Importantly a crucial role of
Importantly, a crucial role of transcription factor SP1 in mediating the anti-lung cancer effect of XJD was found in this study. Our results suggested that the inhibition of SP1 was required to mediate the inhibitory effect of XJD on the growth of NSCLC cells. Nuclear transcription factors SP1 is associated with a variety of essential biological functions, such as cell growth, differentiation, apoptosis, and carcinogenesis, thereby having multiple influences on downstream gene Triptolide (Cho et al., 2015; Chuang et al., 2014; Zhao et al., 2015). Studies have reported the overexpression of HOTAIR in the majority of human cancers, suggesting that HOTAIR promotes cancer cell growth and progression, and may be a critical player in lung cancer development. Our results demonstrated that inhibition of HOTAIR is involved in XJD-inhibited NSCLC cell growth. Interestingly, we observed a reciprocal interaction between SP1 and HOTAIR affected by XJD. Highly expressed HOTAIR blocked XJD-inhibited SP1 protein expression and vice versa, implying a mutual regulatory axis between SP1 and HOTAIR that might mediate the anti-lung cancer effect of XJD. So far, there is limited information regarding links of HOTAIR and SP1. One study reported the regulation of HOTAIR and SP1 in affecting miR-326 expression and reversing chemoresistance of lung cancer, further highlighting the importance of targeting the HOTAIR/SP1 pathway in therapeutics of lung cancer (Li et al., 2016a). More experiments are warranted to validate and expand these findings, and to further dissect the importance of these interactions. Our results also observed a critical role of EP4 in the anti-lung cancer effect of XJD, implying that EP4 could also be a potential target in the treatment of lung cancer. As a PGE2 receptor subtype, EP4 has been associated with multiple signaling pathways leading to diverse cellular functions, including tumor cell growth, invasion, metastasis, and resistance to therapy (Chen et al., 2017; Hiken et al., 2017; Tonisen et al., 2017). Several studies have showed that the inhibition of EP4 suppressed the growth of several cancer cell types, suggesting an critical role of this prostanoid receptor (Parida et al., 2015; Takahashi et al., 2015; Tonisen et al., 2017). Our current findings indicated that the inhibition of SP1 and HOTAIR was required to mediate the effect of XJD to inhibit EP4 expression and suppress NSCLC growth. Although the connections between HOTAIR and EP4 have not been shown, links of EP4 and SP1 signaling have been observed in other studies (Hann et al., 2013; Kambe et al., 2008). An early study demonstrated that the human EP4 gene promoter contains SP1 DNA binding sites and regulates EP4 expression (Kambe et al., 2008). The overexpression of SP1 has been found to reverse the inhibitory effect of curcumin, a well-known diarylheptanoid constituent of turmeric, on EP4 promoter activity and protein expression in other cancer cell types (Hann et al., 2013). Our current data suggested that SP1 and HOTAIR, act downstream of ERK1/2 and upstream of EP4, and regulate and interact with EP4 affect the overall responses of XJD to control the growth of lung cancer cells. This is the first report showing the connection between lncRNA HOTAIR and EP4 expression. Nevertheless, the molecular interactions and regulations among SP1, HOTAIR, and EP4 require further elucidation. Additionally, we observed the potential synergy of XJD and gefitinib. As a first generation EGFR-TKI and first-line treatment for some NSCLC patients, gefitinib was reported to inhibit growth, metastasis, and angiogenesis, and significantly prolong survival time for patients with tumors containing an activated EGFR mutation. However, most patients showed limited effects after treatment due to intrinsic and acquired drug resistance (Chen et al., 2013; Mitsudomi et al., 2010). Strategies to overcome the resistance to EGFR-TKIs in clinical practice are urgently needed. Our results suggest that XJD may enhance the therapeutic effects of gefitinib on suppression of lung cancer cells through the suppression of HOTAIR/SP1 and EP4 signaling pathways. Consistent with our findings, previous studies reported that EGFR-TKIs such as gefitinib are involved in the inhibition of HOTAIR, SP1, and EP4 signaling pathways, allowing cancer therapeutic gain (Jang et al., 2016; Tveteraas et al., 2012; Wang et al., 2015). Thus, we reasoned that there could be potential therapeutic targets for cancer therapy. The current findings uncovered additional molecular mechanisms contributing to the anti-lung cancer effect of XJD. Because of the multiple compounds, and the underlying molecular mechanisms by which XJD can sensitize the inhibition effect of gefitinib in lung cancer may be complicated than we can thought. More in-depth experiments are required to elucidate the details of the synergistic mechanism.