To determine whether resveratrol is effective in inhibiting uveal melanoma tumor growth, we tested mouse xenograft models of human uveal melanoma cell lines. Dose–response experiments were performed with C918 (originally derived from a primary uveal melanoma) and Mum2b (originally derived from a metastatic lesion) cells. The mice were treated daily with resveratrol, starting 5 days after inoculation when tumors were smaller than 50 mm3. After 3 to 5 weeks of oral drug treatment, tumor growth was strongly inhibited in the C918 xenograft model at the 2- to 50-mg/kg doses and at the 5- to 50-mg/kg doses in the Mum2b model (). Tumor growth was inhibited proportionately less at the lowest dose (0.4 mg/kg) compared with the control group that received vehicle alone (). Tumor volume was approximately 50% less at doses more than 2 mg/kg in both mouse models compared with the control group (P < 0.0001 for all groups). The 0.4-mg/kg group showed a significant decrease in response compared with the 50-mg/kg group in both mouse models (P = 0.03). Growth kinetic data show that significant differences between the treated and untreated tumors were apparent almost immediately after initiation of drug treatment (). However, no tumor regression was observed.
Inhibition of Uveal Melanoma Tumor Growth
Figure 1 Inhibition of uveal melanoma tumor growth. Tumor growth kinetics during resveratrol treatment of C918 (A) and Mum2b (B) xenograft models by oral gavage. Data show the average tumor growth in resveratrol- and vehicle-treated groups of mice. *P < (more ...)
Toxicity was assessed by survival, activity and, twice weekly, by body weight. All animals survived the treatment without evidence of toxic effects and animals gained 10% to 15% of weight compared with their pretreatment values. On autopsy, no changes were observed in the major organs after treatment with the highest dose of resveratrol. This is supported by toxicity studies of resveratrol (reviewed in Ref. 17
Bioavailability of resveratrol in serum was determined 30 minutes after oral administration of 0.4, 2, 10, and 50 mg/kg of resveratrol and found to be 0.6 ± 0.28, 1.4 ± 0.49, 1.3 ± 0.21, and 4.3 ± 0.14 μM, respectively. No accumulation of resveratrol was found in tumor tissue. Our experiments showed that these relatively low levels were nonetheless sufficient to inhibit tumor growth. To determine whether the inhibitory effects of resveratrol on tumors can be enhanced, especially given the proapoptotic effects of the drug observed on cells in vitro (described later), we assessed the effect of higher doses of resveratrol on tumor growth and viability. To this end, the drug was directly injected next to the tumor in a series of three injections. This resulted in a decrease in tumor volume in four of five mice compared with the vehicle-treated control animals, all of which increased in volume (). Vehicle-treated tumor tissue showed the presence of healthy tumor cells with clear borders and the presence of many mitotic figures (). In the resveratrol-treated tumors, there were areas of necrosis, increased presence of apoptotic cells interspersed with residual surviving tumor cells, and calcified tumor cells (). Cell borders and nuclear detail were less clear, and, together with cytoplasmic swelling, these changes are consistent with cell damage. Adjacent normal tissue was relatively unaffected. Inflammatory cells were present in the subdermal fat layer and to a lesser degree in the epidermis ().
Figure 2 Tumor regression after peritumor injection of resveratrol in the C918 xenograft model. (A) Tumor size before and after three injections of vehicle or 20 mg resveratrol. H&E-stained sections of vehicle-treated tumor (B), 20 mg resveratrol-treated (more ...)
To understand the mechanism of the drug's antitumor activity, we performed in vitro studies on the uveal melanoma cell lines M619, C918, Mum2b, and 92.1. The viability of these cell lines was sharply decreased in response to resveratrol treatment in a time- and concentration-dependent manner (). Additional cell lines (Ocm1, Ocm3, Mel270, and Mel290) showed similar effects (data not shown). At the highest drug concentration, cell viability decreased approximately 85% to 90% compared with untreated cells after 5 days of treatment. The IC50 of resveratrol after 48 hours of treatment was: M619, 80 μM; C918, 70 μM; Mum2b, 130 μM; and 92.1, 25 μM. If treatment was continued, all cells died after approximately 8 to 10 days. In addition, dense cultures of uveal melanoma cells (>90% confluent) were treated with resveratrol as a function of time and concentration and their viability measured. Under these conditions, cell viability decreased in a concentrationdependent manner over 5 days, further indicating the occurrence of cell death ().
Figure 3 Resveratrol decreased the cell viability of uveal melanoma cell lines by inducing apoptosis. (A) The cell viability of M619, C918, Mum2b, and 92.1 cells was measured as a function of resveratrol concentration and time after drug addition and plotted as (more ...)
To determine whether resveratrol treatment results in enhanced apoptosis, we treated uveal melanoma cells with 100 μM resveratrol for up to 72 hours, and then stained them with Hoechst 33528 dye, to visualize apoptotic nuclear morphology (). The percentage of apoptotic M619 and Mum2b cells steadily increased over 72 hours, up to 39% and 36%, respectively. The fraction of apoptotic C918 and 92.1 cells reached a maximum at 48 hours and then leveled off. These results suggest that the decrease in cell viability is at least in part due to an increase in apoptosis.
