Secondary Logo

The role of the globular heads of the C1q receptor in paclitaxel-induced human ovarian cancer cells apoptosis by a mitochondria-dependent pathway

Lv, Kang-Taia,*; Gao, Ling-Juanb,*; Hua, Xiangdongb; Li, Fengshanb; Gu, Yunb; Wang, Weib

doi: 10.1097/CAD.0000000000000567
PRECLINICAL REPORTS
Open
SDC

As a mitochondrial membrane protein, globular C1q receptor (gC1qR) can mediate a variety of biological responses. Our study aims to investigate the role of gC1qR in paclitaxel-induced apoptosis of human ovarian cancer cells and to elucidate its potential molecular mechanism. The level of gC1qR was examined using real-time PCR and western blot analyses. Human ovarian cancer cells viability, migration, and proliferation were detected using the water-soluble tetrazolium salt (WST-1) assay, the transwell assay, and 3H-thymidine incorporation into DNA (3H-TdR) assay, respectively. Apoptosis in cells was assessed using flow cytometric analysis. The intracellular reactive oxygen species was estimated by the fluorescence of H2DCFDA and the mitochondrial membrane potential was tested using a JC-1 probe. The expression of the gC1qR gene decreased significantly in human ovarian cancer tissues relative to the surrounding non-neoplastic ovarian tissues. Cells treated with paclitaxel showed increased gC1qR gene expression, cell apoptosis, and mitochondria dysfunction, and the effects on these cells could be abrogated by the addition of gC1qR small-interfering RNA or α-lipoic acid that was used to protect the mitochondria function. In summary, these data support a mechanism that gC1qR-induced mitochondria dysfunction was involved in the paclitaxel-mediated apoptosis of ovarian cancer cells.

aDepartment of Gynaecology and Obstetrics, Qixia District Maternity and Child Health Care Hospital

bDepartment of Pathology, Nanjing Maternity and Child Health Care Hospital, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, People’s Republic of China

* Kang-Tai Lv and Ling-Juan Gao contributed equally to the writing of this article.

Correspondence to Wei Wang, MD, Department of Pathology, Nanjing Maternity and Child Health Care Hospital, Tianfei Alley, Nanjing Mochou Road, 210004 Nanjing, People’s Republic of China Tel: +86 255 222 6160; fax: +86 258 446 0507; e-mail: gaolingjuan@njmu.edu.cn

This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/

Received June 26, 2017

Accepted September 11, 2017

Back to Top | Article Outline

Introduction

Until the 1990s, paclitaxel was used to resolve the problem of resistance to cisplatin in the treatment of malignant ovarian tumors and its unique anticancer mechanism 1,2. Paclitaxel inhibits the formation of tumor cell microtubule to cause cell cycle arrest in the G2/M phase; therefore, paclitaxel is used widely for the treatment of recurrent ovarian cancer and drug resistance 3,4. Paclitaxel is the preferred chemotherapy in the treatment of ovarian cancer, which has a better curative effect, especially for long-term chemotherapy, which leads to multidrug resistance in patients 5,6. According to the report, the efficiency is 69.3%, with mild adverse reactions and wide clinical application potential 7,8.

As a mitochondrial membrane protein, the globular C1q receptor (gC1qR) is originally isolated from Raji cells 9. gC1qR has complex biological functions and plays an important role in the physiological and pathological processes of antivirus and anti-infection of organisms. gC1qR genes are involved in the process of cell cycle, transcription and translation of proteins, cell senescence, and apoptosis 10–13. In particular, gC1qR is involved in the regulation of cell apoptosis by the anchor on the surface of the mitochondrial membrane, which has generated considerable attention among researchers. Studies have shown that the gC1qR gene can induce the expression of p53 and cause apoptosis of cervical cancer cells 14. Therefore, the role of gC1qR gene in the apoptosis of ovarian cancer cells induced by paclitaxel deserves further investigation. This study aims to comprehensively identify the important role of the gC1qR gene in the apoptosis of ovarian cancer cells induced by paclitaxel and the related mechanism of mitochondrial dysfunction.

Back to Top | Article Outline

Materials and methods

Chemicals and reagents

Human ovarian cancer cell line SKOV3 and CAOV-3 were provided by Hangzhou Hibio Bio-tech Co. Ltd (Hangzhou, Zhejiang, China). Dulbecco’s modified Eagle’s medium powder, fetal bovine serum, and trypsin were purchased from the Gibco (Grand Island, New York, USA); T4DNA ligase and various restriction endonucleases were provided by Invitrogen (Grand Island, New York, USA); and Lipofectin transfection reagent was purchased from Santa Cruz (Santa Cruz, California, USA). Antibodies targeting gC1qR and actin were purchased from Santa Cruz and Cell Signalling Technology (Beverly, Massachusetts, USA). Diethylpyrocarbonate was obtained from Tianjin chemical reagent two factory (Tianjing, China); the annexin V-FITC/propidium iodide Flow Cytometry Assay Kit was obtained from Invitrogen. gC1qR small-interfering RNA (siRNA) and negative siRNA were synthesized by Wuhan Genesil Biotechnology Co. Ltd (Wuhan, China). siRNA was directed toward an unrelated gene as a negative control. gC1qR and actin gene primers were synthesized by Shanghai Boya Biotechnology Co. Ltd (Shanghai, China).

