Identification of Antitumor Constituents in Toad Venom by Spectrum-Effect Relationship Analysis and Investigation on Its Pharmacologic Mechanism

Abstract

(1) Background: Toad venom (Bufonis Venenum, known as ‘Chansu’ in Chinese), the secretion of the ear-side gland and skin gland of Bufo gargarizans cantor or Duttaphrynus melanostictus Schneider, has been utilized to treat several diseases in China for thousands of years. However, due to the chemical variability of the components, systematic chemical composition and the key pharmacophores in toad venom have not yet fully understood. Besides, it contains a variety of effective compounds with different physiological activity and chemotypes, mainly including alkaloids, bufogenins, bufotoxins, and so on. The recent pharmacological researches have demonstrated that several bufogenins have remarkable pharmacological effects, such as anti-inflammatory, analgesic effects, and anti-tumor effects. Aim of the study: To identify the bioactive compounds and pharmacophores originating from toad venom based on analyzing spectrum-effect relationship by chemometrics and to explore the anti-cancer mechanism primarily. (2) Materials and methods: Fingerprint of the 21 batches of samples was established using HPLC (High Performance Liquid Chromatography). The anti-tumor activity of extracts were determined by in-vitro assays. Chemometric analysis was used to establish the spectrum-effect model and screen for active ingredients. Pharmacodynamic tests for the screened active compound monomers were conducted with in-vitro assays. Further anti-tumor mechanisms were investigated using western blot and flow cytometry. (3) Results: The established spectrum-effect model has satisfactory fitting effect and predicting accuracy. The inhibitory effect of major screened compounds on lung carcinoma cells A549 were validated in vitro, demonstrating that arenobufagin, telocinobufogenin, and cinobufotalin had significant anti-tumor effects. Through further investigation of the mechanism by western blotting and flow cytometry, we elucidated that arenobufagin induces apoptosis in A549 cells with the enhanced expression of cleaved PARP (poly (ADP-ribose) polymerase). These results may provide valuable information for further structural modification of bufadienolides to treat lung cancer and a method for discovery of anti-tumor active compounds. Conclusions: Our research offers a more scientific method for screening the principal ingredients dominating the pharmacodynamic function. These screened compounds (arenobufagin, etc.) were proven to induce apoptosis by overactivation of the PARP-pathway, which may be utilized to make BRCA (breast cancer susceptibility gene) mutant cancer cells more vulnerable to DNA damaging agents and kill them.

Toad venom bufadienolides and bufotoxins: An updated review

“…Arenobufagin and telocinobufagin revealed similar anticancer effects on A549 (human lung adenocarcinoma) and H157 (squamous cell carcinoma) cells. But several‐fold differences were observed by cinobufotalin in the MTT assay carried out on these cells at doses of 50–100 ng/mL (Figure 5) (Wu et al, 2020).…”

“…It was found to promote the phosphorylation of Yes‐associated proteins (Deng et al, 2018), an oncoprotein responsible for cell division (Abylkassov & Xie, 2016). Arenobufagin was elucidated to induce apoptosis by enhanced cleavage of PARP (Wu et al, 2020), a DNA‐repairing enzyme that is activated by DNA breaks (Zaremba et al, 2009). The Molecular docking results demonstrated the affinity of arenobufagin for the transmembrane α‐subunit of Na + /K + ‐ATPase which was in agreement with the experimental results carried out in HeLa cells (Yue et al, 2016).…”

The effect of anti‐alcoholic gastric ulcer before and after vinegar‐processed Yuanhu Zhitong prescription based on spectral‐effect relationship

“…Gray system theory discovers the influence degree between the two factors through the similarity of the development trend or curve shape, which can provide reference for decision-making and judgment of main factors (Wu et al, 2020). In this study, the gray correlation degree was calculated between the fingerprint information of YZP and its efficacy index of anti-alcoholic gastric ulcer (ATGU), and the contribution of each component to pharmacological action was evaluated by the gray correlation coefficient.…”

Arenobufagin causes ferroptosis in human gastric cancer cells by increasing rev-erbα expression

“…Arenobufagin (ArBu, C 24 H 32 O 6 ) is a bufadienolide compound that is considered as one of the major bioactive ingredients in the extracts. 17 These extracts are clinically practiced in patients with non-small cell lung cancer, esophageal cancer, hepatocellular carcinoma, or pancreatic cancer. 11 , 12 , 13 , 14 , 15 , 16 In addition to the cardiotonic and analgesic effects, ArBu is also a broad-spectrum anti-tumor active compound against liver cancer, esophageal squamous cell carcinoma, and non-small cell lung cancer.…”