Mechanism of andrographis paniculata on lung cancer by network pharmacology and molecular docking

1. Introduction

Lung cancer is considered to be a malignant tumor with high incidence worldwide. An estimated 220 million new cases and 179 million deaths per year [1]. Lung cancer is one of the most common cancers in the world, and it is also the main cause of cancer-related death. The incidence of lung cancer accounts for 11.4% of all cancers and 18% of total cancer deaths [2]. In the past decade, people have strengthened research and introduced molecular-targeted drugs and immune checkpoint inhibitors, thereby enhancing the survival rates of patients with advanced lung cancer [3]. However, lung cancer remains a sticking point in the malignancy landscape [4]. Lung cancer is mainly treated by surgery [5]. However, when there are residual cancer cells after surgery, it may recur. Clinical treatment of lung cancer most commonly includes chemotherapy and radiotherapy, although both are always accompanied by side effects. The occurrence of side effects may aggravate the development of the disease and deteriorate the patient’s condition, thus producing a counterproductive effect, causing more pain to patients [6]. Nowadays, traditional Chinese medicine (TCM) can be used as an important adjuvant drug for the treatment of diseases, and has been widely recognized and accepted worldwide to provide favorable therapeutic effects for cancer patients [7].

Andrographis paniculata (Burm. F.) Nees (Acanthaceae) represents a key herb that is extensively utilized across Southeastern Asia [8]. The herb contains diterpenoids, flavonoids and polyphenols as the major bioactive components [9]. Studies have demonstrated that the extract of Andrographis paniculata has several pharmacological effects, including antioxidation, anti-inflammation, and anti-tumor [10]. One study demonstrated that andrographolide, one of the main diterpene lactones in Andrographis paniculata, could prevent the cell cycle by inhibiting the overexpression of vascular endothelial growth factor (VEGF), which induces pulmonary tumors in transgenic mice. This indicated that andrographolide could treat lung cancer through anti-angiogenesis [11]. However, the mechanism of Andrographis paniculata bioactive components against lung cancer remains unclear.

Network pharmacology is based on the network perspective as a new research method, which is used to explore the mechanism of multi-molecule co-regulation that is systemically targeted by TCM components [12]. This approach can analyze the multi-component and multi-target mechanisms, which may be consistent with the treatment hypotheses of complex diseases [13]. By constructing a multi-level, multi-faceted, and multi-angle network model composed of components, targets, pathways, and diseases, as well as further studying the multiple signaling pathways, key targets, and biological processes involved in the treatment of diseases by TCM, network pharmacology aims to explain the mechanism of TCM on disease treatment at the molecular level [12]. In this study, the anti-cancer effect of Andrographis paniculata was analyzed by network pharmacology based on the Gene Expression Omnibus (GEO) database. Subsequently, the potential mechanism of Andrographis paniculata against lung cancer was further analyzed by analyzing the interaction and enrichment pathways between differential expression genes (DEGs) in lung cancer and critical targets for the main components of Andrographis paniculata. Finally, molecular docking (MD) analysis was conducted to further validate the results, which provided theoretical support for an in-depth study on the application of Andrographis paniculata in treating lung cancer (Fig. 1).

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3. Results

4. Discussion

Based on the theory of system biology, network pharmacology integrates system biology, pharmacology and computer analysis technology to explore the complex relationship between active ingredients, diseases and targets, and select specific signal nodes for multi-target drug molecular design. The new discipline helps us analyze the mechanism of TCM in treating diseases [16]. In practice, network pharmacology ensures the reliability of predicted results through the process of network construction, search and network analysis of key compounds and targets [17]. Therefore, by combining TCM and network pharmacology, the priority of disease-related genes is distinguished, thereby revealing the correlation between drug-related target genes and diseases, and further illustrating the network regulation of TCM and its formula. Molecular docking is a method to design drugs by simulating the interaction mode between receptors and drugs. A theoretical simulation method for studying intermolecular (such as ligand and receptor) interactions and predicting their binding modes and affinity. Many studies have used a combination of network pharmacology and molecular docking techniques to explore the material basis and mechanism of action of TCM in the treatment of certain diseases [18], which can provide new insights into the screening of active compounds and the exploration of mechanisms.