Back to Top | Article Outline

Tissue procurement and preparation

This study was approved by the Ethical Committee of the Chinese Academy of Sciences and the Nanjing Maternity and Child Health Care Hospital. From January 2013 to December 2015, 30 patients (median age 52.1 years, age range between 33 and 67 years) with ovarian cancer were recruited into this study at the Nanjing Maternal and Child Health Care Hospital of Nanjing Medical University; the patients were confirmed by pathology and cytology, and the patient’s personal information was complete. Twenty-five patients had serous cystadenocarcinoma, four patients had mucinous cystadenocarcinoma, and one patient had endometrioid carcinoma. The research specimens were divided into two groups: (a) 30 patients with ovarian cancer, whose pathological diagnosis was definite and (b) 30 patients in the paracancerous tissue group (normal tissue group), in whom no tumor cell infiltration was confirmed by pathology. These specimens were stored at −79°.

Back to Top | Article Outline

Cell culture

Human ovarian cancer cells were cultured in Dulbecco’s modified Eagle’s medium complete culture medium containing 10% calf serum, 1% nonessential amino acids, 2 mmol/l glutamine, and antibiotics (100 U/ml penicillin and streptomycin). SKOV3 and CAOV-3 cells were placed in a 37°C, 5% CO2 saturated moisture incubator. Ovarian cancer cells growth density was greater than 90% and can continue to be passaged.

Back to Top | Article Outline

Cloning and transfection of the gC1qR plasmids

The full length of the gC1qR gene was cloned in-frame into the pcDNA3.1 expression plasmid (Invitrogen) using the BamHI and EcoRI restriction sites by PCR amplification. Each sample included the following primer sets: gC1qR, primer-F (5′-AAT CAC ACG GTA GAC ACT GAA ATG CC-3′), primer-R (5′-CAT CAT CCC ATC TAA AAT GTC CCC TG-3′), and probe sequence (5′-TGC TCC AGT TCA ACC AAC GTC CTT CTC-3′) were used to clone the gC1qR gene. Cells were chosen at 6, 9, and 12 passages and starved for an additional 24 h until the cells became quiescent. The resulting gC1qR vectors were then transfected into cells using lipofectamine 2000 according to the manufacturer’s protocol. The transfection efficiency of plasmids was determined by the expression of reporter gene (enhance green fluorescent protein) in the pcDNA3.1 vector and the optimal time of harvesting ovarian cancer cells was determined.

Back to Top | Article Outline

gC1qR siRNA-expressing plasmid construction

The gC1qR gene was obtained from the NCBI GeneBank. The serial number is Genebank, accession, #U55762. The gC1qR siRNA sequence was as follows: 5′-AAC AAC AGC AUC CCA CCA ACA-3′. An unrelated gene siRNA was used as a negative control; the negative siRNA sequence was as follows: 5′-AAA GTC GAC CTT CAG TAA GGC-3′. The siRNA was synthesized, annealed, and ligated into the BamHI and HindIII restriction sites of the pGenesil-1 expression vector using enhance green fluorescent protein as the reporter gene.

Back to Top | Article Outline

Electron microscopy

The cultured SKOV3 and CAOV-3 cells were digested by trypsin to create a suspension and placed in a centrifuge tube; the cells were centrifuged and compressed at 2000 r/min for 10 min. After the supernatant was removed, 2.5% glutaraldehyde and 1% osmium acid were added and fixed for 10 min; the cell mass was subjected to Epon (Agar Scientific; Stansted, Essex, UK) 812 embedding and ultrathin sectioning. The ultrastructural changes in the mitochondria were observed under a JEM-1010 transmission electron microscope.

Back to Top | Article Outline

Western blot analysis

Ovarian cancer tissues and cells were added to protein lysate and protease inhibitor cocktail on ice for 30 min. 100 μg of total proteins were placed on a 12% SDS-polyacrylamide gel and then transferred onto a PVDF membrane. The membranes were blocked with 5% nonfat milk dissolved in PBST (PBS containing 0.05% Tween 20). After blocking, primary antibodies specific to gC1qR and actin were applied to the membranes. An HRP-conjugated secondary antibody was used at a 1 : 500 dilution. The protein bands were visualized using the enhanced chemiluminescence Western Detection System and gray-scale scanning was performed on a Bio-Rad (Hercules, California, USA) gel imaging analyzer; the expression of the target protein was analyzed.

