Withanolide Metabolites Inhibit PI3K/AKT and MAPK Pro-Survival Pathways and Induce Apoptosis in Acute Myeloid Leukemia Cells

Abstract

Acute myeloid leukemia (AML) is an aggressive disease and, despite advances, its treatment remains challenging. Therefore, it remains important to identify new agents for the management of this disease. Withanolides, a group of steroidal lactones found in Solanaceae plants are of potential interest due to their reported anticancer activities in different settings. In this study we investigated the anti-proliferative effects and mode of action of Solanaceae-derived withanolides in AML cell models; these metabolites include withametelin (WTH) and Coagulansin A (CoA) isolated from Datura innoxia and Withania coagluanse, respectively. Both withanolides inhibited the proliferation of AML cells and induced cell death, with WTH being more potent than CoA in the AML models tested. Quantitative label-free proteomics and phosphoproteomics were employed to define the mechanism of action of the studied withanolides. We identified and quantified 5269 proteins and 17,482 phosphosites in cells treated with WTH, CoA or vehicle control. Withanolides modulated the expression of proteins involved in regulating key cellular processes including cell cycle, metabolism, signaling, protein degradation and gene expression. Enrichment analysis of the phosphoproteomics data against kinase substrates, kinase-kinase relationships and canonical pathways showed that the withanolides decreased the activity of kinases such as phosphoinositide 3-kinase (PI3K), protein kinase B (PKB; also known as RAC-alpha serine/threonine-protein kinase or AKT), mammalian target of rapamycin (mTOR), extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and the serine/threonine-protein kinase A-Raf (ARAF), while increasing the activation of DNA repair kinases. These results indicate that withanolide metabolites have pleiotropic effects in the modulation of oncogenic pro-survival and pro-apoptotic signaling pathways that regulate the induction of apoptosis. Withanolide mediated apoptosis was confirmed by immunoblotting showing increased expression of cleaved PARP and Caspases 3, 8 and 9 as a result of treatment. Overall, our results suggest that WTH and CoA have therapeutic potential against AML with WTH exhibiting more potent effects and should be explored further.

Natural withanolides, an update

“…For example, withanolide A exerted anti-cancer properties against adrenocortical carcinomas and hepatocellular carcinoma by increasing MAPK (ERK, p38) activation; 171,172 meanwhile, withaferin A induced apoptosis by up-regulating the expression of phosphorylated p38 in MCF-7 cells. 173 Withanolide D 174 and withametelin 175 modulated the MAPK pathway to inhibit tumor cell growth. Besides, our previous studies demonstrated that (20 S ,22 R ,24 R ,25 S ,26 R / S )-15α-acetoxy-5,6β:22,26-diepoxy-4β,24,25,26-tetrahydroxyergost-2-en-1-one, a withanolide isolated from Physalis Pubescens L., inhibited the proliferation of human melanoma A375 cells by inducing G2/M cell cycle arrest via the EGFR/p38 pathway.…”

“…franchetii significantly induced apoptosis in two androgen-independent cell lines via JNK and/or ERK activation. 177 In addition to MAPK, p53, 178 Akt, 175,179–182 and c-Met 183 were also found to be activated or inactivated in the antitumor effects of withanolides. However, whether these kinases are the molecular targets of withanolides remains to be identified.…”

Anticancer Mechanisms of Bioactive Compounds from Solanaceae: An Update

“…In a different approach to study the role of two other withanolides, proteomic profiling was carried out on acute myeloid leukaemia cell lines (Kasumi-1 and P31/FUJ) that had been treated with withametelin and coagulansin A, extracted from Datura innoxia and Withania coagluanse , respectively [ 55 ]. The use of proteomics techniques such as liquid chromatography in conjunction with mass spectrometry (LC-MS) permitted the identification of several proteins that were either up- or downregulated after treatment with these secondary metabolites.…”

“…The use of proteomics techniques such as liquid chromatography in conjunction with mass spectrometry (LC-MS) permitted the identification of several proteins that were either up- or downregulated after treatment with these secondary metabolites. Overall, there were reductions in the expression of 33 and 52 proteins in cells treated with coagulansin A and withametelin, respectively [ 55 ]. Thereafter, confirmatory functional assays were conducted on a few target proteins, which showed that the compounds exerted their inhibitory effect on the acute myeloid leukaemia cells by downregulating signalling proteins, including those that mediate the PI3K and MAPK pathways.…”

Toxicity evaluation induced by single and 28-days repeated exposure of withametelin and daturaolone in Sprague Dawley rats

“…Withametelin possess antifungal ( Singh et al, 2001 ) cytotoxicity against lung ( Rao et al, 2016 ), liver, prostate and mammary cell lines ( Baig et al, 2020 ). Its antileukemia effects are attributed to the inhibition of the spread of acute myeloid leukemia by targeting the signalling pathways of oncogenic kinase, which regulate the progression and survival of the cell cycle, thus resulting in an induction of apoptosis ( Akhtar et al, 2020 ). It relieves vincristine-induced neuropathic pain by analyzing results from behavior, biochemistry, histopathology and computational data, the molecular mechanism being associated with the suppression of TRPV1/TRPM8/P2Y nociception and MAPK signalling in mice ( Khan et al, 2021b ).…”