Andrographis paniculata belongs to Family Acanthaceae. Traditionally, it has been used in folk medicine that can treat various diseases. Since the 1950s, more than 80 chemical constituents have been isolated and identified from Andrographis paniculata, such as terpenoids, flavonoids and iridoids. Andrographolide is the most common terpene in Andrographis paniculata [19]. Nowadays, it is used in various herbal preparations. Studies have shown that andrographolide has many pharmacological activities, which can be widely used in the treatment of fever, cold, inflammation, and other diseases in clinics [20]. It also has anti-cancer and immune-regulating activities. Therefore, it may be developed as a new chemotherapeutic drug [21].

Medical studies have shown that andrographolide and neoandrographolide in Andrographis paniculata can treat inflammatory reactions caused by pathogenic bacteria invasion. Based on its anti-inflammatory activity, some studies reported the effects of andrographolide [22] and flavonoids [23]. Zhu et al. predicted and verified the potential molecular mechanism of Andrographis paniculata in treating inflammation by combining network pharmacology with molecular biology experiments [24]. Gu et al., through network pharmacology combined with molecular docking technology and experimental verification, stated that the neuroprotective effect of Andrographis paniculata may be related to the regulation of APP-BACE1-GSK3 β signaling axis and inflammatory response, revealing the molecular mechanism of Andrographis paniculata in the treatment of Alzheimer’s disease [25].

Previous chemical studies have shown that flavonoids are the main chemical constituents of Andrographis paniculata and are considered to play a key role in the pharmacological activity of this medicinal plant [23]. Wogonin belongs to flavonoids and exists in aerial parts of Andrographis paniculata. Studies have shown that it has anticancer activity [26]. Chen et al. studied the apoptosis-inducing activity of wogonin on A549 cells and its mechanism. The experimental results showed that wogonin could be used as an effective inhibitor of A549 cell activity [27]. However, there are few studies on the anti-lung cancer effect of wogonin extracted from Andrographis paniculata, the specific mechanism of its action against lung cancer remains unclear. In this study, we found that the active component of wogonin may be used as a compound for the treatment of lung cancer by network pharmacology combined with molecular docking technology, but further research is needed.

In this study, the key active components of Andrographis paniculata were collected by data mining. Based on network pharmacology combined with the GEO database, the anti-lung cancer mechanism of Andrographis paniculata was explored from a microscopic perspective. There were 19 core effective components of Andrographis paniculata, with 23 intersections between its targets and lung cancer action targets. The results of this study showed that the mechanism of Andrographis paniculata in the treatment of LC may be mainly related to these 23 core targets. Upon KEGG analysis, these key targets were mostly enriched into cell senescence, cell cycle, and p53 signaling pathway. For most species, aging promotes a series of degenerative diseases characterized by loss of tissue or cell function [28]. Human aging is the result of cell aging, in which more and more cells reach aging [29]. However, especially in vertebrates, aging can also promote proliferative lesions, the most lethal being cancer [28].

Stable cell cycle arrest and regulation of secretions into the tissue microenvironment are the characteristics of cell senescence [30]. From a physiological point of view, aging inhibits tumor [31], stalls the expansion of precancerous cells [32], and favors wound healing [33]. In future cancer treatment, the discovery of aging factors may provide alternative options [34]. Neganova et al. found that cyclin dependent kinase 1 (CDK1) plays an important role in human pluripotent stem cells, such as regulating mitosis, G2/M checkpoint maintenance and apoptosis [35]. Huang et al. revealed that CDK1 enhanced Sox2 transcription activity by interacting with Sox2, thereby promoting stem cells of lung cancer cells. This CDK1/Sox2 axis may be a therapeutic target for lung cancer [36]. Barbara Kunar-Kamińska et al. reported that the overlapping role of cell senescence in chronic obstructive pulmonary disease (COPD) and lung cancer might suggest common pathogenesis. As cell senescence is known to activate carcinogenesis, it is necessary to further confirm its significance in the progression of lung cancer in patients with COPD [37]. This suggests that cell senescence may inhibit the development of lung cancer.