Back to Top | Article Outline

Cell viability analysis

The effects of different treatments on cells’ viability were detected using the water-soluble tetrazolium salt (WST-1) assay. Cells were cultured for 48 h at 5% CO2, 37°C, and the MTT solution was added to each pore (5 mg/ml, prepared with PBS, i.e., 0.5% MTT) 20 μl. After 4 h, the cell cultures were terminated and the culture supernatant was discarded. DMSO (150 μl) was added to the culture cells and shocked for 10 min to completely dissolve the crystal. Sample absorbance was measured using an enzyme-linked immunosorbent assay at OD 490 nm wavelength. All of the experiments were repeated 3–5 times.

Back to Top | Article Outline

Cell migration analysis

The effects of different treatments on cell migration were measured using the transwell (BD Falcon, New Jersey, USA) assay. Before the experiment, ovarian cancer cells were starved for 24 h and the cells were collected. The ovarian cancer cell suspension (100 μl) was placed in the transwell chamber and to the lower chamber 600 μl culture medium containing 20% FBS was added. The chambers were incubated for 48 h at 37°C, 5% CO2. The migration ability of ovarian cancer cells was calculated by staining the adherent cells directly with 0.1% crystal violet staining. The cells that migrated were counted under a microscopic (×400) in five different fields per filter. Experiments were repeated 3–5 times.

Back to Top | Article Outline

Cell proliferation assay

Cells were seeded into 96-well plates and serum starved for 24 h. The cells were treated with different experimental agents at the indicated times and DNA synthesis was detected by 3H-thymidine incorporation (3H-TdR) for the final 18–24 h. After trypsin digestion of the cells, these media were collected onto a glass fiber filter paper. The radioactivity retained on the dried filters was measured by the addition of 5 ml scintillation solution and counted in a TopCount NxT scintillation counter (LKB Wallac; San Francisco, California, USA).

Back to Top | Article Outline

Detection of apoptotic cells

Cells were treated with different treatments at the indicated times; the cells were washed and resuspended in 250 μl binding buffer. Staining was performed with annexin V-FITC/propidium iodide with flow cytometry analysis. Results: normal cells were found in the Q1-LL region, and the early and late apoptotic cells were distributed in the Q1-LR and Q1-UR regions, respectively. The necrotic cells were located in the Q1-UL region. The staining results of the early and late apoptotic cells were determined by a flow cytometer on a FACS Calibur instrument (BD Biosciences; Franklin Lakes, New Jersey, USA).

Back to Top | Article Outline

Assay of intracellular ROS

The cells were treated with different experimental agents at the indicated times and incubated with 10 μmol/l H2DCFDA for 20 min in the dark. The cells were then lysed with RIPA buffer under ice-cold conditions. The production of intracellular reactive oxygen species (ROS) was detected by inverted fluorescence microscopy at an excitation light wavelength of 488 nm and an emission wavelength of 530 nm. These data were obtained according to the increase in the fluorescence intensity with respect to normoxic untreated controls by subtracting the basal fluorescence levels.

Back to Top | Article Outline

Measurement of the mitochondrial membrane potential (Δψm)

The loss of mitochondrial membrane potential (Δψm) was measured in SKOV3 and CAOV-3 cells after treatments under varying conditions at different time intervals using the fluorescent cationic dye JC-1, which is a mitochondria-specific fluorescent dye. JC-1 was added to ovarian cancer cells at a final concentration of 10 μmol/l; changes in JC-1 monomers were detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm under fluorescence microscopy. The data are representative of 10 individual experiments.

Back to Top | Article Outline

Statistical analysis

The data were expressed as mean±SD. Differences between the various datasets were tested for significance using Student’s t-test and P values less than 0.05 were considered significant (P<0.05; <0.01; >0.05).

Back to Top | Article Outline

Results

The expression of the gC1qR gene in human ovarian tissues

To investigate the relationship between the expression of gC1qR and human ovarian carcinoma, the gC1qR level was detected in 30 cases of human ovarian cancer tissue (T) and 30 cases of surrounding non-neoplastic ovarian tissues (N). Real-time PCR results showed that the expression of gC1qR mRNA was downregulated in 22 ovarian cancer tissues (73.3%) and two surrounding non-neoplastic ovarian tissues (6.66%) (Fig. 1a). Meanwhile, the expression of gC1qR protein was detected by western blot, and the results indicated that the protein expression was downregulated in 27 ovarian cancer tissues (90.0%) and only three cases (10.0%) showed downregulation in paracancerous ovarian tissues (Fig. 1b). Immunohistochemistry was used to localize the gC1qR protein expression. Fig. 1c showed that gC1qR protein is predominantly expressed in the cytoplasm. These above findings suggested that the expression of the gC1qR gene in ovarian cancer tissues was significantly lower than that in its surrounding non-neoplastic ovarian tissues and the difference was significant (P<0.001).

