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. 2018 Feb 21;8(1):3398.
doi: 10.1038/s41598-018-21768-1.

The novel dithiocarbamate, DpdtC suppresses HER2-overexpressed cancer cells by up-regulating NDRG1 via inactivation of HER2-ERK 1/2 signaling

Yun Yang  1 , Youxun Liu  2 , Rui Guo  3 , Yun Fu  2 , Ziheng Zhang  2 , Pengfei Zhang  2 , Pingxin Zhou  2 , Tingting Wang  2 , Tengfei Huang  2 , Xiaotong Li  4 , Changzheng Li  5
Affiliations

The novel dithiocarbamate, DpdtC suppresses HER2-overexpressed cancer cells by up-regulating NDRG1 via inactivation of HER2-ERK 1/2 signaling

Yun Yang et al. Sci Rep. .

Abstract

Dithiocarbamate has been tested for its effective anti-tumor activity, but the underlying mechanism remains unclear. We previously prepared a novel diththiocarbamate derivative, DpdtC with an ability of catalase inhibition. Here, we for the first time investigated the growth inhibition effects of DpdtC on HER2-amplified cancer cells and elucidated its mechanism of action. Results showed that DpdtC exerted the potent anti-tumor effects against HER2-overexpressed SK-OV-3 and SK-BR-3 cells, especially on SK-OV-3 cells with a higher NDRG1 level, which was also confirmed in the SK-OV-3 xenograft model. Interestingly, we observed that NDRG1 was up-regulated, while membrane expression of HER2 was regressed in SK-OV-3 cells upon DpdtC treatment. In agreement, silencing endogenous NDRG1 also increased the expression of HER2 in SK-OV-3 cells, while overexpressing NDRG1 decreased HER2 expression in SK-BR-3 cells. Furthermore, our results showed the formation of the EGFR/HER2 heterodimer was attenuated and phosphorylation of ERK1/2 was inhibited in SK-OV-3 cells when treated with DpdtC. Collectively, these observations demonstrated that NDRG1 plays an important role in mediating the inhibition effects of DpdtC in HER2-overexpressed cancer cells via selective _targeting of the HER2-ERK1/2 pathway. Hence, our investigation suggests that up-regulation of NDRG1 by DpdtC is a promising therapeutic approach in HER2-overexpressed cancers.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
DpdtC inhibited the growth of HER2-overexpressed cancer cells in vitro. (A) Chemical structure of dipyridylhydrazone dithiocarbamate (DpdtC). (B) Inhibitory effect of increasing concentrations of DpdtC on the proliferation of SK-OV-3 or SK-BR-3 cells. The data are shown as the mean ± SD. IC50 for SK-OV-3 cells is 0.244 μM (95% CI, 0.211–0.283 μM), whereas IC50 of achieving ~50% growth inhibition for SK-BR-3 cells is at ~100 μM. Data were obtained from 3 independent experiments.
Figure 2
Figure 2
In vivo efficacy of DpdtC in the SK-OV-3 xenograft tumor model. (A) Mean tumor volumes of mice xenografted with SK-OV-3 cells and treated with DpdtC (5 mg/kg). There were 6 animals per treatment group. DpdtC treatment started as indicated in the graphs (black arrows). Error bars show ± SD. (***P < 0.001). (B) On day 24, xenograft tumor from each group were removed and photographed. Representative tumors in each group were shown. (C) Effect of DpdtC on nude mice body weight was determined using SK-OV-3 tumor-bearing nude mice. Mice were weighed at regular intervals during the whole period to monitor therapy-related toxicity. (D) Histological examination was conducted in nude mice post injection with DpdtC (5 mg/kg) for two times. Images (magnification, ×400) of liver from nude mice (n = 3) injected with PBS (−) or DpdtC (5 mg/kg) for two times were obtained by staining with hematoxylin and eosin. Scale bars, 50 μm.
Figure 3
Figure 3
