Therapeutic Targets of Bufalin on Renal Carcinoma and Mechanisms: Experimental Validation of Network Pharmacology Analysis

Abstract

The possible targets underlying the activity of bufalin on renal cell carcinoma (RCC) were investigated using network pharmacology and experimental approaches. PharmMapper and other databases were explored for predicting the bufalin targets and RCC-related targets. Finally, the enriched pathways and the targets were analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analyses. Furthermore, in vitro cell experiments were used to verify bufalin activation of AKT and MAPK signaling pathways in human mesangial cells. The therapeutic targets related to bufalin were identified via 35 intersecting targets. GO analysis identified 29 molecular functions, 16 cellular components, and 91 biological processes. KEGG pathway annotation identified 15 signal transduction pathways and 4 tumor-related pathways.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Bufalin (C₂₄H₃₄O₄), a major monomer component of toad venom, is extracted from toads for use in traditional Chinese medicine. It is derived from traditional Chinese medicine and includes a single component and is milder than Western medicines [1]. Bufalin has received increasing attention of researchers and clinicians due to its anticancer, heart-strengthening, analgesic, and other effects [2, 3]. Bufalin has been shown to have antitumor efficacy against a variety of tumors, including mouth cancer, esophageal cancer, and bladder cancer [4–6]. Bufalin’s anticancer action is distinguished by its low dose and highly selective nature. At the molecular level, studies employing bufalin have investigated the occurrence and growth of cancer cells, cell cycle, apoptosis, and gene expression. This study aimed to look into the biological signaling pathways and the predicted therapeutic targets of bufalin for treating RCC as well as providing experimental data and bioinformatics for basic clinical research on RCC treatment, using a scientific strategy based on network pharmacology.

2. Materials and Methods

2.1. The Target of Bufalin Resistance to RCC Was Obtained and Predicted

The PubChem website and PharmMapper server were used to predict potential targets for all the predicted targets of bufalin [7]. Additionally, the Draw Venn Diagram website (https://Bioinformatics.psb.ugent.be/webtools/Venn/) in the GeneCards database (https://www.Genecards.org/) [8] was used for intersection comparison, and the possible anti-RCC targets of bufalin were acquired from the GeneCards database (Figure 1).

[figure omitted; refer to PDF]

The expression levels of proteins implicated in the MAPK and PI3K/Akt signaling pathways were evaluated when Caki-1 cells were exposed to bufalin (10, 100, 500, and 1000 nM) for 24 hours to study the signal transduction route through which bufalin influences tumor growth. We observed that the level of MAPK and PI3K was decreased after 24 h in a dose-dependent manner, leading to a decrease in its downstream molecule p-Akt (Figure 12).

[figure omitted; refer to PDF]

4. Discussion

Toxicity and resistance to currently available chemotherapeutic drugs are important barriers to cancer therapy. As a result, the ideal method would be to search for new and effective anticancer drugs with fewer side effects [13]. Bufalin is the main ingredient of the toad venom, causing cell death by inducing cell apoptosis and blocking the cell cycle in human cancer cells [14, 15]. In human liver cancer, prostate cancer, and multiple myeloma, bufalin has been shown to induce apoptosis and decrease proliferation [16–18]. However, it causes apoptosis and suppresses proliferation in Caki-1 cells via a molecular mechanism that is yet to be discovered. The study screened and discovered bufalin’s therapeutic targets in RCC as well as the biological mechanisms that underpin them.

The experiments showed that bufalin exerted an antiproliferative effect on Caki-1 cells in a concentration-dependent manner. The cells underwent morphological changes, including reduced cell proliferation, circular shrinkage, and reduced adherence, which may be caused by the destruction of the cell membrane, cytoskeleton, and some internal cell structures by bufalin, resulting in significant morphological changes.

