Secalonic acid D induces cell apoptosis in both sensitive and ABCG2-overexpressing multidrug resistant cancer cells through upregulating c-Jun expression

Abstract

Secalonic acid D (SAD) could inhibit cell growth in not only sensitive cells but also multidrug resistant (MDR) cells. However, the molecular mechanisms need to be elucidated. Here, we identified that SAD possessed potent cytotoxicity in 3 pairs of MDR and their parental sensitive cells including S1-MI-80 and S1, H460/MX20 and H460, MCF-7/ADR and MCF-7 cells. Furthermore, SAD induced cell G2/M phase arrest via the downregulation of cyclin B1 and the increase of CDC2 phosphorylation. Importantly, JNK pathway upregulated the expression of c-Jun in protein level and increased c-Jun phosphorylation induced by SAD, which was linked to cell apoptosis via c-Jun/Src/STAT3 pathway. To investigate the mechanisms of upregulation of c-Jun protein by SAD, the mRNA expression level and degradation of c-Jun were examined. We found that SAD did not alter the mRNA level of c-Jun but inhibited its proteasome-dependent degradation. Taken together, these results implicate that SAD induces cancer cell death through c-Jun/Src/STAT3 signaling axis by inhibiting the proteasome-dependent degradation of c-Jun in both sensitive cells and ATP-binding cassette transporter sub-family G member 2 (ABCG2)-mediated MDR cells.

Keywords: ABCB1, ATP-binding cassette subfamily B member 1; ABCG2; ABCG2, ATP-binding cassette transporter sub-family G member 2; AP-1, activating protein-1; Apoptosis; CHX, cycloheximide; HUVEC, human umbilical vein endothelial cells; JNKs, c-Jun N-terminal kinases; MAPKs, mitogen-activated protein kinases; MDR, multidrug resistance; MTT, 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide; Multidrug resistance; NCM460, human normal colon epithelial cells; RT-PCR, Real-time polymerase chain reaction; SAD, Secalonic acid D; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SP, side population; Secalonic acid D; c-Jun.

Graphical abstract

Figure 1

The structure and cytotoxic activity of secalonic acid D (SAD). (A) The chemical structure of SAD. (B)–(F) Cytotoxicity of SAD to S1 and S1-MI-80, H460 and H460/MX20, MCF-7 and MCF-7/ADR, NCM460 and HUVEC were determined by MTT assay as described in Methods. Each point represents the mean±standard deviations (SD) of three independent experiments performed in triplicate.

Figure 2

Effect of SAD on cell cycle and apoptosis. (A) The cell cycle analysis was determined by PI staining and flow cytometry cell quest software. S1 and S1-MI-80 cells were treated with 4 μmol/L SAD for 12, 24, 48, and 72 h, respectively. The content of G2/M phase was increased in a time-dependent pattern. (B) Histograms of cell cycle distribution in non-treated and treated S1 and S1-MI-80 cells. (C) S1 and S1-MI-80 cells were treated with SAD (4 μmol/L) for four different time points. Western blot analysis was used to detect the levels of CDC2, p-CDC2 and cyclin B1 protein after SAD treatment. (D) SAD-mediated cell apoptosis in S1 and S1-MI-80 cells were detected by flow cytometer. (E) Cells were incubated for 0, 24, 48 and 72 h in the presence or absence of SAD. The induction of cell apoptosis was detected by flow cytometry. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs. the control. Data were presented as mean±SD from triplicate experiments.

Figure 3

Effect of SAD on c-Jun expression and transportation. (A) After S1 and S1-MI-80 cells were treated with 0, 1, 2, 4, and 8 μmol/L SAD for 48 h, upregulation of c-Jun and p-c-Jun protein were observed. (B) After S1 and S1-MI-80 cells were treated with indicated concentrations of SAD for 48 h, JNK and phosphorylated JNK were up-regulated. (C) S1 and S1-MI-80 cells were treated with 4 μmol/L SAD for 48 h, respectively. c-Jun protein was measured by laser confocal microscopy as described in Methods. (D) Histograms of c-Jun fluorescence intensity in non-treated and treated S1 and S1-MI-80 cells. Each value represents the mean ± standard deviation of three independent experiments. ^***P < 0.001, compared to the control group. (E) S1 and S1-MI-80 cells were treated with 4 μmol/L SAD for 12, 24, 48 and 72 h, respectively. Cytoplasmic and nuclear extract were separated on SDS-PAGE. c-Jun protein was detected by Western Blot assay as described in Methods. GAPDH and Histone H3 were used as a loading control to indicate cytoplasmic or nuclear protein respectively.

Figure 4

SAD stimulating cancer cells apoptosis by upregulation of c-Jun. (A) and (C) Western blot assay was used to detect c-Jun protein expression. (A) Transient transfection of c-Jun and pcDNA3.1 vector served as control was carried out in S1 and S1-MI-80 cells for 48 h. (C) Downregulation of c-Jun expression with stable transfection of their cognate shRNAs was confirmed with Western blot. (B) and (D) After 4 μmol/L SAD treatment, the apoptotic rate of cells was enhanced with c-Jun overexpression, while knockdown c-Jun with shRNA could attenuate SAD effects on programmed cell death.

Figure 5

SAD regulated the expression of c-Jun protein in post-translation levels. (A) and (B) The mRNA levels of c-Jun were determined by RT-PCR and Real-time quantitative PCR. The expression of GAPDH was used as a loading control. (C) S1 cells were pre-incubated with 20 μg/mL CHX for 2 h. Then, S1 cells were treated with or without 4 μmol/L SAD. At different time points, cells were harvested and detected by Western blotting. (D) and (E) MG132 and chloroquine were used as the specific inhibitors for proteasome and lysosome, the expression of c-Jun and phosphor-c-Jun were detected after SAD treatment with or without MG132 (10 μmol/L) and chloroquine (50 μmol/L) at least 6 h, GAPDH was used as a loading control. (F) and (G) The graph demonstrates relative intensity of c-Jun compared to the untreated control and normalized against the loading control. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, N.S (non-significant) vs. the control. Data were presented as mean ± SD from triplicate experiments.

Figure 6

SAD regulated apoptosis through c-Jun/SRC/STAT3. (A) Western blot showed the protein levels of SRC and STAT3 in SAD mediated S1 and S1-MI-80 cells. (B) Overexpression of c-Jun decreased SRC and STAT3 expression as the similar effect of SAD. GAPDH was used as loading control.