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*Corresponding Author: Yepeng Li, Department of Oncology, The Affiliated Hospital of Youjiang Medical University for Nationalities, 18 Zhongshan Second Road, Baise, Guangxi Zhuang Autonomous Region 533000, P.R. China.
Citation: J Zhao, B Yang, Yepeng Li. (2022). Effect Of Puerarin on The Growth of Nasopharyngeal Carcinoma Cells and Its Impact on Angiogenesis. J. Cancer Research and Cellular Therapeutics. 6(2); DOI:10.31579/2640-1053/114
Copyright: © 2022 Yepeng Li, this is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 21 March 2022 | Accepted: 04 April 2022 | Published: 11 April 2022
Keywords: cne1 cells; puerarin; cell proliferation; rna sequencing
Objective
Puerarin is a form of isoflavones obtained from Pueraria lobata. It stimulates hepatic metabolic function and lowers serum ALT, AST, and total-bilirubin level. The purpose of this study was to examine the effect of puerarin on nasopharyngeal carcinoma (NPC) CNE1 cells and preliminarily explore its possible mechanism.
Materials and Methods
CCK8 method was used to detect the proliferation activity of puerarin on NPC CNE1 cells and IC50 was calculated. CNE1 cells were treated with 0 μmol/L puerarin (containing equal volume of DMSO solution) as control group and 1000 μmol/L puerarin (IC50 concentration) as experimental group. Colony formation assay, Scratch-wound test and Transwell invasion assay were used to detect the clone formation ability, migration and invasion ability of puerarin on CNE1 cells. Then, RNA Sequencing was used to detect the changes of differentially expressed genes (DEGs) and signaling pathways after puerarin was applied to CNE1 cells.
Results
The inhibitory effect of puerarin on the proliferation activity of CNE1 cells was enhanced with the increase of concentration, and IC50 was calculated as 1000 μmol/L. Compared with the control group, the treatment of CNE1 cells with 1000 μmol/L puerarin could inhibit the clone formation, migration and invasion of CNE1 cells (P<0.05). A total of 379 DEGs were found by RNA sequencing, including 295 down-regulated genes and 84 up-regulated genes (padj<0.05). The significant differences in biological functions of differentially expressed genes were mainly distributed in “negative regulation of growth”, “angiogenesis”, “regulation of peptidase activity”, “positive regulation of vasculature development”, “digestion”, “positive regulation of angiogenesis”, “negative regulation of peptidase activity”, “extracellular matrix” and “Golgi lumen” (padj<0.05).
Conclusion
Puerarin could inhibit the proliferation, migration and invasion of NPC CNE1 cells, and its mechanism might be related to the inhibition of angiogenesis and cell growth.
Nasopharyngeal Carcinoma (NPC) is an epithelial cancer that occurs in the nasopharyngeal mucosa. It is one of the common malignant tumors of head and neck. It has obvious geographical distribution characteristics and high incidence in Southeast Asia and southern China [1,2]. In southern China, the incidence rate is as high as 25/100,000-50/100,000 [3], and non-keratinizing NPC accounts for more than 95%, mainly related to Epstein-barr Virus (EBV) infection [4]. Due to the particularity of NPC anatomy and radiotherapy sensitivity, intensity-modulated radiotherapy (IMRT) has become an important treatment for NPC. NPC patients are difficult to be found in the early stage, and 75 % of the patients are diagnosed in the late stage [5]. Distant metastasis in locally advanced NPC patients is the main reason for treatment failure, and the 5-year survival rate is still only 50-60% in metastatic patients [6, 7].
