N-Hydroxyphthalimide exhibits antitumor activity by suppressing mTOR signaling pathway in BT-20 and LoVo cells

Abstract

Background: N-Hydroxyphthalimide (NHPI), an important chemical raw material, was found to have potent and selective anti-proliferative effect on human breast carcinoma BT-20 cells, human colon adenocarcinoma LoVo and HT-29 cells during our screening for anticancer compounds. The purpose of this study is to assess the antitumor efficacy of NHPI in vitro and in vivo and to explore the underlying antitumor mechanism.

Methods: Cell cytotoxicity of NHPI was evaluated using MTS assay and cell morphological analysis. After NHPI treatment, cell cycle, apoptosis and mitochondrial membrane potential were analyzed using flow cytometer. The subcellular localization of eukaryotic initiation factor 4E (eIF4E) was analyzed by immunofluorescence assay. The antitumor efficacy of NHPI in vivo was tested in BT-20 xenografts. The underlying antitumor mechanisms of NHPI in vitro and in vivo were investigated with western blot analysis in NHPI-treated cancer cells and tumor tissues. Statistical significance was determined using Student's t-test.

Results: In vitro, NHPI selectively inhibited the proliferation and induced G2/M phase arrest in BT-20 and LoVo cells, which was attributed to the inhibition of cyclin B1 and cdc2 expressions. Furthermore, NHPI induced apoptosis via mitochondrial pathway. Of note, NHPI effectively inhibited mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2) signaling, and overcame the feedback activation of Akt and extracellular signal-regulated kinase (ERK) caused by mTORC1 inhibition in BT-20 and LoVo cells. In vivo, NHPI inhibited tumor growth and suppressed mTORC1 and mTORC2 signaling in BT-20 xenografts with no obvious toxicity.

Conclusions: We found for the first time that NHPI displayed antitumor activity which is associated with the inhibition of mTOR signaling pathway. Our findings suggest that NHPI may be developed as a promising candidate for cancer therapeutics by targeting mTOR signaling pathway and as such warrants further exploration.

Fig. 1

NHPI selectively decreases cell viability in BT-20, LoVo and HT-29 cells. a Chemical structure of NHPI. b All the tested cancer cells were treated with 40 μM NHPI for 48 h. Cell viability was determined by MTS assay and represented with relative viability versus control. Results were presented as mean ± SD (n = 3)

Fig. 2

NHPI concentration-dependently decreases cell viability in BT-20, LoVo and HT-29 cells. a BT-20, LoVo, HT-29 and MCF-10A cells were treated with indicated concentrations of NHPI for 48 h. Cell viability was determined by MTS assay and represented with relative viability versus control. Results were presented as mean ± SD (n = 3). b IC₅₀ values of NHPI on cell proliferation were shown with mean ± SD (n = 3). c The morphology of BT-20, LoVo, HT-29 and MCF-10A cells treated with NHPI at 5 and 10 μM for 24 h were observed under a phase-contrast microscope and photographed (100×)

Fig. 3

NHPI induces G₂/M phase cell cycle arrest by inhibiting the expressions of cyclin B1 and cdc2. a BT-20 cells were treated with NHPI at 2.5, 5 and 10 μM for 24 h. LoVo cells were incubated with NHPI at 5, 10 and 15 μM for 24 h. Cells were stained with PI and subjected to flow cytometry analysis. Representative images were shown. b Quantitative analysis of cells in each cell cycle phase was performed. Results were presented as mean ± SD (n = 3). *p < 0.05 and ***p <0.001, difference versus 0 μM control group. c The inhibition of cyclin B1 and cdc2 expressions contributed to NHPI-induced G₂/M phase arrest. Cells were treated with indicated concentrations of NHPI for 24 h, and the expression levels of G₂/M phase regulatory proteins were analyzed by western blot analysis. β-actin was used as a loading control

Fig. 4

NHPI induces apoptosis via mitochondrial pathway. a BT-20 cells were treated with NHPI at 2.5, 5 and 10 μM for 48 h. LoVo cells were incubated with NHPI at 5, 10 and 20 μM for 48 h or 72 h. Cell apoptosis was analyzed by flow cytometry using the Annexin V-FITC and PI double staining. Representative images were presented. b Quantification of flow cytometry analysis of apoptosis. Results were presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p <0.001, difference versus 0 μM control group. c NHPI induced MMP loss in BT-20 cells. BT-20 cells were treated with NHPI at 2.5, 5 and 10 μM for 48 h, stained with JC-1 and subjected to flow cytometry analysis. The dot-plot representation of the flow cytometry analysis shows the distribution of JC-1 aggregates (cells emitting red fluorescence detected in the FL2 channel) and JC-1 monomers (cells emitting green fluorescence detected in the FL1 channel). d Histograms showing the percentage of JC-1 aggregate-positive and JC-1 monomer-positive cells. Results were presented as mean ± SD (n = 3). **p < 0.01 and ***p <0.001, difference versus 0 μM control group. e Effect of NHPI on the expressions of apoptosis-related proteins. Cells were treated with indicated concentrations of NHPI for 24 h, followed by western blot analysis with indicated antibodies. β-actin was used as a loading control

Fig. 5

NHPI inhibits both mTORC1 and mTORC2 signaling in BT-20 and LoVo cells. a BT-20, LoVo and MDA-MB-231 cells were treated with indicated concentrations of NHPI for 24 h, followed by western blot analysis with indicated antibodies. β-actin was used as a loading control. b BT-20 and LoVo cells were treated with indicated concentrations of NHPI for 24 h, followed by western blot analysis with indicated antibodies. β-actin was used as a loading control. c NHPI induced the eIF4E nuclear translocation in BT-20 cells. BT-20 cells were incubated with indicated concentrations of NHPI for 6 h. Then the cells were analyzed by immunofluorescence assay labeling with eIF4E antibody and DAPI. eIF4E and nucleus were recognized by the red and blue fluorescence, respectively

Fig. 6

NHPI inhibits tumor growth in human breast xenografts in association with suppression of both mTORC1 and mTORC2 signaling. 6.8 × 10⁶ BT-20 cells in Matrigel were subcutaneously implanted into nude mice. When tumor size reached around 100 mm³, tumor-bearing mice were randomized into two groups and treated daily by intraperitoneal injection with 40 mg/kg NHPI or vehicle control for 53 days. a NHPI significantly inhibited tumor growth in BT-20 xenografts. The individual tumor volume was measured at indicated time (days) and presented as mean ± SE (n = 9). **p < 0.01 and ***p <0.001, difference versus vehicle-treated control group. b The individual body weight was measured at indicated time (days) and presented as mean ± SE (n = 9). c Tumors were removed from mice and tumor weight was measured at the end of treatment. Representative tumor images were shown and tumor weight was presented as mean ± SE (n = 9). *p < 0.05, difference versus vehicle-treated control group. d NHPI inhibits both mTORC1 and mTORC2 signaling in BT-20 tumor tissues. The phosphorylation levels of mTOR at Ser2448, S6 at Ser235/236, 4E-BP1 at Ser65 and Akt at Ser473 in tumor tissues were determined by western blot analysis. β-actin was used as a loading control. e Relative densitometric quantification of protein expression detected in (d). Results were presented as mean ± SE (n = 5). *p < 0.05 and **p < 0.01, difference versus vehicle-treated control group