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. 2016 Apr 22;17(4):610.
doi: 10.3390/ijms17040610.

Smad3 Sensitizes Hepatocelluar Carcinoma Cells to Cisplatin by Repressing Phosphorylation of AKT

Hong-Hao Zhou  1 , Lin Chen  2 , Hui-Fang Liang  3 , Guang-Zhen Li  4 , Bi-Xiang Zhang  5 , Xiao-Ping Chen  6
Affiliations

Smad3 Sensitizes Hepatocelluar Carcinoma Cells to Cisplatin by Repressing Phosphorylation of AKT

Hong-Hao Zhou et al. Int J Mol Sci. .

Abstract

Background: Heptocelluar carcinoma (HCC) is insensitive to chemotherapy due to limited bioavailability and acquired drug resistance. Smad3 plays dual roles by inhibiting cell growth initially and promoting the progression of advanced tumors in HCC. However, the role of smad3 in chemosensitivity of HCC remains elusive.

Methods: The role of smad3 in chemosensitivity of HCC was measured by cell viability, apoptosis, plate colony formation assays and xenograft tumor models. Non-smad signaling was detected by Western blotting to search for the underlying mechanisms.

Results: Smad3 enhanced the chemosensitivity of HCC cells to cisplatin. Smad3 upregulated p21(Waf1/Cip1) and downregulated c-myc and bcl2 with the treatment of cisplatin. Moreover, overexpression of smad3 repressed the phosphorylation of AKT, and vice versa. Inhibition of PI3K/AKT pathway by LY294002 restored chemosensitivity of smad3-deficiency cells to cisplatin in HCC.

Conclusion: Smad3 sensitizes HCC cells to the effects of cisplatin by repressing phosphorylation of AKT and combination of inhibitor of AKT pathway and conventional chemotherapy may be a potential way to solve drug resistance in HCC.

Keywords: AKT; LY294002; cisplatin; drug resistance; hepatocelluar carcinoma; smad3.

