Background. Cervical cancer is the 2nd most frequently diagnosed gynecological cancer. Therefore, it is clinically significant to discover an effective anti-cancer approach.
Objectives. This study aimed to investigate the effects of low-intensity ultrasound irradiation (USI) on cervical cancer cells and associated mechanisms of cell death.
Materials and methods. Normal human cervical HaCaT cells and cervical cancer cell lines C33A, Hela and Siha were cultured and γ-rays applied at a dosage of 2.0 Gy/min. The MTT assay was then used to assess viability (proliferation) of HaCaT, C33A, Hela, and Siha cells. Small interfering RNA (siRNA) sequences that silence the glucose-related protein (GRP78) gene were synthesized. Structural changes to cells exposed to USI were observed with scanning electron microscopy. Immunocytochemistry and western blotting were utilized to examine GRP78, C/EBP-homologous protein (CHOP), phosphorylated JNK (p-JNK), and caspase-12 expression in cervical cancer cells.
Results. Ultrasound irradiation reduced the viability of cervical cancer cells and increased apoptosis, compared to untreated tumor cells (p < 0.05). This effect was not apparent on HaCaT cells. Ultrasound irradiation also induced formation of apoptotic bodies compared to untreated tumor cells (p < 0.05), and activated endoplasmic reticulum (ER) stress-associated apoptosis compared to untreated tumor cells (p < 0.05), by triggering GRP78, CHOP and caspase-12 expression. Moreover, USI triggered ER stress by upregulating GRP78 expression. Remarkably, USI triggered phosphorylation of JNK compared to untreated tumor cells (p < 0.05). Ultrasound irradiation initiated phosphorylation of JNK by increasing GRP78 expression. Silencing of GRP78 further enhanced the effect of USI on tumor cells.
Conclusions. Ultrasound irradiation significantly inhibited proliferation and induced apoptosis of cervical cancer cells by initiating ER stress associated with apoptosis signaling pathways and triggering phosphorylation of JNK.
Key words: apoptosis, cervical cancer, ER stress, ultrasound irradiation
Cervical cancer is the 2nd most frequently diagnosed gynecological cancer and the 4th leading cause of tumor-associated death for females worldwide.1, 2 About 275,100 women are diagnosed with cervical cancer and die every year, which accounts for 9% of all cancer-associated female mortality.3 Among the deaths from cervical cancer, approx. 85% occurs in developing countries,4 where patients lack the effective therapy resources for cervical cancer treatment. Therefore, it is clinically significant to discover an effective anti-cancer approach and clarify the associated mechanisms of cervical cancer proliferation.
According to previous investigations,5, 6 non-invasive surgery for cancer treatment that induces cancer cell apoptosis, is an effective approach for killing the cancer cells. In recent years, ultrasound irradiation (USI) has been proven to induce apoptosis of some tumor cell lines, such as human leukemia, ovarian carcinoma and lung cancer cells.9 Actually, low-intensity USI exhibits great potential to induce apoptosis as it is easily applied, and therefore should be considered to be the optimal ultrasound for cancer treatment in clinic.10 As low-intensity USI triggers apoptosis in various cancers, it could be employed as a promising strategy to eliminate tumor cells.11 Meanwhile, a study by Yang et al.12 also reported that USI induced apoptosis in numerous cancer cells by activating different apoptotic signaling pathways, including endoplasmic reticulum (ER) stress-associated and mitochondrial pathways.
By considering the potential effects of USI on cancer cell apoptosis, we hypothesized that the USI treatment might play a critical role for inducing cervical cancer cell apoptosis. Thus, the present research aimed to clarify the effects of low-intensity USI on apoptosis of cervical cancer cell lines and the apoptosis-associated mechanisms.
Materials and methods
The human normal cervical cell line (HaCaT) and the cervical cancer cell lines C33A, Hela and Siha were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Beyotime, Shanghai, China) in the humidified atmosphere with 5% CO2 at 37°C.
Ultrasound irradiation administration
and trial grouping
The C33A, Hela and Siha groups were subdivided into HaCaT group (only referring to the MTT and flow cytometry assay), untreated tumor cells (i.e., tumor cells not treated with USI) group, tumor cells exposed to USI and then cultured for 6 h (6 h group), tumor cells exposed to USI and then cultured for 12 h (12 h group), and tumor cells exposed to USI and then cultured for 24 h (24 h group). The USI was conducted using the JC200 Haifu ultrasound system (Chongqing Haifu Medical Technology Co. Ltd., Chongqing, China). The USI processes were assigned based on a previous study.13 Cells were treated with USI at a transducer frequency of 10.0 MHz and with mechanical index (power) of 0.25 W/cm2 for 10 s.
