Role of APOA1 in the resistance to platinum-based chemotherapy in squamous cervical cancer

Human cervical squamous carcinoma cell lines SiHa was purchased from the Type Culture Collection of the Chinese Academy of Sciences(Shanghai, China)and Caski was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The following materials were used in this study: APOA1-overexpression (OE) and control lentivirus-transfected (negative control, NC) (GeneChem,Shanghai, China), DMEM (HyClone, Utah, USA), puromycin(Thermo Fisher Scientific, Waltham, USA), fetal bovine serum (Four Seasons Green, Hangzhou, China), carboplatin (CBP) (CST, Boston, USA), Triton X-100 (Sigma, St. Louis, USA), TMR red (Roche, Basel, Switzerland), Transwell kit (Corning, New York, USA), GIEMSA staining solution (Sigma, St. Louis, USA) and MTT(Genview, Genview, USA).

APOA1-overexpression (OE) and control lentivirus-transfected (negative control, NC) with puromycin were purchased from GeneChem (Shanghai, China). SiHa and Caski cells with lentivirus with puromycin were co-cultured in DMEM containing 10% fetal bovine serum at 37℃ in a 5% CO2 incubator. The medium was changed every 2–3 days with 3.00 μg/ml puromycin, until all SiHa and Caski cells in the Blank group died, finally SiHa and Caski cell lines stably overexpressing APOAl were screened. Cells in logarithmic growth phase were used in the experiments.

SiHa and Caski OE and NC cells were collected, trypsinized, and counted; then, the cell suspensions were diluted and inoculated into 6-well plates at a concentration of 800 cells per well. The cells were cultured in a cell culture incubator for 14 days, until visible cell clones appeared. The NC/OE + CBP groups were placed in complete medium containing carboplatin (CBP) (30 μg/ml), and incubated for 48 h. Clones were counted with the naked eye, and the number of clones with > 50 cells were counted under a microscope.

SiHa and Caski OE and NC cells were collected, trypsinized, and counted. The density of the cell suspensions was adjusted to 2 × 10³ cells/ml, and the cells were inoculated into 96-well plates and incubated for 1–5 days, the carboplatin groups were treated with carboplatin (30 μg/ml) for 48 h (day 4 and 5). After incubation, the medium was discarded, and 20 μL of MTT (5 mg/mL) was added to each well. Then, 100 μL of DMSO was added to dissolve the formazan crystals, and the OD at 490 nm was measured with a microplate reader. Finally, the relative cell viability was calculated based on the OD, and a growth curve was generated.

SiHa and Caski OE and NC cells were collected, trypsinized, and counted. The cells were then washed, placed in complete medium containing carboplatin (CBP) 30 ug/ml, and incubated for 48 h. Cells were fixed with 4% paraformaldehyde in PBS solution (pH 7.4) at 15–25 °C for 1 h and then washed with PBS. Cells were permeabilized using a sodium citrate solution containing 0.1% Triton X-100 and incubated in an ice bath (2–8 °C) for 2 min. Then, 50 μl of prepared DNase I was added, and the cells were incubated at 20 °C for 10 min. TUNEL reaction mix was prepared by mixing 50 μl of TdT with 450 μl of fluorescein-labeled dUTP solution. The prepared TUNEL reaction mix (50 μl) was added to each specimen, which was incubated at 37 °C for 1 h. The negative control contained fluorescein-labeled dUTP solution only. Nuclei were stained with 50 μl of 5 μg/ml DAPI for 5 min at 20℃ and rinsed thrice in PBS. Finally, TUNEL and DAPI-labeled cells were counted separately under a fluorescence microscope.

We chose the common squamous cervical cell type SiHa cells for further tests. The samples were divided into four group (OE, NC, NC + CBP and OE + CBP), four group labeled with different isotopes and three independent trials were conducted. The types of isotope used in the analysis were following: NC1-126, NC2-127 N, NC3-127C, NC + CBP1-128 N, NC + CBP2-128C, NC + CBP3-131 N, OE1-131C, OE2-132 N, OE3-132C, OE + CBP1-133 N, OE + CBP2-133C, OE + CBP3-134 N. Biological analyses included Gene Ontology (GO) term analysis (http://​geneontology.​org/​), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and enrichment analysis, subcellular localization analysis, domain annotation and enrichment analysis, and clustering and interaction network analysis. Different expressed proteins between groups that met the screening criteria, i.e., expression > 1.2 times higher or lower and a p value less than 0.05 by t-test, were regarded as differentially expressed proteins.

We choose the keyword according to literatures about chemotherapy resistance pathway and progression of cancer. The IPA analysis was performed with the following keywords as input: JAK stat, Notch signaling, p38 MAPK signaling, PI3K signaling in B lymphocytes, tumor progression, chemotherapy, relapse, drug resistance, and metastasis.

SPSS 20.0 was used for all statistical analyses and for generating graphics. Measurement data were expressed as mean ± standard deviation (x ± s). Statistical t-test was used to compare the means between two groups, ANOVA was used to compare the means among multiple groups, and the chi square test was used to compare rates between groups, A p value less than 0.05 was considered statistically significant. Three independent trials were conducted in each assay. The dose of carboplatin used in clone formation assay, MTT assay and TUNEL was 30μg/ml which was according to the pre-experiment. We preformed 3–5 concentration gradients in pre-experiment, and choose the most suitable one for the formal experiment.

