MeiYuan Zhang*, Shukui Qin
Nanjing University of Traditional Chinese Medicine,
Email:zhangmeiyuan1972@163.com, qinsk@csco.org.cn
Abstract
Hepatocellular carcinoma is one of the cancers with low cure rate and high incidence. At present, surgical treatment, chemotherapy and targeted therapy have achieved certain results, and natural compounds are also effective. OroxylinA is a Radix Scutellariae extract that has anti-inflammatory, anti-cancer effects so that it is applied clinically. In this paper, through the analysis of public databases, the target and functional enrichment pathways of oroxylin A treating hepatocellular carcinoma were found. Our study not only validated the therapeutic effect of Oroxylin A but also explored the molecular basis of the drug, including the molecular docking experiments and flow cytometry experiments. Those experiments demonstrated the internal mechanism and practical function of oroxylin A treating hepatocellular carcinoma.
Introduction
Among cancers, liver cancer is the second killer of public health in the world. and there is no fully effective treatment for it1-3 [1,2,3]. In spite of progress in medical, local, and surgical therapy, hepatocellular carcinoma is still one of the primary reasons of cancer-associated mortality worldwide. The etiology and molecular mechanism of primary hepatocellular carcinoma are not completely clear. The pathogenesis of primary hepatocellular carcinoma, affected by both environmental and dietary factors is thought to be a complex procedure with various factors and steps4,5 [4,5]. Epidemiological and experimental research data show that liver cancer has are relevant to hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, aflatoxin and drinking water pollution and so on. The clinical manifestations of hepatocellular carcinoma are varied. Early hepatocellular carcinoma usually has no specific symptoms, while middle and advanced hepatocellular carcinoma has more symptoms6,7 [6,7]. The common clinical features consist of liver pain, abdominal distension, poor appetite, tiredness, emaciation, ascensive liver enlargement or upper abdominal mass. Certain suffers had low fever, icterus, diarrhea and above gastrointestinal bleeding. Liver cancer can lead to some symptoms such as acute abdomen. These symptoms may not be obvious or exist only as metastases8-11 [8,9,10,11]. No clear active marks or only analogous to liver cirrhosis are showed in the early period of liver cancer usually has no obvious positive signs or only similar to liver cirrhosis. Liver amplification, icterus, ascites and other signs are often present in enhanced liver cancer. Besides, the sick who have liver cirrhosis often has liver palm, spider nevus, male mammary gland increases, lower extremity edema to wait. While extrahepatic metastases happen, signs in accordance with each metastasis site may occur. The ordinary complications are above gastrointestinal bleeding, liver cancer fracture bleeding, liver and kidney deficiency. Individualized comprehensive therapy in different phases of liver cancer is the crucial step to improve the sanative effect11-13 [11,12,13]. There are a lot of therapy approachers such as surgery, hepatic artery ligation, transcatheter arterial CHEMoembolization and radiotherapy. Biological treatment, traditional Chinese medicine therapy of liver cancer is also widely used. Surgery is the first and most efficient way to cure liver cancer. Surgical approaches include acute hepatectomy, anetic hepatectomy. As to unresectable liver cancer can be used according to the specific situation14-16 [14,15,16]. Primary liver cancer is also an indication for liver transplantation. Regional chemoembolization with hepatic artery and/or portal vein pump could be utilized for patients with unresectable cancer after laparotomy or as follow-up treatment for palliative resection of tumor. For resectable liver cancer after estimation, interventional radiotherapy is also feasible. Optional cannulation of the femoral artery to the hepatic artery is performed, and embolization agents (commonly used as iodized oil) and anticancer drugs are injected for chemoembolization17 [17]. Better to general condition, liver function is still good, do not accompany cirrhosis of liver, without jaundice, ascites, without hypersplenism and oesophageal varices, canceration is more finite, do not have remote metastasis and not appropriate for surgery excision or recurrence after surgery, use the comprehensive treatment that radiation gives rise to. Radiotherapy is divided into external radiotherapy and internal radiotherapy. External radiotherapy includes stereotactic radiotherapy, three-dimensional conformal radiotherapy, intensity modulated radiotherapy and image-guided radiotherapy. Internal radiotherapy includes Yttrium 90 microsphere selective internal radiotherapy, monoclonal antibodies, radioiodized oil,etc. Immunoribonucleic acid, interferon, interleukin-2 and thymosin are commonly used, which can be used in combination with chemotherapy18,19 [18,19].