Suspensions of HaCaT, C33A, Hela, and Siha cells were seeded into 96-well cell culture plates (Corning-Costar, Corning, USA) at a dosage of 1 × 106 cells/mL and cultured for 6–24 h at 37°C. In order to evaluate the cell viability (cell proliferation), 20 μL of MTT solution (5 mg/mL in phosphate-buffered saline (PBS; Beyotime) was added to each well and cultured for another 4 h at 37°C. After the incubation of MTT, a total of 200 μL of dimethyl sulfoxide (DMSO; Amresco Inc., Solon, USA) was added to the wells for 10 min to dissolve any formed formazan crystals. Finally, the absorbance of each well was examined at wavelength of 490 nm using a Multiskan Mk3 micro-plate reader (Thermo Scientific Pierce, Shanghai, China).
Flow cytometry assay
Apoptosis of cervical cancer cells, including HaCaT, C33A, Hela, and Siha, were assessed with Annexin V-PE/
7-AAD Apoptosis Detection regent (Cat. No. 559763; Becton Dickinson Biosciences, San Jose, USA) based on the manufacturer’s protocol. Briefly, cells were collected and treated with Annexin V-PE for 15 min and then propidium iodide (PI) for 15 min in the dark at 25°C. Next, cells were analyzed using a FACS Vantage SE (Becton Dickinson Biosciences) flow cytometer, using the 546/647 band-pass filter (for monitoring PI signals) and the 530/578 band-pass filter (for monitoring Annexin-V signals), and the data were analyzed with the Cell Quest software v. 5.1 (Becton Dickinson Biosciences).
Small interfering RNA synthesis and identification
The Small interfering RNA (siRNA) synthesis was conducted according to a previous study.14 The candidate sequences for the siRNA synthesis, and the optimal siRNA sequences for silencing the glucose-related protein (GRP78) gene identified using quantitative real-time polymerase chain reaction (qRT-PCR) analysis with specific primer for GRP78 are listed in Table 1. The siRNA was transfected into the above cell lines using Lipofectamine 2000 regent (Invitrogen/Life Technologies, Carlsbad, USA). Briefly, RNAs in all tumor cells were extracted using Trizol kits (Beyotime). Complementary DNAs (cDNAs) were then synthesized with the cDNA Synthesis Kit (Cat. No. 18080200; Thermo Fisher Scientific, Rockford, USA) following the manufacturer’s protocol. The mRNA levels of GRP78 gene were determined with SYBR-Green I PCR system, purchased from Western Biotech (Chongqing, China), using specific primers (Table 1). Amplified products for GRP78 gene were analyzed using GDS8000 Gel-Scanning equipment (UVP, Sacramento, USA) based on equipment instructions. Finally, the GRP78 gene expression was calculated and analyzed using 2−ΔΔct method.
Scanning electron microscopy
The structural changes of the cervical cancer cells when exposed to USI were observed using scanning electron microscopy. Briefly, cervical cancer cells were adjusted to the concentration of 106 cells/well on six-well plates and cultured for 24 h to confirm adhesion. Cells were washed with PBS and the suspensions were dropped onto glass coverslips for 30 min and then incubated using 4% formalin (Beyotime) for 5 min. Then, cells were fixed using the 1% osmic acid (Beyotime) for 30 min, washed with ice-cold PBS solution and then soaked in 2% tannin (Beyotime) overnight at 4°C. Finally, cervical cancer cells were coated with a vacuum spray-plating equipment and the image was captured using scanning electron microscope S-3000N (Hitachi, Tokyo, Japan).
All cells were cultured onto slides and fixed with 4% paraformaldehyde (Beyotime) at room temperature for 10 min. Cells were washed with PBS and endogenous peroxidase inactivated using 3% hydrogen peroxide (Beyotime) at 25°C for 5 min. Cells were subsequently blocked using 5% bovine serum albumin solution (BSA; Beyotime) at 25°C for 15 min, then incubated with rabbit anti-human GRP78 monoclonal antibody (1 : 3000, Cat. No. ab108615; Abcam, Cambridge, USA), rabbit anti-human C/EBP-homologous protein (CHOP) polyclonal antibody (1 : 3000, Cat. No. MBS000292; MyBioSource, Vancouver, Canada), rabbit anti-human phosphorylated C-Jun N-terminal kinase (p-JNK) monoclonal antibody (1 : 3000, Cat. No. ab124956; Abcam), and rabbit anti-human caspase-12 polyclonal antibody (1 : 2000, Cat. No. ab62484; Abcam) overnight at 4°C. Subsequently, cells were washed using PBS and incubated using Biotin-labeled goat anti-rabbit antibody (1 : 1000, Cat. No. ab6720; Abcam) for 60 min at 37°C. Finally, cells were observed and images captured using a professional CKX41 inverted fluorescence microscope (Olympus Corp., Tokyo, Japan).
