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. 2020 Sep 18;25(18):4269.
doi: 10.3390/molecules25184269.

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

Ji-Heng Wu  1 , Yue-Ting Cao  1 , Hong-Ye Pan  1 , Long-Hu Wang  1
Affiliations

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

Ji-Heng Wu et al. Molecules. .

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.

Keywords: apoptosis; chemometrics; spectrum-effect relationship; toad venom.

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Conflict of interest statement

The authors declared that there is no conflict of interest.

Figures

Figure 1
Figure 1
HPLC fingerprints of the 21 batches of toad venom extracts.
Figure 2
Figure 2
The reference atlas of the toad venom extracts.
Figure 3
Figure 3
In vitro anticancer activities of 21 batches of toad venom extracts (A549 cells were incubated with 100 ng/mL toad venom extracts for 72 h, and cell viability was examined by MTT assay). Ordinary one-way ANOVA, **** p < 0.00001 indicates a significant difference versus the control group.
Figure 4
Figure 4
Graphical representation of OPLSR model—calibration model.
Figure 5
Figure 5
Graphical representation of OPLSR model—VIP plot.
Figure 6
Figure 6
The structure of compounds identified in toad venom extract.
Figure 7
Figure 7
The inhibitory effects of arenobufagin, telocinobufagin, and cinobufotalin on A549 cells (a) and H157 cells (b) analyzed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. ** Indicates statistical significance (p < 0.01).
Figure 8
Figure 8
Arenobufagin induces apoptosis in A549 cells. A549 cells were incubated with arenobufagin for 48 h. (a) The effects of arenobufagin on apoptosis were analyzed by flow cytometry. (b) Detecting the protein expression levels of PARP by Western blotting.
Figure 8
Figure 8
Arenobufagin induces apoptosis in A549 cells. A549 cells were incubated with arenobufagin for 48 h. (a) The effects of arenobufagin on apoptosis were analyzed by flow cytometry. (b) Detecting the protein expression levels of PARP by Western blotting.
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