HPLC Analysis of PLX.

<p>Chromatograms of <i>Piper longum</i> extract (PLX) used for this study (10 mg/mL at 2 µL/Sample) (B) compared to Piperamides Standard mix (1 mg/mL at 1 µL/standard) (A).</p

PLX is Well-Tolerated in Mice Models.

<p>Balb/C mice were divided into three groups (3 animals/control (untreated), 3 animals/gavage control (vehicle treatment) and 4 animals/treatment group). The control untreated group was given plain filtered water, while the second and third group was given 50 mg/kg/day vehicle (DMSO) or PLX, respectively. Mice were assessed for toxicity with protein urinalysis by Bradford Assay and dipstick analysis (A) and weight changes (B). (C) Hematoxylin and Eosin stained tissue sections of the liver, heart and kidney of control versus PLX treated group. Images were obtained on a bright field microscope at 63× objective.</p

Quantification of Cell Death Induction Following PLX Treatment.

<p>Image-Based cytometry was used to quantify apoptotic induction (% Annexin V positive), followed by necrosis (% PI positive) in E6-1 and HT-29 cells following PLX treatment. The lack of annexin V or PI staining was used as an indication of live cells following treatment (%Annexin V/PI negative cells) (*P<0.05, ** P<0.003, ***P<0.0001). (E) To further confirm the induction of apoptosis.</p

PLX Selectively Induces Cell Death in Human Cancer Cells in a Dose & Time Dependent Manner.

<p>(A) Following treatment of Human pancreatic (BxPc-3) cancer and T cell leukemia cells with PLX, at indicated time points, cells were incubated with propidium iodide and assessed for the induction of cell death by image-based cytometry. (B) Similar experiments were carried out in human colon cancer cells (HT-29) and normal colon epithelial cells (NCM460). Fluorescence microscopy was used to assess the induction of cell death as characterized by presence of propidium iodide positive cells. Images were taken at 400× magnification on a fluorescent microscope. Scale bar = 15 µm.</p

PLX Selectively Targets Cancer Cells for Apoptosis Induction.

<p>Subsequent to treatment with PLX, cells (Ovarian; OVCAR-3, Melanoma; G-361 and Normal Colon Epithelia cells (NCM460) were stained with Hoechst to characterize nuclear morphology and Annexin-V to detect apoptotic cells (A) and cellular morphology by phase contrast microscopy (B); Images were taken at 400× magnification on a fluorescent microscope. Scale bar = 15 µm. (C) Following PLX treatment, HT-29 colorectal cancer cells and non-cancerous NCM460 cells were incubated with WST-1 cell viability dye for 4 hours and absorbance was read at 450 nm and expressed as a percent of the control. Values are expressed as mean ± SD of 3 independent experiments. **P<0.0001.</p

Summary of Histological Lesions in Balb/C Mice on PLX regimen.

<p>Summary of Histological Lesions in Balb/C Mice on PLX regimen.</p

PLX Induces Double-stranded DNA Breaks in Cancer Cells.

<p>TUNEL labeling was used to detect DNA fragmentation. Following PLX and VP16 (as a positive control for DNA damage) treatment, cells were labelled with DNA staining solution and quantified by image-based cytometry. Treated cells were compared to the control untreated cell sample. (****P<0.0001).</p

PLX Causes but is Not Dependent on the Production of Reactive Oxygen Species (ROS).

<p>(A) Colon cancer (HT-29), Normal Colon Epithelial (NCM460) and Normal Human Fibroblast (NHF) cells were treated with PLX for 48 hours, following which, they were incubated with H₂DCFDA and fluorescence results were obtained using an image based cytometer. Results were quantified using Graphpad prism 6.0 (B). (C) HCT116 colon cancer cells were treated with 3 mM N-acetylcysteine for an hour prior to PLX treatment. Cells were then treated PLX at indicated concentrations for 72 hours, following which the WST-1 assay was performed. Absorbance readings were taken at 450 nm and expressed as a percent of the control. Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. *p<0.05.</p

Crude Ethanolic Extract of Long Pepper (PLX) Effectively Reduces the Percentage of Viable Cancer cells in a Dose & Time Dependent Manner.

<p>Colon (HCT116), Ovarian (OVCAR-3), Pancreatic (BxPC-3) cancer and Melanoma (G-361) cells were treated with a crude ethanolic extract of long pepper (PLX), following which they were incubated with WST-1 cell viability dye for 4 hours. Absorbance was read at 450 nm and expressed as a percent of the control. Values are expressed as mean ± SD from quadruplicates of 3 independent experiments. **P<0.0001.</p

Analysis of five well-known piperamides and crude long pepper extract at a flow rate of 1.0 mL/min with a mobile phase constituted of H₂O and methanol.

<p>Analysis of five well-known piperamides and crude long pepper extract at a flow rate of 1.0 mL/min with a mobile phase constituted of H₂O and methanol.</p