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. 2016 May 10;7(19):27021-32.
doi: 10.18632/oncotarget.8395.

"VSports手机版" The novel JNK inhibitor AS602801 inhibits cancer stem cells in vitro and in vivo

Masashi Okada  1 , Kenta Kuramoto  1 , Hiroyuki Takeda  1   2 , Hikaru Watarai  1   3 , Hirotsugu Sakaki  1   4 , Shizuka Seino  1   5 , Manabu Seino  1   4 , Shuhei Suzuki  1   2 , Chifumi Kitanaka  1   5
Affiliations

"V体育ios版" The novel JNK inhibitor AS602801 inhibits cancer stem cells in vitro and in vivo

Masashi Okada et al. Oncotarget. .

Abstract

A phase 2 clinical trial investigating the efficacy and safety of AS602801, a newly developed JNK inhibitor, in the treatment of inflammatory endometriosis is complete. We are now examining whether AS602801 acts against human cancer cells in vitro and in vivo. In vitro, AS602801 exhibited cytotoxicity against both serum-cultured non-stem cancer cells and cancer stem cells derived from human pancreatic cancer, non-small cell lung cancer, ovarian cancer and glioblastoma at concentrations that did not decrease the viability of normal human fibroblasts. AS602801 also inhibited the self-renewal and tumor-initiating capacity of cancer stem cells surviving AS602801 treatment VSports手机版. Cancer stem cells in established xenograft tumors were reduced by systemic administration of AS602801 at a dose and schedule that did not adversely affect the health of the tumor-bearing mice. These findings suggest AS602801 is a promising anti-cancer stem cell agent, and further investigation of the utility of AS602801 in the treatment of cancer seems warranted. .

Keywords: c-Jun N-terminal kinase; cancer initiating cells; drug repositioning; serial transplantation assay; xenograft. V体育安卓版.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. AS602801 induces selective cytotoxicity in serum-cultured human cancer cells
A. PANC-1, A2780, and A549 human cancer cells and IMR90 human normal fibroblasts were treated without (Control) or with the indicated concentrations of AS602801 for 3 days. The number of viable cells (left panels) and the percentage of dead cells (right panels) were determined using trypan blue as a vital dye. B. Cells were subjected to cell death analysis using propidium iodide (PI) as a vital dye after treatment without (Control) or with 7.5 μM AS602801. Left, the percentage of PI-positive (dead) cells relative to Hoechst-positive (total) cells was determined. Right, representative fluorescence images of PI- (upper rows) and Hoechst-positive cells (lower rows) are shown. Values in the graphs represent the mean + SD from three independent experiments. *P < 0.05.
Figure 2
Figure 2. AS602801 has cytotoxic activity against human cancer stem cells
A. Human cancer stem cell lines (PANC-1 CSLC, A2780 CSC, A549 CSLC, and GS-Y01) were treated without (Control) or with the indicated concentrations of AS602801 for 3 days. Numbers of viable cells (left panels) and percentages of dead cells (right panels) were determined using trypan blue as a vital dye. B. Cells were treated without (Control) or with 7.5μM AS602801 for 3 days and then subjected to cell death analysis using propidium iodide (PI) as a vital dye. Left, the percentage of PI-positive (dead) cells relative to Hoechst-positive (total) cells was determined. Right, representative fluorescence images of PI- (upper rows) and Hoechst-positive (lower rows) cells are shown. Values in the graphs represent the mean + SD from three independent experiments. *P < 0.05.
Figure 3
Figure 3. AS602801 treatment causes loss of stem cell marker expression in cancer stem cells
A. Cells cultured without (Control) or with the indicated concentrations of AS602801 for 6 days were subjected to flow cytometric analysis of the cell-surface expression of CD133. Representative flow cytometric plots together with the percentages of CD133-positive cells are shown. B. Cells cultured as described in A. were subjected to immunoblot analysis of the indicated protein levels.
Figure 4
Figure 4. AS602801 induces loss of sphere formation ability in cancer stem cells
Cells cultured without (Control) or with 7.5 μM AS602801 for 6 days were subjected to a sphere formation assay in the absence of AS602801. Right panels show photographs of the representative wells. The graphs show the percentage of the wells where a tumor sphere was formed from a single cell. Values in the graphs represent the mean + SD from three independent experiments. *P < 0.05.
Figure 5
Figure 5. AS602801 treatment inhibits the tumor-initiating capacity of cancer stem cells
A., B. Mice (3 for each group) were implanted subcutaneously with the indicated number of viable PANC-1 CSLCs A. or A2780 CSLCs B. that had been treated with or without AS602801 (7.5 μM for PANC-1 CSLCs, 4 μM for A2780 CSLCs) for 6 days. C. Two groups of mice (2 mice/group) were given daily intraperitoneal AS602801 injections (40 mg/kg/day) for 10 consecutive days, and their body weight was monitored at the indicated time points. The results are expressed relative to the body weight values recorded at the initial measurement, and represent the mean + SD of the 2 mice in each group. D., E. Mice implanted subcutaneously with PANC-1 CSLCs (1 × 106 viable cells) were randomized into 2 treatment groups (4 mice per group) 3 weeks after implantation when the average tumor volume had reached approximately 500 mm3, and received a daily intraperitoneal injection of the control vehicle or AS602801 (40 mg/kg/day) for 10 consecutive days. One day after the final drug treatment, the subcutaneous tumors (primary tumors) were excised and dissociated, and the serial dilutions of the dissociated tumor cells were transplanted subcutaneously into new mice. The volumes of the secondary tumors (n = 3 for each group) formed by transplantation of the indicated numbers (Left: 1 × 106, Middle: 5 × 105, Right: 2 × 105) of viable cells from the primary tumors treated without (control group) or with AS602801 are presented as mean + SD in the graphs D.. Alternatively, the dissociated cells were subjected to immunoblot analysis to measure levels of the indicated proteins E.
Figure 6
Figure 6. Transient inhibition of c-Jun phosphorylation by AS602801
PANC-1 CSLCs treated with 7.5 μM AS602801 for the indicated times or with 20 μM SP600125 for 1 h were subjected to immunoblot analysis to measure levels of the indicated proteins.
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