Berberine (BBR), a promising phytochemical drug and isoquinoline alkaloid in nature, has been shown to exhibit anti-proliferation or cytotoxic effects against cancer cells of different origins, especially in lung cancer [19–21]. However, the mechanisms by this drug in control of NSCLC cell growth have not been well elucidated. In this study, we confirmed that BBR inhibited NSCLC cell proliferation and induced apoptosis. Moreover, BBR can arrest cell cycle in G0/G1 phase in A549 cells. The concentrations of BBR used here were consistent with or even lower then those reported by others demonstrating significant growth inhibition in different cell systems [21–23]. We realized that a higher dose was needed to inhibit different cancer cell growth, but this was within the range of those reported by others and showed no toxicity [21, 22, 24].
Induction of cell cycle arrest and apoptosis is regulated by a large number of molecules. In our study, we found that activation of p38α MAPK, but not ERK1/2, was mediated the effect of BBR on cell cycle arrest and induction of p53 and FOXO3a protein expression. Of notes, we demonstrated the unique role of p38α isoform played in this process, whether other p38 isoforms, such as p38γ or p38δ MAPK were also involved in this response required to be determined in the future studies. Consistent with this, the role of p38 MAPK pathway in mediating the cancer cell growth inhibition and induction of apoptosis has been established and reported [25–27]. The p38 MAPK pathway negatively regulated cell proliferation and tumorigenesis. Inactivation of the p38 pathway enhanced cellular transformation and rendered mice prone to tumor development with concurrent disruption of the induction of senescence. Conversely, persistent activation of p38 inhibited tumorigenesis, suggesting a tumor-suppressing function of the p38 pathway . Our results suggested that activation of p38 MAPK was required in mediating the effect of BBR on induction of tumor suppressors p53 and FOXO3a, and lung cancer cell cycle arrest. Note that activation of ERK/12 by BBR played no role in this process, which were different or even opposite reported by others [28, 29]. The discrepancy remained unclear; different cell lines and culture conditions may account for this, which needs to be determined with more experiments in the future. The cross-talk between ERK and p38 signaling pathways was reported in other studies [30, 31]. However, in this study we have not observed this link. Thus, more experiments may require to confirm this.
In this study, we demonstrated the important role of tumor suppressor p53 in mediating the effect of BBR on cell proliferation and cell cycle arrest, which were consistent with other studies [24, 32] suggesting that a p53-dependent pathway was required in this process. Tumor suppressor p53 plays a significant role in the regulation of cell growth, cell cycle arrest, and apoptosis in various cancers [33, 34]. p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts . Increased expression of wild-type p53 arrested cells late in the G1 stage of the cell cycle by stimulating the synthesis of inhibitors of cyclin-dependent kinase p21 (CIP1/WAF1) . Consistent with this, we found that BBR increased p21 protein expression in human lung cancer A549 cells, which was eliminated (not observed) in cells silencing of p53 gene. This suggested that BBR-induced p21 (CIP1/WAF1) was through p53-dependent pathway. P21 (CIP1/WAF1) is a direct target of p53 , thus, p53 mediated induction of p21 (CIP1/WAF1) at least contributed to the inhibitory effect of BBR on cell proliferation and cell cycle arrest.
On the other hand, our results suggested that activation of p38 MAPK mediated the BBR-induced FOXO3a protein expression and the latter also contributed to the BBR-inhibited cell growth and -induced apoptosis. It is possible that the inhibition of proliferation can be in part a consequence of increased cell apoptosis or vise versa. The FOXO3a is an important tumor suppressor and is under-expressed in many cancers. There are a number of parallels between FOXO3a and p53, both play a pivotal role in regulating the cellular response to stress and damage signals, inducing cell cycle arrest, apoptosis, and DNA repair . Several studies showed that FOXO3a interacts with p53, and that FOXO3a is a p53 target gene [15, 38]. In this study, we demonstrated that the potential interaction and mutually exclusive events of p53 and FOXO3a may contribute to enhance BBR-induced apoptosis and -inhibited cell proliferation. However, the detailed mechanism underlining the regulation of these transcriptional networks in mediating the effect of BBR on the control of lung cancer cell survival needs to be elucidated.
Our results also demonstrated a causative role of FOXO3a in mediated the effect of BBR on p21 (CIP1/WAF1) expression. We showed that the knockdown of FOXO3a blocked, while overexpression of FOXO3a augmented the increase in p21 (CIP1/WAF1) protein expression in BBR-treated cells. These, together with the observation from silencing of p53 experiments indicated that p21 (CIP1/WAF1) is not only the direct target of p53 but also function as FOXO3a downstream effector, which may be through the p53-independent way . p53 and FOXO3a share similar target genes including p21(CIP1/WAF1), FOXO factors bind to the promoter of p21 to induce cell cycle arrest at the G1/S transition . Given the fact that p21 (CIP1/WAF1) is involved in regulation of fundamental cellular processes, such as cell proliferation, differentiation, regulation of gene transcription and apoptosis [40, 41]. BBR-induced FOXO3a expression may contribute to induce cell apoptosis, which could be in part a consequence of inhibition of NSCLC cell growth. Of note, the dual function of p21 (Cip1/Waf1) was observed in cancerogenesis. On the one hand, p21 (Cip1/Waf1) acts as a tumor suppressor; on the other hand, it prevents apoptosis and acts as an oncogene [40, 42]. Therefore, precise understanding the role of p21 (Cip1/Waf1) and relevant signaling pathways involved would help to develop better cancer-treatment strategies.
Study showed that activation of p38 MAPK reduced protein expression of cyclin D1, another cell cycle regulator . Cyclin D1 actives cyclin dependent kinase 4 and 6 (Cdk4/6) and this active complex is essential for the transition to S-phase and further stimulates cell proliferation . In our study, we showed that BBR decreased the cyclin D1 protein expression, but this was not through the p53- or FOXO3a-dependent pathway, which consistent with other studies  although opposite results were observed [12, 46]. Thus, more studies are required to elucidate the truly connections and precise mechanism underlining this. In addition, whether the BBR-induced pro-apoptotic signaling by p38 MAPK is also activated and the functions of FOXO3a are regulated by p38 MAPK in cells silencing of p53 need to be determined. This may further elucidate pleiotropic anti-cancer mechanisms of this medicinal phyto-chemical compound.