The role of traditional Chinese medicine in tumor treatment has become increasingly prominent. Traditional Chinese medicine can alleviate adverse reactions such as nausea and vomiting caused by chemotherapy [8]. In addition, some traditional Chinese medicine components can play an anti-tumor role by improving the immunity of the body [9]. Flavonoids are one of the active components of traditional Chinese medicine, which can play a role in anti-tumor. Puerarin is an isoflavone compound extracted from Puerariae Lobatae Radix. Its molecular formula is C21H20O9 and its relative molecular mass is 416, which can be dissolved in organic solvents such as dimethyl sulfoxide (DMSO). It has been reported that puerarin has the effects of protecting myocardial cells, lowering blood pressure, anti-oxidation and reducing inflammatory response [10-12]. Studies have found that puerarin can lead to cancer cell death by regulating different mechanisms, including oxidative stress, cell cycle, Phosphatidylinositol 3-Kinase/ Pmtein Kinase B (PI3K / AKT) pathway, Mitogen- activated Protein Kinases/ Extracellular Signal-regulated Kinases (MAPK/ERK) pathway, Nuclear Factor κB (NF-κB) pathway, etc. [13]. The role of puerarin in non- small cell lung cancer showed that puerarin inhibited the proliferation of NCI-H441 and NCI-H460 cells, and induced autophagy through PI3K/Akt and MAPK/Erk signaling pathways [14]. Puerarin can inhibit the proliferation and promote apoptosis of bladder cancer T24 cells, and can inactivate NF-κB signaling pathway [15]. Puerarin can also act on liver cancer [16], ovarian cancer [17], cervical cancer [18], esophageal cancer [19], and gastric cancer [20] through different mechanisms. However, there are few studies on puerarin in NPC. In this study, the effects of puerarin on the proliferation, migration and invasion of NPC CNE1 cells were observed in vitro, and RNA sequencing (RNA-seq) was used to detect the changes of differentially expressed genes and possible pathways after puerarin was applied to CNE1 cells, providing a theoretical basis for the clinical application of puerarin in NPC.
Cell Culture
Nasopharyngeal carcinoma CNE1 cells obtained from Cell Institute of Shanghai Academy of Life Sciences were cultured in RPMI Medium 1640 containing 10
Each experiment in this study was repeated three times independently, represented by mean±sd. Graph Pad Prism 9.0 software was used to count and draw pictures. The mean of the two samples (control vs treated) was compared by unpaired Student's test, P<0>
Puerarin inhibits the proliferation of NPC CNE1 cells.
CCK8 assay showed that puerarin decreased the proliferation activity of CNE1 cells (Figure. 1A), and IC50 was calculated as 1000 μmol/L. The colony formation assay demonstrated that puerarin reduced(P<0>)
Puerarin inhibits migration and invasion of NPC CNE1 cells
Scratch-wound test results showed that compared with the control group, 1000μmol/L puerarin group had no significant difference in healing at 24h and 48h, but decreased (P<0>
Analysis of the DEGs.
The hierarchical clustering heat map of DEGs is shown in Fig. 3A. Compared with the control group, a total of 379 DEGs, 295 downregulated and 84 upregulated were detected in CNE1 cells treated with puerarin(padj<0>
Enrichment Analysis of DEGs.
GO enrichment analysis was performed on DEGs of control group and puerarin group (Figure. 4A). It indicates that DEGs are mainly enriched in BP, such as “negative regulation of growth”, “angiogenesis”, “regulation of peptidase activity”, “positive regulation of vasculature development”, “digestion”, “positive regulation of angiogenesis”, “negative regulation of peptidase activity”. The top 7 entries of BP were annotated (Table II).
About CC, DEGs enriched in “extracellular matrix” and “Golgi lumen”. KEGG analysis showed DEGs enriched in “Complement and coagulation cascades” (padj<0>
and invasion of CNE1 cells. Studies have found that when puerarin acts on bladder cancer T24 and EJ cells, puerarin can block the G0/G1 phase cell cycle and reduce the proliferation activity of bladder cancer cells [21]. Puerarin can also inhibitthe proliferation of chronic myeloid leukemia K562 cells and promote apoptosis by inducing autophagy in K562 cells [22]. Uncontrolled cell proliferation is the basic feature of cancer. The main mechanism is that the disorder of cell cycle leads to excessive proliferation and less apoptosis. Whether puerarin can inhibit the proliferation of CNE1 cells by blocking cell cycle remains unknown.Migration and invasion of tumor cells play an important role in tumor metastasis [23]. Studies have shown that puerarin can inhibit the migration and invasion of lipopolysaccharide--stimulated breast cancer cells by acting on the NF-κB pathway and Erk phosphorylation [24]. Puerarin can also inhibit the invasion and migration of ovarian cancer HO-8910 cells [25]. The incidence of NPC lymph node metastasis is high, and the cervical lymph node metastasis is found to be 70%-80% or more. Distant metastasis remains the leading cause of death in NPC patients [26]. Therefore, effective inhibition of tumor metastasis is an important part of treatment. Can puerarin control NPC metastasis by inhibiting cell migration and invasion? And the mechanism by which puerarin inhibits the migration and invasion of CNE1 cells needs further verification.
