Prostaglandin F2α synthase promotes oxaliplatin resistance in colorectal cancer through prostaglandin F2α-dependent and F2α-independent mechanism

Figure 1 Prostaglandin F_2α synthase is upregulation in patients with colorectal cancer. A: Prostaglandin F_2α synthase (PGFS) protein expression was verified using western blot in 37 pairs of colorectal cancer (CRC) and adjacent normal tissues; B: Real-time polymerase chain reaction analysis of PGFS mRNA expression in 37 pairs of CRC and adjacent normal tissues; C: Hematoxylin and eosin staining and immunohistochemistry for PGFS expression (scale bar, 50 μm); D: PGFS expression in non-response and response patients analyzed by the Cancer Treatment Response gene signature DataBase (http://ctrdb.ncpsb.org.cn/). ^aP < 0.05, ^bP < 0.01. T: Tumor; N: Adjacent normal tissues; PGFS: Prostaglandin F_2α synthase; HE: Hematoxylin and eosin.

Figure 2 Prostaglandin F_2α synthase resists the inhibitory effect of oxaliplatin on colorectal cancer cell proliferation. A: The effects of prostaglandin F_2α synthase (PGFS) overexpression on cell viability in HCT116 and HCT8 cells; B: The effects of PGFS knockdown on cell viability in HCT116-OxR and HCT8-OxR cells; C and D: Western blot analysis showed proliferating cell nuclear antigen levels in parental and oxaliplatin-resistant (OR) colorectal cancer (CRC) cells; E and F: Plate colony formation assay in parental and OR CRC cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dNo significance. PCNA: Proliferating cell nuclear antigen; PGFS: Prostaglandin F_2α synthase; Oxa: Oxaliplatin; IC₅₀: Half-inhibitory concentration.

Figure 3 Prostaglandin F_2α synthase suppresses apoptosis induced by oxaliplatin in colorectal cancer cell lines. A and B: Western blot analysis showed cleaved poly ADP-ribose polymerase and cleaved caspase-3 were assessed in colorectal cancer (CRC) cells or in oxaliplatin-resistant (OR) CRC cells; C and D: Apoptosis of parental and OR CRC cells evaluation by terminal deoxynucleotidyl transferase dUTP nick end labeling assay, scale bar 100 μm; E and F: Apoptosis was measured by the Annexin V-PI staining in parental and OR CRC cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Oxa: Oxaliplatin; PARP: Poly ADP-ribose polymerase.

Figure 4 The protective effect of prostaglandin F_2α synthase on DNA damage induced by oxaliplatin in colorectal cancer cells. A and B: DNA damage marker protein γ-H2A histone family member X (γ-H2AX) was determined using western blot in HCT116 and HCT8 cells and HCT116-OxR and HCT8-OxR cells; C: γ-H2AX fluorescent spots were detected using immunofluorescence staining; D and E: Detection of DNA damage using single-cell gel electrophoresis (comet assay) in parent and drug-resistant cells (scale bar, 50 μm). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. PGFS: Prostaglandin F_2α synthase; Oxa: Oxaliplatin; γ-H2AX: γ-H2A histone family member X.

Figure 5 Prostaglandin F_2α synthase knockdown improves oxaliplatin efficiency in vivo. A: Morphologies of collected tumors in subcutaneous HCT8-OxR xenografts in nude mice; B: Curves of tumor growth in each group; C: Tumor weights were measured after collection; D: Hematoxylin-eosin (upper panel; magnification, × 200) and immunohistochemical staining for proliferating cell nuclear antigen (bottom panel; magnification, × 200) using xenograft tumor samples from each group. ^aP < 0.05, ^bP < 0.01. PCNA: Proliferating cell nuclear antigen; PGFS: Prostaglandin F_2α synthase; HE: Hematoxylin and eosin; Oxa: Oxaliplatin.

Figure 6 The inhibitory effect of prostaglandin F_2α on oxaliplatin-induced cytotoxicity. A: Western blots showed the effect of prostaglandin F2α synthase (PGF_2α) (PGFS) on the expressions of proliferating cell nuclear antigen, cleaved-poly ADP-ribose polymerase, and γ-H2A histone family member X (γ-H2AX) in PGFS knockdown, oxaliplatin-resistant (OR) cells; B: The effect of PGF2α on apoptosis in PGFS knockdown, OR cells evaluated by immunofluorescence. scale bar 100 uM; C: The effect of PGF2α on the cleavage of γ-H2AX protein expressions in PGFS knockdown, OR cells; D: The effect of PGF2α on DNA damage in PGFs knockdown, OR cells evaluated by single-cell gel electrophoresis. scale bar 50 μm). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dNo significance. PCNA: Proliferating cell nuclear antigen; PARP: Poly ADP-ribose polymerase; γ-H2AX: γ-H2A histone family member X; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 7 Prostaglandin F_2α synthase may suppresses the formation of platinum-DNA adducts. A: The effect of Prostaglandin F_2α synthase (PGF2α) (PGFS) overexpression on the formation of platinum-DNA adducts was determined using inductively coupled plasma mass spectrometry in colorectal cancer (CRC) cells; B: The effect of PGFS knockdown on the formation of platinum-DNA adducts in oxaliplatin-resistant (OR) CRC cells; C: The effect of PGF2α on the formation of platinum-DNA adducts in OR CRC cells; D: The reactive oxygen species (ROS) content were significantly decreased in OR CRC cells; E: PGFS overexpression significantly decreased ROS content in CRC cells; F: PGFS knockdown significantly decreased ROS content in OR CRC cells; G: PGFS knockdown significantly decreased ROS content in OR CRC cells (scale bar, 50 μm). ^aP < 0.05, ^bP < 0.01; ^cNo significance. PGF_2α: Prostaglandin F_2α; Oxa: Oxaliplatin.