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Year : 2021  |  Volume : 17  |  Issue : 5  |  Page : 1253-1260

Proteomic analysis of the influence of CO2 pneumoperitoneum in cervical cancer cells

Department of Obstetrics and Gynecology, Shandong Qianfoshan Hospital, Cheeloo College of Medicine, Shandong University, Jingshi Road, Jinan, Shandong Province, China

Date of Submission21-Apr-2021
Date of Acceptance23-May-2021
Date of Web Publication17-Nov-2021

Correspondence Address:
Fengnian Rong
16766 Jingshi Road, Jinan, Shandong
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.jcrt_638_21

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 > Abstract 

Objective: The effect of CO2 pneumoperitoneum (CDP) on the oncology outcomes of patients undergoing laparoscopic radical hysterectomy for cervical cancer remains unclear. In this study, we investigated the effects of CDP on the proliferation of cervical cancer cells and examined the molecular mechanism.
Materials and Methods: We established an in vitro CDP model to study the effects of CDP on the proliferation of cervical cancer cells by Cell Counting Kit-8 (CCK-8) assay, xenografted tumor assay. Tandem mass tag-based quantitative proteomics were used to study the proteomic changes in HeLa cells after CDP treatment. Western blot assay was used to detect the expressions of PI3K/Akt signaling pathway proteins.
Results: CDP increased cell proliferation after a short period of inhibition in vitro and promoted tumorigenesis in vivo. Proteomic analysis showed that the expression levels of 177 and 309 proteins were changed significantly 24 and 48 h after CDP treatment, respectively. The acidification caused by CO2 inhibited the proliferation of cervical cancer cells by inhibiting the phosphorylation of PI3K and Akt.
Conclusions: CDP promoted the proliferation of human cervical cancer cells after a short time of inhibition. The mechanism of which is related to the inhibition of phosphorylation of the PI3K/Akt signaling pathway.

Keywords: Cervical cancer, CO2 pneumoperitoneum, laparoscopy, PI3K/Akt Signaling pathway, proteomics

How to cite this article:
Lv H, Zhou T, Rong F. Proteomic analysis of the influence of CO2 pneumoperitoneum in cervical cancer cells. J Can Res Ther 2021;17:1253-60

How to cite this URL:
Lv H, Zhou T, Rong F. Proteomic analysis of the influence of CO2 pneumoperitoneum in cervical cancer cells. J Can Res Ther [serial online] 2021 [cited 2023 Jan 27];17:1253-60. Available from: https://www.cancerjournal.net/text.asp?2021/17/5/1253/330587

 > Introduction Top

During the last two decades, laparoscopic surgery has become widely used in radical hysterectomy in patients with early-stage cervical cancer.[1] However, recent evidence has suggested that laparoscopic radical hysterectomy is associated with a worse prognosis than open abdominal procedures for the management of early-stage cervical cancer.[2] The use of CO2 pneumoperitoneum (CDP) during laparoscopy may be a factor responsible for this poorer outcome. It is currently unclear how CDP affects tumor cells, as previous studies have shown conflicting results.[3],[4],[5]

In this study, we carried out cell proliferation assay, xenografted tumor assay, TMT-based quantitative proteomics, and western blot analysis to perform an evaluation of the safety of laparoscopy for the treatment of cervical cancer and to explore the molecular mechanism for understanding the influence of CDP on cervical cancer.

 > Materials And Methods Top

Cell culture and reagent

The cervical cancer HeLa cells were purchased from GeneChem Co. Ltd (Shanghai, China). The cells were cultured in RPMI-1640 culture solution (Gibco, CA, USA) containing 10% fetal bovine serum (Gibco) and antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin) (Beyotime, Jiangsu, China). The cells were incubated in an incubator at 37°C supplemented with 5% CO2.