In traditional Chinese medicine, some drugs can improve immunity or kill tumor cells directly, but the underlying mechanism is still unclear20 [20]. Therefore, it is a good way to study the extract, which is more accurate. Among the chief positive ingredients Oroxylin A is obtained from Radix Scutellariae. Oroxylin A has been displayed to have a lot of pharmacological effects, containing anticancer, anti-inflammatory, neuroprotective, anticoagulant, etc. The pharmacological activity of formylurea A was researched in vitro and in animal models, reflecting its potential for disease treatment. Oroxylin A is the primary bioactive flavonoid, with tough anti-liver cancer effect, but less toxicity to standard tissues21,22 [21,22]. Some findings suggest that OA inhibits the diffusion and induces the variation of HCC in vitro and in vivo by producing a high ratio of PKM1 to PKM2. Particularly, OA displayed responsible anticancer effects on primary hepatocellular carcinoma cells and hepatocellular carcinoma xenograft models for suffers, and delayed the progress of primary hepatocellular carcinoma, indicating that OA could be improved as a new inducer of hepatocellular carcinoma differentiation. Meanwhile, in many disease, such as breast cancer, OA has also shown some positive anticancer effects23,24 [23,24].
However, as a drug that has not been widely used in clinical practice, the specific targets of the drug action needs to be further explored. Therefore, we explored public databases, further explored disease and drug-related targets, and performed molecular docking analysis to study the underlying mechanisms of drugs.
Methods and Results
1. Acquisition of Oroxylin A related information
In order to obtain oroxylin A related data ,according to the search keyword OroxylinA from pubchem we download structured. According to the structured figure in swisstarget, pharmmapper, batman – TCM, TCMSP, we download the data from the three database. In order to make the data more accurate, the data screening standard of SwisStarget database is Probability*>0. Pharmmapper, batman-TCM and TCMSP databases are not screened. The data obtained from Pharmmapper and TCMSP databases were changed into corresponding genes through Uniprot database.
2. Obtain hepatocellular carcinoma related data
We searched the data in GeneCards and OMIM databases according to the search keyword: hepatocellular carcinoma. In order to obtain more accurate data, we set data screening criteria. GeneCards database was selected based on Relevance score>=10. In the OMIM database we did not do data screening.
3. Screening of intersection genes and drawing of Venn diagram
The number of genes for oroxylin A obtained from the three databases is 612, and the number of genes related to diseases obtained is 900. The intersection genes of drugs and diseases obtained by us were 102, and the number of genes of drugs in diseases was the intersection made by deleting duplicates after merging. We plotted a Venn diagram based on the number of these genes. (Figure 1)
Figure 1.Venn plots were plotted from hepatocellular carcinoma and Oroxylin A
4. Protein-protein interaction network
The data of common goals of diseases and drugs were input into the STRING database to get the network connection of target protein mutual effect. The needed connection score in minimum was set 0.4 and then we set the outliers to be deleted. The downloaded data is named string_interactions_short.tsv, and the protein-target interaction network is obtained. Then we used Cytoscape3.7.2 software to input PPI data (string_interactions_short.tsv) to construct PPI network, and the graph obtained was protein interaction network (Figure 2B). In these images, brighter colors and larger graphs represent higher degree values, suggesting the corresponding nodes are more significant in the meshwork. R software was made use of to input PPI data (string_interactions_short-tsv) to know the amount of connecting nodes of key genes. Figure 2C shows the bar chart of the top 30 core genes. The names and scores of the 30 core genes are shown in the table.
| Number | Targets | Degree | Number | Targets | Degree |
| 1 | AKT1 | 81 | 16 | IGF1R | 44 |
| 2 | ALB | 75 | 17 | KDR | 43 |
| 3 | EGFR | 74 | 18 | MAPK14 | 43 |
| 4 | ESR1 | 72 | 19 | MAPK8 | 42 |
| 5 | HRAS | 71 | 20 | MAP2K1 | 41 |
| 6 | CASP3 | 70 | 21 | NRAS | 41 |
| 7 | SRC | 69 | 22 | PGR | 41 |
| 8 | HSP90AA1 | 66 | 23 | PPARG | 41 |
| 9 | TNF | 66 | 24 | JAK2 | 40 |
| 10 | IL6 | 64 | 25 | MMP2 | 40 |
| 11 | IGF1 | 57 | 26 | RELA | 40 |
| 12 | MMP9 | 55 | 27 | AR | 39 |
| 13 | BCL2L1 | 52 | 28 | IL2 | 38 |
| 14 | PTGS2 | 52 | 29 | MCL1 | 38 |
| 15 | RHOA | 45 | 30 | PIK3R1 | 38 |
Table 1. 30 core targets.
