Inhibition of polypyrimidine tract-binding protein 3 induces apoptosis and cell cycle arrest, and enhances the cytotoxicity of 5- fluorouracil in gastric cancer cells

Abstract

Background: Human polypyrimidine tract binding protein 3 (PTBP3) was first discovered in 1999 and has been well characterised as a differentiation regulator. However, its role in human cancer has rarely been reported. Our previous study revealed increased PTBP3 protein level in gastric cancer tissues. Downregulation of PTBP3 suppressed the proliferation and differentiation of gastric cancer cells in vivo.

Methods: PTBP3 mRNA levels in human gastric cancer and adjuvant non-tumour tissues were detected. Apoptosis and 5-FU effect were determined in PTBP3-silenced gastric cancer cells. Underlying molecular mechanisms were investigated.

Results: MRNA expression of PTBP3 was upregulated in gastric cancer tissues, especially in those at an advanced stage. PTBP3 silencing led to apoptosis, under which modulation of PTB and thereby switch of Bcl-x pre-mRNA splicing pattern might be an important mechanism. Further research found that inhibition of PTBP3 expression enhanced the chemosensitivity of gastric cancer cells towards 5-FU treatment. This was mediated by reduced expression of histone deacetylase 6 (HDAC6), which further inhibited the phosphorylation of Akt and the expression of thymidylate synthase (TYMS), the critical determinant of 5-FU cytotoxicity.

Conclusions: PTBP3 might serve as a biomarker of gastric cancer or potential target for anti-cancer therapy.

Figure 1

Expression of PTBP3 in gastric cancer tissues, adjuvant non-tumour (NT) tissues and gastric cancer cell lines were detected. (A) Mann–Whitney test showed higher expression of PTBP3 in tumour tissues than in NT tissues. (B) PTBP3 was upregulated in 88.5% cases (23/26) involved in this study. (C) PTBP3 levels in gastric cancer cell lines.

Figure 2

PTBP3 inhibition induced apoptosis and cell cycle arrest in gastric cancer cells. (A) Cleavage of caspase-3, PARP-1, and caspase-9 were activated upon PTBP3-silencing. (B) Higher apoptotic subpopulations in gastric cancer cells upon PTBP3 silencing were detected via Annexin-FITC/PI staining and FACS analyses (***P<0.001, χ²-test). (C) PI staining and FACS analyses showed cell cycle arrest at S-phase in PTBP3-silenced gastric cancer cells (**P<0.01, ***P<0.001, Student's t-test). KD, cells transfected with PTBP3-targeting pSilencer plasmid; NC, cells transfected with negative control pSilencer 2.1-U6 neo plasmid; si-NC, cells transfected with non-targeting siRNA; si-1 and si-2, cells transfected with different PTBP3-targeting siRNAs.

Figure 3

PTBP3 knockdown switched the splicing pattern of Bcl-x via modulating PTB expression. Inhibition of PTBP3 (A) in gastric cancer cells promoted the generation of Bcl-xS (B) and upregulated the expression of PTB (C, D) (*P<0.05, **P<0.01, ***P<0.001, Student's t test). (E) PTB knockdown reversed the increase of Bcl-xS/Bcl-xL ratio resulting from PTBP3-silencing. * indicates the difference between the NC group and each of the rest groups; ^# indicates the difference between the PTBP3 KD and the PTB/PTBP3 KD group (*P<0.05, **P<0.01, ***P<0.001, ^##P<0.01, Student's t test).

Figure 4

PTBP3 modulated the chemosensitivity of gastric cancer cells. (A) PTBP3 inhibition enhanced the cytotoxicity of 5-FU in gastric cancer cells (**P<0.01, ***P<0.001, ^#P<0.05, ^##P<0.01, two-way ANOVA). (B) Ectopic expression of PTBP3 enhanced the resistance of SGC7901 cells against 5-FU (NC: cells transfected with pcDNA 3.1(+) empty vector; OE: cells transfected with PTBP3-pcDNA 3.1(+) plasmid, **P<0.01, ***P<0.001, ^#P<0.05, two-way ANOVA). (C) Lower expression of TYMS, MRP2, and p-gp was observed in PTBP3-silenced gastric cancer cells, while MRP1 level was not affected by PTBP3 modulation. Similar mRNA expression trends of TYMS (D), MRP2 (E), and p-gp (F) were detected (**P<0.01, ***P<0.001, Student's t-test).

Figure 5

PTBP3 knockdown inhibited HDAC6/Akt pathway. (A) PTBP3 inhibition enhanced the cytotoxicity of L-OHP in gastric cancer cells (**P<0.01, ***P<0.001, ^#P<0.05, ^##P<0.01, two-way ANOVA). (B) Inhibition of PTBP3 led to downregulation of HDAC6 protein and dephosphorylation of Akt in gastric cancer cells. (C) HDAC6 mRNA expression were downregulated in PTBP3-silenced cells as well (**P<0.01, ***P<0.001, Student's t test). (D) TYMS and HDAC6 mRNA expression were upregulated in PTB-silenced MKN45 cells as well (**P<0.01, Student's t-test). (E) Inhibition of PTB led to upregulation of HDAC6 protein and phosphorylation of Akt in MKN45 cells.