Previous studies have shown that the influencing factors of CDP on tumors include hypoxia and acidification;[6],[7] therefore, in some experiments, we included hypoxia (5% CO2, 0.1% O2, 37°C) and acidification (RPMI-1640 medium pH 6.2, regulated with hydrochloric acid, 5% CO2 37°C) groups.


A total of 12 female BALB/c nude mice (4–5 weeks old) were obtained from Weitonglihua Bioscience (Beijing, China). All animal experiment protocols were reviewed and approved by the Ethics Committee of Shandong Qianfoshan Hospital. The animals were housed in a specified sterile chamber with controlled air conditions and free access to sterile food and water.

In vitro CO2 pneumoperitoneum model

We punched two holes on two sides of a plastic container. One was connected to the automatic pneumoperitoneum machine (Olympus, UH4, Japan). After cells were placed in the container, we injected 100% CO2 at a flow rate of 1 L/min for 5 min to flush out the air. Then, the outlet was closed. After exposure at 13 mmHg for 4 h, the cells were transferred to a normal cell culture incubator until subsequent experiments. Cells in the control group were cultured under routine conditions.

Evaluation of pH

An acidometer (Sartorius, PB-10, Germany) was used to measure the extracellular pH. HeLa cells were seeded in a 6-well plate at 5 × 104/well in 3 mL media. After the cells fully adhered to the well, the medium was replaced and the pH of the media was assessed before CDP treatment and 5 min, 30 min, 1 h, 2 h, 3 h, and 4 h after CO2 exposure. After the cells were transferred to normal cell culture incubator, the pH of the media was assessed 5 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h post-CO2 exposure.

Cell proliferation

Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan) was used to investigate the effect of hypoxia and acidification induced by CDP and the effect of CDP and LY294002 (MedChemExpress, NJ, USA; dissolved in DMSO) alone or combination on the proliferation of HeLa cells.

Cells were seeded in a 96-well culture plate at a density of 500 cells per well and cultured overnight. In one experiment, cells were then divided into the control group, CO2 group, hypoxia group, acidification group. In another experiment, LY294002 (25 μM) was added for 1 h before stimulation with CDP.

After the cells were incubated with CCK-8 at 37°C for 1 h, the absorbance was measured at a wavelength of 450 nm with a microplate reader (BioTek, VT, USA).

Xenografted tumor model

HeLa cells (2 × 106) of the CO2 and control group in 200 μl PBS were implanted subcutaneously into two opposite buttocks of the same nude mouse. The tumor volume was evaluated twice a week by two cross-sectional measurements, and tumor size was calculated using the following formula: tumor volume = 0.52 × length × width2. After excision from the mice, the xenografted tumors were weighed.

TMT-based proteome analysis

Protein extraction and digestion

HeLa cells were harvested 24 h and 48 h after CDP stimulation. Total protein was extracted by SDT lysis buffer (4% Sodium dodecyl sulfate [SDS], 1 mM Dithiothreitol, pH 7.6, 100 mM Tris-HCl). Protein digestion by trypsin was performed according to the filter-aided sample preparation procedure.[8]

TMT protein labeling and high-performance liquid chromatography fractionation

100 μg of peptide was labeled using a TMT Kit (Thermo Scientific, MA, USA) according to the manufacturer's instructions. After the labeled peptides were mixed, a high pH reversed-phase peptide fractionation kit (Agilent, USA) was used for fractional separation.

Liquid chromatograph-tandem mass spectrometry analysis

Liquid chromatograph-tandem mass spectrometry analysis was performed on a Q exactive mass spectrometer (Thermo Scientific) that was coupled to Easy nLC (Thermo Scientific). The peptides were loaded onto a C18 reverse-phase trap column (Thermo Scientific) connected to a C18 reverse-phase analytical column (Thermo Scientific) in 0.1% formic acid and separated with a linear gradient of buffer (acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min.

