In this study, we investigated the anti-tumor promoting effects of β-tan and Sal A, isolated from Achillea falcata and Centaurea ainetensis, respectively, using the JB6 epidermal cell model of tumor promotion and cell transformation. In the multi-stage model of carcinogenesis, the tumor promotion phase is a rate limiting step that is responsible for the clonal expansion of initiated cells and is largely reversible , offering a practical approach for identifying potential inhibitors of cancer development .
Herein, we report that treatment with either Sal A or β-tan preferentially inhibited the growth of murine neoplastic keratinocytes, whilst sparing normal cells. The promotion-sensitive JB6P + cells were the most sensitive to β-tan treatment at concentrations that did not affect the growth of PMKs. Treatment with Sal A was relatively less potent on JB6P + cells, compared to β-tan, where 10 μg/ml β-tan inhibited cell growth by 74 ± 7%, whereas 10 μg/ml Sal A inhibited by 51 ± 4%. Although both belong to the SL guaianolide family, it seems that β-tan, with its relatively open ring structure, possesses higher flexibility, possibly enhancing β-tan diffusion across the cell membrane; in contrast to Sal A which bears a closed ring structure (Figure 1). In addition to the bioactive –α-methylene-γ-lactone ring present in Sal A and β-tan, the latter harbors an additional alkylating center, the cyclopentenone. Moreover, the presence of two hydroxyl (OH) groups within Sal A renders the molecule less lipophilic, possibly decreasing cell membrane penetration and may explain its reduced toxicity to JB6P + cells compared to β-tan.
In studying the anti-tumor promoting properties of these two purified SL molecules, it was essential to assess their effect on TPA-induced JB6P + cell transformation. In this study, we found that both β-tan and Sal A inhibited TPA-induced JB6P + cell transformation, at concentrations not cytotoxic to normal nor to the non-tumorigenic JB6P + cells. A hallmark of cell transformation is the ability of malignant cells to grow in soft agar in an anchorage-independent manner [18, 23, 36]. Our results show that β-tan and Sal A, at concentrations that did not inhibit JB6P + cell proliferation, were effective in reducing TPA-induced proliferation and inhibiting TPA-induced colony formation. These results suggest that β-tan and Sal A may have promising chemopreventive properties in epidermal carcinogenesis. Future in vivo experiments are required to confirm the chemopreventive properties of these purified SL molecules. However, a limiting step for in vivo studies will be the availability of large quantities of these molecules.
The activation of the transcription factors AP-1 and NF-κB is essential for tumor promotion and neoplastic transformation, and are highly expressed in the promoter-sensitive JB6P + cells, and the inhibition of both or either one of these signaling pathways is sufficient to inhibit neoplastic transformation [19, 23, 25]. To study the modulation of tumor promoter-induced AP-1 and NF-κB transcriptional activities by β-tan and Sal A in JB6P + cells, concentrations that inhibited JB6P + cell transformation and did not affect normal cell growth were used. Interestingly, both SL molecules decreased basal and TPA-induced NF-κB activities, but not of TPA-induced AP-1 activity. This suggests that β-tan and Sal A primarily inhibit NF-κB signaling in tumor cells. In fact, it is well established that NF-κB is a vital molecular target for various SL, and some of them, such as parthenolide, artimisinin and thapsigargin are currently in cancer clinical trials [6, 9, 43]. This can be attributed to the presence of the α-methylene-γ-lactone functional group, which directly alkylates cysteine residues of the p65 subunit, interfering with DNA binding [6, 44]. In fact, elevated NF-κB signaling is sufficient to induce epidermal tumor transformation . This prompted us to study the effect of these SL molecules on the protein levels of one of the main NF-κB inhibitors, IκBα. Previous studies have shown that the expression of non-degradable mutants of IκBα and antisense RNA inhibition of NF-κB, result in tumor regression [29, 45–47]. Interestingly, only pre-treatment with β-tan restored IκBα protein levels after 15 minutes of TPA-treatment, suggesting that Sal A and β-tan differentially mediate their inhibition of NF-κB signaling. This differential regulation of IκBα proteins by the SL molecules can be attributed to their differences in alkylating centers and lipophilicity, thus, affecting their interaction with the IκBα proteins. Nevertheless, β-tan also significantly increased basal AP-1 levels in JB6P + cells at concentrations that decreased cell growth. This may implicate the dual role of AP-1 in increased cell proliferation and cell death .
Since earlier studies have shown that AP-1 and NF-κB can interact together , we assessed how both SL molecules modulated key downstream target genes, containing TPA response elements (TREs) common to both AP-1 and NF-κB. Metalloproteinases are essential for tumor promotion, progression, and invasion and AP-1 and NF-κB play a dominant role in the transcriptional activation of the majority of MMPs [50, 51] including MMP-9 and MMP-2. In fact, it was shown in mice lacking MMP-9 that this gene is functionally involved in the regulation of oncogene-induced keratinocyte hyperproliferation, progression to invasive cancer, and end-stage malignant grade epithelial carcinomas . Treatment of TPA-promoted JB6P + cells with β-tan or Sal A, abrogated MMP-9, but not MMP-2, protein levels. This implies that the two SL molecules differentially modulate MMP protein levels suggesting the regulation of MMP2 by factors other than AP-1 and NF-κB.
Another important AP-1 and NF-κB target gene is the CDKI p16. Both SL molecules noticeably up regulated p16 that was reduced upon TPA treatment, which suggests that β-tan and Sal A inhibit cell cycle progression that is induced by tumor promoters. Furthermore, AP-1 and NF-κB components also regulate apoptotic proteins such as the pro-apoptotic Bax and the anti-apoptotic Bcl-2 proteins [38, 51]. SL are known to be inducers of apoptosis in a variety of cancer cells, and this is considered one of the important mechanisms by which SL exert their anti-tumor properties . Our results show that both β-tan and Sal A increase the Bax:Bcl-2 ratios in TPA-promoted JB6P + cells and suggest that Bcl-2 family members are involved in the growth suppressive effects of β-tan and Sal A.