Bersaldegenin-1,3,5-orthoacetate induces caspase-independent cell death, DNA damage and cell cycle arrest in human cervical cancer HeLa cells

Abstract

Context: Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. Objective: The cytotoxic effect of bersaldegenin-1,3,5-orthoacetatea bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. Materials and methods: The cytotoxic activity of the compound (at 0.1-20.0 lg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/ 7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. Results: The compound had strong effect on the cells (IC 50 ¼ 0.55 lg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. Conclusions: Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides.

An In Vitro Anticancer, Antioxidant, and Phytochemical Study on Water Extract of Kalanchoe daigremontiana Raym.-Hamet and H. Perrier

“…In this process, any of the tested caspases were involved in cell death, which indicates that the non-apoptotic process occurs in SKOV-3 cells treated with the water extract. These results are similar to those from our previous study on bersaldegenin-1,3,5-orthoacetate and HeLa cells [ 41 ]. This was also confirmed in flow cytometry analysis, whereby mostly dead cell populations significantly increased after incubation with the extract, while the number of early apoptotic cells was not higher than in the control.…”

“…To determine the cell death pathway in SKOV-3 cells treated with K. daigremontiana extract, the expression levels of 92 related death genes were tested with a qPCR human apoptosis array [ 40 , 41 ]. The expression levels of seven genes were significantly downregulated (their values were ≤ 0.5-fold lower than the control value), including BAD (Bcl2-associated agonist of cell death), BCL3 (BCL3 transcription coactivator), CASP6 (caspase 6), CASP9 (caspase 9), LRDD (PIDD-p53-induced death domain protein 1), NFKBIZ (NF-kappa-B inhibitor zeta), and TNFSF10 (tumor necrosis factor ligand superfamily member 10).…”

“…Our phytochemical study revealed that this extract has twenty bufadienolide compounds, with the highest contents of daigremontianin, bryophyllin A (bryotoxin C), and bersaldegenin-1,3,5-orthoacetate. In our previous study, we showed that the main bufadienolide, bersaldegenin-1,3,5-orthoacetate, may be responsible for the cytotoxic action of the plant [ 41 ]. However, the whole extract, which is the most commonly used option in therapeutic applications, has not been evaluated.…”

Potential of Kalanchoe pinnata as a Cancer Treatment Adjuvant and an Epigenetic Regulator

“…Bersaldegenin-1,3,5-orthoacetate plays a role in cell cycle arrest and caspase-independent cell death in HeLa cells [ 8 ]. Xiuzhen et al [ 93 ] reported its potent cytotoxicity in human lung carcinoma A-59 and colon HTC-8 cell lines, which could be attributed to the presence of bersaldegenin-1,3,5-orthoacetate in chloroform extract from a whole fresh plant of Bryophyllum pinnatum collected in Taipei, Taiwan.…”

“…For example, quercetin has been reported to inhibit the proliferation of human breast cancer cells [ 7 ]. Bufadienolides have been shown to have antiangiogenic activity, cause inhibition of cell growth and proliferation, and induce cell death [ 8 ]. Antiproliferative activity has also been reported for triterpenes [ 9 ] and kalanchosides [ 5 ].…”