Cervical cancer is the fourth leading cause of cancer death in women, and is one of the most common malignant tumors of the reproductive system. However, more effective treatment for cervical cancer is needed. In this study, we aim to investigate whether N-acetyl-l-cysteine (NAC) could inhibit the proliferation of human papillomavirus (HPV)-positive cells, and reduce cervical carcinogenesis.
Methods
The cervical cancer cell lines SiHa, HeLa, HPV-negative cell line C33A, and the immortalized human cervical keratinocyte cells S12 were used. The protein expression was determined using Western blot assay. mRNA expression was determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation was determined by Cell Counting Kit-8 assay. Cell apoptosis was evaluated using Annexin V-FITC apoptosis kits. The numbers of colonies were measured using colony-forming assay. Xenograft tumor necrosis and HPV16 E7 expression were determined using hematoxylin and eosin (H&E) staining and immunohistochemistry.
Results
Our results showed that NAC treatment at the concentration of 1.5 mM significantly promoted cell apoptosis and reduced cell growth by inhibiting HPV16 E7 expression. NAC inhibited HPV16-oncoprotein-induced hypoxia-inducible factor (HIF)-1α protein expression and Akt activation in vitro. Additionally, NAC suppressed tumor growth, as evidenced by the smaller tumor size in the xenograft mouse model and decreased HPV16 E7 expression in tumor tissues.
Conclusion
Our findings demonstrate that NAC exhibits the potential to promote HPV-positive cell apoptosis, and suppress the proliferation of HPV-positive cells by inhibiting cell inhibitor of apoptosis protein 2 and HIF-1α.
1. NAC regulates the expression of HPV16 E7 and inhibits the proliferation of HPV-positive cells.
2. NAC restores cell inhibitor of apoptosis protein 2 (c-IAP2)-mediated apoptosis.
3. NAC exerts an inhibitory effect on HPV16-positive cells by inhibiting HIF-1α.
1 Introduction
Cervical cancer, as one of the most common malignant tumors of the reproductive system in women, is the fourth leading cause of cancer death [1, 2]. Human papillomavirus (HPV) infection has been clearly identified as the main cause of cervical cancer. Several studies have confirmed that persistent infection with high-risk HPV types is the main cause of cervical lesions and is necessary for the pathogenesis of cervical cancer [3‐5]. More than 200 subtypes of HPV have been identified, of which about 40 or more can infect the cervix and are recognized as the most common pathogens of sexually transmitted infections internationally [6, 7]. According to its carcinogenic risk, it can be divided into two categories: low-risk types mainly cause ectopic warts and low-grade cervical intraepithelial neoplasia (CIN), such as HPV42 and 81; high-risk types are also associated with external genital cancer, CIN, and cervical cancer. Among the cervical cancer population, HPV16 accounts for the highest proportion [8].
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HPV oncogenes E6/E7 can evade host immune surveillance through various pathways, leading to persistent viral infection and the development of cervical lesions, which eventually lead to the development of cervical cancer [9, 10]. E6 and E7 are widely believed to cause cervical cancer proliferation and progression. Numerous basic studies and clinical trials have targeted the E6/E7 genes to search effective treatments for cervical cancer. For example, Jin et al. genetically engineered T cells to target the HPV16 E7 gene, leading to regression of HPV16-infected cervical cancer in a xenograft mouse model [11].
N-acetylcysteine (NAC) treatment has been shown to inhibit chronic obstructive pulmonary disease progression and prevent contrast-induced kidney damage and pulmonary fibrosis. NAC can also be used as a chemopreventive agent for cancer and as an adjunct to Helicobacter pylori eradication, as well as to prevent gentamicin-induced hearing loss in renal dialysis patients [12]. More effective approaches to manage and treat HPV infection-induced cervical cancer are needed. In this study, we investigated whether NAC could inhibit HPV-positive cell proliferation using HPV cell lines and HPV model mice.
2 Methods
2.1 Cell Culture
The cervical cancer cell lines SiHa, HeLa, and HPV-negative cell line C33A (ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY) and 100 U/ml penicillin and streptomycin (Invitrogen, Waltham, MA) in a humidified incubator at 37 °C and the presence of 5% CO2. S12 cells, as an immortalized human cervical keratinocyte cell line containing the integrated HPV16 genome, were cultured in a 1:3 mix of DMEM and Ham F12 medium supplemented with 5% FBS, 8.4 ng/mL cholera toxin, 5 µg/mL insulin, 24.3 µg/mL adenine, 0.5 µg/mL hydrocortisone, and 10 µg/ml of epidermal growth factor.
