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.