput screening (HTS) of 2640 approved drugs and bioactive compounds resulted in the identification of cytotoxic agents with potent activity toward both the Y79 and RB355 human retinoblastoma cell lines. Subsequent profiling of the drug candidates was performed in a panel of ocular cancer cell lines. Induction of apoptosis in Y79 cells was assessed by immunofluorescence detection of activated caspase-3. Therapeutic effect was evaluated in a xenograft model of retinoblastoma.

Results: Several FDA-approved drugs were identified that showed potent cytotoxic activity toward retinoblastoma cell lines in vitro. Among them were several cardiac glycosides, a class of cardenolides historically associated with the prevention and treatment of congestive heart failure. Caspase-3 activation studies provided an insight into the mechanism of action of cardenolides in retinoblastoma cells. When tested in a xenograft model of retinoblastoma, the cardenolide ouabain induced complete tumor regression in the treated mice.

Conclusions: Cardenolides were identified as a new class of antitumor agents for the treatment of retinoblastoma. Members of this class of cardiotonic drugs could be repositioned for retinoblastoma if administered locally via direct intra-arterial infusion.

Figure 1

Overall comparative analysis of the Y79 and RB355 screens. The percentage loss of cell viability induced by each tested compound in both screens is represented as a heat map to identify those compounds potent toward both cell lines.

Figure 2

Scatter plot comparative analysis of the Y79 and RB355 screens. The percentage loss of cell viability induced by each tested compound in both screens is represented as a scatter plot. The 29 positives at a threshold of 90% inhibition in both screens are highlighted in red, and the 19 cardenolides present in the library are highlighted in green to assess the proportion of cardenolides among all positives as well as their potency.

Figure 3

Summary of the structures for the 11 positives selected at a threshold of 95% inhibition in both screens. (A) Cardenolides. (B) Non-cardenolides.

Figure 4

Structure-activity relationship study for a collection of 35 cardenolides in a panel of four ocular cancer cell lines. (A) Heat map and numerical summary of calculated IC₅₀s for the 35 cardenolides in the ocular cancer cell line panel. The structure of identified chemical scaffolds is highlighted. (B) Representative dose response curves generated for Derivative-1 in the panel of ocular cancer cell lines. (C) Representative dose response curves generated for the drug ouabain in the panel of ocular cancer cell lines.

Figure 5

Compared potency of the drug ouabain toward (A) Y79 cells and (B) RB355 cells with clinical agents and experimental drugs. (C) Summary of the calculated IC₅₀s.

Figure 6

Immunofluorescence detection of activated Caspase-3 in Y79 cells treated with (A) 1% DMSO (v/v); (B) 100 µM vincristine, 1% DMSO (v/v); (C) 10 µM etoposide, 1% DMSO (v/v); (D) 0.5 µM ouabain, 1% DMSO (v/v).

Figure 7

In vivo antitumor effect of the drug ouabain evaluated by bioluminescent imaging of tumor burden in a mouse xenograft model of retinoblastoma. Images for one representative mouse per group treated with either vehicle only (10% DMSO v/v) or 15 mg/kg ouabain, 10% DMSO v/v over 19 days are shown.

Figure 8

(A) In vivo antitumor effect of the drug ouabain evaluated by tumor volume measurement in a mouse xenograft model of retinoblastoma. The average tumor volume per group over 19 days treatment is plotted. (B) Monitoring of animal weight. The average animal weight per group over 19 days treatment is plotted.