2.2 Western Blot
Protein was collected and quantified at 48 h after transfection of the CRISPR plasmid. Same amount of total proteins were loaded to the sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The primary antibodies against HPV16-E7 (1:1000; Biorbyt, Shanghai, China), retinoblastoma (RB; 1:800; Proteintech, Rosemont, IL), cyclin-dependent kinase 2 (CDK2; 1:500; Proteintech), E2F transcription factor 1 (E2F1; 1:1000; Proteintech), total-Akt (t-Akt; 1:800; Abcam, Shanghai, China), phosphorylated Akt (p-Akt; 1:800; Abcam), total extracellular signal-regulated kinase 1/2 (t-ERK1/2; 1:500; Abcam), or phosphorylated-ERK1/2 (p-ERK1/2; 1:500; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2000; Proteintech) were used. Then, blots were incubated with horseradish-peroxidase-conjugated secondary antibodies, and the bands were visualized using chemiluminescence. The experiments were repeated at least 3 times.
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2.3 RT-PCR
Total mRNA was extracted using RNeasy Kit (Qiagen, Valencia, CA). Reverse transcription polymerase chain reaction (RT–PCR) was performed using Super Script One Step RT-PCR kit (Invitrogen). The primers listed below were used: cell inhibitor of apoptosis protein 2 (c-IAP2) forward, 5´-CTACGTATTCCACTTTTCCT-3´; c-IAP2 reverse, 5´-AAGTACTCACACCTTGGAAACCA-3´. cIAP2 was normalized to GAPDH. The relative density was determined using image J analysis software (NIH Image 1.61).
2.4 Cell Proliferation Assay
Cell proliferation assay was performed to assess cell growth using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan). S12, SiHa, C33A, and HeLa cells were seeded in a 96-well plate with 2 × 103 cells per well. Cells were treated with 1.5 mM NAC or phosphate-buffered saline (PBS) for 48 h. Briefly, 10 μL of CCK-8 dye and 90 μL of DMEM was added to each well and incubated for 3 h according to the manufacture protocol. The absorbance was read at 450 nm.
2.5 Apoptosis Assay
SiHa, C33A, HeLa, and S12 cells were pretreated with 1.5 mM NAC or PBS. Following that, cells were collected and washed 3 times with PBS. Cell apoptotic fractions were determined using Annexin V-FITC apoptosis kits (KenGen Biotech, Nanjing, China).
2.6 Colony-Forming Assay
After 48 h treatment with NAC or PBS, SiHa and S12 cells were washed with PBS and stained with 4% crystal violet and scanned. The colony numbers were counted using ImageJ.
2.7 Xenograft
All animal experiments were approved by the Institutional Animal Care and Use Committee of Peking University International Hospital. Four-week-old Balb/c-nu female mice (weighing 15–16 g) were used. Animals were housed in a pathogen-free environment with a consistent temperature and 12-h light/dark cycles. They received unlimited amounts of water and the diet. Balb/c-nu mice were injected subcutaneously in the right flanks with 5 × 106 of S12 cells. Then, mice were treated with saline or NAC when the xenografts reached approximately 50 mm3. The xenografts were measured every 6 days after treatment with saline and different doses of NAC (1.0, 1.5 or 3.0 mM). Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining of HPV16 E7 in the tissues of S12 xenografts were conducted. Tumor volume was measured individually every 2 days. Tumor volume was calculated using the formula: Volume = (width2 × length)/2.
2.8 Immunohistochemistry
The transplanted tissues (xenografts) in nude mice were removed and fixed with 4% paraformaldehyde. Paraffin-embedded sections (5 μm) were subjected to antigen for 30 min and then treated with 3% hydrogen peroxide for 20 min. Then, the sections were incubated with the antibody overnight at 4 °C (rabbit anti-HPV16 E7 (1:100; Biorbyt), followed by incubation of sections with rabbit secondary antibodies at room temperature. Lastly, the sections were developed with 3,3´-diaminobenzidine (DAB) reagent kit and finally photographed. The intensity was quantified using ImagePro Plus. Using the threshold calculation function of ImageJ, colored areas were selected for area quantification. Then the quantification result of HPV16 E7 was compared with the result of H&E to obtain the percentage of necrotic area.
