Novel Etoposide Analogue Modulates Expression of Angiogenesis Associated microRNAs and Regulates Cell Proliferation by Targeting STAT3 in Breast Cancer.

Tumor microenvironment play role in angiogenesis and carcinogenesis. Etoposide, a known topoisomerase II inhibitor induces DNA damage resulting in cell cycle arrest. We developed a novel Etoposide analogue, Quinazolino-4β-amidopodophyllotoxin (C-10) that show better efficacy in regulating cell proliferation and angiogenesis. We evaluated its role on expression of microRNAs-15, 16, 17 and 221 and its targets Bcl-2, STAT3 and VEGF that dictate cell proliferation and angiogenesis. Docking studies clearly demonstrated the binding of Etoposide and C-10 to STAT3. We conclude that combination of Etoposide or C-10 with miR-15, 16, 17 and 221 as a new approach to induce apoptosis and control angiogenesis in breast cancer

[<i>O</i>-<i>methyl</i>-¹¹C]<i>N</i>-(4-(4-(3-Chloro-2-methoxyphenyl)-piperazin-1-yl)butyl)-1<i>H</i>-indole-2-carboxamide ([¹¹C]BAK4-51) Is an Efflux Transporter Substrate and Ineffective for PET Imaging of Brain D₃ Receptors in Rodents and Monkey

Selective high-affinity antagonists for the dopamine D3 receptor (D3R) are sought for treating substance use disorders. Positron emission tomography (PET) with an effective D3R radioligand could be a useful tool for the development of such therapeutics by elucidating pharmacological specificity and target engagement in vivo. Currently, a D3R-selective radioligand does not exist. The D3R ligand, N-(4-(4-(3-chloro-2-methoxyphenyl)piperazin-1-yl)butyl)-1H-indole-2-carboxamide (BAK4-51, 1), has attractive properties for PET radioligand development, including full antagonist activity, very high D3R affinity, D3R selectivity, and moderate lipophilicity. We labeled 1 with the positron-emitter carbon-11 (t1/2 = 20.4 min) in the methoxy group for evaluation as a radioligand in animals with PET. However, [11C]1 was found to be an avid substrate for brain efflux transporters and lacked D3R-specific signal in rodent and monkey brain in vivo

Chemical structure and anti-cancer activities of Etoposide and C-10 on breast cancer cells.

<p>(A) Chemical structure of Etoposide and C-10 [4β-[6-(3, 4-dichlorophenyl) 3, 4-dihydro-2-methyl-4-oxoquinazolin-6-yloxy) hexanamide]-4-desoxy-podophyllotoxin]. (B) Trypan blue assay for cell viability on MCF-7 and MDA-MB-231 cells showing gradual decrease in viability after treatment with Etoposide or C-10 at 1–16 μM or Cisplatin at 5–40 μM and incubated for 24 h. (C) HUVEC cells were grown on EGM media and treated with Cisplatin, Etoposide or C-10 compounds after tube formation. Drastic inhibitory effect was observed upon C-10 treatment. (D) Wound healing assay on MCF-7 and MDA-MB-231 cells showing antiangiogenic and antimigratory effects after treatment with C-10 for 48 h. Images were obtained at 0 h, 24 h and 48 h by using an inverted microscope with 4X objective lens.</p

Combinatorial effect of C-10 and microRNAs on its target genes.

<p>(A and B) MCF-7 and MDA-MB-231 cells were transfected with microRNA-15, 16, 17 and 221 for 24 h followed by treatment with Cisplatin, Etoposide or C-10 and incubated for 24 h. Western blot analysis showing decrease in levels of Bcl-2, STAT3 and VEGF proteins. GAPDH was used as loading control in all combinations separately.</p

C-10 modulates microRNA expression and its biogenesis.

<p>(A) Endogenous microRNA expression studies in compound treated MCF-7 and MDA-MB-231 cells showing significant upregulation of miR-15 and miR-16 in Etoposide or C-10 treated cells compared to miR-17 and miR-221. (B) The C-10 compound enhanced the expression of Drosha, Dicer, TRBP and Ago-1 enzymes that involved in synthesis and processing of matured microRNAs. (C) Computational analysis of miRNA prediction shows the possible binding sites in 3’UTR of Bcl-2, STAT3 and VEGFA for each miRNA-15, 16, 17 and 221 along with miSVR scores as depicted by miRanda software.</p

C-10 physically interacts with STAT3.

<p>(A and B) The panel of poses represent the molecular docking interactions of C-10 and Etoposide ligands on STAT3 at SH2 pocket. The backbone of protein has been shown as <i>wheat</i> colored cartoon and the ligands that have docked are dipicted as <i>green</i> colored sticks model. The interacting amino acid residues are shown in magenta sticks and potential inter molecular hydrogen bondings were displayed in <i>red</i> dashes. (C) The poses with black background indicate C-10 with residue in megenta colored significant residues. (D) Image represents C-10 superimposes on Etoposide (yellow stick) suggestive of the same site in the SH2 domain. PyMol programme has been employed for the visualization of docked images. (E) STAT3 protein was separated from total cell protein by immunoprecipitation method and western blotting was performed to analyze the expression of STAT3. Immunoglobulin-G (IgG) was used as negative control.</p

Caspase-9 assay.

<p>(A and B) Apoalert Caspase-9 fluorescent assay showing the apoptotic inducing ability of Etoposide or C-10 separately and in combination with miR-15, 16, 17 and 221. Results represent mean ± SD of three independent experiments. * represents p-value < 0.05, ** represents p-value < 0.01 and *** represents p-value < 0.001.</p

Combinatorial effect of C-10 and microRNAs on cell cycle and apoptosis.

<p>(A and B) MCF-7 and MDA-MB-231 cells transfected with 2 μg each of precursor microRNAs (miR-15, 16, 17 and 221) for 24 h followed by treatment with Etoposide or C-10 at 4 μM concentration for 24 h. FACS analysis data showed an increase in percentage of apoptosis, in combination of microRNA and C-10.</p