Manish Debnath

A nucleus-imaging probe that selectively stabilizes a minor conformation of c-MYC G-quadruplex and down-regulates c-MYC transcription in human cancer cells

The c-MYC proto-oncogene is a regulator of fundamental cellular processes such as cell cycle progression and apoptosis. The development of novel c-MYC inhibitors that can act by targeting the c-MYC DNA G-quadruplex at the level of transcription would provide potential insight into structure-based design of small molecules and lead to a promising arena for cancer therapy. Herein we report our finding that two simple bis-triazolylcarbazole derivatives can inhibit c-MYC transcription, possibly by stabilizing the c-MYC G-quadruplex. These compounds are prepared using a facile and modular approach based on Cu(I) catalysed azide and alkyne cycloaddition. A carbazole ligand with carboxamide side chains is found to be microenvironment-sensitive and highly selective for “turn-on” detection of c-MYC quadruplex over duplex DNA. This fluorescent probe is applicable to visualize the cellular nucleus in living cells. Interestingly, the ligand binds to c-MYC in an asymmetric fashion and selects the minor-populated conformer via conformational selection.

A small molecule peptidomimetic that binds to c-KIT1 G-quadruplex and exhibits antiproliferative properties in cancer cells.

A modular synthesis of l-proline derived peptidomimetics has been developed using the Cu(I) catalyzed Huisgen cycloaddition between an azido prolinamide with pyridine and benzene dicarboxamide containing dialkynes. Förster Resonance Energy Transfer (FRET) melting assay provided an initial indication that the pyridyl analogue can stabilize the c-KIT1 quadruplex DNA. A competitive FRET-melting assay and Fluorescent Intercalator Displacement (FID) assay suggest that the pyridyl ligand shows excellent selectivity for c-KIT1 quadruplex over duplex DNA and other investigated G-quadruplexes. Molecular docking studies indicate that the pyridyl ligand can adopt unique conformations upon binding to c-KIT1 quadruplex due to the presence of intramolecular hydrogen bonds. The pyridyl ligand can perturb cell cycle progression and induce necrotic cell death of human hepatocellular liver carcinoma HepG2 cells.

Environment‐Sensitive Probes Containing a 2,6‐Diethynylpyridine Motif for Fluorescence Turn‐On Detection and Induction of Nanoarchitectures of Human Telomeric Quadruplex

Bis(phenylethynyl)pyridylcarboxamides with amide side chains at the para position of the NH2 group possess strong solvatochromic properties compared with the meta analogues. Fluorescence binding titrations show that these probes exhibit remarkable fluorescence turn-on responses upon interacting with the human telomeric G-quadruplex (h-TELO). Förster resonance energy transfer melting analysis shows the high selectivity of these probes for h-TELO over duplex DNA. Isothermal titration calorimetry, as well as UV/Vis and fluorescence spectroscopy studies, show that the meta analogue has a twofold binding affinity for h-TELO over the para analogue. The noncovalent interaction of these small-molecule probes with h-TELO has been used to regulate the assembly of novel supramolecular nanoarchitectures.

Poly-L-Lysine Inhibits Tumor Angiogenesis and Induces Apoptosis in Ehrlich Ascites Carcinoma and in Sarcoma S-180 Tumor

Background: This study focuses on the role of Poly-L-lysine (PLL), an essential amino acid, on molecular changes of tumor angiogenesis suppression, pro-apoptotic and anti-apoptotic gene expression after treatment on Ehrlich ascites carcinoma (EAC) and solid sarcoma-180 tumor cells bearing mice. Materials and Methods: The cell viability was carried out using MTT assay. The antitumor activity was evaluated by treatment with PLL at 20 and 40mg/kg/b.w doses for 14 days in EAC ascites tumor and 21 days for Sarcoma-180 solid tumor model. Several tumor evaluation studies, haematological and biochemical parameters were estimated. Importantly, the tumor cell apoptosis was assessed using microscopic observations, DNA fragmentation assay, Flow cytometric analysis, cell-cycle and electron-microscopic study, following which, the expression of several signal proteins related to pro-apoptosis, anti-apoptosis and tumor angiogenesis were quantified using western blotting and immunohistochemistry study. Results: Precisely, PLL had cytotoxic effect on K562; A549; U937 and B16F10 cancer cells. Significant decreases in liquid and solid tumors and increased life span of treated mice were observed (P<0.05). Typical morphological changes, apoptosis bleb phenomenon and sub-G1 cell cycle arrests revealed that PLL promoted apoptotic cell death. Western blot and immunohistochemistry confirms, PLL activated apoptotic signalling cascades through down regulation of Bcl-2 and CD31 protein and up-regulation of Bax and p53 proteins. The anti-angiogenic effects were also accompanied with decreased VEGF expression and reduced peritoneal-angiogenesis and microvessel density. Conclusions: The antitumor and antitumor-angiogenic activity of PLL was confirmed from all the results via up and down regulation of relevant signal proteins reported in this publication.

