TCM has been used to treat various diseases for thousands of years. Numerous studies have indicated that the combination of TCM and modern medicine can play an indispensable role in improving efficacy and reducing toxicity associated with cancer treatment[]. KLTi is a medicinal herb extracted from coix lacryma-jobi, which acts by supplementing Qi and nourishing Yin and removing stasis. TCM focuses on ‘syndrome differentiation and treatment.’ KLTi has demonstrated clinical effect in patients with PDAC with splenasthenic hygrosis and deficiency of both Qi and Yin. In previous studies, KLTi monotherapy has shown antitumor activity in mouse models of PDAC by inhibiting tumor cell proliferation and inducing apoptosis[]. In addition, a meta-analysis showed that the combination of KLTi and radiochemotherapy is more effective than radiochemotherapy alone in the treatment of advanced PDAC. The combination treatment not only improved the 1-year overall survival rate, overall response, and disease control rate, but also enhanced the quality of life and relieved pain. In addition, it alleviated the adverse events caused by chemoradiotherapy, such as gastrointestinal side effects, nephrotoxicity, leukopenia, thrombocytopenia, and myelosuppression[].
TCM injection is a preparation composed of multiple compounds corresponding to many targets. Further, there may be synergistic or antagonistic effects between targets. Hence, it is difficult to elucidate the mechanism of action of KLTi using conventional approaches that use drug-target-disease framework. Network pharmacology is based on various types of biological information databases. Through the network analysis of drugs and diseases, it is possible to explain the overall mechanism of action and compound information of TCM and related compounds. Network pharmacology emphasizes the study of multi-target pathways, which is consistent with the overall concept of TCM.
Potential Active Compounds
The top four compounds obtained from the compound-target network of KLTi were stigmasterol, mandenol, sitosterol alpha1, and isoarborinol. The degree values of stigmasterol and mandenol were found to be significantly superior compared to others. Moreover, these compounds exhibited a high degree of biological activity and could map multiple drug targets. Hence, they were designated as the core compounds in KLTi. Stigmasterol and sitosterol alpha1 belong to the class of phytosterols, which are chemicals with tumor prevention and anticancer effects, and are present in a variety of medicinal plants. Phytosterols can also induce significant reduction in cell viability and induce apoptotic death of tumor cells[].
Stigmasterol has demonstrated anti-inflammatory, anticancer, anti-allergic, and immune regulation effects. Stigmasterol inhibited lipopolysaccharide (LPS)-induced innate immune responses in murine models[], significantly reduced the transcript level of TNF-α, destroyed tumor angiogenesis, and reduced the chance of metastasis[]. Studies have reported that stigmasterol induces mitochondrial-mediated apoptosis to inhibit tumor cell proliferation by interfering with Bax and Bcl-2 expression. It also inhibits tumor cell metastasis and induces G2/M cell cycle arrest in a dose-dependent manner[].
Although there is insufficient data on sitosterol alpha1, related subclasses such as β-sitosterol and γ-sitosterol have demonstrated obvious anticancer effects. Several in vitro and in vivo studies of β-sitosterol combined with gemcitabine have revealed that β-sitosterol could increase the expression of Bax protein and reduce the expression of Bcl-2 protein, induce G0/G1 phase arrest and apoptosis, and suppress NF-κB activity to effectively inhibit PC cell growth. It also inhibits the invasion and metastasis of anti-tumor cells, and the combination with gemcitabine showed a significant synergistic effect[]. In addition, γ-sitosterol exerts anticancer activity by inhibiting the growth of tumor cells by blocking them in the G2/M phase and inducing apoptosis[].
Modern pharmacological studies have confirmed that isoarborinol can be used to improve anxiety, depression, and pain. Moreover, it has an auxiliary effect on the clinical symptoms which are prone to occur during the development of cancer treatment[]. In case of mandenol, there is currently no relevant clinical or experimental research data available.