Fig. 1

Fig. 1

Back to Top | Article Outline

The effect of the gC1qR gene on the biological function of SKOV3 and CAOV-3 cells

In this experiment, the effect of gC1qR gene on the proliferation viability of ovarian cancer cells was evaluated, and the pcDNA3.1–gC1qR plasmid and the pcDNA3.1 null carrier plasmid were transfected into cells. The results showed that compared with the PBS group, after transfection of the pcDNA3.1–gC1qR plasmid for 72 h, the proliferation viability rate of cells decreased significantly (P<0.01). The difference between the pcDNA3.1 null carrier group and the PBS group was not statistically significant (P>0.05, Fig. 2a and Supplementary Fig. 1a, Supplemental digital content 1, http://links.lww.com/ACD/A222).

Fig. 2

Fig. 2

Ovarian cancer cell migration ability was assessed by a transwell assay. The data showed that after transfection of the pcDNA3.1–gC1qR plasmid for 72 h, the number of migrating cells was only 47.3% of that of the PBS group, and there was a significant difference between the two groups (P<0.01); the pcDNA3.1 null vector acted on ovarian cancer cells and the results showed no significant difference compared with the PBS group (P>0.05, Fig. 2b and Supplementary Fig. 1b, Supplemental digital content 1, http://links.lww.com/ACD/A222).

The effect of the gC1qR gene on the proliferation of ovarian cancer cells was evaluated using a 3H-TdR incorporation assay. The results showed that compared with the PBS group, after transfection of the pcDNA3.1–gC1qR plasmid for 72 h, the proliferation of SKOV3 and CAOV-3 cells decreased by 70.7%; on comparison of these two groups, a significant statistical difference was found (P<0.001). However, the proliferation capacity of pcDNA3.1 null carriers in ovarian cancer cells in-vitro did not change; compared with the PBS group, the difference was not statistically significant (P>0.05, Fig. 2c and Supplementary Fig. 1c, Supplemental digital content 1, http://links.lww.com/ACD/A222).

In this study, flow cytometry was used to evaluate the effect of overexpression of the gC1qR gene on apoptosis in cells. The data showed that, compared with the PBS group, the apoptosis in ovarian cancer cells increased significantly in the pcDNA3.1–gC1qR plasmid group; the difference was statistically significant (P<0.001). However, in the pcDNA3.1 empty vector group, the apoptosis of ovarian carcinoma cell number showed no change compared with the PBS group, and no statistical significance was found (P>0.05, Fig. 2d and Supplementary Fig. 1d, Supplemental digital content 1, http://links.lww.com/ACD/A222).

Back to Top | Article Outline

Effect of gC1qR on the mitochondrial function of SKOV3 and CAOV-3 cells

This study aimed to explore the gC1qR gene-induced apoptotic process of ovarian cancer cells; there is a close relationship between mitochondrial dysfunction and apoptosis in ovarian cancer cells. The functional parameters of mitochondria were examined. Alterations in mitochondrial oxidative stress in ovarian cancer cells were assessed using changes in mitochondrial ROS content and mitochondrial membrane potential. The experimental data showed that the overexpression of the gC1qR gene could induce ROS production in cells compared with the PBS control group (P<0.001, Fig. 3a and Supplementary Fig. 2a, Supplemental digital content 2, http://links.lww.com/ACD/A223). At the same time, overexpression of gC1qR could significantly reduce the mitochondrial membrane potential of cells (P<0.001, Fig. 3b and Supplementary Fig. 2b, Supplemental digital content 2, http://links.lww.com/ACD/A223). The pcDNA3.1 vector group, ROS production and mitochondrial membrane potential compared with the PBS group, there was no statistical difference (P>0.05).

Fig. 3

Fig. 3

The morphological changes in mitochondria in ovarian cancer cells were observed by transmission electron microscopy. The mitochondrial morphology of ovarian cancer cells was normal in the pcDNA3.1 null carrier group and the PBS group, the mitochondrial cristae were clearly visible, and no significant change was observed. However, mitochondrial morphology showed a significant change in the gC1qR gene plasmid transfection group; the mitochondrial crista were broken, shortened and sparse, the stroma presents a loose state. Also, some mitochondria appeared to be markedly swollen and some mitochondria even appeared to be vacuolated (Fig. 3c and Supplementary Fig. 2c, Supplemental digital content 2, http://links.lww.com/ACD/A223).

Back to Top | Article Outline

Effects of paclitaxel on the expression of the gC1qR protein in ovarian cancer cells

SKOV3 and CAOV-3 cells were treated with paclitaxel for 0, 12, 24, and 36 h, respectively. Real-time PCR analysis showed that paclitaxel could significantly induce the expression of gC1qR mRNA, which began to increase at 12 h; its value was 1.89 times that of 0 h (P<0.05). After 24 h, the expression continued to increase and the value was 2.67 times 0 h (P<0.01); the gC1qR mRNA level peaked at 36 h: up to 4.12 times that of 0 h (P<0.001). The expression of the gC1qR mRNA showed significant time dependence (Fig. 4a and Supplementary Fig. 3a, Supplemental digital content 3, http://links.lww.com/ACD/A224).

