Inhibition of Twist1-mediated invasion by Chk2 promotes premature senescence in p53-defective cancer cells.

Twist1, a basic helix-loop-helix transcription factor is implicated as a key mediator of epithelial-mesenchymal transition (EMT) and metastatic dissemination in p53-deficient cancer cells. On the other hand, checkpoint kinase 2 (Chk2), a major cell cycle regulatory protein provides a barrier to tumorigenesis due to DNA damage response by preserving genomic stability of the cells. Here we demonstrate that Chk2 induction proficiently abrogates invasion, cell scattering and invadopodia formation ability of p53-mutated invasive cells by suppressing Twist1, indicating Chk2 confers vital role in metastasis prevention. In addition, ectopic Chk2, as well as its (Chk2) induction by natural podophyllotoxin analog, 4'-demethyl-deoxypodophyllotoxin glucoside (4DPG), strongly restrain Twist1 activity along with other mesenchymal markers, for example, ZEB-1, vimentin and Snail1, whereas the epithelial markers such as E-cadherin and TIMP-1 expression augmented robustly. However, downregulation of endogenous Chk2 by siRNA as well as Chk2 selective inhibitor PV1019 implies that 4DPG-mediated inhibition of Twist1 is Chk2-dependent. Further, mechanistic studies unveil that Chk2 negatively regulates Twist1 promoter activity and it (Chk2) interacts steadily with Snail1 protein to curb EMT. Strikingly, Chk2 overexpression triggers premature senescence in these cells with distinctive increase in senescence-associated ß-galactosidase (SA-ß-gal) activity, G2/M cell cycle arrest and induction of senescence-specific marker p21waf1/Cip1. Importantly, stable knockdown of Twist1 by shRNA markedly augments p21 expression, its nuclear accumulation, senescence-associated heterochromatin foci (SAHF) and amplifies the number of SA-ß-gal-positive cells. Moreover, our in vivo studies also validate that 4DPG treatment significantly abrogates tumor growth as well as metastatic lung nodules formation by elevating the level of phospho-Chk2, Chk2 and suppressing Twist1 activity in mouse mammary carcinoma model. In a nutshell, this report conceives a novel strategy of Twist1 suppression through Chk2 induction, which prevents metastatic dissemination and promotes premature senescence in p53-defective invasive cancer cells.

4'-Demethyl-deoxypodophyllotoxin glucoside isolated from Podophyllum hexandrum exhibits potential anticancer activities by altering Chk-2 signaling pathway in MCF-7 breast cancer cells.

We investigated the root of Podophyllum hexandrum as a potential source of lead bioactive metabolites with anticancer activity. The present study led to the isolation of six known aryltetralin-type lignans designated as 4'-demethyl-deoxypodophyllotoxin (1), podophyllotoxin (2), 4'-demethyl-podophyllotoxin (3), podophyllotoxin-4-O-ß-d-glucopyranoside (4), 4'-demethyl-deoxypodophyllotoxin-4-O-ß-d-glucopyranoside (5), 4'-demethyl-podophyllotoxin-4-O-ß-d-glucopyranoside (6), along with three known flavones Kaempferol (7), Quercetin (8), Astragalin (9) from the root of P. hexandrum. Compounds (1-9) exhibited the remarkable cytotoxic potential in diverse cancer cell lines. 5 therapeutic potential was extensively studied first time which exhibiting antiproliferative and ROS generating activity than its non-glycoside analogue 1. Furthermore, 5 augmented the apoptotic cascades in MCF-7 breast cancer cells, viz. nuclear condensation, membrane blebbing, probably by destabilizing the micro-tubular protein tubulin. Strikingly, our docking study and in vitro assays demonstrate that 5 binds to and modulate checkpoint kinase-2, a key cell cycle regulatory protein in normal and cancer cells.

A convergent synthesis of alkyne-azide cycloaddition derivatives of 4-α,ß-2-propyne podophyllotoxin depicting potent cytotoxic activity.

A facile synthetic approach to construct the O-propargyl derivatives of 4α and 4ß-(1,2,3-triazol-4-yl)-podophyllotoxin (9a-k & 10a-k) and 4'-Demethyl-4'-4ß-(1,2,3-triazol-4-yl)-epipodophyllotoxin (12a-d) were synthesized by means of click chemistry. The chemical structures were confirmed by (1)H, (13)C, 2D NMR and HRMS spectral analysis and their cytotoxicities were measured against diverse human cancer cell lines viz. PC-3, PANC-1, COLO-205 and A-549 by MTT assay. Some of the compounds were found more potent than the parent molecule Podophyllotoxin, like; 9a &10a, 9h &10h, 9k &10k, 10d, 8 and 12a. The most potent molecule discovered was compound 9k that exhibited the highest cytotoxicity on all the four cancer cell lines with IC50 values of 3.8-22 nM. The compound further found to induce apoptosis and strongly hindered the motility of aggressive prostate cancer PC-3 cells.

Bioactive metabolites from an endophytic Cryptosporiopsis sp. inhabiting Clidemia hirta.

An endophytic Cryptosporiopsis sp. was isolated from Clidemia hirta and analyzed for its secondary metabolites that lead to the isolation of three bioactive molecules. The compounds were purified from the culture broth of the fungus and their structures were determined by spectroscopic methods as (R)-5-hydroxy-2-methylchroman-4-one (1), 1-(2,6-dihydroxyphenyl)pentan-1-one (2) and (Z)-1-(2-(2-butyryl-3-hydroxyphenoxy)-6-hydroxyphenyl)-3-hydroxybut-2-en-1-one (3). Compound 1 exhibited significant cytotoxic activity against the human leukemia cell line, HL-60 with an IC50 of 4 µg/ml. This compound induced G2 arrest of the HL-60 cell cycle significantly. In addition, out of these compounds, 2 and 3 were active against several bacterial pathogens. Compound 2 was active against Bacillus cereus, Escherichia coli and Staphylococcus aureus with IC50 values varying from 18 to 30 µg/ml, and compound 3 displayed activity against Pseudomonas fluorescens with an IC50 value of 6 µg/ml. Compounds 2 and 3 are novel whereas compound 1 was reported earlier but the stereochemistry of its C-2 methyl is established for the first time.