Nowadays, people also often believe that cancer is a cellular periodic disease. The cell cycle is a successive and well-organized process followed by cell replication. It is necessary to strictly control this process to guarantee cell division if required. Thus, precise copying of the information transmitted to the next generation can be guaranteed [38]. Several articles have suggested that cell cycle defects exist in most tumors, cell cycle arrest causes DNA repair and repair unsuccessful cells enter apoptosis process [39]. When the regulators that control the correct entry and progression of cell cycles change, they may be involved in the development of tumors [40]. Cyclin B1 (CCNB1) is an important promoter and controller for mitosis. This gene product can interact with p34 (CDC2) to become a mature promoter, which is considered to regulate the G2/M transition period of cell cycle and has been confirmed [41]. Studies have shown that CCNB1 overexpression is closely related to the proliferation and tumorigenesis of many cancer cells [42]. Studies have shown that CCNB1 in non-small cell lung cancer (NSCLC) cell lines is essential for cell cycle progression and cloning [43]. The cyclin A2 encoded by CCNA2 is also a member of the cyclin family and a cell cycle regulator. It can bind and activate cyclin-dependent kinase 2, thereby promoting G1/S and G2/M conversion [44]. Gong et al. found that the expression levels of CCNB1 and CCNA2 were up-regulated in patients with NSCLC. These genes may be meaningful diagnostic biomarkers [41]. Regulation of the cell cycle has a critical effect on treating tumors.

The tumor suppressor gene p53, known as the “genomic guardian and oncogene police”, has an important effect on sensing of and responding to carcinogenic signals and DNA injury [45]. Considerable evidence demonstrates that post-translational modifications can regulate the stability and activity of p53 [46]. The stability and activity of p53 are also regulated by a variety of protein kinases [47]. When p53 is activated, it promotes the transcription levels of target genes [48]. Checkpoint kinase I (CHK1; CHEK1) reacts to DNA damage by initiating cell cycle arrest, and its expression constitutes a defense mechanism that can avoid the toxicity of DNA damage agents [49]. The activated CHEK1 can phosphorylate and regulate the activity of many proteins, including p53. P53 can regulate BAX, Cyclin-dependent kinase inhibitor 1A (CDKN1A) and other genes involved in cell survival and proliferation regulation [50]. CDKN1A plays a central role in cell cycle progression. CDKN1A was instantly recruited to the target site to promote its repair of DNA damage. However, CDKN1A activity depends on p53 status [51]. Zamagni et al. showed that CDKN1A played a certain role in the response to cisplatin-pemetrexed combination therapy in advanced NSCLC with KRAS mutation, indicating that it may be used as a promising predictor [52]. Regulation of the p53 signaling pathway may be a key factor in the treatment of LC [53]. Mutation of the p53 gene is the most common gene change related to cancer [54, 55]. Its mutation may result in the loss of tumor inhibition, thus enhancing cell growth and suppressing their apoptosis [56]. Mutations of the p53 gene may include deletion, point mutation, and overexpression [57]. Therefore, the abnormal expression of p53 is greatly correlated with the occurrence of lung cancer.