It was found by RNA-seq that the effect of puerarin on NPC may be related to the inhibition of angiogenesis and the inhibition of proliferation. It is well known that the growth of tumors requires blood vessels to provide nutrition, and blood vessels provide pathways for tumor metastasis and invasion. Vascular dysplasia exists in various types of tumors [27]. Excessive angiogenesis promotes the rapid growth of tumors, which is one of the main causes of tumor death [28]. Hypoxia is the main driver of tumor angiogenesis [29]. Radiotherapy, chemotherapy and immunotherapy can reduce the efficacy of hypoxia [30-32]. Angiogenesis and the production of vascular endothelial growth factor (VEGF) can also promote tumor growth, leading to increased expression of oncogenes or loss of tumor suppressor genes [33]. VEGF induces the expression of matrix metalloproteinases (MMPs) in NPC, which not only participates in the formation of new blood vessels by degrading the extracellular matrix of endothelial cells, but also regulates the invasion and metastasis of cancer, leading to the progress of NPC [34]. In addition, VEGF was overexpressed in nearly 70% of EBV-positive NPC patients and was associated with lymph node metastasis, recurrence and overall survival [35]. Studies have shown that Chinese herbal medicine can reduce the expression level of VEGF by inhibiting the expression of Hypoxia-inducible factor-1α (HIF-1α), and ultimately inhibit tumor angiogenesis [36,37]. NPC01 is an ancient Chinese herbal medicine prescription modified by Liang Gesang. NPC01 has the effect of inhibiting the growth of NPC cells. By inhibiting the PI3K/Akt signaling pathway, NPC01 reduces the expression of angiogenesis-related factors, including HIF-1α and VEGF [38]. Puerarin is also a kind of Chinese herbal medicine. This study found that puerarin acting on CNE1 cells may be related to inhibiting angiogenesis. Therefore, whether puerarin can play a role in NPC by inhibiting angiogenesis deserves further study.
Current cancer research efforts focus on epigenetic alterations that may be related to Sirtuin (SIRT) 1-7 [39]. In recent years, SIRT1 is considered to be the most characteristic sirtuin in seven members. And SIRT1 can be widely involved in cell processes, such as cell division, autophagy and senescence [40]. SIRT1 plays an important role in aging, metabolism and cancer [41]. SIRT1 is related to tumor cell proliferation, migration, invasion and angiogenesis [42,43]. A study found that after puerarin treatment of ovarian cancer cells, the expression of SIRT1 decreased and inhibited Wnt/β-catenin signaling pathway, thereby increasing the apoptosis of platinum-resistant ovarian cancer cells [44]. At present, many SIRT1 inhibitors have been reported, and whether puerarin can be used as a SIRT1 inhibitor related to the inhibition of NPC cells remains to be studied.
In summary, the results of this study showed that puerarin could inhibit the proliferation, migration and invasion of CNE1 cells in vitro, which might be related to the inhibition of angiogenesis and cell growth. The inhibitory mechanism of puerarin on NPC still needs further verification. In addition, in vivo investigation is highly recommended. This study provides a theoretical basis for the study of puerarin in nasopharyngeal carcinoma.
Not applicable.
The present work was supported by Doctorate Awarding Unit Funding of the Affiliated Hospital of Youjiang Medical University for Nationalities [grant no. (2021)29]. The funders had no role in the design of the study, the collection, analyses or interpretation of data, the writing of the manuscript or the decision to publish the results.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
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