After treating Caki-1 cells with bufalin for 24 hours, the fraction of cells in the G0/G1 phase of the cell cycle was reduced significantly, while the fraction of cells in the G2/M phase continued to increase which is essentially consistent with the findings of Suehiro et al. and Ni et al. [10, 19]. This study also revealed that the number of cells in the G2/M phase increased as the bufalin concentration increased which is extremely significant, and when cycling cells face stress, they are arrested at one of the two cell cycle checkpoints, G2/M or G1/S.

Here, a “compound-target-pathway-disease” network was built using the network pharmacology approach to identify possible bufalin anti-RCC targets. Through KEGG pathway annotation and GO enrichment analysis, the target protein genes of bufalin contain anti-RCC including MAPK10 and PI3K. These target protein genes are involved in multiple tumor pathways and signal transduction pathways [20, 21]. In this study, the predicted potential targets were annotated through the KEGG pathway to obtain signal transduction pathways related to RCC, such as MAPK and PI3K signaling pathway. These pathways are linked to the development and occurrence of tumors.

One of the most significant routes in the eukaryotic signal transmission network is the mitogen-activated protein kinase (MAPK) chain, which is involved in gene expression control and cytoplasmic functional activities [22]. The MAPK pathway is one of the common intersections of signal transduction pathways, such as stress, inflammation, cell proliferation, differentiation, functional synchronization, transformation, and apoptosis, and the extracellular signal by the receptor, G/G of protein, protein kinase, and transcription factors, such as signal network, passed to the intracellular, involved in cell proliferation, differentiation, cancerous, transfer, and apoptosis [23]. In this study, we found that the levels of PI3K and MAPK were reduced after 24 hours in a dose-dependent manner. Therefore, we suggest that bufalin may inhibit the proliferation of Caki-1 cells by inhibiting the MAPK and PIK signaling pathways. These findings support bufalin’s clinical use as an anticancer drug due to its therapeutic effects in renal cell carcinoma. However, we believe that more animal experiments are needed to better evaluate bufalin’s therapeutic potential.

5. Conclusion

The possible targets of the bufalin on renal cell carcinoma were investigated using network pharmacological and experimental approaches. The bufalin-related therapeutic targets were identified via 35 intersecting targets. GO analysis identified 29 molecular functions, 16 cellular components, and 91 biological processes. KEGG pathway annotation identified 15 signal transduction pathways and 4 tumor-related pathways.

Acknowledgments

This work was supported by the previous research project of the Heilongjiang Postdoctoral Talent Program (grant no. LBH-Q19190) and basic scientific research operating expenses of Provincial Universities in Heilongjiang Province (grant nos. QN2021003, 1355ZD006, 1354MSYTD007, and 202110233034).

References

[1] Y.-G. Fang, P.-F. Wang, H.-D. Zhu, L. M. Chen, Z. M. Wang, H. M. Gao, X. T. Fu, J. Nie, "Study on quality standard of Bufonis Venenum and its processed slice, toad venom powder," Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica, vol. 45, pp. 1726-1733, 2020.

[2] L. L. Wen, X. B. Xie, W. Huang, J. Yin, W. Lin, Q. Jia, W. Zeng, "Bufalin’s anti-inflammatory and analgesic effects on NF-B signaling pathway," Journal of Sun Yat-Sen University(Medical Science Edition), vol. 35 no. 05, pp. 680-684 + 667, 2014.

[3] W.-W. Huang, J.-S. Yang, S.-J. Pai, P.-P. Wu, S.-J. Chang, F.-S. Chueh, M.-J. Fan, S.-M. Chiou, H.-M. Kuo, C.-C. Yeh, P.-Y. Chen, M. Tsuzuki, J.-G. Chung, "Bufalin induces G0/G1 phase arrest through inhibiting the levels of cyclin D, cyclin E, CDK2 and CDK4, and triggers apoptosis via mitochondrial signaling pathway in T24 human bladder cancer cells," Mutation Research: Fundamental and Molecular Mechanisms of Mutagenesis, vol. 732 no. 1-2, pp. 26-33, DOI: 10.1016/j.mrfmmm.2011.09.010, 2012.

