The Antitumor Activity of the Novel Compound Jesridonin on Human Esophageal Carcinoma Cells

<div><p>Jesridonin, a small molecule obtained through the structural modification of Oridonin, has extensive antitumor activity. In this study, we evaluated both its in vitro activity in the cancer cell line EC109 and its in vivo effect on tumor xenografts in nude mice. Apoptosis induced by Jesridonin was determined using an MTT assay, Annexin-V FITC assay and Hoechest 33258 staining. Apoptosis via mitochondrial and death receptor pathways were confirmed by detecting the regulation of MDM2, p53, and Bcl-2 family members and by activation of caspase-3/-8/-9. In addition, vena caudalis injection of Jesridonin showed significant inhibition of tumor growth in the xenograft model, and Jesridonin-induced cell apoptosis in tumor tissues was determined using TUNEL. Biochemical serum analysis of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (GGT), total protein (TP) and albumin (ALB) indicated no obvious effects on liver function. Histopathological examination of the liver, kidney, lung, heart and spleen revealed no signs of JD-induced toxicity. Taken together, these results demonstrated that Jesridonin exhibits antitumor activity in human esophageal carcinomas EC109 cells both in vitro and in vivo and demonstrated no adverse effects on major organs in nude mice. These studies provide support for new drug development.</p></div

JD and Oridonin inhibits the growth of EC109 monoclonal cells.

<p>A. Cells were grown in a 6-well plate at a concentration of 1000 cells/well. After the cells became adherent, JD or Oridonin (0.5 μM, 1 μM and 2 μM) was added to the media, and the plates were incubated for approximately 7 days. Control colonies reached a typical density of 50 cells/colony after the 7-day incubation. Colonies were stained with crystal violet and images were obtained. This figure is a representative result of 3 independent experiments. The clonogenicity assay was quantified using Image J software. Inhibition rate = (1- number of treatment/number of control)*100% B. The rate of inhibition induced by JD or Oridonin was expressed as the Mean ± SD. * p < 0.05 versus control; **p < 0.01 versus control.</p

Effect of JD on PUMA, Bid, Bak, Bcl-XL and Mcl-1 protein expression in EC109 cells.

<p>A. Western blot of protein extracted from EC109 cells following 24 h treatment with JD (15μM and 30μM). A representative result of 3 independent experiments is shown. B. The PUMA/GAPDH ratio is displayed as Mean ± SD. C. The Bid/GAPDH ratio is displayed as Mean ± SD. D. The Bak/GAPDH ratio is shown as Mean ± SD. E. The Bcl-XL/GAPDH ratio is displayed as Mean ± SD. F. The Mcl-1/GAPDH ratio is displayed as Mean ± SD. *p < 0.05 versus control; **p < 0.01 versus control.</p

JD treatment in vivo induces cancer cell apoptosis.

<p>When the mice were sacrificed, the tumors were collected and fixed in 4% buffered paraformaldehyde and paraffin embedded for H&E and TUNEL staining. Pictures were original captured at 200× and 400× magnification. The bar represents 100μm in 200× and 200μm in 400×.</p

Jesridonin synthesis.

<p>Jesridonin synthesis.</p

The IC₅₀ of JD or Oridonin on various cell lines.

<p>Human esophageal cells (EC109, EC9706, KYSE450, KYSE750, TE-1) were incubated with JD or oridonin for 24 h, 48 h, or 72 h and analyzed via MTT assay to determine cell viability. Normal cells (GES-1, HL7702) were incubated with JD for 24 h, 48 h, or 72 h and analyzed via MTT assay to determine cell viability. The data is shown by Mean ± SD of 3 independent experiments.</p><p>The IC₅₀ of JD or Oridonin on various cell lines.</p

Cell viability treated by JD and Oridonin.

<p>A. EC109 cells were treated for 24 h, 48 h and 72 h with 5-FU as a positive control. B-F. JD treatment of 24h, 48h and 72h to human esophageal cell lines EC109, EC9706, KYSE450, KYSE750 and TE-1, respectively. G-K. Oridonin treatment of 24h, 48h and 72h to human esophageal cell lines EC109, EC9706, KYSE450, KYSE750 and TE-1, respectively. L. JD treatment of 24h, 48h and 72h on normal cell line GES-1. M. JD treatment of 24h, 48h and 72h on normal cell line HL7702. Cell viability was determined by MTT assay and results are shown as the Mean ± SD of 3 independent experiments.</p

JD treatment of nude mice bearing EC109 tumor xenografts.

<p>NS refers to NaCl (0.9%) and β-CD refers to β-cycloamylose. A. Image of EC109 xenograft tumor tissues after the mice were sacrificed. B. Body weight changes of the mice during treatment. C. Mean tumor weight prior to when the mice were sacrificed. The tumor weight was expressed as the mean ± SD. Statistical analysis was performed using Student’s t-test, *p < 0.05 versus control; **p < 0.01 versus control. D. Tumor volume changes during treatment.</p

Effect of JD and oridonin treatment on cell morphology and nuclei of EC109 cells.

<p>EC109 cells were treated with JD or Oridonin (15 μM or 30 μM) for 16 h and observed for changes in cell morphology and nuclei. A. An inverted microscope (200X) was used to observe the morphology of EC109 cells treated by JD or Oridonin. B. EC109 cells treated by JD or Oridonin were stained with Hoechst 33258 and observed under a fluorescence microscope (200X). A representative result of 3 independent experiments is shown.</p

Effect of caspase-8/9 inhibitor on JD induced cell apoptosis.

<p>A. Apoptosis induced by JD or/with Caspase-8 inhibitor (Z-IETD-FMK) or Caspase-9 inhibitor (Z-LEHD-FMK). A representative result of 3 independent experiments is shown. B. The percentage of FITC-positive cells of 3 independent experiments is shown as Mean ± SD. *p < 0.05 versus control; **p < 0.01 versus control.</p