Zinc oxide nanoparticles reduce the chemoresistance of gastric cancer by inhibiting autophagy

Abstract

Background: Gastric cancer (GC) is a common malignancy that results in a high rate of cancer-related mortality. Cisplatin (DDP)-based chemotherapy is the first-line clinical treatment for GC therapy, but chemotherapy resistance remains a severe clinical challenge. Zinc oxide nanoparticle (ZnO-NP) has been identified as a promising anti-cancer agent, but the function of ZnO-NP in GC development is still unclear.

Aim: To explore the effect of ZnO-NP on chemotherapy resistance during GC progression.

Methods: ZnO-NP was synthesized, and the effect and underlying mechanisms of ZnO-NP on the malignant progression and chemotherapy resistance of GC cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, colony formation assays, transwell assays, wound healing assays, flow cytometry, and Western blot analysis in GC cells and DDP-resistant GC cells, and by tumorigenicity analyses in nude mice.

Results: Our data revealed that ZnO-NP was able to inhibit proliferation, migration, and invasion and induce apoptosis of GC cells. Meanwhile, ZnO-NP significantly reduced the half maximal inhibitory concentration (IC₅₀) of DDP for the inhibition of cell proliferation of DDP-resistant SGC7901/DDP cell lines. Autophagy was increased in DDP-resistant GC cells, as demonstrated by elevated light chain 3-like protein 2 (LC3II)/LC3I and Beclin-1 expression and repressed p62 expression in SGC7901/DDP cells compared to SGC7901 cells. Mechanically, ZnO-NP inhibited autophagy in GC cells and treatment with DDP induced autophagy, which was reversed by ZnO-NP. Functionally, ZnO-NP attenuated the tumor growth of DDP-resistant GC cells in vivo.

Conclusion: We conclude that ZnO-NP alleviates the chemoresistance of GC cells by inhibiting autophagy. Our findings present novel insights into the mechanism by which ZnO-NP regulates the chemotherapy resistance of GC. ZnO-NP may serve as a potential therapeutic candidate for GC treatment. The potential role of ZnO-NP in the clinical treatment of GC needs clarification in future investigations.

Keywords: Autophagy; Chemoresistance; Gastric cancer; MTT assays; Progression; Zinc oxide nanoparticle.

Figure 1

Zinc oxide nanoparticle inhibits proliferation and induces apoptosis of gastric cancer cells. SGC7901 and BGC823 cells were treated with the zinc oxide nanoparticle (ZnO-NP, 5 μg/mL) or an equal volume of saline. A and B: Cell viability was analyzed by the MTT assay; C and D: Cell proliferation was assessed by the colony formation assay; E and F: Cell apoptosis was measured by flow cytometry. Data are presented as the mean ± SD. Statistically significant differences are indicated: ^aP < 0.05; ^bP < 0.01. FITC: Fluorescein isothiocyanate.

Figure 2

Zinc oxide nanoparticle reduces the invasion and migration of gastric cancer cells. SGC7901 and BGC823 cells were treated with zinc oxide nanoparticle (5 μg/mL, ZnO-NP) or an equal volume of saline. A and B: Cell migration and invasion were determined by transwell assays; C and D: Migration and invasion were examined by wound healing assays. The wound healing proportion is shown. Data are presented as the mean ± SD. Statistically significant differences are indicated: ^bP < 0.01.

Figure 3

Zinc oxide nanoparticle attenuates the chemotherapy drug resistance of gastric cancer cells. A: SGC7901/cisplatin (DDP) cells were treated with DDP at the indicated doses and treated with zinc oxide nanoparticle (ZnO-NP, 5 μg/mL) or an equal volume of saline. Cell viability was measured by the MTT assay; B and C: SGC7901/DDP cells were treated with DDP or co-treated with DDP and ZnO-NP (5 μg/mL). Cell apoptosis was assessed by flow cytometry. Data are presented as the mean ± SD. Statistically significant differences are indicated: ns, no significance, ^aP < 0.05. FITC: Fluorescein isothiocyanate.

Figure 4

Autophagy is increased in chemotherapy-resistant gastric cancer cells. A: Expression of light chain 3B-II (LC3B-II), LC3B-I, Beclin-1, p62, and β-actin was measured by Western blot analysis in SGC7901 and SGC7901/cisplatin (DDP) cells; B: Ratio of LC3II/LC3I; C: Ratio of Beclin-1/β-actin; D: Ratio of p62/β-actin. The results of Western blot analysis were quantified by ImageJ software. Data are presented as the mean ± SD. Statistically significant differences are indicated: ^bP < 0.01.

Figure 5

Zinc oxide nanoparticle inhibits autophagy in gastric cancer cells. A-H: SGC7901 and BGC823 cells were treated with zinc oxide nanoparticle (ZnO-NP, 5 μg/mL) or an equal volume of saline. The expression of light chain 3B-II (LC3B-II), LC3B-I, Beclin-1, p62, and β-actin was measured by Western blot analysis. The results of Western blot analysis were quantified by ImageJ software; I and J: SGC7901 and BGC823 cells were treated with cisplatin (DDP, 10 μmol/L) or co-treated with DDP (10 μmol/L) and ZnO-NP (5 μg/mL). The expression of LC3B-II, LC3B-I, Beclin-1, p62, and β-actin was analyzed by Western blot analysis. Data are presented as the mean ± SD. Statistically significant differences are indicated: ^bP < 0.01.

Figure 6

Zinc oxide nanoparticle attenuates chemotherapy drug resistance by inhibiting the autophagy of gastric cancer cells. SGC7901 and BGC823 cells were treated with cisplatin (DDP), DDP and zinc oxide nanoparticle (ZnO-NP, 5 μg/mL), co-treated with DDP, ZnO-NP (5 μg/mL) and 3-methyladenine (3-MA, 5 mmol/L). A and B: Cell viability was determined by the MTT assay; C and D: Cell apoptosis was analyzed by flow cytometry. Data are presented as the mean ± SD. Statistically significant differences are indicated: ^bP < 0.01. FITC: Fluorescein isothiocyanate.

Figure 7

Zinc oxide nanoparticle reduces the tumor growth of chemoresistant gastric cancer cells in vivo. The impact of zinc oxide nanoparticle (ZnO-NP) on tumor growth of cisplatin (DDP)-resistant gastric cells in vivo was analyzed by the nude mice tumorigenicity assay (n = 5). SGC7901/DDP cells were treated with ZnO-NP (5 μg/mL) or an equal volume of saline. A: Representative images of dissected tumors from nude mice are shown; B: The average tumor volume was calculated; C: The average tumor weight was calculated. Data are presented as the mean ± SD. Statistic significant differences are indicated: ^bP < 0.01.