Luteolin (3, 4, 5, 7- tetrahydroxylflavone) is a flavonol-type flavonoid ubiquitously present in plant-derived foods. Luteolin-rich vegetables and fruits include celery, parsley, broccoli, onion leaves, carrots, peppers, cabbages, apple skins and chrysanthemum flowers (Miean and Mohamed, 2001; Sun et al., 2007;
Xie et al., 2009).
Luteolin exhibits a wide range of biological activities in the prevention and treatment of chronic diseases due to their anti-oxidant, anti-inflammatory, anti- microbial and anti-cancer activities (reviewed by (Lopez-Lazaro, 2009). In our laboratory, luteolin has been found to enhance TNF--induced apoptosis in human colorectal cancer COLO205, HCT116 and cervical cancer Hela cells via suppression of NF-B (Shi et al., 2004). In a second study, pre-treatment of TRAIL-sensitive cancer cells like Hela and TRAIL-resistant cancer cells like CNE1, HT29 and Hep G2 with a non-cytotoxic concentration of luteolin was able to enhance TRAIL-induced apoptosis mediated by caspase -8 and -3 activation (Shi et al., 2005).
At present, the anti-cancer potential of luteolin is mainly based on its ability to induce apoptosis in cancer cells (Lin et al., 2008). However, relatively little is known about the effects of luteolin on cell cycle regulation. Several earlier reports have demonstrated that luteolin induces cell cycle arrest either at G1 by down-regulating cellular protein levels of CDK4 and CDK2 (Casagrande
Page | 137 and Darbon, 2001; Lim do et al., 2007) or G2/M arrest by the inhibition of cdc2 and up-regulation of p21CIP1 (Wu et al., 2008).
In this study, we also focussed on the effect of luteolin on cell cycle regulation in human nasopharyngeal carcinoma cells. We identified a different molecular mechanism leading to cell cycle arrest by luteolin. Luteolin inhibits the Akt-GSK-3-cyclin D1 signalling pathway in the NPC line, HK1 by promoting cyclin D1 phosphorylation and subsequent proteasomal degradation. In the absence of cyclin D1, pRb is maintained in the hypophosphorylated form and this prevents the activation of E2F-1 transcription activity.
Progression from G1 to S phase of the cell cycle is controlled by cyclin Ds and their kinases, namely CDK4 and CDK6, which act by phosphorylating and inactivating Rb, thus liberating E2F-1 transcriptional activity to drive the cells into S phase (Genovese et al., 2006; Malumbres and Barbacid, 2009). In this study, we first defined the critical role of cyclin D1 in luteolin-induced cell cycle arrest. It is well known that cyclin D1 is important in the development of numerous cancers including NPC (Gladden and Diehl, 2005; Tashiro et al., 2007;
Xie et al., 2000) (reviewed in Chapter 1.3 of this thesis). Moreover, cyclin D1 over-expression is a common event in cancer and is usually a result of defective regulation at the post-translational level (Kim and Diehl, 2009). Therefore, regulation of the cyclin D1 protein level is one of the critical aspects in cell proliferation and tumour development. An earlier study by Diehl et al (Diehl et al., 1997) demonstrated that cyclin D1 degradation is dependent on Thr286 phosphorylation by GSK-3 and ubiquitin-dependent proteasomal degradation.
Interestingly, we found that luteolin acts on this signalling pathway in HK1, resulting in the induction of cell cycle arrest. An increase in the phospho form of
Page | 138 cyclin D1 Thr286 was observed when HK1 was treated with luteolin. As GSK-3
regulates cyclin D1 degradation, a GSK-3-specific inhibitor, LiCl, suppressed luteolin-induced down-regulation of this protein, suggesting the involvement of the GSK-3 pathway in luteolin-mediated cell cycle arrest.
Frequent activation of Akt has been reported in many human cancers (Altomare and Testa, 2005; Tokunaga et al., 2008) and GSK-3 has been identified as one of Akt’s molecular targets. Akt inactivates GSK-3 kinase activity by site-specific phosphorylation at Ser9 which leads to subsequent reduction in cyclin D1 phosphorylation and an increase in its protein stability (Diehl, 2002). In this study, we found that luteolin is capable of inhibiting Akt phosphorylation and activation. It remains to be investigated how luteolin may target PI3K, as suggested by earlier reports (Bagli et al., 2004; Lee et al., 2006).
Interestingly in our study luteolin was unable to induce apoptosis in HK1 and CNE2 (although these cells are responsive to camptothecin-induced apoptosis) when many studies have demonstrated the apoptotic effects of luteolin on numerous cancer cell lines. The exact reason for this lack of apoptotic response to luteolin by HK1 and CNE2 is not clear. One possible explanation is that this group of cells have higher basal level of Akt activation (as demonstrated in Chapter 3 of this thesis), thus making them more susceptible to the inhibitory effect by luteolin on cell cycle via the Akt-GSK-3-cyclin D1 pathway. It would be of interest to elucidate the underlying mechanisms responsible of the different response by different types of cancer cells.
Thus, this study (Chapter 3 of this thesis) demonstrates that luteolin is able to suppress NPC cells proliferation via cell cycle arrest by targeting the Akt-GSK- 3-cyclin D1 signalling axis. Since Akt is often over-activated in many human
Page | 139 cancers including NPC, it is thus believed that the data from this study support the potential application of luteolin as a chemotherapeutic or chemopreventive agent in human cancer.