DAPI Staining Studies Research Articles

Regioselective synthesis and in vitro cytotoxicity evaluation of 3-thiooxindole derivatives: Tubulin polymerization inhibition and apoptosis inducing studies

Herein, regiospecific nucleophilic ring-opening of spiroaziridine oxindoles has been established to afford 3-substituted-thiooxindole derivatives as anticancer agents. Among the new series, compounds 7d and 9c exhibited promising cytotoxic activity toward HCT-116 cells with IC50 values of 6.73 ± 0.36 and 6.64 ± 0.95 µM, respectively. Further, AO/EB, DCFDA, and DAPI staining studies were executed to establish the underlying apoptosis mechanism which displayed significant nuclear and morphological alterations. JC-1 staining and annexin V binding assay inferred the loss of mitochondrial membrane potential in HCT-116 cancer cells. Cell cycle analysis showed the treatment of 9c against HCT-116 cells, arrested the cell cycle in G2-M phase. In addition, tubulin binding assay revealed that compound 9c exhibited tubulin polymerase inhibition with IC50 value of 9.73 ± 0.18 μM. This inhibition of tubulin polymerase was further supported by binding interactions of 9c with tubulin through docking studies on PDB ID: 3E22.

Synthesis and Biological Evaluation of Novel Heterocyclic Imines Linked Coumarin- Thiazole Hybrids as Anticancer Agents.

Human Galectin-1, a protein of lectin family showing affinity towards β-galactosides has emerged as a critical regulator of tumor progression and metastasis, by modulating diverse biological events including homotypic cell aggregation, migration, apoptosis, angiogenesis and immune escape. Therefore, galectin-1 inhibitors might represent novel therapeutic agents for cancer. A new series of heterocyclic imines linked coumarin-thiazole hybrids (6a-6r) was synthesized and evaluated for its cytotoxic potential against a panel of six human cancer cell lines namely, lung (A549), prostate (DU-145), breast (MCF-7 & MDA-MB-231), colon (HCT-15 & HT-29) using MTT assay. Characteristic apoptotic assays like DAPI staining, cell cycle, annexin V and Mitochondrial membrane potential studies were performed for the most active compound. Furthermore, Gal-1 inhibition was confirmed by ELISA and fluorescence spectroscopy. Among all, compound 6g {3-(2-(2-(pyridin-2-ylmethylene) hydrazineyl) thiazol-4-yl)-2H-chromen-2- one} exhibited promising growth inhibition against HCT-15 colorectal cancer cells with an IC50 value of 1.28 ± 0.14 µM. The characteristic apoptotic morphological features like chromatin condensation, membrane blebbing and apoptotic body formation were clearly observed with compound 6g on HCT-15 cells using DAPI staining studies. Further, annexin V-FITC/PI assay confirmed effective early apoptosis induction by treatment with compound 6g. Loss of mitochondrial membrane potential and enhanced ROS generation were confirmed with JC-1 and DCFDA staining method, respectively by treatment with compound 6g, suggesting a possible mechanism for inducing apoptosis. Moreover, flow cytometric analysis revealed that compound 6g blocked G0/G1 phase of the cell cycle in a dose-dependent manner. Compound 6g effectively reduced the levels of Gal-1 protein in a dose-dependent manner. The binding constant (Ka) of 6g with Gal-1 was calculated from the intercept value which was observed as 1.9 x 107 M-1 by Fluorescence spectroscopy. Molecular docking studies showed strong interactions of compound 6g with Gal-1 protein. Our studies demonstrate the anticancer potential and Gal-1 inhibition of heterocyclic imines linked coumarin-thiazole hybrids.

Synthesis of substituted biphenyl methylene indolinones as apoptosis inducers and tubulin polymerization inhibitors

A new series of biphenyl methylene indolinones has been designed, synthesized and evaluated for their in vitro antiproliferative activity against various cancer cell lines like DU-145 (prostate cancer cell line), 4T1 (mouse breast cancer cell line), MDA-MB-231 (human breast cancer cell line), BT-549 (human breast cancer cell line), T24 (human urinary bladder carcinoma cell line), and HeLa (cervical cancer cell line). Among the series, compound 10e showed potent in vitro cytotoxic activity against HeLa and DU-145 cancer cell lines with IC50 value of 1.74 ± 0.69 µM and 1.68 ± 1.06 µM respectively. To understand the underlying mechanism of most potent cytotoxic compound 10e, various mechanistic studies were carried out on DU-145 cell lines. Cell cycle analysis results revealed that these conjugates affect both G0/G1 and G2/M phase of the cycle, tubulin binding assay resulted that compound 10e interrupting microtubule network formation by inhibiting tubulin polymerization with IC50 value of 4.96 ± 0.05 μM. Moreover, molecular docking of 10e on colchicine binding site of the tubulin explains the interaction of 10e with tubulin. Clonogenic assay indicated inhibition of colony formation by compound 10e in a dose dependent manner. In addition, morphological changes were clearly observed by AO/EB and DAPI staining studies. Moreover, ROS detection using DCFDA, JC-1, and annexin V-FITC assays demonstrated the significant apoptosis induction by 10e.