Potential Genetic Targets
From the "gene-pathway" network, it was found that RELA, NFKB1, IKBKG, JUN, MAPK1, TP53, and AKT1 were the genes with the highest interactions and were identified as potential gene targets for KLTi intervention in PDAC. Among these genes, RELA, NFKB1, and IKBKG are all components of the NF-kappa B signaling pathway. The NF-kappa B signaling pathway is one of the major signaling pathways linking cancer to inflammation. This classical pathway is activated when the cells are exposed to inflammatory cytokines, such as TNFα and IL-1 or in response to inflammatory signals, such as LPS[]. In addition to inhibiting tumor cell proliferation and metastasis, NF-κB also interferes with inflammation[]. Specifically, for PDAC with pancreatitis, intervention with NF-kappa B signaling pathway can simultaneously result in tumor and tumor-related inflammation. NF-κB also helps TNF-α to induce epithelial-mesenchymal transition (EMT) and complete angiogenesis and metastasis. Thus, KLTi could curb tumor progression by inhibiting NF-kappa B signaling pathway.
RELA can promote PDAC progression by activating proliferation or migration-related gene expression. This suggests that the overexpression of RELA promotes proliferation and metastasis of PDAC cells. Binding of miR-302a-3p to RELA, inhibited RELA expression as well as PDAC cell proliferation and migration[]. Tumor suppression, mediated by oncogene-induced senescence (OIS), is thought to play a protective role in the development of PDAC. In the Kras-driven PDAC mouse model, Lesina et al demonstrated that RELA reinforced OIS to inhibit carcinogenesis[]. However, genetically disabling OIS can cause RELA to promote tumor proliferation; thus revealing a dual role of RELA in PDAC carcinogenesis.
NFKB1 is part of the NF-kappa B signaling pathway. Low expression of MUC4 inhibited the expression of NFKB1, thereby down-regulating the NF-kappa B signaling pathway to inhibit the migration and invasion of PDAC cells and impaired the migration of PDAC cells along the nerve[]. As a transcription factor, NFKB1 is responsible for regulating immune response, inflammatory processes and cell growth, and is closely related to the risk of tumor occurrence and disease prognosis[]. NFKB1 also affects susceptibility to tumors, and its polymorphisms significantly increase susceptibility to cancer in Asians[]. Several researchers have pointed out the important role of NFKB1 as an inhibitor of inflammation, cancer, and aging[].
IKBKG binds and regulates IκB kinase (IKK), which is required for NF-kappa B signaling pathway activation. The IKBKG -binding domain peptide (NBDP) of IKK has been found to inhibit NF-κB activation and increase the cleavage of PARP and Caspase 3 in the apoptotic pathway to promote apoptosis in PDAC cells. It has also been reported that NBDP promotes sensitivity of PDAC to gemcitabine. Therefore, regulating the activity of IKBKG can help in controlling PDAC, and also be used to increase the sensitivity to chemotherapy[].
Studies have shown that JUN may be involved in the growth of PDAC cells[]. Inhibition of JUN also inhibits histone deacetylase inhibitors (HDACi)-induced inflammatory gene expression and tumor-supported response in fibroblasts. This indicates that the combination of HDACi and chemical inhibitors of the JUN attenuates the inflammatory phenotype of fibroblasts, and may improve the anti-tumor efficacy of HDACi in PDAC, and even in other solid tumors[]. In addition, it plays an important role in regulating the K-Ras pathway by interfering with JUN and exerting anti-inflammatory activity and inhibiting PDAC metastasis[].
MAPK1 is an important protein kinase and regulatory factor in the MAPK signaling pathway which can enhance the invasion and proliferation of PDAC cells[], and also reverse the inhibitory effect of miR-212 on tumor cell proliferation and apoptosis[]. MAPK1 can also induce PDAC EMT, which greatly promotes the migration and invasion of cancer cells[]. Therefore, inhibition of MAPK1 expression is also a potential target for future oncological research.
AKT1, a serine/threonine protein kinase, has been identified as an oncogene in a variety of cancers, including PDAC. Studies have confirmed that activated AKT1 accelerates the occurrence and development of PDAC. It mediates proliferation inhibition and induces apoptosis of PDAC cells by inhibiting AKT1 expression[]. Phosphorylation of AKT1 also increases the risk of cachexia in the PDAC population, which is strongly associated with disease prognosis[].
