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Arecoline, the major alkaloid of areca nut, has been shown to cause strong genotoxicity and is considered a potential carcinogen. However, the detailed mechanism for arecoline-induced carcinogenesis remains obscure. In this study, we noticed that the levels of p21 and p27 increased in two oral squamous cell carcinoma cell lines with high confluence. Furthermore, when treated with arecoline, elevated levels of p21 and p27 could be downregulated through the reactive oxygen species/mTOR complex 1 (ROS/mTORC1) pathway. Although arecoline decreased the activity of mTORC1, the amounts of autophagosome-like vacuoles or type II LC3 remained unchanged, suggesting that the downregulation of p21 and p27 was independent of autophagy-mediated protein destruction. Arecoline also caused DNA damage through ROS, indicating that the reduced levels of p21 and p27 might facilitate G 1/S transition of the cell cycle and subsequently lead to error-prone DNA replication. In conclusion, these data have provided a possible mechanism for arecoline-induced carcinogenesis in subcytolytic doses in vivo. (Cancer Sci 2012; 103: 1221–1229)
Oral cancer is one of the most common cancers, resulting in 128 000 deaths worldwide in 2008. Oral squamous cell carcinoma (OSCC) is frequently found on the tongue and buccal, as well as gingival areas and accounts for more than 90% of oral cancer incidence. In addition to cigarettes and alcohol, betel quid is a risk factor of oral cancer. Betel quid is composed of areca nut, lime, and Piper betle leaf. Being a major alkaloid in areca nut, arecoline has long been considered a potential carcinogen. Several reports showed that arecoline may increase reactive oxygen species (ROS), retard the cell cycle, and induce apoptosis.[3, 4] Arecoline was also proved to enhance unscheduled DNA synthesis that caused strong genotoxicity in mouse germ cells. In addition, arecoline impeded DNA repair and mitotic spindle assembly.[6-8] Recently, arecoline was suggested to regulate the expression of certain genes through epigenetic control.
Cell cycle progression is dependent on sequential coordination among multiple proteins. In particular, cyclin levels can oscillate in different stages and interact with cyclin-dependent kinases (CDKs), forming the cyclin/CDK complexes for promoting cell cycle progression. In contrast, p21 and p27 belong to the kinase inhibitor protein family and regulate the cell cycle in response to various stresses, such as DNA damage, hypoxia, and confluence stress.[10-13] For example, once activated by DNA damage sensors like ataxia telangiectasia mutated, p53 enhances p21 transcription to arrest cell cycle before G1/S transition for DNA repair.
Reactive oxygen species consists of various radicals that are triggered by a range of agents and may exert different effects on downstream signaling pathways, including the levels of p21 and p27.[14, 15] Deregulated ROS levels are strongly implicated in cancers. Reactive oxygen species can induce DNA damage that results in genomic instability and contributes to carcinogenesis.[17, 18]
The mTOR complex 1 (mTORC1) is composed of mTOR, raptor, and GβL and is controlled by multiple pathways, such as PI3K-Akt signaling. Activated mTORC1 facilitates protein synthesis through controlling various downstream factors including 4E-BP1, p70S6K, and eEF2K, thus allowing release of eIF4E for translation initiation and activation of elongation factor 2 (eEF2) for translation elongation. Another important role of mTORC1 is to regulate autophagy. In conditions like energetic stress, mTORC1 may be inhibited by AMP-activated protein kinase (AMPK) to allow autophagy induction.
This study was initiated from an interesting observation: upregulation of p21 and p27 in OSCC cells with high confluence could be downregulated by arecoline treatment. In 2003, the International Agency for Research on Cancer announced that betel quid and areca nut chewing were carcinogenic to humans. Arecoline is one of the most abundant alkaloids of areca nut, suggesting its potential role in carcinogenesis. Here, we show the mechanism behind arecoline-induced downregulation of p21 and p27, which may be important for betel quid chewing oral carcinogenesis.