Synthesis and Biological Evaluation of Thieno[2, 3‐d]pyrimidine‐amides as Potential Anticancer Agents

AbstractIn our search towards the development of potential anticancer agents, a new class of N‐(3‐(thieno[2, 3‐d]pyrimidin‐4‐ylamino)phenyl)amides (10 a−x) were synthesized and examined for their anticancer activity towards different human cancer cell lines. Of the derivatives tested, 10 g, 10 p and 10 w displayed potent growth inhibition on some of the cell lines, peculiarly 10 w exhibited selective and promising activity on MDA‐MB‐453 and MCF‐7 cell lines. In comparison to the standard ZSTK474, nearly 3 fold growth inhibitory effect was noticed with 10 w against MCF‐7 cell line. Notably, the morphology and long term clonogenic survival of MCF‐7 cells were affected by compound 10 w. Moreover, the results from AO/EB and DAPI staining studies as well as analysis of mitochondrial membrane potential divulged that the growth of MCF‐7 cells was inhibited by 10 w through the induction of apoptosis. From flow‐cytometry analysis, it is notable that 10 w shows G0/G1 cell cycle arrest in MCF‐7 cells. Overall, compound 10 w could be considered as a promising lead for the further exploration and identification of thieno[2, 3‐d]pyrimidine‐based anticancer agents.

Metal complexes of naphthoquinone based ligand: synthesis, characterization, protein binding, DNA binding/cleavage and cytotoxicity studies

Protein binding, DNA binding/cleavage and in vitro cytotoxicity studies of 2-((3-(dimethylamino)propyl)amino)naphthalene-1,4-dione (L) and its four coordinated M(II) complexes [M(II) = Co(II), Cu(II), Ni(II) and Zn(II)] have been investigated using various spectral techniques. The structure of the ligand was confirmed by spectral and single crystal XRD studies. The geometry of the complexes has been established using analytical and spectral investigations. These complexes show good binding tendency to bovine serum albumin (BSA) exhibiting high binding constant values (105 M−1) when compared to free ligand. Fluorescence titration studies reveal that these compounds bind strongly with CT-DNA through intercalative mode (Kapp 105 M−1) and follow the order: Cu(II) > Zn(II) > Ni(II) > Co(II) > L. Molecular docking study substantiate the strength and mode of binding of these compounds with DNA. All the complexes efficiently cleaved pUC18-DNA via hydroxyl radical mechanism and the Cu(II) complex degraded the DNA completely by converting supercoiled form to linear form. The complexes demonstrate a comparable in vitro cytotoxic activity against two human cancer cell lines (MCF-7 and A-549), which is comparable with that of cisplatin. AO/EB and DAPI staining studies suggest apoptotic mode of cell death, in these cancer cells, with the compounds under investigation.

Synthesis and biological evaluation of curcumin inspired imidazo[1,2-a]pyridine analogues as tubulin polymerization inhibitors

With an aim to develop new curcumin inspired analogues as potent anticancer agents, we synthesized a series of (1E,4E)-1-phenyl-5-(3-phenylimidazo[1,2-a]pyridin-2-yl)penta-1,4-dien-3-ones (12a–t) as tubulin polymerization inhibitors. An initial screening was carried out to evaluate their cytotoxic potential on a panel of six cancer cell lines namely, cervical (HeLa), gastric (HGC-27), lung (NCI-H460), prostate (DU-145 and PC-3) and breast (4T1), using MTT assay. Among the compounds tested, compounds 12e, 12r and 12t showed potent growth inhibition and 12t {(1E,4E)-1-(3-(3,4-difluorophenyl)imidazo[1,2-a]pyridin-2-yl)-5-(2,4,6-trimethoxyphenyl)penta-1,4-dien-3-one} being the most active member of the series inhibited the growth of all the tested cell lines with IC50 values varying from 1.7 – 2.97 μM. Moreover, 12t showed promising cytotoxicity on PC-3, HGC-27 and HeLa cell lines with IC50 values of 2.11 ± 0.27 μM, 2.21 ± 0.25 μM and 2.53 ± 0.01 μM respectively. The results from aqueous solubility test showed that compounds 12e and 12t have 1.7 and 2.8 times more aqueous solubility than curcumin. Interestingly, the most active compound 12t was found to be nearly 2 times more selective on PC-3 cells as well as safe on normal human prostate (RWPE-1) cells. In addition, compound 12t efficiently inhibited tubulin polymerization with IC50 value of 8.44 ± 0.13 μM and molecular modelling studies disclosed that 12t binds at the colchicine binding site of the tubulin. Cell cycle analysis revealed that 12t arrests PC-3 cells in G2/M phase in a dose dependant manner. Further, treatment of PC-3 cells with 12t showed typical apoptotic morphology, also led to the impairment of mitochondrial membrane potential (DΨm) and increased levels of reactive oxygen species (ROS). Altogether, the results from acridine orange/ethidium bromide (AO-EB) and DAPI staining studies, annexin V-FITC/propidium iodide staining assay, analysis of mitochondrial membrane potential (DΨm) and reactive oxygen species (ROS) levels undoubtedly demonstrated the induction of apoptosis in PC-3 cells by compound 12t.

Synthesis of different heterocycles-linked chalcone conjugates as cytotoxic agents and tubulin polymerization inhibitors

A series of new heterocycles-linked chalcone conjugates has been designed and synthesized by varying different alkane spacers. These conjugates were tested for their in vitro cytotoxic potential against a panel of selected human cancer cell lines namely, lung (A549 and NCI-H460), prostate (DU-145 and PC-3), colon (HCT-15 and HCT-116), and brain (U-87 glioblastoma) by MTT assay. Notably, among all the tested compounds, 4a exhibited potent cytotoxicity on NCI-H460 (lung cancer) cells with IC50 of 1.48±0.19µM. The compound 4a showed significant inhibition of tubulin polymerization and disruption of the formation of microtubules (IC50 of 9.66±0.06μM). Moreover, phase contrast microscopy and DAPI staining studies indicated that compound 4a can induce apoptosis in NCI-H460 cells. Further, the flow-cytometry analysis revealed that compound 4a arrests NCI-H460 cells in the G2/M phase of the cell cycle. In addition, molecular docking studies of the most active compounds 4a and 4b into the colchicine site of the tubulin, revealed the possible mode of interaction by these new conjugates.