Fig. 4

Fig. 4

Meanwhile, the expression of the gC1qR protein was detected by western blot; the experimental results indicated that paclitaxel induced the expression of gC1qR protein at 12, 36, and 48 h, and its value was 1.91, 3.82, and 6.18 times that at 0 h, respectively, (P<0.05; <0.01; <0.001), showing significant dependence on time (Fig. 4b and Supplementary Fig. 3b, Supplemental digital content 3, http://links.lww.com/ACD/A224).

Back to Top | Article Outline

Effects of paclitaxel on the biological function of human ovarian cancer cell lines SKOV3 and CAOV-3

This study aimed to investigate the effect of paclitaxel on the proliferation viability (Fig. 5a, Supplementary Fig. 4a, Supplemental digital content 4, http://links.lww.com/ACD/A225 and Supplementary Fig. 7, Supplemental digital content 7, http://links.lww.com/ACD/A226) and migration ability (Fig. 5b and Supplementary Fig. 4b, Supplemental digital content 4, http://links.lww.com/ACD/A225) of ovarian cancer cells using a WST-1 assay and a transwell assay, respectively. The results showed that compared with the paclitaxel group, cell proliferation viability and migration ability showed a significant increase in the paclitaxel+gC1qR siRNA group; the difference between the two groups was statistically significant (P<0.01). The paclitaxel+negative siRNA group did not show a statistically significant difference compared with the paclitaxel group (P>0.05). The proliferation viability and migration ability were significantly increased in the paclitaxel+α lipoic acid group compared with paclitaxel group; the difference was statistically significant (P<0.01).

Fig. 5

Fig. 5

The effect of paclitaxel on the proliferation of ovarian cancer cells was detected using the 3H-TdR assay (Fig. 5c, Supplementary Fig. 4c, Supplemental digital content 4, http://links.lww.com/ACD/A225 and Supplementary Fig. 6, Supplemental digital content 6, http://links.lww.com/ACD/A227); the results showed that compared with the paclitaxel group, cell proliferation showed a significant increase in the paclitaxel+gC1qR siRNA group and the difference between these two groups was statistically significant (P<0.001). The paclitaxel+negative siRNA group did not show a statistically significant difference compared with the paclitaxel group (P>0.05). The proliferation was significantly increased in the paclitaxel+α lipoic acid group compared with the paclitaxel group; the difference was statistically significant (P<0.001).

Flow cytometry was used to evaluate the effect of paclitaxel on apoptosis in SKOV3 and CAOV-3 cells. The data showed that, compared with the paclitaxel group, the paclitaxel+gC1qR siRNA group, and the paclitaxel+α lipoic acid group, the number of apoptosis of ovarian cancer cells decreased significantly; the difference was statistically significant (P<0.001). However, in the paclitaxel+negative siRNA group, the apoptosis of ovarian carcinoma cell numbers showed no change compared with the paclitaxel group, and there was no statistical significance (P>0.05, Fig. 5d). Electron microscopy images showing typical morphologic changes in ovarian cancer cells, including nuclear chromatin condensation and formation of apoptotic bodies in paclitaxel-treated and paclitaxel+negative siRNA-treated CAOV-3 cells (Supplementary Fig. 4d, Supplemental digital content 4, http://links.lww.com/ACD/A225).

Back to Top | Article Outline

The effect of paclitaxel on mitochondrial function in SKOV3 and CAOV-3 cells

The aim of this study is to investigate whether mitochondrial dysfunction induced by the gC1qR gene plays an important role in the apoptosis of ovarian cancer cells induced by paclitaxel. Alterations in mitochondrial oxidative stress in ovarian cancer cells were determined using changes in the mitochondrial ROS content and the mitochondrial membrane potential. In the paclitaxel+gC1qR siRNA group and the paclitaxel+α lipoic acid group, the intracellular ROS decreased significantly compared with the paclitaxel group; the difference was statistically significant (P<0.01). However, on comparison of the paclitaxel+negative siRNA group and the paclitaxel group in terms of ovarian cancer cell ROS content, there was no statistically significant difference (P>0.05, Fig. 6a and Supplementary Fig. 5a, Supplemental digital content 5, http://links.lww.com/ACD/A228).

Fig. 6

Fig. 6

At the same time, the paclitaxel gC1qR siRNA group and the paclitaxel+α lipoic acid group showed a significant increase in the mitochondrial membrane potential of cells (P<0.01, Fig. 6b and Supplementary Fig. 5b, Supplemental digital content 5, http://links.lww.com/ACD/A228). On comparison of the paclitaxel+negative siRNA group and the paclitaxel group in terms of mitochondrial membrane potential, there was no statistically significant difference (P>0.05).

