IP10-CDR3 Reduces The Viability And Induces The Apoptosis Of Ovarian Cancer Cells By Down-Regulating The Expression Of Bcl-2 And Caspase 3

Abstract

Purpose: This study aimed to explore the effects of interferon-γ inducible protein 10 (IP10) and complementarity-determining region 3 (CDR3) of T cells receptor on ovarian cancer cells and the involved mechanisms.

Methods: IP10 and CDR3 were linked with single-chain antibody (scfv) and exotoxin gene muton of Pseudomonas aeruginosa (PE40) to construct IP10-CDR3scfv and IP10-CDR3-PE40scfv. Then, we constructed pcDNA3.1-IP10-CDR3scfv and pcDNA3.1-IP10-CDR3-PE40scfv plasmids which were proved by HindIII/EcoRI digestion. SKOV3 cells and HOSEpiC cells were incubated with fluorescein isothiocyanate (FITC) labeled IP10-CDR3scfv and IP10-CDR3-PE40scfv proteins and protein levels were examined by flow cytometry. After gene transfection, SKOV3 cells were divided into four groups: Control, pcDNA3.1(+) negative control (NC) (pcDNA3.1(+) NC transfection), IP10-CDR3scfv (IP10-CDR3scfv transfection) and IP10-CDR3-PE40scfv (IP10-CDR3-PE40scfv transfection). Levels of IP10, CDR3, Caspase-3, cleaved Caspase-3 and Bcl-2 were determined by RT-PCR and Western blot. Cell viability and apoptosis were investigated by CCK-8 assay and Annexin V-FITC/PI assay, respectively.

Results: The levels of FITC-labeled IP10-CDR3scfv and IP10-CDR3-PE40scfv proteins in the SKOV3+IP10-CDR3scfv group and the SKOV3+IP10-CDR3-PE40scfv group were remarkably higher than that in the SKOV3 group (P<0.05). So was the HOSEpiC related groups. There was no obvious difference in the levels of IP10, CDR3, Caspase-3, cleaved Caspase-3 and Bcl-2 between the control group and the pcDNA3.1(+) NC group. However, compared with the control group, the levels of Caspase-3 and Bcl-2 were reduced notably and the levels of IP10, CDR3 and cleaved Caspase-3 were elevated sharply in the IP10-CDR3scfv and IP10-CDR3-PE40scfv groups (P<0.05). The control group and the pcDNA3.1(+) NC group demonstrated similar cell viability and apoptosis. However, compared with the control group, cell viability in the IP10-CDR3scfv and IP10-CDR3-PE40scfv groups decreased significantly and cell apoptosis increased (P<0.05).

Conclusion: IP10-CDR3 could reduce the viability and induce the apoptosis of ovarian cancer cells by down-regulating the expression of Bcl-2 and Caspase-3.

Keywords: Bcl-2; CDR3; Caspase-3; IP10; cell apoptosis; ovarian cancer.

Figure 1

Electrophorograms of pcDNA3.1-IP10-CDR3scfv (A) and pcDNA3.1-IP10-CDR3-PE40scfv (B) plasmids. Lane 1: pcDNA3.1-IP10-CDR3scfv plasmid or pcDNA3.1-IP10-CDR3-PE40scfv plasmid; Lane 2: pcDNA3.1-IP10-CDR3scfv plasmid or pcDNA3.1-IP10-CDR3-PE40scfv plasmid digested by HindIII/EcoRI. Abbreviation: M, marker.

Figure 2

The protein levels after FITC-labeled IP10-CDR3scfv protein and FITC-labeled IP10-CDR3-PE40scfv protein were added to SKOV3 cells and HOSEpiC cells for 48 hrs which were determined by flow cytometry. *P<0.05 vs. SKOV3 or HOSEpiC; ^#P<0.05 vs. SKOV3+IP10-CDR3scfv or HOSEpiC+IP10-CDR3scfv.

Figure 3

The mRNA levels of IP10, CDR3, Caspase-3 and Bcl-2 in SKOV3 cells of various groups which were determined by RT-PCR. *P<0.05 vs. Control; ^#P<0.05 vs. IP10-CDR3scfv. Control: without any transfection; NC: pcDNA3.1(+) negative control transfection.

Figure 4

The protein levels of Caspase-3, Bcl-2 (A) and cleaved Caspase-3 (B) in SKOV3 cells of various groups which were evaluated by Western blot. *P<0.05 vs. Control. Control: without any transfection; NC: pcDNA3.1(+) negative control transfection.

Figure 5

The viability of SKOV3 cells in various groups which was expressed as absorbance and examined by CCK-8 assay. *P<0.05 vs. Control; ^#P<0.05 vs. IP10-CDR3scfv. Control: without any transfection; NC: pcDNA3.1(+) negative control transfection.

Figure 6

The apoptosis of SKOV3 cells in various groups which was assessed by Annexin V-FITC/PI assay. *P<0.05 vs Control; ^#P<0.05 vs IP10-CDR3scfv. Control: without any transfection; NC: pcDNA3.1(+) negative control transfection.