2.9 Statistical Analysis
Comparisons were conducted by two-way analysis of variance (ANOVA) and followed Tukey’s multiple comparisons test or Student’s t test. Data were presented as mean ± standard deviation (SD). A significant difference was regarded when p < 0.05.
3 Results
3.1 NAC Inhibits the Expression of HPV16 E7 and Recovers the Expression of the Related Protein in HPV16-Positive Cell Lines
It has been revealed that E7 is a key factor in HPV16 pathogenesis, and its expression reflects the activity of HPV16-positive [13, 14]. To investigate whether the function of HPV16-positive cells is affected by NAC, different concentrations of NAC were obtained. Our pilot study showed that NAC at concentrations below than 1 mM had no obvious effect on E7 protein expression (data now shown). It was further found that, when treated cells with NAC at a concentration of 1.5 mM, NAC could effectively inhibit E7 protein expression in both S12 and SiHa cells. With the treatment of 1.5 mM NAC, the protein expression of HPV16 E7 was significantly reduced when compared with the control (Fig. 1A, B, S1). Notably, downstream target RB was increased relative to control, whereas CDK2 and E2F1 were decreased in S12 and SiHa cells (Fig. 1C, D). Consistently, NAC treatment significantly inhibited HPV16 E6 protein expression in both S12 and SiHa cells (Fig. 1E, F, S1). These findings imply that NAC effectively inhibits HPV16-positive cells function.
Fig. 1
NAC inhibits the expression of HPV16 E7. HPV16 E7/RB/CDK2/E2F1 expression of S12 (A, C) and SiHa (B, D) cells, and HPV16 E6 expression in S12 (E) and SiHa (F) cells 48 h after treatment with PBS and NAC at the indicated concentrations
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3.2 NAC Induces Apoptosis in HPV16-Positive Cells
The HPV E2, E6, and E7 have been shown to inhibit cell growth and lead to cycle arrest of the associated tumor cells [15, 16]. HPV oncogene E7 can interact with the tumor suppressor gene RB and thus inhibit apoptosis. To evaluate the effects of NAC on apoptosis, we measured apoptotic fraction in different cell lines after NAC treatment. We found that NAC promoted apoptosis in S12 and SiHa cells expressing HPV16 gene when compared with the control (Fig. 2A, B). In contrast, there was no significant change between NAC and the control in the C33A (without HPV gene) and HeLa cells (containing integrated HPV18 gene) (Fig. 2C, D).
Fig. 2
NAC induces apoptosis in HPV16-positive cells. The apoptosis rate of S12 (A), SiHa (B), C33A (C), and HeLa (D) cells 48 h after treatment with PBS and 1.5 mM NAC. n = 3 replicates. ****p < 0.0001. ns indicates p > 0.05. D RT–PCR analysis. RT–PCR was performed using SuperScript One Step RT–PCR kit. GAPDH mRNA was measured as a loading control. The numbers represent the relative density of c-IAP2 PCR product after normalization against GAPDH
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It is also known that the HPV16 mediated inhibition of apoptosis via the transcription of c-IAP2 [16], so the transcript levels of c-IAP2 were examined. We found that c-IAP2 was significantly repressed by NAC in S12 and SiHa cells, while no change was observed in C33A and HeLa cells (Fig. 2E). This implies that NAC was able to promote apoptosis via specific repressive effects on HPV16.
3.3 NAC Induces Inhibition of Cell Growth of HPV16-Positive Cells In Vitro
To determine whether the growth of HPV16-positive cells is affected by NAC, HPV16-positive cell lines S12 and SiHa, HPV18-positive cell line HeLa, and HPV16-negative cell lines C33A were treated with PBS or 1.5 mM NAC for 48 h. We found that the growth of HPV16-positive cells (S12 and SiHa cells) was decreased with the treatment of NAC compared with the control (Fig. 3A, B). Cell growth did not change in HPV16-negative cell line C33A and HPV18-positive cell line HeLa cells with or without NAC treatment (Fig. 3C, D). The number of cell clones was detected by colony-forming assay and showed that NAC reduced the number of cell clones significantly when compared with the control (Fig. 3E). These findings indicate that NAC can inhibit HPV16 in vitro, whereas HPV18 is nonresponsive to NAC treatment.