The morphological changes in the mitochondria of ovarian cancer cells were observed by transmission electron microscopy. The mitochondrial morphology of ovarian cancer cells was normal in the paclitaxel+gC1qR siRNA group and the paclitaxel+α lipoic acid group, the mitochondrial cristae were clearly visible, and no significant change was observed. However, mitochondrial morphology showed a significant change in the paclitaxel group and the paclitaxel+negative siRNA group; the mitochondrial crista were broken, shortened and sparse, the stroma presents a loose state. Also, some mitochondria were markedly swollen and some mitochondria even appeared to be vacuolated (Fig. 6c and Supplementary Fig. 5c, Supplemental digital content 5, http://links.lww.com/ACD/A228).

Back to Top | Article Outline

Discussion

Cell apoptosis, cell proliferation, and cell differentiation are important physiological processes for multicellular organisms to maintain the dynamic balance of cell numbers 15. In mature multicellular organisms, ovarian epithelial cells maintain a dynamic balance between cell production and cell apoptosis 16. Studies have shown that, on the one hand, the absence of genes regulating ovarian epithelial cell growth and differentiation is the reason for the uncontrolled number of cells; on the other, the rate of epithelial cell death is an important reason for the uncontrolled number of cells 17.

Ovarian malignant tumors can be regarded as the inactivation of apoptosis-related gene or overexpression of apoptosis suppressor genes in ovarian epithelial cells. Cell mortality was significantly decreased, resulting in an imbalance of proliferation and apoptosis, which in turn induced malignant ovarian tumors 18. Among a number of genes that induce apoptosis in ovarian epithelial cells, the c-myc gene and the transforming growth factor-β (TGF-β) 1 gene, under certain conditions, can not only stimulate ovarian epithelial cell proliferation but also induce cell apoptosis. In fact, this seeming contradiction fully explains the universal balance mechanism in nature 19. The c-myc gene can promote the ovarian epithelial cell proliferation but also cause ovarian epithelial cells is sensitive to apoptosis. TGF-β 1 can induce apoptosis of ovarian cancer cells, but high concentrations of ovarian epithelial cells can inhibit the induction function of TGF-β 1 20.

gC1qR protein anchors on the surface of the mitochondrial membrane, which has multidirectional regulation functions, such as involvement in cell cycle progression, cell proliferation, protein translation processing, and physiological functions such as cell senescence and apoptosis 9. Moreover, the uncertainty of the distribution of the gC1qR protein at different stages and in different cells makes gC1qR gene have a diverse biological function 14. As a complement C1q receptor, gC1qR can mediate the apoptosis of monocytes and affect the biological function of monocytes under the action of exogenous chemical toxins 21. Our experimental data show that the expression of the gC1qR gene in ovarian cancer tissues (mainly stage I/II patients) was significantly lower than that in its surrounding non-neoplastic ovarian tissues, and the difference was significant, but some studies 22 have focused on the III/IV stages of ovarian cancer patients. These data indicated that gC1qR gene overexpression in ovarian carcinomas is related to a decrease in overall survival and progression-free survival. The inconsistencies in the conclusions may be attributable to inconsistencies in the phases of specimen collection. Moreover, our experimental data show that when the pcDNA3.1–gC1qR vector was transfected with ovarian cancer cells for 72 h, its proliferation viability, migration, and proliferation capacity decreased significantly, whereas the number of apoptotic cells in ovarian cancer cells increased significantly. These results suggest that the gC1qR gene can induce significant apoptosis in ovarian cancer cells.

Mitochondria are the major organelles responsible for triggering endogenous apoptotic pathways in cells; the permeability of the mitochondria membrane increases under stress and the release of Cyt C and Apaf-1 binding to activate the caspase family and trigger the apoptosis program 23. When exogenous substances stimulated cells, mitochondria are the main site of ROS production 24. High levels of ROS cause mitochondrial damage, and then lead to changes in the mitochondrial membrane potential, release of p21, Bax, and caspase-3 and other apoptosis-inducing factors to induce apoptosis 25. In our study, overexpression of gC1qR as a membrane protein anchored on the mitochondria could significantly induce apoptosis in ovarian cancer epithelial cells and gC1qR could also effectively induce mitochondrial function changes. The experimental results show that, first of all, overexpression of the gC1qR gene could significantly induce the production of ROS in ovarian cancer cells. Second, JC-1 fluorescence data showed that the gC1qR gene could significantly reduce the mitochondrial membrane potential of ovarian cancer cells. Finally, mitochondrial cristae were broken and disappeared, the matrix presents a loose state, part of the mitochondrion swelling, around the nucleus was densely arranged, or even mitochondrial vacuoles were present. The results of this study are in good agreement with those reported in the literature.