5. GO function analysis to explore the target function.
We used R software and its background database to acquire gene IDs of latent targets (entrezID). “DOSE”, “clusterProfiler” and “PathView” program packages were utilized to conduct GO functional enrichment analysis for underlying goals. Functional enrichment consists of three ways: Biological procedure enrichment analysis (BP), Cellular Component enrichment analysis and Molecular Function enrichment analysis (MF). To get more accurate data, we set pvalueCutoff = 0.05, qvalueCutoff = 0.05. We ranked each category according to importance, and the top 10 enriched items were presented by bar charts and bubble charts (Figure 3A, Figure 3B). In the GO graph, the circle size or line length indicates the amount of genes enhanced in GO, and the color means the importance of enrichment. We found that a series of physiological processes such as glandular development, oxidative stress and stress response are related to OA. At the same time, lipid metabolism, transmembrane transport and protein kinase complex are also related. It has been displayed in studies oxidative stress is closely associated with cancer, and superoxide dismutase also has many potential anti-cancer and cancer-promoting mechanisms. Therefore, we can know the potential role of OA.
Figure 3.GO analysis chart, (A) Bar chart. (B) Bubble chart.
6. Functional analysis of KEGG.
R software was utilized and its background database to acquire gene IDs of latent targets, and “DOSE”, “clusterProfiler”, and the “PathView” package was utilized to present KEGG functional enrichment analysis for latent goals. And we set pvalueCutoff = 0.05, qvalueCutoff = 0.05. In order of significance, the top 20 enriched items are presented by bar charts and bubble charts (Figure 4A, B).We found that in the bar graph, EGFR kinase inhibition resistance, hepatitis B virus, chemical carcinogenesis and other related pathways were more enriched. Surprisingly, most of these are also risk factors for hepatocellular carcinoma, thus confirming the accuracy of our study.
Figure 4.KEGG analysis chart, (A) Bar chart. (B) Bubble chart.
7. Molecular docking analysis of key targets.
We downloaded the structure formula of the composition from PubChem database and converted it into. Pdbqt format by AutoDockTools software. The file was saved for later use. Protein ceptors were then downloaded from the PDB database, and water molecules and small molecule ligands were deleted by PyMOL software. The protein was hydrotreated by AutoDockTools-1.5.6 software, and the protein in. pdbqt formwas exported. The active pocket of the protein was determined by AutoDockTools-1.5.6 software, and molecular docking was performed by VINA software. See pictures below. (Figure 5)
A.
B.
C.
D.
E.
Figure 5.Molecular docking.(A)AKT1.(B)ALB.(C)EGFR.(D)ESR1.(E)HRAS.
- Experimental varification by flow cytometry
Flow cytometry was performed to verify the effect of Oroxylin A on apoptosis. Three types of liver cancer cells, namely Bel-7402, Huh-7 and SMMC-7721 were obtained to perform the experiment. The prepared cell plates were placed into 6-well plates and cultured overnight at a inoculation density of about 2*106/mL at 37℃ and 5% CO2. Then, according to the calculated volume of Oroxylin A, it was added into 2 mL culture solution, mixed upside down, and Oroxylin A solution was configured. Then the original culture medium was discarded, washed twice with 1*PBS (pH 7.4), and treated with Oroxylin A working solution for 48 hours. After that, the culture medium was discarded, washed with 1*PBS (pH 7.4) twice, and 0.25% pancreatic enzyme was added for digestion, centrifugation, and cell collection. After the supernatant was discarded, 1x×binding buffer was added to adjust the cell concentration, and 5μl Annexin V-FITC and 5μl PI Staining Solution were added and gently blown well. Incubate the solution at room temperature and away from light for 10 min. Add 400μl 1 × Binding Buffer and mix gently. The samples were detected by flow cytometry within 1 h after staining. As shown in the results, in Bel-7402 cells, it can be seen that with the increase of oroxylin A concentration (0-120uM), the apoptosis rate also gradually increased, almost reaching more than 23% of the apoptosis rate. When the concentration of oroxylin A was 0, the apoptosis rate was 7.77%, and when the concentration of oroxylin A was increased to 120uM, the apoptosis rate could reach 43.88%. In Huh-7 cells, the rate of apoptosis gradually increased with the addition of oroxylin A concentration (0-120um), and when the concentration of oroxylin A was 0, the rate of apoptosis was 8.22%. When oroxylin A concentration increased to 120uM, the apoptosis rate reached 21.7%. In SMMC-7721 cells, with the increase of oroxylin A concentration (0-120uM), the apoptosis rate also gradually increased, and the data showed statistical significance. When the concentration of oroxylin A was 0, the apoptosis rate was 6.14%. When the concentration of oroxylin A increased to 75uM, the apoptosis rate reached 23.61%. It can be seen that after different concentrations of oroxylin A treatment, the apoptosis level of tumor cells increased, and the concentration was positively correlated. Compared with Huh-7 and SMMC-7721 cells, Bel-7402 cells were more sensitive to oroxylin A concentration and had a higher apoptosis rate.
Figure 6. Apoptosis rate of hepatocellular carcinoma cells treated with different concentrations of oroxylinA.