Protein kinase B (AKT) is composed of 480 amino acids and is the intersection of multiple signaling pathways [58]. Studies have demonstrated that AKT controls the key processes of cells, such as glucose metabolism, cell cycle, and apoptosis. As PKB can stimulate cell proliferation and inhibit apoptosis, it has an important effect on carcinogenesis [59]. Active AKT is associated with several human malignancies, such as gastric cancer (GC), brain cancer, breast cancer (BC), LC, prostate cancer, and colon cancer [60]. Interestingly, AKT1, an important subtype of AKT, can affect cell proliferation and apoptosis and modulate the invasion and migration of different cancer cells [61]. AKT1 is an efficient therapeutic target for research and exploration. Wu et al. demonstrated that miR-377–5p targeted the AKT1 signal, cell cycle control, inhibition of cell development, and epithelial-mesenchymal transition (EMT) to delay the progression of lung cancer providing the theoretical foundation for future research on lung cancer treatment [62]. Kim MJ and colleagues demonstrated that AKT1 gene polymorphism independently predicted the prognosis of NSCLC cases receiving surgical treatment. NSCLC is a common LC subtype with a prevalence of about 80%. The researchers also observed that the AKT1 gene polymorphism-mediated AKT1 expression was the possible determining factor for LC survival. Therefore, beyond the pathological stage, the detection of gene polymorphism helps the high-risk groups of patients with diseases to improve the treatment of NSCLC [63]. AKT1 has a critical effect on the progress of cancers by regulating the downstream genes of the signaling pathway. As reported in previous studies, AKT1 modulates tumor development, migration, and metastasis [64]. According to Yoo et al., miR-9500 suppressed the growth and invasion of LC cells via Akt1 [65]. The results of this study indicated that the active ingredient wogonin might participate in cellular senescence by regulating AKT1, thereby exhibiting effects on lung cancer.

MAPK signal is a cascade organization; when the receptor activates upstream kinase, it will sequentially activate MAPKs (MAPK and MAPKK). In mammals, four genes encoding p38 MAPK have been identified: MAPK11 (p38 β ), MAPK12 (p38 γ ), MAPK13 (p38 δ ), and MAPK14 (p38 α [66]). p38 α and p38 β are closely related and possibly have common activities [67]. p38 MAPK belongs to the serine/threonine kinase family, which is classified as a “stress-activated” kinase [67]. The p38 pathway has a critical effect on regulating the cell cycle, cell proliferation, differentiation, and apoptosis. p38 MAPK exerts a complex effect on tumors, and results from different studies are inconsistent due to the differences in conditions and systems. Zarredar et al. reported that Kirsten rat sarcoma (KRAS), p38 α , and nuclear factor- κ B (NF- κ B), possibly had a vital effect on controlling the apoptosis, development, and proliferation of human lung cancer cells. They may become potential targets for the treatment of LC [68]. p38 α is involved in G2-M arrest under various stimuli. A possible mechanism is that p38 α phosphorylates p53, induces the transactivation of Gadd45a, and inhibits the G2-M process [69]. In a study by Ventura et al., it was observed that the deletion of p38 α in mice could lead to increased differentiation of lung progenitor and stem cell (LPC and LSC) both in vitro and in vivo. p38 α modulates the activity of LPC and LSC. Therefore, it contributes to antagonizing the carcinogenic effect of K-Ras G12V (K-Ras G12V can destroy the growth and differentiation of normal LPC and LSC to initiate lung cancer). Since p38 α is an important target for inflammatory diseases, its anti-lung cancer effect is also crucial [70]. Vitos-Faleato et al. studied the relationship between p38 α signal in lung cancer epithelial cells and tumor development. They observed that the high expression of p38 α decreased the survival of lung adenocarcinoma, while the genetic or chemical inhibition of p38 α could prevent tumor growth in mouse models of lung cancer [71].

