Ultramicroscopic nanoparticles are used to facilitate novel state-of-the-art therapeutic regimens and targeted drug delivery systems in an attempt to improve treatment efficacy (30
). They are also used in cosmetics and other consumer products (13
). However, the increased surface to volume ratio of the miniscule nanoparticles increases reactivity and may result in intrinsic toxicity. Despite the wide application of nanoparticles, knowledge of their adverse effects, especially on carcinogenesis, is lacking. One of the most common entry routes for nanoparticles is inhalation, and early reports indicate that workers involved in aluminum production may be at increased risk of developing lung cancer (32
). In vivo
studies have demonstrated lung inflammation due to exposure to nanoparticles (33
). Systemic distribution of nanoparticles has been reported into the blood stream and lymphatic pathways (34
). Because aluminum distributes evenly in plasma and blood cells, aluminum concentrations in plasma and whole blood have similar value in assessing toxicity (35
). Aluminum is excreted predominantly via the kidneys and therefore accumulates in patients with renal failure (36
). Long-term exposure could lead to accumulation of aluminum, even in workers having normal renal function.
Another important route for nanoparticle entry is the skin, from accidental exposure and use of cosmetics and other topical applications. Although the outer layer of the epidermis, the stratum corneum, protects against environmental insults, TiO2
has been shown to penetrate the stratum corneum and even hair follicles (37
). Penetration of nanosized titanium dioxide (5–20 nm) into the skin and interaction with the immune system has been demonstrated (38
). Studies have also demonstrated that, in conjunction with motion, nanoparticles penetrate into the stratum corneum of human skin reaching to the epidermis and dermis (39
Recently, aluminum has been found to be a potential pro-oxidant in sunscreens and sunblocks (40
). Several studies report permeability and accumulation of aluminum salts in antiperspirants with dispersion to systemic sites (41
). In addition, aluminum has been categorized as a metalloestrogen that can interfere with estrogen receptors and that has a potential role in breast cancer (31
). Further, aluminum could function as a pro-oxidant increasing oxidative damage to the skin (42
). However, whether and how nanoparticles of alumina exert a carcinogenic effect on skin epithelial cells are unknown.
Our results show that alumina is internalized and significantly increases MnSOD protein levels, indicating that the effect of alumina may occur, in part, via alteration of cellular redox status. Our results also indicate that nanoparticle exposure can cause increased proliferation and anchorage-independent transformation in JB6 cells. PCNA is a well-established indicator of cell proliferation, actively involved in DNA replication and repair (43
). The increase in PCNA levels further validates the role of alumina in cell proliferation. Our results also demonstrate that treatment with alumina enhances MnSOD protein and activity as well as the levels of total cellular ROS. These results suggest that the observed increase in MnSOD levels is an adaptive response to alumina-induced oxidative stress. The finding that the total cellular ROS is also increased in the presence of higher MnSOD activity is consistent with this possibility. Further support for this possibility includes the finding that alumina activates AP-1, a redox-sensitive transcription factor. The major components of AP-1 are the ‘Jun’ (c-Jun, JunB and JunD) and ‘Fos’ (c-Fos, FosB, Fos-related antigen-1 and Fos-related antigen-2) family of proteins (44
). They are characterized by the leucine zipper regions that allow the different components to form homodimers or heterodimers and bind to specific DNA-binding elements called TPA response elements (45
). Unlike the Jun family of proteins, Fos proteins cannot form homodimers. They form heterodimers with the Jun family of proteins (46
). Activation of AP-1 is essential for the neoplastic transformation of mouse epithelial JB6 cells (47
). Inhibition of c-Jun or AP-1 represses transactivation of AP-1 and transformation of JB6 cells (48
). Thus, AP-1 transcriptional activity instigated by alumina may result from redox-mediated events that lead to cell proliferation and neoplastic transformation.
Mammalian silent information regulator 2 homolog (SIRT1) has been recently identified as a prosurvival factor against stress-induced DNA damage (18
). SIRT1 promotes cell survival by negatively regulating the tumor suppressor protein p53 (18
). Previous studies have shown that the deacetylase activity of SIRT1 is responsible for gene silencing, DNA recombination, increase in survival and longevity in response to oxidative stress and other stress factors (18
). Our studies indicate that alumina exposure to mouse epithelial cells increases SIRT1 protein and activity levels. Interestingly, we also observed interaction of SIRT1 with the AP-1 components c-Jun and JunD in alumina-exposed cells. This is the first study to demonstrate SIRT1 as a component of AP1-mediated transcription. Our results also show that SIRT1 is an essential modulator of AP-1 mediating cell proliferation and neoplastic transformation as the use of siRNA to block SIRT1 attenuates AP-1 transcriptional activity, protein levels of c-Jun, JunD and Bclxl, as well as PCNA levels, cells in S phase and cell viability.
The Bcl-2 family of proteins consists of both antiapoptotic and proapoptotic members and the ratio of these proteins often determines the life/death fate of cells (55
). The Bcl-2
gene was originally identified as an oncogene involved in human follicular B cell lymphoma (57
). Bcl-2 and BclxL
prevent apoptosis by sequestering to death-inducing procaspases and/or preventing release of cytochrome c
and apoptosis-inducing factor into the cytoplasm (55
). In contrast, proapoptotic Bax and Bak trigger the release of cytochrome c
that initiates the caspase signaling cascade (55
). The BclxL
protein is localized within the mitochondrial membrane (60
) and inhibits apoptosis (61
). An AP-1 consensus sequence was found at −267 of the promoter region of the mouse bcl-xL
). Our findings that alumina increases the prosurvival AP-1 target gene BclxL
and that suppression of SIRT1 reverses BclxL
levels further support the possibilities that AP-1 activation is a mechanism by which nanoparticles of alumina can cause transformation.
The mechanisms by which SIRT1 participates in the carcinogenesis process are unknown. Although the precise mechanism by which SIRT1 modulates AP1 activity is unclear, our study indicates that SIRT1 may contribute to the carcinogenesis potential of alumina, at least in part, by interacting with AP-1 and modulating the expression of AP-1 target genes. These results reveal a novel mechanism involving the positive role of SIRT1 on transcription, leading to enhanced proliferation in alumina-treated cells. Further study in an animal model will be needed to establish this novel observation. Our initial observations in a cellular model suggest that alteration of cellular longevity and metabolic regulator should be considered in tandem with the evolving new opportunities using engineered nanoparticles to ensure the safety of nanomaterials.