Fig. 3
NAC inhibits cell growth of HPV16-positive cells in vitro. A Growth curves of PBS- and NAC-treated S12 cell. B Growth curves of PBS- and NAC-treated SiHa cell. C Growth curves of PBS- and NAC-treated C33A cell. D Growth curves of PBS- and NAC-treated HeLa cell. E Quantification of the number of colonies in S12 and SiHa cells of different treatment groups (n = 3 replications). The experiments were repeated independently 3 times
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3.4 NAC Inhibits HPV16-Oncoprotein-Induced HIF-1α Protein Expression and Akt Activation in S12 and SiHa Cells
To further investigate the mechanism of NAC inhibition in HPV16-positive cells, we assessed the protein expression of HIF-1α, phosphorylated Akt (p-Akt), and phosphorylated ERK (p-ERK1/2). NAC treatment effectively suppressed the upregulation of HIF-1 and p-Akt, while the p-ERK was not affected (Fig. 4A, D; S2). These results suggest that NAC may inhibit the growth of HPV16-positive cells by reducing HIF-1 protein expression and Akt activity.
Fig. 4
NAC inhibits HPV16-oncoprotein-induced HIF-1α protein expression and Akt activation in S12 and SiHa cells. HIF-1α expression of S12 (A) and SiHa (B) cells 48 h after treatment with PBS and NAC. Phosphorylated Akt (p-Akt), and phosphorylated ERK1/2 (p-ERK1/2) levels of S12 (C) and SiHa (D) cells 48 h after treatment with PBS and NAC
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3.5 NAC Induces Inhibition of Cell Growth of HPV16-Positive Cells In Vivo
To investigate whether the effect of NAC on HPV16-positive cells is also present in vivo, we established xenografts models by inoculating S12 cells in Balb/c nude mice subcutaneously. A concentration gradient experiment was first done to find the optimal in vivo dosing concentration. We found that NAC effectively reduced tumor size in a dose-dependent manner (Fig. 5A). NAC 3.0 mM exhibited the most significant effect, which was chosen for IHC staining. Following this, H&E staining and immunohistochemistry staining were carried out on the xenograft tumor sections with HPV16 E7 antibody. The results showed that necrosis and necrotic area were significantly reduced by the treatment of NAC when compared with the control (Fig. 5B, C). Correspondingly, HPV16 E7 expression was significantly reduced by NAC treatment compared with control (Fig. 5B, D). These findings suggest that NAC plays a key role in inhibiting the pathogenic process of HPV in vivo.
Fig. 5
NAC inhibits cell growth of HPV16-positive cells in vivo. Balb/c-nu mice were injected subcutaneously in the right flanks with 5 × 106 of S12 cells. Then, mice were treated with PBS or NAC when the xenografts reached approximately 50 mm3. The xenografts were measured every 6 days after treatment with PBS and NAC. n = 3 at each time point for each group. A Tumor size at indicated time points. B Representative pictures of H&E staining and IHC staining of HPV16 E7. Necrosis area (C) and relative expression of HPV16 E7 (D) in the control and NAC-treated groups. Scale bars: 50 μm. The experiments were independently repeated 3 times
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4 Discussion
Cervical cancer is one of the top four common gynecologic diseases worldwide, usually caused by persistent infection with HPV [1]. More than 80% of women may become infected with HPV during their lifetime [17]. Previous studies have found that integrating the HPV genes into the human genome greatly exacerbates the incidence and development of cervical cancer [18]. Currently, effective treatment for HPV-induced cervical cancer is limited. Here, experiments were conducted using HPV cell lines and HPV mouse model to investigate whether NAC can promote apoptosis and inhibit proliferation in HPV-positive cells. We found that NAC can inhibit the proliferation of HPV-positive cells in vitro and in vivo by inhibiting HIF-1α.