TP53 mutations occur in more than 75% of PC patients, and mutated TP53 promotes EMT and tumor cell invasion[]. TP53 is a driver gene that is essential for the proliferation and metastasis of PDAC, and the expression of TP53 results in shorter disease free survival (HR: 1.33; 95% CI, 1.02–1.75; P = 0.04)[]. In addition, some studies have found that the mutation of TP53 is closely related to the occurrence of malignant intraductal papillary mucinous neoplasms (IPMNs)[]. Therefore, silencing or inactivation of TP53 will prevent the further development of IPMN to PDAC and improve the prognosis of patients.
Gene Ontology
The GO molecular function of the core genes showed that these genes are mainly enriched in enzyme binding, transcription factor binding, and receptor binding. There are many transcription factors in the gene targets of KLTi for the treatment of PDAC, as well as in the binding processes of enzyme, transcription factor, and receptor protein. These factors play an important role by intervening the binding of these proteins. For example, myc-associated zinc-finger protein (MAZ) is the transcription factor involved in the transcription initiation and termination. Deregulation of MAZ expression is associated with the progression of PDAC and increases CRAF-ERK signal. It is mediated through p21-activated protein kinase (PAK) and protein kinase B (AKT/PKB) signaling cascades to enhance the invasion of PDAC cells[]. There are also some transcription factors that promote the malignant transformation of PDAC[]. Thus, intervention of the binding of transcription factors might have some interference effects on PDAC.
The GO biological process of core genes was mainly enriched in apoptotic signaling pathway, cell cycle, and metabolic process. As far as the function of potential gene targets in this study is concerned, KLTi inhibited the proliferation and induced apoptosis in human PC xenografts through various mechanisms, such as cell cycle arrest in G2/M phase, down-regulating the expression of phosphorylated Akt and mTOR, and regulating the PI3K/Akt/mTOR signaling pathway[].
The GO cellular components of core genes were mainly enriched in nuclear chromosomes, perinuclear regions, and ribosomes. Previous studies have shown that KLTi reduces NF-κB levels in the nucleus. Additionally, it reduces the expression of IκBα, IKK, and EGFR in tumor cells and the overall cytoplasm. This corresponds to the role of KLTi at the nuclear and cytoplasmic levels[]. Enzyme receptors and transcription factors rely on protein to function, and proteins are, in turn, synthesized by ribosomes. Thus, intervening the functioning of the ribosomes that synthesize the corresponding protein may have some benefits in controlling the disease. For example, connexins are a group of tumor suppressor genes, amongst which Connexin 43 (Cx43) is the most widely expressed gene. The up-regulation of Cx43 expression can improve the sensitivity of many tumors to chemotherapy and radiotherapy, and this site of action may be present in cytoplasmic ribosomes[].
KEGG pathway
Results from KEGG analysis showed that the regulatory pathways of KLTi for PDAC are mainly enriched in viral carcinogenesis, cancer pathways, cell cycle, MAPK signaling pathway, PI3K-Akt signaling pathway, and neurotrophin signaling pathway. It is worth noting that KLTi can act directly on PC, in addition to intervening in signaling pathways that affect tumor development. This observation is sufficient to confirm that KLTI has an adequate scientific basis in the treatment of PDAC.
It has been proven that many malignant tumors are associated with virus infection. Virus produce prion protein that induce reprogramming and genomic instability, including the accumulation of mutations, aberrations, and DNA damage[]. Most DNA oncogenic viruses are often involved in cell proliferation and regulation of the cell cycle by binding to p53 and pRB tumor suppressor proteins, thereby inducing tumor formation[]. Studies have shown a correlation between HBV infection and the occurrence of PC,and poor prognosis. The X protein released by HBV significantly enhances cell proliferation and migration, induces EMT, up-regulates PI3K-Akt and MAPK signaling pathways, render PDAC malignant, and promotes disease progression[].