Of the 29 screened downstream proteins, which were subjected to quantitative PRM protein analysis, 13 had no statistically significant expression differences between the OE and NC groups but did have statistically significant expression differences between the OE + CBP and NC + CBP groups (Table 2 marked in bold), suggesting that these 13 proteins, which are downstream factors of APOA1, may be involved in platinum-based chemoresistance in cervical cancer. Literature searches were conducted to find APOA1 downstream proteins and mechanisms affecting resistance to platinum-based chemotherapy in cervical cancer. Our search identified the following: 1) TOP2A, CDKN2A, and APOE, which are involved in tumor growth, recurrence, and metastasis; 2) STAT1, which may be involved in metastasis of tumor through the p38 MAPK signaling pathway; 3) CD81, C3, and RAC1, which may promote tumor progression through the PI3K signaling pathway; and 4) other proteins, such as ALB, AFP, GC, LTF, SOD2, and NDRG1, which are involved in tumor metastasis.

As the main structural protein of HDL, APOA1 has anti-atherosclerotic, anti-inflammatory, antioxidant, and anti-endotoxic effects [11], and chronic inflammation, oxidative stress, lipids, and cholesterol are associated with tumor development. Several studies have found that serum APOA1 levels are reduced in patients with different malignancies [12, 13]. In gynecological tumors, it has been reported that APOA1 levels are significantly downregulated in patients with epithelial ovarian cancer serum, and APOA1 is a potential tumor marker for epithelial ovarian cancer [14, 15]. APOA1 was shown to inhibit tumor development, and low serum levels of APOA1 are associated with poor prognosis. In studies of advanced ovarian cancer, high APOA1 mRNA levels in body fluids were found to be an independent diagnostic factor for clinically longer overall survival (OS) [16].

In a large European prospective study, APOA1 was found to be negatively associated with the risk of colon cancer [17]. In hepatocellular carcinoma, low levels of APOA1 were associated with poorer progression free survival (PFS), thus APOA1 may be a useful predictor of recurrence in hepatocellular carcinoma [18]. In metastatic nasopharyngeal carcinoma, higher serum levels of APOA1 at pre-treatment were associated with higher OS [19]. In breast cancer, low expression of APOA1 predicted a higher risk of breast cancer development and recurrence [20]. The results of some meta-analyses suggest that low levels of APOA1 may be a poor prognostic indicator for various malignancies, including non-small cell lung cancer, gallbladder cancer, and gastric cancer [21].

The mechanism by which APOA1 inhibits tumors is currently unclear. Studies have shown that APOA1 exerts anti-tumor effects mainly by inhibiting tumor cell proliferation and promoting apoptosis [22, 23]. APOA1 has been reported to capture circulating tumor cells and promote tumor cell apoptosis by downregulating that MAPK pathway, thereby inhibiting tumor cell proliferation [24]. APOA1 also regulates inflammation signals through the STAT3 signaling pathway and reduces matrix metalloproteinase-9 (MMP-9) levels, which is an important factor that promotes tumor proliferation and metastasis [25]. It has also been reported that a short peptide on APOA1 regulates the phosphorylation of c-Src through the c-Src/ERK signaling pathway, thereby inhibiting tumor growth and angiogenesis [26].

APOA1 is easy to detect clinically and has great value as a marker of tumor prognosis. However, there are few studies on the correlation between APOA1 and tumor chemotherapy. Some studies have reported that APOA1 can be used as a predictive marker for platinum-based chemotherapy resistance in ovarian cancer [27]. In the field of gynecological oncology, proteomic studies have shown that APOA1 is highly expressed in platinum-based chemotherapy-resistant ovarian cancer when compared to the levels in paracancerous tissue. A screen for differentially expressed proteins in serum samples from platinum-resistant and platinum-sensitive patient groups identified APOA1 [28], indicating its association with platinum-based chemoresistance. Cruz et al. [27] searched for proteins related to the proteasomal ubiquitination resistance signaling pathway in platinum-resistant ovarian cancer cells and tissues and found that APOA1 was a significantly upregulated platinum chemoresistance-associated protein and could be used as a predictive marker of platinum chemoresistance in ovarian cancer. However, whether APOA1 is related to chemotherapy in cervical cancer has not yet been reported. In this study, we found that APOA1-overexpressing (OE) and normal control (NC) SiHa and Caski cervical squamous carcinoma cells showed smaller differences in the change of clones after administration of chemotherapy (p < 0.05), indicating that overexpression of APOA1 induced resistance to carbopaltin chemotherapy in cervical cancer cells and reduced the chemotherapy-induced reduction of cell cloning. In contrast, there were no significant differences in cell proliferation and apoptosis.

Through bioinformatic analysis and IPA bioanalysis, we found that the factors downstream of APOA1 that are involved in carboplatin chemoresistance in cervical cancer are mainly located in the extracellular matrix and nucleus and may enhance chemoresistance to carboplatin through multiple molecular mechanisms involved in cell composition, cell function, and biological processes. A literature review revealed downstream genes that may be related to chemoresistance: AFP, APOE, B2M, C3, GC, and TOPA2. Our study found seven downstream factors that may be associated with chemoresistance, LTF, SOD2, STAT1, CDKN2A, CD81, RAC1, and NDRG1 and five factors that may be associated with tumor development, CCN1, DCTN3, MUC1, H1-5, and SLC1A5. The specific mechanisms involving these genes need to be further investigated.

We identified and quantitatively validated the proteins downstream of APOA1 by PRM, which indicated that APOA1 may exert its carboplatin chemoresistance effects in cervical squamous carcinoma by 1) promoting tumor growth, recurrence, and metastasis through TOP2A, CDKN2A, and APOE; 2) regulating the p38 MAPK signaling pathway through STAT1 to promote in tumor growth and metastasis; 3) regulating the PI3K signaling pathway through CD81, C3, and RAC1 to promote tumor progression; and 4) promoting metastasis through ALB, AFP, GC, LTF, SOD2, and NDRG1. Our group investigated and validated APOA1-regulated downstream factors involved in the mechanism of platinum-based chemoresistance in cervical squamous carcinoma.