The data were searched using the MASCOT engine (version 2.2, Thermo Scientific) embedded into Proteome Discoverer software (version 1.4, Thermo Scientific) for identification and quantitation analysis. Benjamini–Hochberg multiple hypothesis testing was used to adjust the statistical confidence measures based on the number of tests performed. Proteins with a fold change larger than 1.2 (CO2:Control ratio ≥1.2 or CO2:control ratio ≤0.83) and P < 0.05 were considered significantly differentially expressed.

Bioinformatic analyses

Subcellular localization and analysis

The CELLO method (http://cello.life.nctu.edu.tw/) was used to predict protein subcellular localization.

Cluster analysis of differentially expressed proteins

Cluster 3.0 (https://www.encodeproject.org/software/cluster/) and Java TreeView (http://jtreeview.sourceforge.net/) were used for hierarchical clustering analysis. The Euclidean distance algorithm for similarity measure and average linkage clustering algorithm for clustering were selected when performing hierarchical clustering.

Annotation and gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses

To determine the functional properties of the proteins identified, NCBI Blast + and InterProScan software were used to search the selected proteins and find homologous sequences. Then, Blast2GO software was used to draw the gene ontology (GO) terminology map and annotate the sequences. The results of the GO annotation were plotted by R scripts. Moreover, the studied proteins were blasted against the online Kyoto Encyclopedia of Genes and Genomes (KEGG) database to retrieve their KEGG orthology identifications. Fisher's exact test was used to compare the distribution of each GO classification or KEGG pathway in the target protein set and the overall protein set, and enrichment analysis of the GO annotation or KEGG pathway annotation was carried out on the target protein set. Functional categories and pathways with P values under a threshold of 0.05 were considered significant.

Western blot

The cells were harvested 24 h after treatment and lysed with RIPA buffer (Beyotime, Shanghai, China) containing 1% phenylmethanesulfonyl fluoride (Beyotime) on ice. Protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, MA, USA). After blocking with 5% nonfat milk for 1 h, the PVDF membranes were incubated with primary antibodies against p-Akt (1:1000; CST, MA, USA), p-PI3K (1:1000; CST) or GAPDH (1:1000, Proteintech, Chicago, IL, USA) overnight at 4°C. The membranes were incubated with secondary antibodies coupled with horseradish peroxidase–conjugated secondary antibodies (1:5000; CST) for 1 h. The membranes were incubated with chemiluminescent substrate (Thermo Scientific), and images were acquired with the ChemiDocTM Imaging System (BIO-RAD, WA, USA). ImageJ software was used to analyze bands.

Statistical analysis

Data were presented as mean ± standard deviation. Statistical analyses were performed using SPSS 22.0 software (IBM, NY, USA) and PRISM 8.0 (GraphPad, CA, USA). Data comparison was performed using t-test. P <0.05 was considered to be significant.

 > Results Top

CO2 pneumoperitoneum leads to acidification of the extracellular environment

A CDP model was constructed [Figure 1]a. Pressure was control by pneumoperitoneum machine [Figure 1]b. CDP caused a rapid decrease in extracellular pH which stabilized at 6.173 ± 0.006 at 2 h after the introduction of CO2 [Figure 1]c. It takes 5 h for pH to return to the same level as the control group [Figure 1]d.
Figure 1: The effect of CO2 pneumoperitoneum on extracellular pH. (a) CO2 pneumoperitoneum model in vitro (b) Pneumoperitoneum machine: control flow rate and pressure. (c) The extracellular pH value decreased after ventilation. (d) 5 h after cessation of ventilation, extracellular pH slowly returned to normal level

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CO2 pneumoperitoneum increases proliferation after a short period of inhibition