Paclitaxel is a first-line chemotherapy regimen for the treatment of ovarian cancer; especially, better efficacy has been found in ovarian cancer patients with long-term resistance to chemotherapy 26. Paclitaxel is a two terpenoid which can inhibit cell division and reproduction by inhibiting the mitosis of ovarian cancer cells, thus exerting antitumor effects 27. According to the literature reports, paclitaxel is approved by clinicians in the treatment of ovarian cancer, whether on its own or in combination with other agents, and has good antitumor efficacy 28. To date, paclitaxel is the only plant medicine that can effectively inhibit the growth of tumor cells. This study showed that paclitaxel could significantly induce apoptosis of ovarian cancer cells in vitro, which is consistent with the reports of Zom 29.

In this study, paclitaxel could induce apoptosis of ovarian cancer cells; it also could significantly induce gC1qR gene expression in the process. The gC1qR expression reached its peak at 36 h and its expression showed significant time dependence. Meanwhile, our experimental results showed that the gC1qR gene could induce apoptosis of ovarian cancer cells; the gC1qR gene-induced apoptosis of ovarian cancer cells by triggering mitochondrial dysfunction. The experimental results showed that the apoptosis of ovarian cancer cells induced by paclitaxel decreased significantly when the gC1qR gene was silenced, which was equivalent to the effect of α lipoic acid on mitochondrial protection. Further observation showed that the gC1qR gene was silenced, paclitaxel in ovarian cancer cells could reduce damage to the mitochondria; mitochondrial morphology, ROS level, and mitochondrial membrane potiential were gradually returning to normal.

In summary, this study describes the paclitaxel-induced apoptosis of ovarian cancer cells and the related molecular mechanism of gC1qR gene-induced mitochondrial dysfunction. The exploration of this scientific problem may provide new clues and potential intervention targets for the prevention and treatment of chemotherapy resistance.

Back to Top | Article Outline

Acknowledgements

The authors thank all the staff in the department of clinical laboratory in Nanjing Maternity and Child Health Care Hospital affiliated to Nanjing Medical University for guidance.

This study was supported by grants from the National Natural Science Foundation of China (contract grant number: 81571437) and the National Natural Science Foundation of Jiangsu Province (contract grant number: BK20151078).

