ABSTRACT
Objective:
To investigate the inhibitory effect of combined strategy of
poly adenosine diphosphate ribose polymerase (PARP) inhibitor and
interleukin-1β (
IL-1β) inhibitor on
homologous recombination deficiency (HRD)-proficient
ovarian cancer cells.
Methods:
(1) HRD-proficient
ovarian cancer cell lines OVCAR3 and CAOV3 were treated with
PARP inhibitor olaparib.
Screening by
RNA sequencing analysis, the expression level of
IL-1β was validated by
enzyme-linked immunosorbent assay (
ELISA) and
western blot. (2) The
dose-response curves of
IL-1β inhibitor diacerein were evaluated by
cell counting kit-8 (
CCK-8) assays in OVCAR3 and CAOV3
cells.
CCK-8 assays were further applied to determine the viabilities of OVCAR3 and CAOV3
cells. (3) To evaluate the synergistic effects of olaparib and
IL-1β inhibitor in vivo, the transplanted
ovarian cancer model was constructed. BALB/c-
nude mice ( n=16) were injected intraperitoneally with 1×10 7 OVACR3
cells labelled with
luciferase (OVCAR3-Luc).
Immunohistochemistry (IHC) assay was performed to determine
nuclear antigen associated with
cell proliferation (Ki-67) expression. (4)
Blood routine tests,
kidney and
liver function tests were performed to analyze the toxic reaction of different
drug treatments. The potential
drug-induced
injuries of vital organs including
heart,
liver,
spleen,
lungs and
kidneys of
nude mice were determined by
hematoxylin-
eosin (HE)
staining.
Results:
(1) The
RNA sequencing results showed that the
mRNA level of
IL-1β was the most significantly increased among the 25 differentially expressed
genes in OVCAR3
cells treated with olaparib, compared to the negative
control group. Olaparib
treatment significantly promoted the
secretion and expression of
IL-1β
protein in both OVACR3 and CAOV3
cells by
ELISA [(36.2±3.5) and (49.5±3.5) pg/ml, respectively; all P<0.001] and western bolt (2.87±0.37 and 2.05±0.08, respectively; all P<0.01). (2) The half maximal inhibitory concentration (IC 50) value of
IL-1β inhibitor was determined as follows 75 μmol/L for OVACR3
cells and 100 μmol/L for CAOV3
cells. The
treatments were divided into four groups including
control group, olaparib monotherapy group,
IL-1β inhibitor monotherapy group and the combination
therapy group. The
cell viabilities of each group in OVCAR3 and CAOV3 were determined by
CCK-8 assay. The data in each group were showed as follows for OVCAR3 and CAOV3
cells (100.0±0.4)% and (100.0±3.5)% in
control group; (63.1±6.2)% and (63.3±3.8)% in olaparib monotherapy group; (61.6±4.7)% and (63.8±3.5)% in
IL-1β inhibitor monotherapy group; and (32.9±5.2)% and (30.0±1.3)% in the combination
therapy group. The viability assay showed that the combined strategy exhibited a significant inhibition effect on OVACR3 and CAOV3
cells, compared to the monotherapy group and the
control group (all P<0.01). (3) All
mice with transplanted
tumors of HRD-proficient
ovarian cancer cells were randomly divided into four groups, and treated with four different
treatments as mentioned above, respectively. After 4 weeks (on day 29), the vivo
fluorescence imaging were determined. The results showed that the amount of
fluorescence of transplanted
tumors was mostly decreased in the combination
therapy group [(0.5±0.4)×10 10 p/s], compared to the
control group [(4.2±1.0)×10 10 p/s] or the groups treated with any
single drug [(3.1±0.9)×10 10, (2.2±0.9)×10 10 p/s; all P<0.05].
Mice were then sacrificed under
anesthesia, and all transplanted
tumors detached and weighed for further investigation. The weight of transplanted
tumors was significantly decreased in the combination
therapy group [(0.09±0.03) g], compared to that in
control group [(0.25±0.05) g] or groups treated with any
single drug [(0.17±0.03), (0.19±0.04) g; all P<0.05]. The measurement of the expression of Ki-67 showed that it was significantly decreased in the combination
therapy group (0.33±0.10), compared to that in the
control group (1.00±0.20) or monotherapy groups (0.76±0.07, 0.77±0.12; all P<0.05). (4) There were no significant differences of
body weights,
blood routine test, renal and
liver function tests among
mice with different
treatments (all P>0.05). Moreover, no significant
injuries were observed in the vital organs among the four groups.
Conclusions:
The combination of olaparib and
IL-1β inhibitor synergistically exhibits significant cytotoxicity in HRD-proficient
ovarian cancer cells. Moreover, the
blood routine and
blood biochemistry results confirmed the
biosafety of the combination of olaparib and
IL-1β inhibitor.