HPV16/18 plays an important role in the development of cervical cancer, and E2, E6, and E7 are the main genes in which HPV16/18 plays an oncogenic role [19, 20]. It has been reported that the prevalence of HPV16 infection among cervical cancer patients is as high as 50–60%, HPV 18 infection is 10–12%, and HPV16/18 detection is as high as 65–72% [21]. Williams et al. reported that the viral load of HPV 16-E6-DNA in cervical lesions of patients with cervical cancer was significantly higher than that of controls and was closely related to the long-term prognosis of patients with cervical cancer [22]. Dong et al. showed that the 10-year cumulative risk of CIN II-III in HPV16-positive patients was 47.5%, and high-risk types of HPV such as HPV31, 33, 52, and 58 also have high infection rates [23]. The protein encoded by E7 gene can degrade RB and release E2F transcription factor, which affects the regulation of cell cycle and induces malignant cell proliferation [24, 25]. Kong et al. found that overexpression of HPV E7 protein can upregulate miR-21 expression, thus promoting the proliferation and invasion of HeLa cells [26]. In contrast, inhibition of HPV16 E7 expression downregulated miR-21 expression in HeLa cells and inhibited cell proliferation and invasion. In agreement with the published work, we found that NAC treatment can effectively inhibit HPV16 E7 expression in a dose-dependent fashion in S12 and SiHa cells. These results were confirmed in xenograft tumor, evidenced by reduced tumor size and decreased HPV16 E7 expression in tumor tissues by NAC treatment.
Cell inhibitor of apoptosis protein 2 (c-IAP2) is a member of the apoptosis inhibitory protein family and is one of the most potent apoptosis inhibitors identified to date, with potent inhibitory functions [16]. c-IAP2 is highly expressed in many tumors, including human kidney cancer and non-small-cell lung cancer [27‐29]. Tamm et al. showed that c-IAP2 expression is low in normal tissues [30]. However, they found that c-IAP2 was highly expressed in many tumor cells and was associated with inhibiting cell apoptosis and resistance to multiple drugs [30]. Li et al. showed that activation of c-IAP2 inhibited caspase-4 activation in the development of sinonasal squamous carcinoma, leading to proliferation of tumor cells [31]. It has been suggested that hypoxia can enhance the anti-apoptotic ability of tumor cells via the anti-apoptotic proteins IAP-2, Bcl-2, and Bcl-xl, which are highly expressed under hypoxic conditions [32, 33]. In addition, the PI3K-AKT signaling pathway plays an important role in cell growth and proliferation [34]. It was found that HIF-1α and vascular endothelial growth factor (VEGF) expression were positively correlated in cervical cancer tissues, suggesting that HIF-1α may activate hypoxic pathways by upregulating VEGF expression, allowing cancer cells to adapt to the hypoxic environment and promoting proliferation of cervical cancer [35, 36]. In this study, we found that NAC treatment significantly inhibited tumor cell growth and promoted cell apoptosis via inhibiting c-IAP2 expression. Interestingly, HIF-1α was significantly reduced by NAC treatment. In parallel, NAC treatment reduced AKT activation, whereas increased EKR activation in S12 and SiHa cells.
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However, the current study has a few limitations. Firstly, we only evaluated the effects of NAC on HPV16 E7. To gain a comprehensive knowledge of NAC in inhibiting cervical cancer cell proliferation, other subtypes of HPV should be included in the future study. Secondly, the HPV16 E7 expression in patients with cervical cancer should be evaluated in the future research. Thirdly, to confirm the role of NAC in inhibiting HPV16 E7, a transfected cell line overexpressing HPV16 E7 should be included. In addition, we only used one cell line for xenograft. The phenotypic trends in S12 and SiHa were consistent. Besides, S12 was more sensitive to lower concentrations of NAC and had a relatively more pronounced phenotype. Therefore, we chose S12 to construct xenograft model.
5 Conclusions
Our results demonstrate that NAC promotes apoptosis in HPV-positive cells. Moreover, our results highlight that NAC effectively inhibits the proliferation of HPV-positive cells by inhibiting cIAP2 and HIF-1α. Our results provide the experimental evidence to support that NAC could serve as a potential pharmacological agent to prevent and treat cervical cancer.
Declarations
Funding
None.
Conflict of Interest
None declared.
Ethical approval
All animal experiments were approved by the institutional animal care and use committee of Peking University International Hospital.
Data Availability
Data would be made available on reasonable request to the corresponding author.
Consent to participate
Not applicable.
Consent for publication
All authors gave consent for publication.
Authors’ Contributions
W.G and W.J conducted the experiments, analyzed the data, and wrote the manuscript. W.G supervised the study.
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