[4] S. Shen, Y. Zhang, Z. Wang, R. Zhang, X. Gong, "Bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress," International Journal of Biological Sciences, vol. 10 no. 2, pp. 212-224, DOI: 10.7150/ijbs.8056, 2014.

[5] Y. Ding, W. Liu, X. Wang, L Zhang, M Zhao, H Deng, Y Liu, "Bufalin induces apoptosis in human esophageal carcinoma ECA109 cells by inhibiting the activation of the mTOR/p70S6K pathway," Oncology Letters, vol. 15 no. 6, pp. 9339-9346, DOI: 10.3892/ol.2018.8526, 2018.

[6] H.-Y. Chou, F.-S. Chueh, Y.-S. Ma, R. S.-C. Wu, C.-L. Liao, Y.-L. Chu, M.-J. Fan, W.-W. Huang, J.-G. Chung, "Bufalin induced apoptosis in SCC-4 human tongue cancer cells by decreasing Bcl-2 and increasing Bax expression via the mitochondria-dependent pathway," Molecular Medicine Reports, vol. 16 no. 6, pp. 7959-7966, DOI: 10.3892/mmr.2017.7651, 2017.

[7] S. F. Zhao, X. J. Zhang, "Anticancer mechanism of ailanthone via reverse molecular docking and network pharmacological technology," Chinese Traditional and Herbal Drugs, vol. 49, pp. 4085-4092, 2018.

[8] H. Li, H. Liu, "Investigating the mechanisms of action of depside salt from salvia miltiorrhiza using bioinformatic analysis," Evidence Based Complement Alternative Medicine, vol. 2017,DOI: 10.1155/2017/5671860, 2017.

[9] L. Zeng, K. Yang, H. Liu, G. Zhang, "A network pharmacology approach to investigate the pharmacological effects of Guizhi Fuling Wan on uterine fibroids," Experimental and Therapeutic Medicine, vol. 14 no. 5, pp. 4697-4710, DOI: 10.3892/etm.2017.5170, 2017.

[10] T. Ni, H. Wang, D. Li, L. Tao, M. Lv, F. Jin, W. Wang, J. Feng, Y. Qian, M. Sunagawa, Y. Liu, "Huachansu Capsule inhibits the proliferation of human gastric cancer cells via Akt/mTOR pathway," Biomedicine & Pharmacotherapy, vol. 118,DOI: 10.1016/j.biopha.2019.109241, 2019.

[11] J. Xie, W. Lin, L. Huang, N Xu, A Xu, B Chen, M Watanabe, C Liu, P Huang, "Bufalin suppresses the proliferation and metastasis of renal cell carcinoma by inhibiting the PI3K/Akt/mTOR signaling pathway," Oncology Letters, vol. 16 no. 3, pp. 3867-3873, DOI: 10.3892/ol.2018.9111, 2018.

[12] S. Yang, C.-C. Duan, R.-Y. Yan, J.-Y. Zhang, "Active-component and integrative mechanism of Scutellaria barbatain treatment of cancer based on network pharmacology method," Chinese Traditional and Herbal Drugs, vol. 49 no. 15, pp. 3471-3482, 2018.

[13] E. Y. Su, Y. L. Chu, F. S. Chueh, Y. S Ma, S. F Peng, W. W Huang, C. L Liao, A. C Huang, J. G Chung, "Bufalin induces apoptotic cell death in human nasopharyngeal carcinoma cells through mitochondrial ROS and TRAIL pathways," The American Journal of Chinese Medicine, vol. 47 no. 1, pp. 237-257, DOI: 10.1142/S0192415X19500125, 2019.

[14] C. Zhang, K. Ma, W.-Y. Li, "Cinobufagin suppresses the characteristics of osteosarcoma cancer cells by inhibiting the IL-6-OPN-STAT3 pathway," Drug Design, Development and Therapy, vol. 13, pp. 4075-4090, DOI: 10.2147/dddt.s224312, 2019.

