Prophylaxis And Mechanism Of Chemically Induced Skin Tumor By Diallyl Disulfide

Bcakground and PurposeThe challenges of conventional anticancer therapies do not only exist in the frequent failure of clinical outcomes, but the out-of-pocket expenditure for patients. Cancer chemoprevention is the use of substances to to slow down or eliminate the progression of intra epithelial neoplastic or precancerous lesions to tumor, and these substances for cancer prevention should be nontoxic, economical, orally active and easily available. Black Garlic, a new garlic processed foods, has demonstrated a variety of pharmacological and biological activities, including anti-inflammatory, antimicrobial, and antitumor activities. DADS has the potential to inhibiting tumor cell proliferation, inducing cancer cell apoptosis and redcuing cancer cell metastasis. The present study focused on DADS-evoked defense mechanism to combat the chemically induced skin carcinogenesis, which might clue the molecular mechanisms that mediate the action of DADS.Research Contents and ResultsThe present study set to utilize a two-stage chemically induced papillomagenesis in the back skin of mice, which involves topically use of DMBA, a tumor initiator that induces DNA damage, followed by TPA, a phorbol ester that promotes tumor development, and anchorage-independent JB6 P+ cell growth assay for characterizing the potential chemopreventive effect of DADS. The results suggested treatment with DADS remarkably reduced both the incidence, multiplicity of skin papilloma formation and papilloma size distribution in a concentration-dependent manner in vivo. DADS suppressed TPA-induced JB6 P+ cells transformation in vitro. Given the antioxidant properties of DADS, we found pre-treatment with DADS significantly inhibited the reactive oxygen species (ROS) accumulation in a dose-dependent manner compared to the DMBA/TPA-treated group. Therefore, we further asked whether DADS could trigger cellular antioxidant response in presence of DMBA/TPA. Moreover, the mRNA levels of antioxidant enzymes were significantly up-regulated after DADS treatment. The next findings demonstrated DADS obviously induced nuclear accumulation of Nrf2 in the epidermis. Consistent with this finding, DADS also increased the nuclear localization of Nrf2 in vitro. To further verify whether Nrf2 was a prerequisite target for the chemoprevention of DADS, we further established DMBA/TPA-induced skin carcinogenesis in Nrf2 global deletion mice. The results shown DADS failed to prevent DMBA/TPA induced skin carcinogenesis in Nrf2-/-mice. We then examined the underlying molecular mechanism of Nrf2 translocation induced by DADS in JB6 P+ cells. The nuclear Nrf2 level was also elevated by DADS treatment in time-dependent.The result was confirmed by immunofluorescence staining and WesternBlot. But DADS failed to induce Nrf2 nuclear translocation through activation of upstream MAPKs and Akt. Similarly, Western blot analysis revealed DADS treatment resulted in a significant increase in the total Nrf2 level. We then mainly focused on the post-translational regulation of Nrf2 which was evidenced by our observation that DADS did not alter the mRNA level of Nrf2. Moreover, we introduced the MG132, a proteasome inhibitor to check whether the DADS-mediated Nrf2 stabilization was the result of blockage of its proteasomal degradation, and we found DADS increased Nrf2 protein levels partially through a proteasome-dependent pathway. We next examined the role of p21 mediating the Nrf2 stability regulation and proposed p21-dependent Nrf2 induction by DADS. Supporting the notion, p21 might participate in the Nrf2 regulation as we observed that DADS elicited a time-dependent induction of p21 which was almost synchronized with the induction of Nrf2. However, we could not observe the change of Keapl expression level. We next investigated the status of the p21 and Nrf2 complex in presence of DADS. As we found in co-IP study, DADS induced the dissociation of Keapl with Nrf2, accompanied by increased interaction of p21 with Nrf2, which implied that DADS-mediated interaction of p21 with Nrf2 rescued Nrf2 from Keapl-mediated ubiquitination and degradation. Moreover, DADS-induced expression of Nrf2 was abrogated in HEK293T cells transfected with si-p21. Collectively, these data suggested that recruitment of p21 to release Nrf2 from Keap1 was a primary mechanism of DADS-mediated Nrf2 stabilization.Conclusion and SignificanceIn conclusion, our discovery are briefly summarized as follows:topical application of DADS inhibits TPA-induced tumor promotion by means of promoting Nrf2 nuclear localization in mouse skin, which seemed to be mediated through suppressing degradation of Nrf2 via up-regulation of p21 protein level. The present study will provide insight into the future application of naturally occurring phytochemicals-DADS in the clinical setting.