There is increasing evidence for an association between a high consumption of fruit and vegetables and reduced risk of oral cancer, suggesting that natural products offer a protective effect against oral cancer [34
]. In addition many substances derived from dietary or medicinal plants are known to be effective and versatile chemopreventive and antitumoral
agents in a number of experimental models of carcinogenesis [36
]. In this regard, Li et al. [37
] showed that curcumin, a natural Hmox1 inducer, present in turmeric and curry and possessing antioxidant properties, appeared to have an inhibitory effect on the progression from dysplasia to SCC.
We describe, in the present study, the effects of pharmacological induction of Hmox1 using Chilean propolis and SnCl2
in KB cells, and how this may impact on KB cell cytotoxicity and proliferation. We demonstrated that Chilean propolis and SnCl2
showed a significant increase in Hmox1 protein expression which was followed by a decrease in cell viability and this effect was reversed by the addition of SnMP, thus suggesting that Hmox1 may play an important role in both propolis and SnCl2
toxicity. These results are consistent with our previous results showing that KB cells are particularly sensitive to propolis [28
]. The possible involvement of the Hmox system was further suggested by the use of CORM-II which showed that, CO, one of the Hmox products, is toxic for KB cells in a dose- and time-dependent manner. We also propose that propolis has different mechanisms of toxicity in KB cells. In fact, we found that this compound caused a significant decrease in cell number as a result of cell necrosis as measured by LDH release. By contrast, SnCl2
showed a significant decrease in cell number unaccompanied by a concomitant cell membrane breakdown. In this case, decreased cell number may be related to the increased expression of Hmox1 which leads to increased CO cellular levels, thus upregulating p21 protein expression. This hypothesis is supported by our experiments with CORM-II, which showed that increased CO levels result in a significant increase in p21 protein expression. These results are in apparent contrast with our recent work showing that the same concentrations of CO releasing molecule resulted in a significant increase of endothelial cell proliferation and angiogenesis, as measured by capillary formation; however high concentrations of CO releasing molecule resulted toxic for endothelial cells and inhibited angiogenesis [38
]. These data obtained on different cell types suggest that CO regulates cell proliferation in a cell-specific, dose- and time-dependent manner. In addition, these results are consistent with our previous work [39
] demonstrating that Hmox1 regulates proliferation in a cell-specific manner by differentially regulating p21 protein expression; in fact, pharmacological Hmox1 induction increased endothelial cell proliferation, but inhibited smooth muscle cell proliferation. Furthermore, we and others demonstrated that the same CORM-II concentrations showed no significant cytotoxic effects in other cell types such as astrocytes, endothelial cells, smooth muscle cells and cardiomyocyte [33
The idea of the involvement of different mechanisms in propolis toxicity, besides Hmox1 induction, is supported also from our ROS formation data showing that propolis, because of its anti-oxidant extract phenolic components activity (galangin, caffeic acid, p-cumaric acid, ferulic acid and CAPE) [22
], resulted in a dramatic reduction in the formation of ROS, a mechanism involved in cancer cell proliferation [41
]. This hypothesis is supported also by previous studies showing that phenolic phytochemicals may scavenge the constitutively high amounts of ROS in cancer cells, thereby blocking MAPK signaling, activation of NFkB and AP-1, and ultimately the expression of responsive genes that stimulate cancer cell proliferation [26
The addition of SnMP, significantly attenuated the anti-oxidant effects of propolis, even though ROS remained significantly low when compared to control, thus confirming that the anti-oxidant properties of this compound are mediated in part by Hmox1 induction and also by the phenolic structure of propolis components. These data are also confirmed by our observations showing that SnCl2
caused a significant decrease in ROS formation eventhough propolis was a more potent ROS scavenger.
Taken all together, our data indicates that KB cells seem to be particularly vulnerable to Hmox1 induction which may represent a mechanism by which these cells regulate their proliferation and cell cycle progression, thus suggesting that the Hmox system may be the Achille’s heel of KB cells. In fact, pharmacological induction of Hmox1 is associated with decreased cell proliferation following p21 upregulation and increased cytotoxicity. These effects seem to be mediated by Hmox derived CO as suggested by the results following CORM-II treatment. Furthermore, other minor sources of CO include the auto-oxidation [42
] of phytochemical phenols which may account, in part, for propolis mediated cytotoxicity. These in vitro results seem to be consistent with recent clinical findings showing that increased Hmox1 expression was associated with reduced lymph node metastasis in patients affected by oral SCC [5
]. These results together with our recent data on angiogenesis strongly suggest that CO may represent an excellent strategy for controlling cancer growth.
In conclusion, our studies demonstrate that Chilean propolis, due to its phenolic components, and SnCl2
not only have cytotoxic and antiproliferative effects in KB cells, but also utilize Hmox1 in exerting their antitumoral effects, thus providing a new and powerful strategy for oral SCC treatments.