Effect of orlistat administration to tumor-bearing mice on apoptotic tumor cell population.

<p>Tumor cells (1x10⁶ cells/ml) harvested from control and orlistat administered tumor-bearing mice were analysed for apoptotic cells by Wright-Giemsa (a) and TUNEL assay (b). Values shown in (a) & (b) are mean ± SD of three independent experiments done in triplicate.*<i>p<0</i>.<i>05 </i><i>vs</i>. values of respective control. </p

Effect of orlistat administration to tumor-bearing mice on BMC count, survival and apoptotic population.

<p>Viable cells in BMC harvested from the femur of control and orlistat-administered tumor-bearing mice were enumerated by trypan blue dye exclusion test (a). A differential count of the BMC population was performed by Leishman staining (b). BMC (1x10⁶ cells/ml) of control and orlistat-administered groups were incubated for 24h in 96 well culture plates followed by estimation of cell survival by MTT assay (c). Induction of apoptosis was estimated by Wright Giemsa staining (d) and TUNEL assay (e). Values are mean ± SD of three independent experiments done in triplicate.* <i>p<0.05 vs</i>. values of respective control.</p

Orlistat augments differentiation of BMDM associated with increased expression of M-CSF and M-CSFR.

<p>BMC (1x10⁴ cells/ml) harvested from control or orlistat-administered tumor-bearing mice were cultured in vitro in the presence of L929 culture medium (20%v/v) as a source of M-CSF, for 10 days to allow the BMC to differentiate into colonies. Colonies were counted based on cellular morphology of each colony forming unit (CFU) displaying features of CFU-M, CFU-GM and CFU-G phenotype (a). CFU-M obtained from the BMC of control group displayed lesser number of Mϕ-like cells compared to orlistat-treated group where the colonies were denser with larger macrophage like cells, as indicated by arrows (b). BMC (1x10⁶ cells/ml) obtained from control or orlistat-administered tumor-bearing mice were also processed for RT-PCR to detect expression of mRNA for MCSF and M-CSFR. Bars shown in (e) are densitometric scan of bands shown in (d), which are from a representative experiments out of 3 experiments with similar results. BMDM grown on glass cover slips in petri-dishes were stained with Wright Giemsa stain (c upper panel) and F4/80 FITC-conjugated antibody (c lower panel). As indicated by arrows BMDM of orlistat administered group showed increased size, spreading and cytoplasmic extensions. Plates shown are from a representative experiment. Values shown in (a) are mean ± SD of three independent experiments done in triplicate.*<i>p<0</i>.<i>05 </i><i>vs</i>. values of respective control.</p

Protocol for administering orlistat to tumor-bearing mice.

<p>Mice were transplanted DL cells (1x10⁵cells/0.5 ml PBS) on day 0 following administration of Vehicle alone (control) or containing orlistat 240mg/kg body weight/day up to day 14 post tumor transplantation. On day 16 BMC were harvested from femurs.</p

Summary of the suggested mechanisms underlying myelopoietic action of orlistat.

<p>Myelopoietic action of orlistat in tumor bearing hosts leads to augmented differentiation of Mϕ with M₁ phenotype. Modulated expression of cytokines, cell survival and differentiation regulatory molecules play a central role.</p

BMDM obtained from BMC of orlistat-administered groups display M1 Mϕ phenotype.

<p>BMDM differentiated from BMC of control or orlistat-administered tumor-bearing mice were incubated <i>in </i><i>vitro</i> for 24h in medium alone or containing IFN- γ (100IU/ml) + LPS (10ng/ml) followed by estimation of NO (a), indicated cytokines by ELISA in culture supernatant (b), assay of ROS expression (d,e), phagocytosis (c), BMDM-mediated tumoricidal activity (f,g) and expression of cell surface functional markers: CD11c and TLR2 (h). Values shown in (a,b,e,f,g) mean ± SD of three independent experiments done in triplicate.*<i>p<0</i>.<i>05 </i><i>vs</i>. values of respective control. *#p<i><</i>0.05 vs. values for orlistat and LPS + IFN-γ treated control groups. Arrows indicates increased phagocytosis (c), expression of ROS (d) and CD11c & TLR-2 (h) in BMDM of orlistat group treated with IFN-γ + LPS.</p

Enumeration of F4/80⁺ TAM following orlistat administration to tumor-bearing host.

<p>Peritoneal exudates cells obtained from control (a) and orlistat administered (b) tumor-bearing hosts were analysed for the number of TAM by flow cytometry using FITC conjugated antibody against macrophage marker F4/80. Data shown is from a representative experiment out of three independent experiments done in triplicate with similar results.</p

Orlistat administration to tumor-bearing mice alters the expression pattern of cell survival regulatory molecules.

<p>Cell lysates of BMC (1x10⁶ cells/ml) harvested from control and orlistat-administered tumor bearing mice were processed for western blot analysis to examine expression pattern of the indicated cell survival regulatory proteins (a,b). Bars shown in (b) are the densitometric scan of bands shown in (a). Data shown is from a representative experiment out of three independent experiments with similar results. Sera harvested from control and orlistat-administered tumor-bearing mice on day 16 post-tumor transplantation were immunodetected by ELISA for the level of the indicated cytokines (c). Values in (c) are mean ± SD of three independent experiments done in triplicate.*<i>p<0</i>.<i>05 </i><i>vs</i>. values of respective control. </p

Molecular docking studies of 3-bromopyruvate and its derivatives to metabolic regulatory enzymes: Implication in designing of novel anticancer therapeutic strategies

<div><p>Altered metabolism is an emerging hallmark of cancer, as malignant cells display a mammoth up-regulation of enzymes responsible for steering their bioenergetic and biosynthetic machinery. Thus, the recent anticancer therapeutic strategies focus on the targeting of metabolic enzymes, which has led to the identification of specific metabolic inhibitors. One of such inhibitors is 3-bromopyruvate (3-BP), with broad spectrum of anticancer activity due to its ability to inhibit multiple metabolic enzymes. However, the molecular characterization of its binding to the wide spectrum of target enzymes remains largely elusive. Therefore, in the present study we undertook <i>in silico</i> investigations to decipher the molecular nature of the docking of 3-BP with key target enzymes of glycolysis and TCA cycle by PatchDock and YASARA docking tools. Additionally, derivatives of 3-BP, dibromopyruvate (DBPA) and propionic acid (PA), with reported biological activity, were also investigated for docking to important target metabolic enzymes of 3-BP, in order to predict their therapeutic efficacy versus that of 3-BP. A comparison of the docking scores with respect to 3-BP indicated that both of these derivatives display a better binding strength to metabolic enzymes. Further, analysis of the drug likeness of 3-BP, DBPA and PA by Lipinski filter, admetSAR and FAF Drug3 indicated that all of these agents showed desirable drug-like criteria. The outcome of this investigation sheds light on the molecular characteristics of the binding of 3-BP and its derivatives with metabolic enzymes and thus may significantly contribute in designing and optimizing therapeutic strategies against cancer by using these agents.</p></div

Summary of docking analysis.

<p>Figure presents summary of the binding of 3-BP to various target enzymes of glycolysis and TCA cycle, indicating the wide spectrum of its targets.</p