A potent Bioorganic azapodophyllotoxin derivative Suppresses tumor Progression in Triple negative breast Cancer: An Insight into its Inhibitory effect on tubulin polymerization and Disruptive effect on microtubule assembly.

Triple negative breast cancer (TNBC) has long been a challenging disease owing to its high aggressive behaviour, poor prognosis and its limited treatment options. The growing demand of new therapeutics against TNBC enables us to examine the therapeutic efficiency of an emerging class of anticancer compounds, azapodophyllotoxin derivative (HTDQ), a nitrogen analogue of podophyllotoxin, using different biochemical, spectroscopic and computational approaches. The anticancer activities of HTDQ are studied by performing MTT assay in a dose depended manner on Triple negative breast cancer cells using MDA-MB-468 and MDA-MB-231 cell lines with IC50 value 937 nM and 1.13 µM respectively while demonstrating minimal effect on normal epithelial cells. The efficacy of HTDQ was further tested in 3D tumour spheroids formed by the human TNBC cell line MDA-MB468 and also the murine MMTV positive TNBC cell line 4 T1. The shrinkage that observed in the tumor spheroid clearly indicates that HTDQ remarkably decreases the growth of tumor spheroid thereby affirming its cytotoxicity. The 2D cell viability assay shows significant morphological alteration that possibly caused by the cytoskeleton disturbances. Hence the binding interaction of HTDQ with cytoskeleton protein tubulin, its effect on tubulin polymerisation as well as depolymerisation of preformed microtubules along with the conformational alternation in the protein itself have been investigated in detail. Moreover, the apoptotic effects of HTDQ have been examined using a range of apoptotic markers. HTDQ-treated cancer cells showed increased expression of cleaved PARP-1 and pro-caspase-3, suggesting activation of the apoptosis process. HTDQ also upregulated pro-apoptotic Bax expression while inhibiting anti-apoptotic Bcl2 expression, supporting its ability to induce apoptosis in cancer cells. Hence the consolidated biochemical and spectroscopic research described herein may provide enormous information to use azapodophyllotoxin as promising anticancer therapeutics for TNBC cells.

Switching of the Polarity-Sensitive Aggregation Pattern of a Thiosemicarbazone-Based Anticancer Luminophore and Its Involvement in Cellular Apoptosis of the Human Lung Cancer Cell Line.

Elucidation of the photophysical and biochemical properties of small molecules can facilitate their applications as prospective therapeutic imaging (theragnostic) agents. Herein, we demonstrate the luminescence behavior of a strategically designed potential therapeutic thiosemicarbazone derivative, (E)-1-(4-(diethylamino)-2-hydroxybenzylidene)-4,4-dimethylthiosemicarbazide (DAHTS), accompanied by the illustration of its solvation and solvation dynamics using spectroscopic techniques and exploring its promising antitumor activities by adopting the necessary biochemical assays. Solvent-dependent photophysical properties, namely UV-vis absorption, fluorescence emission, and excitation profiles, concentration-dependent studies, and time-resolved fluorescence decays, serve as footprints to explain the existence of DAHTS monomers, its excited-state intramolecular proton transfer (ESIPT) product, and dimeric and aggregated forms. The emission intensity progressively intensifies with increasing polarity and proticity of the solvents up to MeOH, but in water, a sudden dip is seen. Solvent polarity and H-bonding modulate the fluorescence behavior of the primary emission peak and significantly influence the formation of the dimer and DAHTS aggregates. The designed luminophore (DAHTS) exhibits significant antiproliferative activity against the human lung cancer (A549) cell lines with inhibitory concentrations (IC50) of 16.88 and 11.92 µM for 24 and 48 h, respectively. DAHTS effectively reduces the cell viability and induces cytotoxicity with extensive morphological changes in A549 cells in the form of spikes when compared to the normal HEK cell lines. More importantly, it increases the p53 expression at the mRNA level that consolidates its potential therapeutic activity. The effect of DAHTS on apoptotic pathways against the A549 cell line has been investigated to determine its probable mechanism of cell death. Thus, the all-inclusive understanding of the photophysical properties and the necessary biochemical assays put forward important steps toward tailoring the thiosemicarbazone core structure for favorable cancer theragnostic applications in academic and pharmaceutical research.

