LACTB induced apoptosis of oxaliplatin-resistant gastric cancer through regulating autophagy-mediated mitochondrial apoptosis pathway

Abstract

Oxaliplatin (OXA), as a third-generation platinum anticancer drug, is a treatment drug for gastric cancer (GC). However, OXA resistance has become the main reason for OXA treatment failure. Serine beta-lactamase-like protein (LACTB), acts as a mitochondrial protein, can affect multiple cancer processes. Here, we aimed to investigate the function and mechanism of LACTB in OXA-resistant GC. After LACTB overexpression or autophagy activator (RAPA) treatment, cell proliferation, reactive oxygen species (ROS), apoptosis, mitochondrial dysfunction were evaluated through CCK-8 assay, Edu staining, flow cytometry and immunofluorescence assay. Moreover, DNA double-stranded damage and autophagy-related proteins were examined via western blot. We revealed that LACTB was downregulated in OXA-resistant MGC-803 cells, and overexpression of LACTB reduced the resistance of GC cells to OXA. Besides, our results uncovered that overexpression of LACTB induced apoptosis, reduced the mitochondrial membrane potential (MMP) and accelerated ROS accumulation in OXA-resistant MGC-803 (MGC-803/OXA) cells. Meanwhile, we verified that overexpression of LACTB decreased glucose uptake and ATP synthesis, induced mitochondria and DNA damages, and inhibited autophagy of MGC-803/OXA cells. Furthermore, our results certified that RAPA could weaken the function of LACTB on apoptosis and mitochondrial morphology and function in OXA-resistant MGC-803 cells with OXA treatment. Therefore, we demonstrated that LACTB could attenuate the resistance of MGC-803/OXA cells to OXA through autophagy-mediated mitochondrial morphological changes, mitochondrial dysfunction, and apoptosis, suggesting that LACTB, functions as a suppressor, is conducive to the therapy of OXA-resistant GC.

Keywords: Gastric cancer; apoptosis; autophagy; mitochondrial dysfunction; oxaliplatin; serine beta-lactamase-like protein.

Figure 1

Overexpression of LACTB repressed proliferation of OXA-resistant MGC-803 cells. A. The mRNA level of LACTB was examined by qRT-PCR analysis in GES-1, MGC-803 and MGC-803/OXA cells, respectively. *P < 0.05; **P < 0.01, ***P < 0.001. B. The protein level of LACTB was determined by western blotting analysis in GES-1, MGC-803 and MGC-803/OXA cells, respectively. *P < 0.05; **P < 0.01. C, D. qRT-PCR and western blot assays were applied to confirm the transfection effect of pcDNA3.0 LACTB plasmid in MGC-803/OXA cells. ***P < 0.001. E-G. CCK-8 assay and Edu staining were utilized to examine the effect of LACTB overexpression on the proliferation of MGC-803/OXA cells. *P < 0.05, ***P < 0.001 vs. vector group. #P < 0.05 vs. blank group.

Figure 2

Overexpression of LACTB enhanced OXA-induced ROS and apoptosis-promoting of OXA-resistant MGC-803 cells. A. After LACTB overexpression in MGC-803/OXA cells, the intracellular level of ROS was examined using DCFH-DA fluorescent probe. B. The apoptosis level of MGC-803/OXA cells after LACTB overexpression was analyzed by flow cytometry, and respective results were also exhibited. ***P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. blank group.

Figure 3

Upregulation of LACTB reduced OXA-induced mitochondrial depolarization, glucose uptake and ATP synthesis of OXA-resistant MGC-803 cells. A. After LACTB overexpression, MGC-803/OXA cells were stained with JC-1 to evaluate mitochondrial depolarization by flow cytometry. B. The degree of mitochondria damage was examined by IF assay. Magnification 200 ×, Scale bars = 20 μm. C, D. The levels of 2-DG uptake and ATP were determined using the respective kits in LACTB overexpressed MGC-803/OXA cells. ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. blank group.

Figure 4

Overexpression of LACTB induced DNA damage and inhibited autophagy of OXA-resistant MGC-803 cells. A. Western blot assay was applied to examine the protein levels of pSer4/Ser8 RPA2, RPA2, γH2AX, LC3I/II, Beclin-1 and P62 in LACTB overexpressed MGC-803/OXA cells. B-G. And the relative expressions were calculated according to the gray values of each group. *P < 0.05, ***P < 0.001 vs. vector group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. blank group.

Figure 5

RAPA reversed the effects of LACTB overexpression on the proliferation, ROS production and apoptosis of MGC-803/OXA cells. After transfection with LACTB plasmid, MGC-803 cells were treated with RAPA or/and OXA, respectively. A. CCK-8 assay was carried out to assess the proliferation changes of MGC-803/OXA cells in each group. ***P < 0.001 vs. vector group; ##P < 0.001, ###P < 0.001 vs. LACTB group; &P < 0.05, &&P < 0.01 vs. LACTB+RAPA group. B. EdU staining was utilized to detect the proliferation changes of MGC-803/OXA cells in each group. C. DCFH-DA fluorescent probe was utilized to confirm the intracellular level of ROS. D. Flow cytometry was applied to analyze the apoptosis of MGC-803/OXA cells. *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 6

LACTB suppressed mitochondrial depolarization, glucose uptake and ATP synthesis, and reduced mitochondria of MGC-803/OXA cells by autophagy pathway. A. The mitochondrial depolarization was examined using flow cytometry with JC-1 staining. B. Mitochondrial damage of MGC-803/OXA cells was evaluated by TEM after treatment with LACTB plasmid, RAPA or/and OXA. C. D. Respective kits were used to determine the levels of 2-DG uptake and ATP in the treated MGC-803/OXA cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

LACTB resulted in OXA-induced DNA damage by inhibiting autophagy pathway in MGC-803/OXA cells. A. IF assay was performed to assess the degree of LC3B. Magnification 200 ×, Scale bars = 20 μm. B. Markers of DNA double-stranded damage and autophagy were examined by Western blot assay in MGC-803/OXA cells after treatment with LACTB plasmid, RAPA or/and OXA. C-H. The relative gray density was counted by comparison with GAPDH. *P < 0.05; **P < 0.01, ***P < 0.001.