4 DISCUSSION
Currently, the therapeutic options for patients with pancreatic cancer include surgical resection, chemotherapy, radiotherapy, and palliative care (Moore & Donahue, 2019). Most patients with pancreatic cancer have no symptoms until the disease reaches the late stage at which tissue invasion and distant metastasis have been occurring. Chemotherapy is the predominant treatment for patients with metastatic pancreatic cancer (Neoptolemos et al., 2018). In the past thirty years, the standard drugs for treating pancreatic cancer were 5-fluorouracil (5-FU) and gemcitabine (Stan, Singh, & Brand, 2010). In 1996, owing to the longer median survival time and higher quality of life, gemcitabine substituted 5-FU for the standard first-line chemotherapy agent (Ying, Zhu, & Liu, 2012). Unfortunately, most of patients with advanced or metastatic pancreatic carcinoma could not gain benefits from gemcitabine monotherapy (Akinleye, Iragavarapu, Furqan, Cang, & Liu, 2015). Furthermore, gemcitabine-based chemotherapy is associated with serious side effects and high resistance to the treatment (Adamska et al., 2018). Therefore, there is an unmet clinical need for effective chemotherapy for managing patients with pancreatic cancer. In this work, we identified a natural compound TA that strikingly inhibited pancreatic tumor growth both in vitro and in vivo , and significantly suppressed pancreatic cancer cell invasion and migration. In a subcutaneous tumor growth xenograft model of PANC-1 cells, we found that gemcitabine hydrochloride (80 mg/kg/3d) was as effective as TA (20 mg/kg/d), which could remarkably inhibit the growth of pancreatic tumor and had no obvious toxicity.
To further investigate the gene expression profiling of human pancreatic cancer cells under TA treatment, we performed RNA-seq after treating PANC-1 cells with or without TA. To validate the results of RNA-seq, significantly differentially expressed genes were selected for qRT-PCR verification. Indeed, as shown in Figure S7, the expression patterns of the tested genes were in accordance with the RNA-seq data, including 4 down-regulated and 6 up-regulated genes. Moreover, we identified 2320 significantly differentially expressed genes, including 894 up-regulated genes and 1426 down-regulated genes (Figure S6a,b). Gene Ontology analysis demonstrated that these genes were primarily involved in basic processes (cell communication, biological regulation, signal transduction, etc.), suggesting that TA exhibited potent anticancer activity in pancreatic cancer by inhibiting critical processes (Figure S6c). KEGG pathway analysis suggested that these genes were mainly participated in cancer-related signaling pathways (metabolic pathways, pathways in cancer, JAK-STAT signaling pathway, etc.), supporting the potential therapeutic use of TA in pancreatic cancer (Figure S6d).
Under normal physiological conditions, STAT3 activation is rapid and lasts for only a few hours during signal transduction. Once activated by various upstream kinases, STAT3 protein is phosphorylated at the conserved tyrosine residue (Tyr705), leading to dimerization and nuclear translocation of p-STAT3, which subsequently binds to specific DNA sequences and induces transcription of STAT3 downstream target genes involved in a variety of biological processes (Avalle & Poli, 2018; Swiatek-Machado & Kaminska, 2020), such as cell cycle regulation (Cyclin D1 and c-Myc), evasion of apoptosis (Bcl-2 and Survivin), invasion and migration (MMP-9 and ICAM-1), and angiogenesis (VEGF and IL-8). Therefore, aberrantly activated STAT3 has been closely associated with the initiation, promotion and progression of various illnesses, especially cancer (Yu & Jove, 2004). Recently, more and more studies have shown that STAT3 and its mediated signal transduction play a prominent role in the occurrence, development, invasion, and metastasis of pancreatic cancer (Huang & Xie, 2012), and are also involved in the drug resistance of pancreatic cancer (Venkatasubbarao et al., 2013). In our research, we found that TA apparently reduced the transcriptional activity of STAT3 in a concentration-dependent manner. Furthermore, TA effectively inhibited the phosphorylation of tyrosine at the 705 site of STAT3 and the expression of downstream functional genes (e.g. Cyclin D1, c-Myc, Bcl-2, Survivin, and ICAM-1) at the protein levels, resulting in the growth inhibitory effects of TA on pancreatic cancer both in vitro and in vivo .
In fact, due to the pivotal role of STAT3 in tumor initiation and progression, a campaign in drug discovery has been launched to identify small molecules that interfere with the activation and function of STAT3 (Debnath, Xu, & Neamati, 2012). Until now, a great quantity of STAT3 small-molecule inhibitors have been discovered and developed for clinical utility in treatment and prevention of malignant tumors (Yang et al., 2018). For instance, BP-1-102 is an orally bioavailable, highly potent and specific STAT3 inhibitor that has a very good clinical application prospect for cancer treatment (Zhang et al., 2012). Although several STAT3 inhibitors have entered the early clinical stage, there currently are no FDA-approved small molecule inhibitors (Beebe, Liu, & Zhang, 2018). Hence it is of great significance to find potential and effective STAT3 inhibitors. First of all, we predicted the theoretical binding mode between the natural compound TA and STAT3 protein by molecular docking, and subsequently demonstrated that TA was able to bind to STAT3 (127-722 AA) protein by SPR assay. More importantly, we found that the cell proliferation activity of TA was much better than that of BP-1-102 in pancreatic cancer.
Although we reported the potential target protein of TA for the first time, its affinity with STAT3 protein needs to be further improved, and the binding site with STAT3 protein also needs to be further studied. In addition, the water solubility of TA should be further improved, and the molecular mechanisms of TA against pancreatic cancer should also be further clarified.
In summary, we screened and characterized the natural compound TA, a potential STAT3 pathway inhibitor that restrained the growth of pancreatic cancer both in vitro and in vivo . TA was efficacious in suppressing the migration and invasion of pancreatic cancer cells via inhibiting the STAT3 pathway. More than anything, we first revealed the underlying molecular mechanisms of TA against pancreatic cancer (Figure 7). TA can be a new potential candidate compound for treating pancreatic cancer, and it is valuable to be further investigated.