Discussion
In traditional Chinese medicine, Ginkgo leaf extract is widely used as medical herb to treat cardiovascular disorders (Xu, Hu, Shen & McQuillan, 2015). It has been reported that GGC isolated from Ginkgo leaf , can exhibit diverse pharmacological properties (Huang et al., 2014; Huang, Chen, Liu, Wu & Liou, 2018; Liou, Lai, Chen, Wang, Wei & Huang, 2015; Zhang et al., 2018). This is the first investigation that has analyzed the potential impact of GGC and its mechanism in NSCLC model. We found that GGC diminished activation of STAT3 as well as that of JAK1, JAK2, and c-Src phosphorylation. This inhibition led to an induction in the expression of phosphatase PTPε. GGC also mitigated proliferation and promoted apoptosis through down-regulating STAT3 signaling cascade. Moreover, it attenuated tumor growth and survival in a xenograft mouse model without displaying any toxicity.
Aberrant STAT3 activation has been associated with various tumors, including head and neck, lung, breast, prostate, kidney, pancreas, liver cancer, lymphomas, and multiple myeloma (Banerjee & Resat, 2016; Bharti, Donato & Aggarwal, 2003; Chai et al., 2016; Hwang et al., 2019; Jung et al., 2018). We first investigated if GGC could alter constitutive and inducible STAT3 phosphorylation in NSCLC cells. We noted that GGC abrogated STAT3 phosphorylation and localization of this protein into the nucleus. Phosphorylation of JAKs has been linked to STAT3 activation (Ahn, Sethi, Sung, Goel, Ralhan & Aggarwal, 2008; Baek et al., 2017; Bowman, Garcia, Turkson & Jove, 2000). Interestingly, GGC mitigated JAK1, JAK2, as well as c-Src phosphorylation substantially and overexpression of STAT3 not only alleviated the inhibitory actions of GGC on p-STAT3 levels but also relieved apoptosis caused by this agent.
Recently, many studies have reported that protein tyrosine phosphatases (PTPs) may negatively control STAT3 signaling pathway (Ahn, Sethi, Sung, Goel, Ralhan & Aggarwal, 2008; Baek et al., 2016a; Baek et al., 2016b; Kim, Morales, Jang, Cho & Kim, 2018). We noted that GGC may have altered STAT3 activation by causing the modulation of PTPs. Interestingly, GGC promoted PTPε expression at protein and mRNA levels. Moreover, GGC could increase PTPε M expression also at mRNA levels. Moreover, the silencing of PTPε expression repudiated GGC-driven attenuation of STAT3 activation and induction of apoptosis. However, how GCC can stimulate PTPε levels and if GCC can also alter, its activity needs additional investigations.
It has been reported that reduction of STAT3 activity may decrease the survival ability of tumor cells (Aoki, Feldman & Tosato, 2003). Hence, we observed if GGC could alter proliferation and promote apoptosis in NSCLC cells. GGC induced apoptotic death was detected by cell cycle, annexin V and TUNEL assays. We demonstrated that GGC caused sub-G1 arrest, early apoptosis, and mediated the activation of caspase-3 and PARP. In addition, we noted that GGC dramatically reduced the expression of oncogenic molecules such as anti-apoptotic and metastasis promoting proteins that may contribute to its varying anti-neoplastic activities.
In a NSCLC preclinical model, GGC significantly suppressed lung tumor growth, altered the levels of p-STAT3, PTPε, and caspase-3 in treated groups. These modifications are consonant with its observed in vitro actions and were noted in the absence of any adverse effects. Thus, our preclinical studies imply that GGC may have a potential as a novel therapeutic agent for the management of NSCLC.
In summary, it was noticed that GGC can exert pleiotropic anti-neoplastic effects through modulating STAT3 signaling pathway by affecting the PTPε expression. Taken together, GGC may act as an effective inhibitor of STAT3 phosphorylation and its function can be investigated further in alleviation of different malignancies.