Discussion
Hepatocellular carcinoma is a ordinary complicating disease of liver sclerosis. Constantly recurring viral hepatitis B and C are risk elements for its progression too. As to suffers with sclerosis, hepatocellular carcinoma is often caused by harmful conversion of regenerating abnormal nodules. The distinct diacrisis from other liver knubs was made by calculated tomography with venous distinction. In some cases, an MRI may help. The only possible method to treat hepatocellular carcinoma is resect knub, which could be done by cut liver resection partially or liver implantation. Only 15% of hepatocellular cancer could be cured surgically25,26 [25,26]. Hepatocellular carcinoma is an ordinary disease with bad prevision, high economic cost and heavy disease weight. Although often symptomless in the previous phases, suffers may suffer from significant unrest due to enhanced disease, opposite reactions to treatment, or potential dyspnea from sclerosis. Enhanced hepatocellular carcinoma (HCC) was regarded as an internally intractable cancer in the age of chemotherapy (1950-2000). Yet, systematic treatment has turned into molecularly aimed treatment and immunotherapy, with nine regimens confirmed in the world in the last two decades. The approved protocols were aimed at cancer angiogenesis or tumor immunity, which are two cancer characteristics. In recent years, the combination of altezolizumab and bevacizumab has modified the effectiveness of scientific treatment of enhanced HCC without extreme toxicity and degradation of quality of life. This article reviews the main progress of scientific treatment and puts forward prospects for the next generation of scientific treatment for enhanced HCC27,28 [27.28]. Relaxative tend has the probability to significantly enhance quality of life, physical and psychological signs in suffers with terminal liver cancer and has been displayed to prolong the chance of living in some suffers with non-hepatocellular malignant tumors. For patients with liver disease, new therapy choices are essential. The adoption of existing mixture in nature can offer better outcomes for suffers with less scientific poisonousness and fewer undesirable effects. Enhanced treatment may result in better outcomes.
Oroxylin A, an active ingredient in traditional Chinese medicine, are proved to possess effects of curing cancers. Now it has been used as an anticancer drug in some places, and some cases have reported good efficacy29,30 [29,30]. However, there are few studies on the intrinsic molecular mechanism of this drug, especially the target of action, so we conducted network pharmacological analysis. In GO and KEGG analysis, we found that oroxylin had a significant relationship with oxidative stress and stress response. This indicated that the drug had a certain effect on the activation and development of reactive oxygen species. Reactive oxygen species have been shown to be significantly associated with cancer, thus confirming the drug’s effect on liver cancer. We found that a series of physiological processes such as glandular development, oxidative stress and stress response are related to OA. At the same time, lipid metabolism, transmembrane transport and protein kinase complex are also related. It has been indicated in research oxidative stress is closely associated with cancer, and superoxide dismutase also has many potential anti-cancer and cancer-promoting mechanisms. At the same time, we found that this drug has a significant correlation with proliferation and kinase activation pathways. EGFR kinase inhibition resistance, hepatitis B virus, chemical carcinogenesis and other related pathways were more enriched. Surprisingly, most of these are also risk factors for hepatocellular carcinoma, thus confirming the accuracy of our study.
We screened out the relevant targets and carried out molecular docking on the targets. These targets have their own characteristics. The serine threonine kinase encrypted by AKT1 gene is catalytically negative in sera-deficient primary and imperishable fibroblasts. AKT1 and relevant AKT2 are stimulated by platelet-derived increase factors in the developing nervous system, and AKT is a key adjuster of growth factor-seduced survival of nerve fiber. Albumin is a dissolvable simplicial protein, accounting for serum protein in part. Albumin acts chiefly as a conveyor protein for steroids and act in making extracellular flowing volume stable. The protein encrypted by EGFR is a transmembrane glucoprotein and belong to the protein kinase superfamily, which is a ceptor for members of the cuticular increase factor family. EGFR is a cell face protein that is combined with cuticular enhancement factor. The protein related with ligand gives rise to ceptor dimerization and tyrosine autophosphorylation and results in cell proliferation.ESR1 encodes an estrogen receptor, a ligand-activated transcription factor, and consists of some domains significant for hormone combination, DNA combination, and transcriptional simulation. HRAS belongs to the Ras carcinogene family, members of which are associated with changing genes of mammalian harmful lump retroviruses. These genes encode products that play an important role in signal conversion ways. In these five common targets, we conducted molecular docking, and also explored the related effects of drugs and targets.
In our study, we explored the interaction between oroxylin A and molecular targets through network pharmacology, and carried out detailed analysis of key targets such as molecular docking. Flow cytometry showed that apoptosis ratio increased with the increase of oroxylin A treated with different concentrations of oroxylin A, proving the function of oroxylin A treating HCC. Our experiment provides ideas for drug development and clinical application.
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