The present study extends earlier reports that documented the ability of the ATF2 peptide to cause apoptosis in mouse and human melanoma cells in culture and in animal models (29
). To identify compounds that mimic the activity of the ATF2 peptide, we screened a small-molecule chemical library consisting of 3,280 compounds. Two compounds, CSL and AIGA, at low micromolar concentrations, were able to (a
) induce melanoma cell death, (b
) inhibit the transcriptional activity of ATF2, and (c
) induce the activity of JNK and transcriptional activity of c-Jun and required active JNK for their ability to affect melanoma viability.
Gambogic acid was originally isolated from the resin of the Garcinia hanburyi
tree, whose cytotoxic properties are recognized (37
). In recent studies, AIGA was shown to possess the capacity to potently induce apoptosis in a breast cancer cell line T47D (38
), in a human hepatoma cell line SMMC-7721 (39
), and in a gastric carcinoma cell line BGC-823, in part through G2
-M phase arrest (40
). Consistent with these findings, we show herein that AIGA elicits potent inhibition of melanoma cell growth in culture. We further show that AIGA elicits these effects in part through activation of JNK and c-Jun and inhibition of ATF2.
CSL, a quinone methide triterpenoid isolated from the Chinese “thunder of god vine” plant, was reported to exhibit immunosuppressive activities (41
). CSL was also shown to induce the heat shock response and to elicit cytoprotection (42
). Consistent with these findings, gene expression signature-based chemical genomic prediction identified CSL as a modulator of the HSP90 pathway (43
). At micromolar concentrations, CSL was shown capable of inhibiting proteasome activity and the growth of prostate cancer cells in nude mice (44
). At the concentrations used in the present studies (0.3–1 µmol/L), CSL did not affect the stability of short-lived proteins, including Mdm2 and p53 (data not shown). CSL was also shown to inhibit NF-κB activation through the inhibition of IκB kinase (36
) and TAK1 activities (45
). Previous studies showed that NF-κB inhibits JNK activities and that inhibition of NF-κB results in elevated JNK activities (46
). Thus, it is likely that the ability of CSL to attenuate NF-κB activity would result in increased JNK activity, which is consistent with our findings. It is the increase in JNK activity, and consequently of its substrate c-Jun, in concert with inhibition of ATF2 activities that brings about the potent cytotoxic effects of both CSL and AIGA on melanoma cells, similar to what was previously shown for the ATF2 peptides (29
The greater apoptotic effects seen with CSL compared with AIGA in cultured cells prompted us to focus on CSL. Examination of the structure of CSL led us to postulate that the quinone methide functional group was responsible for its cytotoxic activity. Along these lines, we reasoned that the highly electrophilic nature of the quinone methide unit could potentially lead to irreversible or pseudo-irreversible binding to proteins, possibly through interaction with cysteinyl residues. Supporting this hypothesis is the fact that dihydrocelastrol, which lacks the quinone methide moiety, is inactive in the cellular assays (). On the other hand, the methyl ester derivative of CSL (pristimerin) is equally efficacious as CSL with respect to its ability to induce apoptosis, suggesting that the presence of the acidic carboxylate functional group in CSL is not required for apoptotic activity but that the quinone methide moiety is responsible for the observed cellular activity in our experiments. Data recently reported by Westerheide et al. (42
) support this hypothesis and further suggest that the carboxylic acid moiety in CSL is responsible for the chemical chaperone activity observed in their experiments. Thus, in the study conducted by Westerheide et al. (42
), three ester derivatives of CSL were inactive as regulators of the heat shock response, whereas dihydrocelastrol was active as a heat shock promoter. These results suggest that the quinone methide moiety is not responsible for the observed chemical chaperone activity. We therefore synthesized a series of CSL derivatives in which the carboxylic acid unit in CSL was converted to an amide or ester derivative with the quinone methide functional group retained intact in the molecules (see Results). Consistent with a role for the quinone methide unit in the cytotoxicity of CSL, some of the CSL derivatives exhibited improved efficacy (). These results led to the identification of the benzyl ester (CA16) and isopropyl ester (CA19) derivatives as potent inducers of apoptosis, which were profiled in detail in in vivo
models of melanoma. The precise role for the carboxylic acid moiety in this modality of CSL remains to be further elucidated, however.
Consistent with its ability to induce apoptosis of melanoma cells in culture is the ability of CSL and its isopropyl ester derivative CA19 to attenuate the growth of melanoma in mouse models. Our analysis, which was done in syngeneic mouse (SW1 cells in C3H mice) and human xenograft (LU1205 in nude mice) models, shows the ability of these compounds to attenuate growth of metastatic lesions, which are the major clinical burden in treatment of this tumor type. The lower activity of CA16 compared with CA19 and CSL on tumor growth and metastasis development may be attributed to stability or permeability of this compound in vivo. Along these lines, in all cases, it was necessary to inject the compounds at high frequency; it is expected that improved formulation will allow prolonged half-life and more efficient delivery of these compounds. The low concentrations of the compounds used in our present studies would be expected to minimize effect on other organs, consistent with our initial maximum tolerated dose studies.
In conclusion, the effects of AIGA and CSL on melanoma cells in culture and in mouse models point to possible new treatment modalities for melanoma. In light of the notion that MAPK signaling is up-regulated in human melanoma, in many cases as a result of mutations in B-RAF or N-RAS, our findings suggest that these compounds could be also considered as a means of complementing the activities of pharmacologic inhibitors developed against MAPK/ERK kinase.