The proliferative viability of HeLa cells in the CO2 group was decreased in the first 2 days compared with the control group (P < 0.05). However, 3 days after treatment, the proliferative viability of the CO2 group was higher than that of the control group (P < 0.05) [Figure 2]a. Cells in acidification group showed a similar inhibition of proliferation as the CO2 group (P < 0.05). Cell proliferation activity of the hypoxia group was significantly higher than that of the control group (P < 0.05) [Figure 2]b.
Figure 2: Effect of CO2 pneumoperitoneum on the proliferation of HeLa cells. (a) CO2 pneumoperitoneum promoted the proliferation of HeLa cells after temporarily inhibiting (*P < 0.05, *** P < 0.001). (b) Acidification inhibit cell proliferation (*P < 0.05), hypoxia promote cell proliferation (*P < 0.05)

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CO2 pneumoperitoneum increased xenograft tumor growth

The effects of CO2 on HeLa cells were evaluated in xenografts in vivo [Figure 3]a and [Figure 3]b. CDP increased the growth of HeLa cells in BALB/c nude mice. The tumor volume [Figure 3]c and weight [Figure 3]d in the CO2 group were larger than those in the control groups (P < 0.05).
Figure 3: CO2 pneumoperitoneum promotes tumor growth in vivo. (a) HeLa cells (2 × 106) of the control and CO2 group in 200 μL PBS were implanted subcutaneously into two opposite buttocks of the same nude mice (n = 6). (b) After excision from the mice, the xenografted tumors were photographed. (c) The tumor volumes and their growth trends (*P < 0.05, ** P < 0.01). (d) The tumor weight (**P < 0.01)

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Quantitative analysis of differentially expressed proteins

To identify the impact of CDP on the whole proteomics of HeLa cells, we collected total proteins from the control group and CO2 groups (24 h and 48 h after treatment). Three samples were analyzed for each group and the complete protocol is summarized in [Figure 4]a. In total, 6815 proteins were identified, of which 6778 proteins were quantified. Compared with the control group, CDP induced 177 (309) differentially expressed proteins 24 (48) hours after treatment, of which 115 (181) proteins were upregulated and 62 (128) were downregulated [Figure 4]b. Clustering of all the differentially regulated proteins indicated that CDP induced large changes in protein levels [Figure 4]c.
Figure 4: Workflow and quantitative analysis. (a) The systematic workflow for quantitative profiling of global proteome of HeLa cell (3 replicates). (b) Quantitative analysis of identified proteins. (c) Cluster analysis of the significantly dysregulated proteins of HeLa cell

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Functional enrichment analysis of differentially quantified proteins

On the basis of the differentially regulated proteins, the subcellular location, GO functions, and KEGG pathways were investigated.

According to the subcellular location annotation data of the proteins identified, the components included nuclear, cytoplasmic, plasma membrane, extracellular region part, mitochondrial, and lysosomal components [Figure 5]a.
Figure 5: Functional enrichment analysis of differentially quantified proteins. (a) Subcellular location classification. (b) The TOP 20 enriched GO terms were analyzed based on Fisher' exact test. (c) The TOP 20 enriched KEGG pathway was analyzed based on Fisher' exact test

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GO analysis showed that the differentially regulated proteins covered a wide range of biological processes, including positive regulation of cell adhesion mediated by integrin, cell adhesion mediated by integrin, regulation of cell adhesion mediated by integrin, myeloid leukocyte activation, extracellular matrix organization [Figure 5]b.

KEGG pathway enrichment analysis showed that the pathways with the highest number of differentially expressed proteins were Parkinson's disease, the PI3K–Akt signaling pathway, Alzheimer's disease, amyotrophic lateral sclerosis, and oxidative phosphorylation. KEGG pathway analysis also found that the differentially expressed genes were significantly enriched in five pathways, including the p53 signaling pathway, beta-alanine metabolism, ECM–receptor interaction, the PI3K–Akt signaling pathway, and oxidative phosphorylation. Notably, the PI3K–Akt and oxidative phosphorylation pathways ranked highly for both number and significance [Figure 5]c.