The consequences of adopting therapeutic luminophore azapodophyllotoxin into BSA: a molecular regulator to control emissive population of two tryptophan residues in carrier protein.

Bovine serum albumin (BSA) is a widely recognized plasma protein for its ubiquitous function as one of the paramount transporter of different drugs and enzymes inside biological systems. HPFQ, a member of azapodophyllotoxin family, has been observed to be highly bioactive against a majority of cancer cell lines; while subsequently showing impressive fluorescent properties throughout the polarity scale. However, further pursuit into compliance of this bioactive fluorophore with carrier protein remains imperative for excavating its suitable transporter inside human body. The present biophysical spectroscopic study attempts to exhibit the adaptability of BSA towards a potential therapeutic fluorophore (HPFQ) by combining in vitro optical spectroscopy and in silico molecular docking. The competitive site-binding studies demonstrated that BSA nurtures neutral anti-cancer fluorophore HPFQ into Sudlow site I, where it experiences varying interactions with surrounding hydrophobic amino acid residues viz. Phe 205, Trp 213, Ala 209, Leu 330, Ala 349, Leu 480 etc. HPFQ gets accommodated at the vicinity of Trp-213 in BSA and initiates operation of FRET between them. Adaptation of HPFQ encourages an allosteric modulation, leading to a minor deformation in secondary protein structure, which probably allows the invading water molecules to increase the micropolarity of the adjacent environment around Trp-213. HPFQ assumes to administer conformational alteration in BSA and regulate emissive population of two tryptophan residues Trp-134 and Trp-213. The amalgamated spectroscopic investigation described herein may encourage design of azapodophyllotoxin based potential therapeutic agents for effective in vivo bio-circulation using BSA-based drug distribution systems.Communicated by Ramaswamy H. Sarma.

Entrapment in micellar assemblies switches the excimer population of potential therapeutic luminophore azapodophyllotoxin.

Azapodophyllotoxin is a new class of anti-tumor agent with brilliant therapeutic activity and understanding its physicochemical nature in bio-mimetic microenvironments may provide substantial importance in context of its intercellular localization, efficacy as well as delivery. The present work epitomizes environment-sensitive fluorescence modulation of a prodigy, 4-(2-Hydroxyethyl)-10-phenyl-3,4,6,7,8,10- hexahydro-1H-cyclopenta[g]furo[3,4-b]quinoline-1-one (HPFQ) from the class of anti-cancer agent Azapodophyllotoxin, in differently charged model bio-mimetic micellar microenvironment of cationic CTAB, anionic SDS and neutral Triton X-100 using UV-visible absorption, steady state fluorescence, time-resolved fluorescence and fluorescence anisotropy studies. As a distinct phenomenon, anticancer HPFQ exhibits prolific fluorescence in solvents of varying polarity, originating from a mixed contribution of locally excited, charge transfer and excimer emission. A dramatic modulation in the photophysics of HPFQ has been observed in two types of surfactant consortiums: pre-micellar and post-micellar at physiological and anoxic pH. On photo-excitation, anti-cancer HPFQ exists in monomer-excimer equilibrium with varying ratios in different polarity regions. The marked enhancement in fluorescence intensity of HPFQ in post-micellar region of the surfactant under study probably arises due to regeneration of the monomer from its excimer. This reoccurrence reduces the possibility of Förster resonance energy transfer (FRET) from monomer to excimer, which essentially increases the desired emission intensity. Localization of HPFQ in micellar systems highly depends on polarity gradient inside the micelle, electrostatic, hydrophobic and intermolecular hydrogen bonding interactions. Further corroboration with the polarity sensitive experiments in dioxane-water mixture indicates towards spatial localization of the probe molecule in the stern layer of cationic CTAB, sheer surface of neutral TX100 and outer Gouy-Chapman layer in anionic SDS micelles. A molecular binary logic gate correlates the dominance of micellization over the polarity factor, which enhances the fluorescence response of HPFQ. The enhancement of the emissive potential of anti-cancer HPFQ in biomimetic environments by switching its excimer population may have an immense importance to achieve the status of a dual therapeutic and imaging agent altogether in progressive biomedical research.