Photoprotective and therapeutic effects of the flavonoid Luteolin in human keratinocytes

The skin is the physical barrier of our inner body with the external environment. As such, the skin faces daily exposure to a variety of environmental, chemical and microbial insults. Although the skin, with its layered structure and a variety of endogenous stress response mechanisms, is very well adapted to perform its protective function, accumulation of damage throughout life can trigger dermatological pathologies, such as skin cancer. Ultraviolet (UV)-radiation, part of sunlight, is the major risk factor for skin cancer, which is the most common cancer in Caucasianpopulation. Notwithstanding the availability of valuable photoprotective measures, the incidence of both non-melanoma and melanoma skin cancer is still rising and is reaching epidemic proportions, which raises the urge for new and better photoprotective strategies. Squamous cell carcinoma (SCC), the most severe non-melanoma skin cancer, has the ability to metastasize and can therefore become lethal. This cancer derived from epidermal keratinocytes occurs more frequent, and shows a more aggressive behavior especially in immune compromised transplant patients. Therefore,the development and investigation of novel therapeutic agents to improve or replace current used therapeutics is required. Flavonoids arebotanical compounds which have protective functions in plants and are abundantly present in the historically known medicinal plants. Since flavonoids are demonstrated to exert pleotropic pharmacological functions in mammalian cells, they are widely proposed and investigated for the treatment of several diseases. Based on a few studies emphasizing the anti-inflammatory and anticarcinogenic effects of Luteolin or Luteolin-rich plant extracts, we hypothesized that this flavone molecule might be a potential photopreventive or photochemotherapeutic agent. In the first part of this thesis, we investigated the photoprotective potential of the flavonoid Luteolin in normal skin cells, and we demonstrated that treatment with Luteolin increased the resistance of normal human keratinocytes (NHK) against subsequent radiation with apoptotic doses of UVB. Luteolin inhibited UVB-induced ROS and interfered with the intrinsic apoptotic pathway resulting in a Luteolin dependent inhibition and delay of apoptotic cell death. While the repair of UVB-induced DNA lesions was not significantly increased by Luteolin, the enhanced survival of NHK after UVB was accompanied by the inhibition of cell proliferation and the induction of differentiation, reducing the risk of carcinogenic transformation. Furthermore, the UVB-induced release of pro-inflammatory mediatorsby NHKs, which is a prominent hallmark of sunburn and may create a tumor-promoting micro-environment, was diminished by Luteolin. Importantly, malignant keratinocytes derived from cutaneous SCC were not protected against UVB-induced apoptosis by Luteolin. Given that the differential response of normal and malignant keratinocytes on Luteolin treatment might entail possibilities for therapeutic application of Luteolin, we investigated possible cytotoxic effects of Luteolin in SCC cells in the second part of this thesis. Indeed, concentrations of Luteolin which werenot toxic for NHK, induced significant amount of caspase dependent apoptosis in several SCC cell lines. However, MET4 cells (derived from a lymph node metastasis) were less sensitive to Luteolin as compared to the isogenic MET1 cells (derived from a primary SCC). We discovered that MET4cells induced autophagy, a lysosomal pathway for protein/organelle degradation, as a survival mechanism in response to Luteolin treatment. Consequently, inhibition of autophagy using an inhibitor of lysosomal degradation reduced the resistance of MET4 cells to Luteolin. Furthermore, ourdata demonstrate that Luteolin inhibited AKT-signaling, an important pathway which modulates both apoptosis and autophagy which is frequently deregulated in SCC and also progressively upregulated in our SCC progression model. Altogether, our data contained in this thesis underscore the potential of the flavonoid Luteolin as part of new photoprotective strategies, in which flavonoids are added to conventional sunscreens or combined with DNA repair stimulating agents. Furthermore, we provided evidence that Luteolin triggers cell death specifically in malignant keratinocytes. Therefore, Luteolin might be suitable for therapeutic applications to treat cutaneous SCC. Nevertheless, further studies, especiallyin an in vivo setting, are required to further elucidate the differential response of normal and malignant keratinocytes after Luteolin treatment.status: publishe