The effects of CO2 pneumoperitoneum on inhibition of the PI3K/Akt signaling pathway

To determine whether the effects of CDP on the proliferation of HeLa cells are related to the PI3K/Akt signaling pathway, we examined the expressions of signaling pathway proteins (p-PI3K and p-Akt) by western blot analysis. The results showed that the expression levels of p-PI3K and p-Akt in the CO2 group declined significantly compared with controls (P < 0.05). Phosphorylation of PI3K and Akt was also down-regulated in the acidification group, which was consistent with the effects of CDP. In addition, p-PI3K expression was slightly up-regulated (P > 0.05) in the hypoxia group, while p-Akt was not significantly changed [Figure 6].
Figure 6: The Western blotting test results. The expression of p-PI3K p-Akt and GAPDH protein and quantitative analysis in each group (*P < 0.05, **P < 0.01)

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The combination of LY294002 and CDP had a stronger inhibitory effect on the proliferation of cervical cancer cells than the CDP treatments [Figure 7]a. In addition, LY294002 enhanced the inhibitory effect of CDP on the PI3K/Akt pathway [Figure 7]b. These results indicate that CDP probably inhibits cervical cancer cell proliferation by inhibiting the PI3K/Akt signaling pathway.
Figure 7: Effect of CO2 and LY294002 combination on proliferation and PI3K/Akt pathway-related proteins. (a) LY294002 (25 μM) enhanced the inhibitory effect of CO2 pneumoperitoneum on HeLa cell proliferation (*P < 0.05, **P < 0.01, *** P < 0.001). (b) LY294002 (25 μM) inhibited the expression levels of p-Akt decreased compare with control and CO2 group (*P < 0.05)

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 > Discussion Top

Cervical cancer accounts for approximately 570,000 and 310,000 new cases and deaths, respectively, worldwide in 2018.[9] Laparoscopic radical hysterectomy is believed to be associated with less intraoperative blood loss, a shorter hospital stays, and a lower risk of postoperative complications, without increasing the recurrence rate.[10],[11] However, recent studies have shown that laparoscopic radical hysterectomy is associated with a worse prognosis than an open abdominal procedure for the management of early-stage cervical cancer.[2] In addition to the use of intrauterine manipulator and intra-abdominal colpotomy, the use of CO2 for the pneumoperitoneum might be a reason for these events.[12] CO2 is the most commonly used pneumoperitoneum medium.[13] Therefore, further investigation of the effects of CDP on tumors and the molecular mechanism underlying the influence of CDP on tumor growth and metastasis is required.

In the present study, we demonstrated that the effect of CDP on cervical cancer is multifactorial, with effects on acidification and hypoxia. The acidification caused by CDP inhibited the proliferation of cervical cancer cells, while hypoxia promoted the proliferation of cervical cancer cells. This result is similar to the study by Riemann et al.,[14] which demonstrated that acidosis and hypoxia have opposing effects on tumor cells. Furthermore, our experiments showed that CDP promoted HeLa cell tumor growth in nude mice. Therefore, CDP may be a factor leading to poor tumor outcome. Both the use of a uterine manipulator and intracorporeal open colpotomy have also been considered as possible reasons for poor outcome.[15],[16] A uterine manipulator could disrupt the tumor by direct crushing, and intracorporeal open colpotomy can increase the chance of tumor spread. Tumor cells scattered in the abdominal cavity are more likely to cause tumor recurrence under the stimulation of a CO2 environment.