Back to Top | Article Outline

Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

References

1. Yin H, Kristensen SG, Jiang H, Rasmussen A, Andersen CY. Survival and growth of isolated pre-antral follicles from human ovarian medulla tissue during long-term 3D culture. Hum Reprod 2016; 31:1531–1539.
2. Hainsworth JD, Thompson DS, Bismayer JA, Gian VG, Merritt WM, Whorf RC, et al. Paclitaxel/carboplatin with or without sorafenib in the first-line treatment of patients with stage III/IV epithelialovarian cancer: a randomized phase II study of the Sarah Cannon Research Institute. Cancer Med 2015; 4:673–681.
3. Li Y, Wan X, Wei Y, Liu X, Lai W, Zhang L, et al. LSD1-mediated epigenetic modification contributes to ovarian cancer cell migration and invasion. Oncol Rep 2016; 35:3586–3592.
4. Choi CH, Kim MK, Park JY, Yoon A, Kim HJ, Lee YY, et al. Safety and efficacy of aprepitant, ramosetron, and dexamethasone for chemotherapy-induced nausea and vomiting in patients with ovarian cancer treated with paclitaxel/carboplatin. Support Care Cancer 2014; 22:1181–1187.
5. Szender JB, Eng KH, Matsuzaki J, Miliotto A, Gnjatic S, Tsuji T, et al. HLA superfamily assignment is a predictor of immune response to cancer testis antigens and survival inovarian cancer. Gynecol Oncol 2016; 142:158–162.
6. Shawky H, Tawfik H, Hewidy M. Weekly dose-dense paclitaxel and carboplatin in recurrent ovarian carcinoma: a phase II trial. J Egypt Natl Canc Inst 2014; 26:139–145.
7. Ciccone MA, Maoz A, Casabar JK, Machida H, Mabuchi S, Matsuo K. Clinical outcome of treatment with serine-threonine kinase inhibitors in recurrent epithelial ovarian cancer: a systematic review of literature. Expert Opin Investig Drugs 2016; 25:781–796.
8. Yoshihama T, Chiyoda T, Kataoka F, Nomura H, Iguchi Y, Hashimoto S, et al. Effectiveness of third-line chemotherapy in recurrent ovarian cancer patients. Eur J Gynaecol Oncol 2015; 36:424–427.
9. Paasonen L, Sharma S, Braun GB, Kotamraju VR, Chung TD, She ZG, et al. New p32/gC1qR ligands for targeted tumor drug delivery. Chembiochem 2016; 17:570–575.
10. Yu Y, Gaillard S, Phillip JM, Huang TC, Pinto SM, Tessarollo NG, et al. Inhibition of spleen tyrosine kinase potentiates paclitaxel-induced cytotoxicity in ovarian cancer cells by stabilizing microtubules. Cancer Cell 2015; 28:82–96.
11. Abedi H, Aghaei M, Panjehpour M, Hajiahmadi S. Mitochondrial and caspase pathways are involved in the induction of apoptosis by IB-MECA in ovarian cancercell lines. Tumour Biol 2014; 35:11027–11039.
12. Pednekar L, Valentino A, Ji Y, Tumma N, Valentino C, Kadoor A, et al. Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy. Mol Immunol 2016; 74:18–26.
13. Kouokam Fotso GB, Bernard C, Bigault L, de Boisséson C, Mankertz A, Jestin A, et al. The expression level of gC1qR is down regulated at the early time of infection with porcine circovirus of type 2 (PCV-2) and gC1qR interacts differently with the Cap proteins of porcine circoviruses. Virus Res 2016; 220:21–32.
14. Chen ZL, Gu PQ, Liu K, Su YJ, Gao LJ. The globular heads of the C1q receptor regulate apoptosis in human cervical squamous carcinoma cells via ap53-dependent pathway. J Transl Med 2012; 10:255.
15. Yu T, Lin W. Small-molecule GSK-3 inhibitor rescued apoptosis and neurodegeneration in anesthetics-injured dorsal root ganglion neurons. Biomed Pharmacother 2016; 23:395–402.
16. Chiang CK, Wang CC, Lu TF, Huang KH, Sheu ML, Liu SH, et al. Involvement of endoplasmic reticulum stress, autophagy, and apoptosis in advanced glycation end products-induced glomerular mesangial cell injury. Sci Rep 2016; 26:34167.
17. Zhang G, Liu Z, Han Y, Wang X, Yang Z. Overexpression of miR-509 increases apoptosis and inhibits invasion via suppression of tumor necrosis factor-α in triple-negative breast cancer Hs578T cells. Oncol Res 2016; 24:233–238.
18. Paul BT, Blanchard Z, Ridgway M, ElShamy WM. BRCA1-IRIS inactivation sensitizes ovarian tumors to cisplatin. Oncogene 2015; 34:3036–3052.
19. Wang X, Duan W, Li X, Liu J, Li D, Ye L, et al. PTTG regulates the metabolic switch of ovarian cancer cells via the c-myc pathway. Oncotarget 2015; 6:40959–40969.
20. Sun W, Gui L, Zuo X, Zhang L, Zhou D, Duan X, et al. Human epithelial-type ovarian tumour marker beta-2-microglobulin is regulated by the TGF-β signaling pathway. J Transl Med 2016; 14:75.
21. Du Q, Huang Y, Wang T, Zhang X, Chen Y, Cui B, et al. Porcine circovirus type 2 activates PI3K/Akt and p38 MAPK pathways to promote interleukin-10 production in macrophages via Cap interaction of gC1qR. Oncotarget 2016; 7:17492–17507.
22. Yu G, Wang J. Significance of hyaluronan binding protein (HABP1/P32/gC1qR) expression in advanced serous ovarian cancer patients. Exp Mol Pathol 2013; 94:210–215.
23. Mohibbullah M, Hannan MA, Choi JY, Bhuiyan MM, Hong YK, Choi JS, et al. The edible marine alga gracilariopsis chorda alleviates hypoxia/reoxygenation-induced oxidative stress in cultured hippocampal neurons. J Med Food 2015; 18:960–971.
24. Gu ZT, Li L, Wu F, Zhao P, Yang H, Liu YS, et al. Heat stress induced apoptosis is triggered by transcription-independent p53, Ca(2+) dyshomeostasis and the subsequent Bax mitochondrial translocation. Sci Rep 2015; 5:11497.
25. Cai J, Cao S, Chen J, Yan F, Chen G, Dai Y. Progesterone alleviates acute brain injury via reducing apoptosis and oxidative stress in a rat experimental subarachnoid hemorrhage model. Neurosci Lett 2015; 600:238–243.
26. Hu L, Lv QL, Guo Y, Cheng L, Wu NY, Qin CZ, et al. Genetic variation of CYP3A5 influences paclitaxel/carboplatin-induced toxicity in Chinese epithelial ovarian cancer patients. J Clin Pharmacol 2016; 56:349–354.
27. Otsuka I, Takaya H, Takagi K, Tanaka A, Kaseki H, Izuta C, et al. Carcinosarcoma of the ovary treated with paclitaxel and carboplatin chemotherapy – a report of 4 cases. Gan To Kagaku Ryoho 2013; 40:1249–1253.
28. Lee HY, Yang BM, Hong JM, Lee TJ, Kim BG, Kim JW, et al. Cost-utility analysis for platinum-sensitive recurrent ovarian cancer therapy in South Korea: results of the polyethylene glycolated liposomal doxorubicin/carboplatin sequencing model. Clinicoecon Outcomes Res 2013; 5:297–307.
29. Zorn KK. Challenges to the paclitaxel/carboplatin algorithm in ovarian cancer treatment. Oncology (Williston Park) 2010; 24:738–740.
Keywords:

apoptosis; gC1qR gene; mitochondria function; ovarian cancer cells; paclitaxel

Supplemental Digital Content

Back to Top | Article Outline
Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.