[15] H.-Y. Qi, X.-J. Qu, J. Liu, K.-Z. Hou, Y.-B. Fan, X.-F. Che, Y.-P. Liu, "Bufalin induces protective autophagy by Cbl-b regulating mTOR and ERK signaling pathways in gastric cancer cells," Cell Biology International, vol. 43 no. 1, pp. 33-43, DOI: 10.1002/cbin.11076, 2019.

[16] F. Qi, A. Li, Y. Inagaki, N. Kokudo, S. Tamura, M. Nakata, W. Tang, "Antitumor activity of extracts and compounds from the skin of the toad Bufo bufo gargarizans Cantor," International Immunopharmacology, vol. 11 no. 3, pp. 342-349, DOI: 10.1016/j.intimp.2010.12.007, 2011.

[17] Y.-Q. Dong, W.-L. Ma, J. Gu, W.-L. Zheng, "Effect of cinobufagin on nuclear factor-kappaB pathway in HepG2 cells," Nan Fang Yi Ke Da Xue Xue Bao = Journal of Southern Medical University, vol. 30, pp. 137-139, 2010.

[18] C.-H. Yu, S.-F. Kan, H.-F. Pu, E. Jea Chien, P. S. Wang, "Apoptotic signaling in bufalin- and cinobufagin-treated androgen-dependent and -independent human prostate cancer cells," Cancer Science, vol. 99 no. 12, pp. 2467-2476, DOI: 10.1111/j.1349-7006.2008.00966.x, 2008.

[19] Y. Suehiro, Y. Takemoto, A. Nishimoto, K. Ueno, B. Shirasawa, T. Tanaka, N. Kugimiya, A. Suga, E. Harada, K. Hamano, "Dclk1 inhibition cancels 5-FU-induced cell-cycle arrest and decreases cell survival in colorectal cancer," Anticancer Research, vol. 38 no. 11, pp. 6225-6230, DOI: 10.21873/anticanres.12977, 2018.

[20] X.-F. Xu, F. Liu, J.-Q. Xin, J.-W. Fan, N. Wu, L.-J. Zhu, L.-F. Duan, Y.-Y. Li, H. Zhang, "Respective roles of the mitogen-activated protein kinase (MAPK) family members in pancreatic stellate cell activation induced by transforming growth factor- β 1 (TGF- β 1)," Biochemical and Biophysical Research Communications, vol. 501 no. 2, pp. 365-373, DOI: 10.1016/j.bbrc.2018.04.176, 2018.

[21] W. Liu, J. Li, "Theaflavin-3, 3′-digallate attenuates rheumatoid inflammation in mice through the nuclear factor- κ B and MAPK pathways," Archivum Immunologiae et Therapiae Experimentalis, vol. 67 no. 3, pp. 153-160, DOI: 10.1007/s00005-019-00536-7, 2019.

[22] E. K. Kim, E.-J. Choi, "Compromised MAPK signaling in human diseases: an update," Archives of Toxicology, vol. 89 no. 6, pp. 867-882, DOI: 10.1007/s00204-015-1472-2, 2015.

[23] L. A. M. D. Santos, G. B. Rodrigues, F. V. B. Mota, M. E. R. D. França, K. P. De Souza Barbosa, W. H. D. Oliveira, S. W. S. Rocha, D. B. Lós, A. K. S. Silva, T. G. D. Silva, C. A. Peixoto, "New thiazolidinedione LPSF/GQ-2 inhibits NF κ B and MAPK activation in LPS-induced acute lung inflammation," International Immunopharmacology, vol. 57, pp. 91-101, DOI: 10.1016/j.intimp.2018.02.011, 2018.

[24] D. Szklarczyk, A. Franceschini, S. Wyder, K. Forslund, D. Heller, J. Huerta-Cepas, M. Simonovic, A. Roth, A. Santos, K. P. Tsafou, M. Kuhn, P. Bork, L. J. Jensen, C. von Mering, "STRING v10: protein-protein interaction networks, integrated over the tree of life," Nucleic Acids Research, vol. 43 no. D1, pp. D447-D452, DOI: 10.1093/nar/gku1003, 2015.