Since resveratrol treatment of tumor cells resulted in ultrastructural damage of the mitochondria before cell death, as indicated by disorganization of the cristae (Sareen D, unpublished observations, 2006), the intrinsic apoptosis pathway, in which the mitochondria play a key role, was studied in uveal melanoma cells. In this pathway, apoptotic stimuli induce mitochondrial dysfunction that in turn signals activation of downstream mediators of cell death. One of the parameters affected when mitochondria become dysfunctional is the mitochondrial transmembrane potential. Therefore, mitochondrial membrane potential was measured by using JC-1 in M619, C918, Mum2b, and 92.1 cells (). These cell lines showed an early and sustained loss of mitochondrial membrane potential in response to resveratrol treatment. Increasing concentrations of the drug led to increased loss of membrane potential. Both resveratrol and the positive control carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonophore, caused a collapse of the membrane potential, as indicated by the sharp decrease in the red-green JC-1 ratio. These results suggest a mechanism for resveratrol action involving mitochondrial function leading to apoptosis via the intrinsic pathway. Further, pretreatment with cyclosporin A did not prevent resveratrol-induced membrane potential collapse after 15 minutes of drug treatment. In the absence of cyclosporin A the red-green ratio of untreated and 100 μM resveratrol- treated samples was 112.4 ± 3.9 and 18.0 ± 1.4, respectively. Similarly, in the presence of cyclosporin A the red-green ratio of untreated and 100 μM resveratrol-treated samples was 84.0 ± 16.1 and 14.8 ± 0.6, respectively. Longer incubation of cells with resveratrol for 6 hours gave similar results, suggesting that, at least initially, resveratrol does not cause mitochondrial permeability transitional (MPT) pore opening.
Figure 4 Resveratrol caused loss of mitochondrial membrane potential in uveal melanoma cells and in isolated mitochondria. Mitochondrial membrane potential in M619, C918, Mum2b, and 92.1 cells (A) and 92.1 mitochondria (B) was measured as a function of resveratrol (more ...)
To test whether the mechanism of resveratrol action involves direct targeting of this organelle, we isolated mitochondria from 92.1 cells, treated with resveratrol and measured the mitochondrial membrane potential with JC-1 dye (). Since no cytoplasmic compartment is present in these experiments, only the red fluorescence was measured. As an additional positive control, mitochondria were treated with sodium azide, a mitochondrial complex IV inhibitor. Membrane potential of isolated mitochondria was decreased after resveratrol treatment, suggesting that resveratrol causes mitochondrial dysfunction by directly targeting the mitochondria without the need to interact with cytosolic components. As expected, the drug concentration needed to attain these effects was less than needed for whole cells, probably due to the lack of diffusion barriers.
In other cell types, loss of mitochondrial membrane potential with resveratrol treatment is followed by release of cytochrome c
and Smac/Diablo from the mitochondria.13
To determine whether this release occurs in uveal melanoma cells, we treated M619, C918, and Mum2b cells with resveratrol, lysed and fractionated the cells, and determined the amount of cytochrome c
and Smac/Diablo found in the cytosolic fraction by Western blot analysis. Cytochrome c
was released into the cytosol in all three cell types 3 hours after initiating drug treatment (). Smac/Diablo was also released from the mitochondria in all three cell lines. The kinetics of Smac/Diablo release compared with cytochrome c
release in response to resveratrol treatment were similar. In the C918 and Mum2b cells, the highest levels of these proteins in the cytoplasm were observed 3 hours after the addition of the drug, after which they tapered off (). In contrast, cytochrome c
and Smac/Diablo levels remained the same at 24 and 48 hours after treatment in the M619 cells ().
Figure 5 Resveratrol causes loss of cytochrome c and Smac/Diablo from the mitochondria and induces activation of caspase-9 and -3. (A) Cytochrome c and Smac/Diablo were measured by Western analysis in the cytoplasmic fractions of M619, C918, and Mum2b cells in (more ...)
To determine whether other components of the mitochondrial cell death pathway were activated, we measured caspase-9 and -3 activities in resveratrol-treated cells in a time-course experiment (). These proteins act as executioner enzymes late in the apoptotic process. All three cell lines demonstrated increased activity of these enzymes after 24 hours, suggesting that these downstream components in the intrinsic apoptosis pathway are activated in response to resveratrol treatment.
In some instances, chemical-induced apoptosis can act via the mitochondria18
and involve caspase-8. To determine whether this pathway is activated in response to resveratrol treatment as well, processing of procaspase-8 was measured. Procaspase-8 was cleaved in the M619, C918, Mum2b, and 92.1 cells in a time-dependent manner indicated by the appearance of 43- and 41-kDa intermediate cleavage products and an 18- kDa active product ().