DpdtC up-regulated NDRG1 expression and down-regulated membrane expression of HER2 on SK-OV-3 cells. (A) SK-OV-3 cells were incubated for 24 h at 37 °C with control media (−) or media containing the DpdtC (2 μM), and NDRG1 level was examined by western blot. And quantification of western blot signal intensity analysis is expressed relative to the β-actin loading control by using Image J software. ***p < 0.001. (B) Representative micrographs of fluorescent immunostaining showed that DpdtC decreased HER2 expression at the cell membrane. Cells were incubated for 36 h at 37 °C with control media (−) or media containing the DpdtC (2 μM), and HER2 localization was examined via immunofluorescence. Original magnification, ×600. Scale bars, 25 μm. (C) Percentage of HER2-positive stained cells reduced markedly in SK-OV-3 cells when incubated with DpdtC. Data are expressed as mean ± SD of the integrated fluorescence signals from 3 fields for each specimen. (D) Flow cytometry assay quantifying the membrane expression of HER2 on SK-OV-3 cells, which was expressed as MFI (Median Fluorescence Intensity) upon treatment control media (−) or media containing the DpdtC (2 μM) for 36 h at 37 °C. **p < 0.01; ***p < 0.001.
Figure 4
Figure 4
NDRG1 expression decreased HER2 level in both SK-OV-3 and SK-BR-3 cells. (A) Western blot indicating the expression of NDRG1 in SK-OV-3 and SK-BR-3 cells. (B) Quantification of western blot signal intensity analysis is expressed relative to the β-actin loading control by using Image J software. (C) Suppression of NDRG1 by siRNA leaded to increase in the level of HER2 in SK-OV-3 cells. SK-OV-3 cells were transiently transfected with nonspecific control siRNA (Ctrl) or NDRG1 siRNA (siNDRG1) for 72 h at 37 °C. (D) NDRG1 expression inhibits HER2 expression in SK-BR-3 cells. The plasmid of pCDNA3.1-NDRG1 or the vector control (Ctrl) was constructed and transfected into SK-BR-3 cells. (E) Quantification of western blot signal intensity analysis in SK-OV-3 cells is expressed relative to the β-actin loading control by using Image J software. (F) Quantification of western blot signal intensity analysis in SK-BR-3 cells is expressed relative to the β-actin loading control by using Image J software. Data show the mean ± SD (3 independent experiments); *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5
Figure 5
Effect of DpdtC on suppressing HER2 expression is dependent on NDRG1 in SK-OV-3 cells. (A) SK-OV-3 cells were transfected with control siRNA or siNDRG1 for 72 h at 37 °C, followed by treatment with DpdtC (2 μM) for 30 h at 37 °C. Then levels of NDRG1 and HER2 were tested by western blot. (B) Quantification of western blot signal intensity analysis is expressed relative to the β-actin loading control by using Image J software. Data show the mean ± SD (3 independent experiments); **p < 0.01; ***p < 0.001.
Figure 6
Figure 6
HER2/EGFR heterodimer formation was inhibited in SK-OV-3 cells when treated with DpdtC. (A and B) SK-OV-3 cells were incubated with control medium (−) or medium with DpdtC (2 μM) for 36 h at 37 °C in the absence or presence of EGF, and tested via co-immunoprecipitation according to “Materials and Methods”. (C and D) Quantification of western blot signal intensity analysis is expressed relative to untreated control cells by using Image J software. Data show the mean ± SD (3 independent experiments); *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7
Figure 7
Up-regulation of NDRG1 inhibited HER2-ERK1/2 downstream signaling pathway in SK-OV-3 cells. (A) SK-OV-3 cells were incubated with control medium(−) or medium containing DpdtC for 36 h at 37 °C followed by treatment with EGF (10 ng/ml) in the last 20 min of 36 h incubation. (B) Vector control (Ctrl) or NDRG1-overexpressing (NDRG1) cells were incubated with control medium(−) or medium containing EGF (10 ng/ml; 20 min/37 °C) and levels of NDRG1, ERK 1/2,pERK 1/2 were tested by western blot. (C and D) Quantification of western blot signal intensity analysis is expressed relative to β-actin by using Image J software. Data show the mean ± SD (3 independent experiments); *p < 0.05; ***p < 0.001.
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