Regulating cell cycle also plays a role in the development of PDAC. A variety of genes and proteins are involved in this process. Usami et al[] observed that the class Ⅱa HDACi could inhibit the activation of FOXO3, thus inhibiting the growth of PC cells. The combination of class Ⅱa HDACi with the proteasome inhibitor, carfilzomib, could have a synergistic effect on the FOXO3 activation, thus resulting in G1/S arrest in AsPC-1 cells.
Peripancreatic nerve invasion is an important oncological feature of PDAC and is closely related to disease prognosis. KLTi can also act on the neurotrophin signaling pathway to intervene nerve invasion. Perineural invasion is associated with a variety of neurotrophic factors produced by neural tissue inside and outside the pancreas which bind to specific receptors resulting in autophosphorylation and activation of multiple signaling pathways, such as MAPK, PI3K-Akt, and NF-kappa B[]. The nerve growth factor promotes the spread of PC cells by autocrine and/or paracrine mechanisms through MAPK-mediated phosphorylation[]. It also activates the ERK/CD133 signaling cascade, resulting in enhanced tumor cell invasion, and plays a key role in perineural invasion of PC[]. Studies have confirmed that regulating NF-kappa B signaling pathway through activating IKK plays an important role in mediating EMT and inducing neural invasion[].
The activation of the MAPK signaling pathway is crucial for PDAC proliferation and metastasis. This pathway is involved in the regulation of various biological activities through three major proteins, ERK1/2, p38MAPK, and MKK4[]. Yan et al[]. compared the expression of p-ERK1/2 between pancreatic cancerous tissues and normal cells. They found that the expression of p-ERK1/2 in pancreatic tissues was significantly increased. In vivo and in vitro experiments confirmed that the inhibition of ERK1/2 expression reduced EMT, activated cancer-related autophagy, and decreased cell proliferation and migration in human PC cells. In another study[], it was shown that the p38MAPK inhibitor, VCP979, could regulate the MAPK/NF-κB signaling pathway, reduce inflammation, and inhibit EMT to exert antitumor effect. Further, MKK4 was associated with high proliferation of tumor cells and promoted rapid proliferation of PDAC cells.
The PI3K-Akt signaling pathway is a key pathway that promotes tumor cell proliferation, invasion, metastasis, and drug resistance. As one of the substrates of Akt, Girdin enhanced phosphorylation of Akt and induced activation. Wang et al[]. found that Girdin showed high expression in PDAC and was involved in the regulation of tumor cell metastasis, angiogenesis, and autophagy. Silencing of the Girdin gene resulted in decreased levels of p-Akt and p-PI3K, and inhibition of the PI3K-Akt signaling pathway, thereby increasing apoptosis and inducing cell cycle arrest in tumor cells. PI3K-Akt signaling pathway is closely related to the abnormal express of lncRNA. Studies[] have shown that the expression of lncRNA small nucleolar RNA host gene1 elevates the expression of PI3K and phosphorylated Akt, which in turn activates the PI3K-Akt signaling pathway to promote cell proliferation, inhibits apoptosis, and enhances invasion in PDAC. The expression of lncRNA AB209630 inhibited the PI3K-Akt signaling pathway in gemcitabine-resistant PDAC cells and reduced the proliferation of resistant cells to improve the sensitivity to chemotherapy[].
It is worth noting that both MAPK and PI3K-Akt are important signaling pathways for the transduction of membrane receptor signals into cells, and there exists an interaction of receptor signals in the two pathways[]. Ras, an upstream molecule of MAPK, can induce the activation of Akt, and p38MAPK can act between PI3K and Akt and thus play an important role in Akt phosphorylation. Similarly, the activity of PI3K has an important induction effect on the activity of the Ras/MAPK pathway, and Akt can also negatively regulate the Ras/MAPK pathway by phosphorylating Raf[].
PI3K-Akt and NF-κB signaling pathways also have an interaction effect. Akt activates Ikk, by phosphorylation, then releases NF-κB from the cytoplasm for nuclear translocation, activates downstream gene expression, and participates in the regulation of NF-κB pathway[].