Abstract
Induction of premature senescence represents a novel functional strategy to curb the uncontrolled proliferation of malignant cancer cells. This study unveils the regiospecific synthesis of novel isoxazoline derivatives condensed to ring A of medicinal plant product Withaferin-A. Intriguingly, the cis fused products with β-oriented hydrogen exhibited excellent cytotoxic activities against proliferating human breast cancer MCF7 and colorectal cancer HCT-116 cells. The most potent derivative W-2b triggered premature senescence along with increase in senescence-associated β-galactosidase activity, G2/M cell cycle arrest, and induction of senescence-specific marker p21Waf1/Cip1 at its sub-toxic concentration. W-2b conferred a robust increase in phosphorylation of mammalian checkpoint kinase-2 (Chk2) in cancer cells in a dose-dependent manner. Silencing of endogenous Chk2 by siRNA divulged that the amplification of p21 expression and senescence by W-2b was Chk2-dependent. Chk2 activation (either by ectopic overexpression or through treatment with W-2b) suppressed NM23-H1 signaling axis involved in cancer cell proliferation. Finally, W-2b showed excellent in vivo efficacy with 83.8% inhibition of tumor growth at a dose of 25 mg/kg, b.w. in mouse mammary carcinoma model. Our study claims that W-2b could be a potential candidate to limit aberrant cellular proliferation rendering promising improvement in the treatment regime in cancer patients.
Highlights
Among the five-membered heterocyclic compounds, 2-isoxazolines have gained tremendous attention from the medicinal chemists as structural building blocks of biologically active molecules and versatile intermediates in organic synthesis[9]
In order to increase the yield of the reaction, we explored various bases in different proportion to find that triethylamine was the most appropriate for this cycloaddition (Fig. 1, entries 1–7)
We checked the effect of W-2b on NM23-H1expression; the results found a steady downregulation in the expression of NM23-H1 in a dose-dependent manner in MCF7 and HCT-116 cells after 48 h of treatment (Fig. 8C,D)
Summary
Among the five-membered heterocyclic compounds, 2-isoxazolines have gained tremendous attention from the medicinal chemists as structural building blocks of biologically active molecules and versatile intermediates in organic synthesis[9]. Though therapeutic agents and small molecule natural products such as doxorubicin, camptothecin, resveratrol, triptolide etc., are reported to induce senescence by augmenting p21 through various mechanisms in human cancer cells[19,20], the effect of WA and its derivatives on induction of premature senescence is yet to be examined. In this endeavour, we sought to examine the potential of fused 2-isoxazoline derivatives of WA to induce cytotoxicity in human cancer cells by abrogating cell proliferation through the induction of premature senescence
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