NF- κ B, initially identified in 1986, is a nuclear factor that binds to the gene regulatory region encoded by the immunoglobulin κ -light chain, which activates B cells (hence called NF- κ B) [72]. It is a widely expressed transcription factor that regulates various biological processes. NF- κ B belongs to the Rel family that consists of five members in mammals, namely RELA (p65), Rel (c-Rel), Rel-B, NF- κ B1 (p50/p105), and NF- κ B2 (p52/p100) [72]. The abnormal expression of NF- κ B has a critical effect on the development of LC [73]. NF- κ B (especially RELA) is continuously activated in lung tumorigenesis, and RELA remains a challenge as a direct anti-LC therapeutic target [74]. The anti-tumor activity of anlotinib, discovered by Li and colleagues, on lung cancer cells may involve the reduction of the phosphorylation levels of I κ B- α and NF- κ B p65 in LSCs [75]. A study of the anti-lung cancer mechanism of cyclovirobuxine D (CVB-D) indicated that it could stimulate mitotic phagocytosis, while the p65/BNIP3/LC3 axis may be related to the CVB-D-mediated mitotic phagocytosis of lung cancer cells [76]. P65 plays a key role in regulating the MAPK signaling pathway. Studies have reported that the chromosome segregation-like 1 protein/CAS (CSE1L) may promote the stability of p65, and the two interact to activate NF- κ B/MAPK pathway, thus enhancing the growth of NSCLC cells and suppressing cell apoptosis [77]. As a key factor in LC, the potential mechanism of RELA in the progression of lung cancer should be investigated further.

Nuclear receptor coactivator 1 (NCOA1, also known as SRC-1) is the founding member of the nuclear receptor coactivator family [78]. NCOA1 can regulate gene transcription by interacting with other transcription factors. Studies have shown that NCOA1 imbalance has a certain effect on the occurrence and development of different cancers [79]. It is reported that NCOA1 overexpression is associated with lymph node metastasis of prostate cancer, which enhances cell proliferation [80]. Studies have shown that increased expression of NCOA1 promotes proliferation [81] and metastasis [82] of breast cancer cells. Other studies have shown that NCOA1 promotes the progression of hepatocellular carcinoma [83]. Interestingly, 14-deoxy-11-oxo-andrographolide, 14-deoxyandrographolide and andrographolide-19- β -D-glucoside_qt in the active components of Andrographolide can be molecularly docked with NCOA1, but there are few studies on the correlation between NCOA1 and lung cancer. This will be the focus of a follow-up study.

5. Conclusion

This work analyzed the component-target network and discovered that wogonin was highly correlated with the targets in the network, thus showing marked anti-cancer effects. Studies have shown that wogonin may coordinate cell senescence by targeting AKT1, MAPK14, and RELA. 14-deoxy-11-oxo-andrographolide, 14-deoxyandrographolide and andrographolide-19- β -D-glucoside_qt can simultaneously target NCOA1 to play the role of anti-lung cancer. But further experimental verification is needed. It is hypothesized that Andrographis paniculata may exhibit its anti-lung cancer activity through this process and needs further experimental verification. However, the GO and KEGG analysis results of the effective components of Andrographis paniculata in this paper are relatively simple, so it will continue to pay attention to and study the therapeutic effect of this drug on pneumonia in the future.

The present work first examined the critical main components, candidate targets, and relevant pathways of Andrographis paniculata against lung cancer based on network pharmacology (NP) technology, and the results have offered a foundation for future experimental investigation. However, considering the restrictions of NP, its therapeutic mechanism can only be predicted by data mining, while the content of each compound, its associations, and in vivo pharmacokinetics have been neglected. Based on reasonable prediction, further research should be continued. The in vivo or in vitro experiments were conducted to explore whether Andrographis paniculata could be used as medicinal components screened by bioinformatics in this paper, so as to regulate the action targets obtained in this paper, and then produce the effect of anti-lung cancer. This can provide more scientific theoretical basis for clinical research and application of Andrographis paniculata. However, our findings also provide a preliminary basis for further understanding the synergistic mechanism of Andrographis paniculata in the treatment of lung cancer.

Author contributions

JL conducted statistical analysis and drafted the manuscript. XL, JL and DH participated in the statistical analysis the manuscript. PG and YL conceived the research, participated in the research design and coordination, and provided suggestions on the writing of the manuscript. All authors read and approved the final manuscript.

Funding

This work was funded by the Ministry of Education Key Laboratory of Harbin Medical University Open Fund Project (No. KF201619), as well as the Heilongjiang University of Chinese Medicine Science and Technology Innovation Research Platform Construction Fund (No. 2018pt05).

Availability of data and materials

All data generated and analyzed during this study are included in this article and the supplementary materials.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/THC-220698.