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In this study, we showed an interesting observation that the levels of p21 and p27 were obviously upregulated, particularly in high confluence of OSCC cells. Several studies have indicated that cell confluence affects protein expression or function in malignant cells. For example, accumulation of HSP27 during high confluence increases resistance against anticancer drugs in colon cancer cells. CD26 also has a confluence-dependent expression pattern in colon adenocarcinoma cell lines HCT-116 and HCT-15. Therefore, the arecoline-induced downregulation of p21 and p27 in confluent cells might play an important role in regulating the mechanism of oral carcinogenesis. Here, we illustrated the potential roles of p21 and p27 in arecoline-induced oral carcinogenesis.
Several factors could affect the cellular levels of p21 and p27, including gene expression, stability, and intracellular localization.[42, 43] For example, active p53 increases the expression of p21 and p27 to arrest cells.[44, 45] In human epithelial cells, arecoline was reported to decrease p53–p21 signaling and repress DNA repair. Given that p53 was either weakly expressed or mutated in the two oral cancer cell lines, it is conceivable that p53 was not the major factor accounting for arecoline-induced downregulation of p21 and p27 (Fig. 1B). Instead, we showed that mTORC1 played an important role in this biological effect.
However, the detailed mechanism by which mTORC1 regulates the levels of p21 and p27 remains unclear. One possible mechanism is through the autophagy degradation pathway. However, 10 nM rapamycin is sufficient to downregulate p21 and p27 but fails to increase autophagosome-like vacuoles or LC3-II simultaneously (data not shown and Fig. 4C, lane 8), highly suggesting that downregulation of p21 and p27 might not be through the mTORC1-dependent autophagy degradation pathway. The other possible mechanism is through protein synthesis, as arecoline affected translational machinery like S6 (Fig. 3A,B) and eEF2, which is known to mediate GTP-dependent translocation of synthesizing peptide chain from A-site to P-site in ribosomes. In addition, mTORC1 was reported to phosphorylate SGK1 and modulate the function of p27.
As important regulators at cell cycle checkpoints, p21 and p27 are frequently considered as tumor suppressors. Reduction of p21 and/or p27 possibly facilitates deregulated proliferation or insensitive to contact inhibition.[23, 26, 47] Several studies have indicated the prognostic potential of p27 in various cancers, including oral cancers.[48-53] It was shown that p27 could induce senescence in cancer cells and was considered a checkpoint for limiting the progression of prostatic intraepithelial neoplasia to invasive cancer. Unlike p27, the role of p21 remains controversial.[55, 56] Previous studies regarding the prognostic application of p21 in different stages of OSCC were inconsistent.[48, 57-60] It remains unclear whether these inconsistencies result from p21 polymorphism, which may be associated with oral malignancy.
Long-term exposure of betel quid, which is composed of areca nut, lime, and Piper betle leaf, is frequently associated with the induction of carcinogenesis in the oral cavity. However, the detailed mechanism behind betel quid chewing carcinogenesis is still elusive. Arecoline is the major alkaloid in areca nut. According to previous studies, the detected concentration of arecoline in betel quid chewers' saliva was approximately 0.3 mM or 0.1–10 μg/mL, with the sudden peak concentration of approximately 100 μg/mL during betel quid chewing.[6, 62] This urged us to propose a model as shown in Figure 6: the arecoline concentration in the oral cavity of betel quid chewers may fluctuate dramatically in vivo. The sudden impact of high concentrations of arecoline may induce DNA damage through the ROS pathway and prevent increases of p21 and p27 through the ROS/mTORC1 pathway simultaneously, leading to a bypass of the G1/S checkpoint. Once betel quid is removed, cells are allowed to continue proliferation through G2/M presumably without genomic integrity. Finally, in combination with other unidentified factors, such as p53 mutation, arecoline leads to the deregulated growth of surviving cells, accounting for the mechanism of arecoline-induced carcinogenesis.
Figure 6. Proposed model for arecoline-induced carcinogenesis. Arecoline induced reactive oxygen species (ROS), followed by downregulation of p21 and p27 through mTOR complex 1 (mTORC1) and increased susceptibility to DNA damage. Low amounts of p21 and p27 may result in a less stringent G 1/S checkpoint and the ensuing error-prone DNA replication, leading to deregulated growth of cells.
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