The results of TMT-based proteomics showed that hundreds of proteins were up- or downregulated significantly, indicating that CDP strongly influences tumor cell physiology. Laparoscopy with CO2 results in a CO2-rich, acidic, and hypoxic local environment.[7] On the one hand, CDP causes hypoxia, which alters mitochondrial fusion and fission, mitophagy, and oxidative phosphorylation[17] and triggers several mechanisms to adapt cells to a low oxygen environment. Our data showed that 11.9% (8%) of differentially expressed proteins were associated with mitochondria 24 (48) hours after CO2 stimulation. Mitochondria serve as metabolic hubs in the cell and are intimately linked with the adaptive response to hypoxia.[18] On the other hand, our data showed that CDP caused severe acidification of extracellular pH which has a wide impact on various biological processes, including cell proliferation, migration, and angiogenesis.[19] Acidosis activates a rescue program preventing necrotic cell death,[20] and extracellular pH can regulate autophagy.[19] Autophagy can eliminate dysfunctional mitochondria damaged by cell stress to restrict cell growth or induce apoptosis in early stages of cancer. Conversely, in the later stages of cancer, autophagy can support tumor growth under hypoxic conditions.[21] This is consistent with the observed trend that CDP first inhibited and then promoted HeLa cell proliferation. In addition, studies suggests that acidosis of the abdominal environment suppresses the peritoneal immune system.[22] Acidic environment also provokes particular damage to the peritoneum, making it easier for tumor cells to adhesion and planting.[23],[24]

In our study, GO function annotation and enrichment analysis showed that the top three enriched GO terms were all related to cell adhesion mediated by integrin 24 h after CO2 treatment, indicating that CDP may influence tumor cells through regulation of integrin-mediated cell adhesion. Cell adhesion molecules often integrate extracellular cues with cell-intrinsic signaling, affecting intracellular responses, intracellular signaling, and gene expression.[25] Integrins are a family of transmembrane glycoprotein adhesion receptors which are the main receptors for sensing the extracellular environment of the cell.[26] Cells are able to sense their surrounding environment through integrin-associated adhesion complexes.[25] Loss of adhesion results in cell cycle arrest[27] and leads to apoptosis.[28]

Moreover, KEGG pathway enrichment analysis showed significant changes in the PI3K-Akt pathway, which is one of the most deregulated pathways in human cancer.[29] Early studies indicated that integrin controls general protein synthesis through the Akt/mechanistic target of rapamycin pathway.[30]

In our study, we further analyzed CDP's mechanism of action in cervical cancer. We demonstrated that CDP induces acidification of the cell microenvironment and inhibits the PI3K/Akt pathway by inhibitory phosphorylation of PI3K and Akt. The combination of LY294002 and CDP significantly inhibited the growth of HeLa cervical cancer cells. The PI3K/Akt pathway is activated in cancer and represents an optimal target for cancer therapy.[31] Indeed, strategies targeting the inhibition of PI3K have been used to reduce cell survival and proliferation in human cancers.[32] For example, LY294002 increases the sensitivity of cervical cancer to paclitaxel and cisplatin.[33] Our results suggest that it may be possible to improve the poor prognosis of cervical cancer patients treated with laparoscopic surgery by applying LY294002. Notably, study shows that CO2 inhibits tumor growth by improving tumor hypoxia through the Bohr effect.[34] The effect of CDP on tumors is very complex in vivo and requires additional in vivo experiments.

In summary, this study provides a reference proteome map to quantify the changes to the whole proteome of HeLa cervical cancer on CDP treatment. CDP causes severe acidosis, which inhibits the PI3K/Akt pathway, thereby inhibiting the proliferation of cervical cancer cells. However, CDP also causes hypoxia of cells, which increases the proliferation activity of cells. The combined effect of the two factors was to promote the proliferation of cervical cancer cells after the temporary inhibition. The application of LY294002 can enhance the inhibitory effect of CDP on cervical cancer cells, which may be a method to reverse the adverse outcome of laparoscopic treatment of early cervical cancer tumors. Further in vivo studies are needed.

 > Conclusion Top

CDP promoted the proliferation of human cervical cancer cells after a short time of inhibition and increased xenograft tumor growth. CDP induced large changes in protein levels. The mechanism of which is related to the PI3K/Akt signaling pathway.

Financial support and sponsorship

The study was financially supported by Jinan Science and Technology Development Plan (Grant number 202019180) and Shandong Medical and Health Technology Development Project (Grant number 2018WS259).

Conflicts of interest

There are no conflicts of interest.

 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]


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