Allyl isothiocyanate-rich mustard seed powder inhibits bladder cancer growth and muscle invasion

Abstract

Allyl isothiocyanate (AITC), which occurs in many common cruciferous vegetables, was recently shown to be selectively delivered to bladder cancer tissues through urinary excretion and to inhibit bladder cancer development in rats. The present investigation was designed to test the hypothesis that AITC-containing cruciferous vegetables also inhibit bladder cancer development. We focused on an AITC-rich mustard seed powder (MSP-1). AITC was stably stored as its glucosinolate precursor (sinigrin) in MSP-1. Upon addition of water, however, sinigrin was readily hydrolyzed by the accompanying endogenous myrosinase. This myrosinase was also required for full conversion of sinigrin to AITC in vivo, but the matrix of MSP-1 had no effect on AITC bioavailability. Sinigrin itself was not bioactive, whereas hydrated MSP-1 caused apoptosis and G(2)/M phase arrest in bladder cancer cell lines in vitro. Comparison between hydrated MSP-1 and pure sinigrin with added myrosinase suggested that the anticancer effect of MSP-1 was derived principally, if not entirely, from the AITC generated from sinigrin. In an orthotopic rat bladder cancer model, oral MSP-1 at 71.5 mg/kg (sinigrin dose of 9 μmol/kg) inhibited bladder cancer growth by 34.5% (P < 0.05) and blocked muscle invasion by 100%. Moreover, the anticancer activity was associated with significant modulation of key cancer therapeutic targets, including vascular endothelial growth factor, cyclin B1 and caspase 3. On an equimolar basis, the anticancer activity of AITC delivered as MSP-1 appears to be more robust than that of pure AITC. MSP-1 is thus an attractive delivery vehicle for AITC and it strongly inhibits bladder cancer development and progression.

Fig. 1.

Characterization of MSP. (A) Four different preparations (from four commercial sources) of MSP, including MSP-4, MSP-3, MSP-2 and MSP-1, were each incubated with exogenous myrosinase in PBS (1 mg powder per ml with 0.1 U myrosinase) for 30 min at room temperature (longer incubation time did not lead to further increase in ITC yield). MSP-1 was also incubated in PBS at room temperature for 0.5–4 h without exogenous myrosinase. At the end of incubation, total ITC levels in each solution were measured by the cyclocondensation assay. Each value is a mean ± SEM (n = 3). The result at the 0 time point was obtained by mixing MSP-1 with DADW, so that the endogenous myrosinase was inactivated and potential conversion from sinigrin to AITC was blocked. (B) MSP-1 was either mixed with DADW or incubated with exogenous myrosinase in phosphate buffer for 30 min before high-performance liquid chromatography. The arrows point to sinigrin and two potential minor glucosinolates of unknown identity. The compound or compounds representing the peak around 4 min has not been characterized, but it is not a glucosinolate, because myrosinase treatment had no effect on the peak. (C) Sinigrin was mixed in water with or without myrosinase in phosphate buffer for 30 min before high-performance liquid chromatography. The results are representative of at least three experiments.

Fig. 2.

Sinigrin hydrolysis and urinary excretion of AITC. Groups of five female F344 rats were administered a single oral dose of sinigrin, MSP-1 or AITC. Both sinigrin and MSP-1 were mixed in water, whereas AITC was mixed in soy oil, which were given to rats within 30 min of preparation. The rats were kept in metabolism cage for 24 h urine collection (1 rat per cage). Urinary levels of ITC equivalents were measured by the cyclocondensation assay. All values were adjusted by background urinary levels of ITC equivalents, which were 8.6 ± 2.3 μM (average 24 h urinary concentration) and 0.05 ± 0.01 μmol (24 h urine), determined in another group of rats. Each value is mean ± SEM (n = 5).

Fig. 3.

The effect of sinigrin and MSP-1 on survival and proliferation of bladder cancer cells. (A) UM-UC-3 cells and AY-27 cells were grown in 96-well plates and treated with MSP-1 (filled sqaures), sinigrin (inverted filled triangles) and sinigrin plus myrosinase (open squares) for 72 h, followed by measurement of cell density by 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (B) Cells were grown in 96-well plates and treated with MSP for 24 h. Apoptosis was measured by an enzyme-linked immunosorbent assay. (C) Cells were grown in 10 cm dishes and treated with MSP-1 for 24 h. Cell cycle distribution was measured by flow cytometry (open bars, G₁; striped bars, S; filled bars, G₂/M). Each value is mean ± SEM (n = 3–8). *P < 0.05 compared with the control.

Fig. 4.

Inhibition of bladder cancer development by MSP-1. Female F344 rats were inoculated with AY-27 cells intravesically via a urethra catheter to initiate development of orthotopic bladder cancer. Oral administration of MSP-1 or vehicle (water) once daily was started 1 day after cancer cell inoculation and ended 3 weeks later. The number of rats per group varied from 11–29. (A) Initial (open bars) and final (filled bars) body weights. (B) Tumor weight was calculated by subtracting the average normal bladder weight from tumor-bearing bladder weight. *P < 0.05. Each value in A and B is mean ± SEM. (C) Percentage of bladder where the tumor invaded the muscle tissue.

Fig. 5.

The effect of MSP-1 on selected anticancer targets. UM-UC-3 cells and AY-27 cells in culture were treated with MSP-1 at the sinigrin concentrations of 13 or 26 μM for 24 h. The results are representative of at least two experiments. The bladder tumors were removed from rats, which were treated with vehicle or MSP-1 at the sinigrin doses of 9 or 90 μmol/kg once daily for 3 weeks, starting 1 day after cancer cell inoculation. The results are representative of tumors from other rats. Cell lysates and tumor homogenates were analyzed by western blotting, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control.