Cell penetrating thiazole peptides inhibit c-MYC expression via site-specific targeting of c-MYC G-quadruplex

Abstract The structural differences among different G-quadruplexes provide an opportunity for site-specific targeting of a particular G-quadruplex structure. However, majority of G-quadruplex ligands described thus far show little selectivity among different G-quadruplexes. In this work, we delineate the design and synthesis of a crescent-shaped thiazole peptide that preferentially stabilizes c-MYC quadruplex over other promoter G-quadruplexes and inhibits c-MYC oncogene expression. Biophysical analysis such as Förster resonance energy transfer (FRET) melting and fluorescence spectroscopy show that the thiazole peptide TH3 can selectively interact with the c-MYC G-quadruplex over other investigated G-quadruplexes and duplex DNA. NMR spectroscopy reveals that peptide TH3 binds to the terminal G-quartets and capping regions present in the 5′- and 3′-ends of c-MYC G-quadruplex with a 2:1 stoichiometry; whereas structurally related distamycin A is reported to interact with quadruplex structures via groove binding and end stacking modes with 4:1 stoichiometry. Importantly, qRT-PCR, western blot and dual luciferase reporter assay show that TH3 downregulates c-MYC expression by stabilizing the c-MYC G-quadruplex in cancer cells. Moreover, TH3 localizes within the nucleus of cancer cells and exhibits antiproliferative activities by inducing S phase cell cycle arrest and apoptosis.

Induction of apoptosis, anti-proliferation, tumor-angiogenic suppression and down-regulation of Dalton’s Ascitic Lymphoma (DAL) induced tumorigenesis by poly-l-lysine: A mechanistic study

PURPOSE The present study, attempts to validate the molecular mechanism(s) of Poly-l-lysine (PLL) induced apoptosis, anti-proliferative and anti-tumorigenic properties in in-vitro HUVECs cells and Daltons Ascitic Lymphoma (DAL) and in in-vivo DAL cell bearing BALB/c mice model. MATERIALS AND METHODS The cell proliferation assay and morphological assay was carried out using the MTT assay and Giemsa staining method. The antitumor activity of PLL was evaluated in BALB/c mice at 20 and 40 mg/kg/b.w doses for 21 days for DAL solid tumor model. Several tumor evaluation endpoints, hematological and biochemical parameters were estimated. Additionally, the tumor apoptosis, anti-proliferative and anti-tumor angiogenesis effects were assessed using western blots and immunohistochemistry. RESULTS PLL significantly decreased cell proliferation in in-vitro HUVECs and DAL cells without significant effects on normal cell growth. PLL also induced alteration in cellular morphology in DAL cells. Therafter, in the BALB/c mouse model, PLL had noticeable inhibition in DAL-induced tumorigenesis. This inhibition was evident through reduced solid tumor volume and weight versus the control group. However, PLL promoted tumor apoptosis and suppressed cell-proliferation and tumor-angiogenesis. PLL also increased hematological markers significantly compared to 5-flurouracil (5-FU). The amount of TdT in the nuclei of DAL cells in mice treated with PLL was significantly increased while in contrast decreases of anti-apoptotic protein Bcl-2 expression were observed. PLL also significantly upregulated the pro-apoptotic protein Bax and activated caspase-3. Measurable decreases of cyclin-D1 were observed through PLL treatments, an indicator of cell-cycle arrest. These studies also indicate PLLs induction and anti-proliferative effects through suppression of the c-Myc and Ki-67 proliferation-indices. Additionally, PLL inhibited tumor-angiogenesis through suppression of VEGF and CD34 protein expression levels and reduction ofmicrovesseldensityversus similar parameters in tumors from control mice. CONCLUSION The present study offers opportunities and hopes for possible anti-tumortherapies with PLL in the near future and warrants further formulation developments.