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Figures

Figure 1
Figure 1
Transforming growth factor-β (TGF-β) signaling is intact in smad3-expressing heptocelluar carcinoma (HCC) cells. (A) The expression of smad3 in HCC cell lines was detected by Western blot; (B) Stable overexpression of smad3 in SMMC-7721 cells and knockdown of smad3 in HCC-LM3 cells were confirmed by Western blot. Smad3 phosphorylation was activated upon TGF-β treatment in smad3-expressing cells. All of the experiments were performed three times and representative pictures are shown.
Figure 2
Figure 2
Smad3 increases the sensitivity of HCC to cisplatin in vitro. SMMC-7721 (A) and HCC-LM3 (B) cells were treated with indicated concentrations of cisplatin for 48 h. The number of viable cells was determined by CCK-8. Relative percentages of live cells were analyzed by comparing with cells without cisplatin treatment; (C) The 50% inhibitionary concentration values (IC50) of 7721 and LM3 cells were calculated and analyzed by Graphpad Prism 5.0; (D–G) Plate colony formation assay was performed to detect cisplatin sensitivity, and the number of colonies was evaluated 14 days after cell plating. The data are presented as the mean ± S.D. from three wells; (H–K) Cell apoptosis assays were examined using Fluorescence activated cell sorting (FACS) 48 h after cisplatin treatment. The percentages of apoptotic cells are presented as the mean ± S.D. from three wells (** p < 0.01, *** p < 0.001).
Figure 2
Figure 2
Smad3 increases the sensitivity of HCC to cisplatin in vitro. SMMC-7721 (A) and HCC-LM3 (B) cells were treated with indicated concentrations of cisplatin for 48 h. The number of viable cells was determined by CCK-8. Relative percentages of live cells were analyzed by comparing with cells without cisplatin treatment; (C) The 50% inhibitionary concentration values (IC50) of 7721 and LM3 cells were calculated and analyzed by Graphpad Prism 5.0; (D–G) Plate colony formation assay was performed to detect cisplatin sensitivity, and the number of colonies was evaluated 14 days after cell plating. The data are presented as the mean ± S.D. from three wells; (H–K) Cell apoptosis assays were examined using Fluorescence activated cell sorting (FACS) 48 h after cisplatin treatment. The percentages of apoptotic cells are presented as the mean ± S.D. from three wells (** p < 0.01, *** p < 0.001).
Figure 3
Figure 3
Smad3 increases the sensitivity of HCC to cisplatin in vivo. SMMC-7721 (A) and HCC-LM3 (B) cells were injected subcutaneously into Balb/c-nu mice and cisplatin was injected i.p. every three days; (C,D) Tumor weight was examined after mice were sacrificed; (E) Subcutaneous tumors were subjected to smad3 and ki67 staining. Representative pictures are shown (100×); (F) The percentage of ki67 stained nuclei was calculated in different groups. All the results are represented as the mean ± S.D. from three independent trials. (*** p < 0.001; n.s. means no significance).
Figure 3
Figure 3
Smad3 increases the sensitivity of HCC to cisplatin in vivo. SMMC-7721 (A) and HCC-LM3 (B) cells were injected subcutaneously into Balb/c-nu mice and cisplatin was injected i.p. every three days; (C,D) Tumor weight was examined after mice were sacrificed; (E) Subcutaneous tumors were subjected to smad3 and ki67 staining. Representative pictures are shown (100×); (F) The percentage of ki67 stained nuclei was calculated in different groups. All the results are represented as the mean ± S.D. from three independent trials. (*** p < 0.001; n.s. means no significance).
Figure 4
Figure 4
Smad3 activates mitogen-activated protein kinases (MAPK) but represses AKT signaling: (A) Extracelluar signal regulated kinase (ERK) and smad3 signaling were examined with the treatment of TGF-β1 (5 ng/mL, 0.5 h) and U0126 (10 µM) in 7721 and LM3 cells; (B) JNK and smad3 signaling were examined with the treatment of TGF-β1 (5 ng/mL, 6 h) and SP600125 (30 µM) in 7721 and LM3 cells; (C) P38 and smad3 signaling were examined with the treatment of TGF-β1 (5 ng/mL, 1 h) and SB203580 (30 µM) in 7721 and LM3 cells; and (D) AKT and smad3 signaling were examined with the treatment of TGF-β1 (5 ng/mL, 3 h) and LY294002 (20 µM) in 7721 and LM3 cells. All of the experiments were performed in triplicate and representative pictures are shown.
Figure 5
Figure 5
Smad3 represses AKT phosphorylation and regulates apoptosis-related proteins in the presence of cisplatin. (A,B) Western blot assay was performed using lysates from SMMC-7721 and HCC-LM3 cells after treating with cisplatin (48 h) for different concentration points. The expression of p15, p21, c-myc, bcl2, bax, p-AKT (S473), AKT, and smad3 were examined; β-actin was used as a loading control. All of the experiments were performed in triplicate and representative pictures are shown.
Figure 6
Figure 6
LY294002 restores chemosensitivity of HCC in smad3-defeciency cells. (A–D) SMMC-7721 and HCC-LM3 cells were treated with indicated concentrations of cisplatin with or without LY294002 for 72 h. The number of viable cells was determined by CCK-8. IC50 values were calculated and analyzed by Graphpad Prism 5.0. Data are presented as mean ± S.D. from triplicate wells; (E,F) Plate colony formation assay was performed to detect the synergistic effects of cisplatin and LY294002, and the number of colonies was evaluated 14 days after cell plating. The data are presented as the mean ± S.D. from three wells; (G) SMMC-7721 cells were treated with cisplatin and/or LY294002 for 48 h and cell lysates were used for Western blot assays. All of the experiments were performed in triplicate (** p < 0.01, *** p < 0.001; n.s. means no significance).
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