Western blot assay
The cervical cancer cells were lysed with RIPA buffer (Beyotime) according to manufacturer’s protocol. Cell lysates were then separated using 15% SDS-PAGE (Beyotime) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, USA). The membrane was blocked using PBS solution containing 5% skim milk and supplementing with 0.05% Tween-20 (Beyotime). Subsequently, PVDF membrane was treated with rabbit anti-human GRP78 monoclonal antibody (1 : 3000; Abcam), rabbit anti-human CHOP polyclonal antibody (1 : 2000; MyBioSource), rabbit anti-human p-JNK monoclonal antibody (1 : 3000; Abcam), rabbit anti-human caspase-12 polyclonal antibody (1 : 2000; Abcam), and rabbit anti-human β-actin polyclonal antibody (1 : 2000; Abcam) overnight at 4°C. Subsequently, PVDF membranes were treated with horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody (1 : 2000; Abcam). The western blot band signals for the isolated proteins were visualized and imaged with ECL Western Blotting Substrate (Cat. No. 32106, Thermo Fisher Scientific). Finally, the images were captured using GDS8000 Gel Scanning System (UVP).
Our data were analyzed using IBM SPSS v. 19.0 software (IBM Corp., Armonk, USA). All data are presented as mean ± standard deviation (SD). The statistical differences between 2 groups were analyzed using the Student’s t test. The differences among multiple groups were also analyzed using the Tukey’s post hoc test-validated analysis of variance (ANOVA). A p-value <0.05 represented a significant difference. At least 6 repeats were conducted for all tests or experiments.
Ultrasound irradiation reduced viability and increased apoptosis of cervical cells
In order to observe effects of USI on the proliferation of HaCaT, C33A cells, Hela cells, and Siha cells, an MTT assay was conducted. The MTT results indicated that cell viabilities were significantly reduced at 6 h, 12 h and 24 h post-treatment compared with the untreated cells (Figure 1A, p < 0.05). Meanwhile, the apoptosis of cervical cancer cells was measured with flow cytometry. Our findings showed that cell apoptosis rates were significantly increased at 6 h, 12 h and 24 h post-treatment compared with untreated cells (Figure 1B, p < 0.05).
Ultrasound irradiation induced the formation of apoptotic body
To confirm the reason that USI caused cervical cancer cell death, apoptotic bodies were evaluated in Hela cells. The results indicated that there were no apoptotic bodies present in the untreated cells (Hela cells) (Figure 2). However, apoptotic bodies in the 6-hours treatment group and 12-hours treatment group were visibly more compared to that in untreated cells (Figure 2). Meanwhile, both C33A and Siha cells displayed numerous apoptotic bodies (data not shown).
Ultrasound irradiation activated
ER stress-associated apoptosis
The ER stress-associated apoptotic signaling molecules,15 including GRP78, CHOP and caspase-12, were examined with immunocytochemistry analysis. Our findings demonstrated that USI significantly increased caspase-12 expression in C33A cells, Hela cells and Siha cells, compared with the untreated cells (Figure 3, p < 0.05). Furthermore, USI significantly upregulated CHOP expression in C33A cells, Hela cells and Siha cells, compared with the untreated cells (Figure 4, p < 0.05). Finally, compared with the untreated cells, GRP78 levels were also significantly enhanced in the C33A cells, Hela cells and Siha cells undergoing the USI treatment (Figure 5, p < 0.05).
Ultrasound irradiation increased phosphorylation of JNK
The phosphorylation of the JNK molecule could reflect apoptosis occurring within cancer cells. Therefore, we examined the expression of p-JNK in C33A, Hela and Siha cells. The present findings showed that USI significantly increased phosphorylation of JNK in both C33A and Siha cells, compared to that in the untreated tumor cells at 6 h, 12 h and 24 h post-treatment (Figure 6, p < 0.05). However, the effects of USI on p-JNK were not found in Hela cells (Figure 6).
Ultrasound irradiation triggered ER stress by modulating GRP78 expression
In this study, we synthesized 3 siRNA sequences, and found that the siRNA sequence GRP78-homo-1548 exhibited the best silencing effects on GRP78 expression (Figure 7), and was therefore used for subsequent studies in cervical cancer cells.
To determine specific mechanisms associated with USI-triggered ER stress, the stress-regulatory protein GRP78 was detected with western blotting analysis (Figure 8A). Our findings demonstrated that USI significantly increased caspase-12 (Figure 8B), CHOP (Figure 8C) and GRP78 (Figure 8D) protein expression in Hela cells at all time points post-treatment, compared to that of the untreated cells (p < 0.05). Meanwhile, inhibition of GRP78 (siRNA treatment) also significantly enhanced the expression of caspase-12 (Figure 8B, 12 h post-USI administration), CHOP (Figure 8C, at 6 h, 12 h and 24 h post-USI treatment) and reduced GRP78 (Figure 8D, at 6 h, 12 h and 24 h post-USI treatment) proteins in Hela cells compared to that in the untreated tumor cells (p < 0.05) at 6 h, 12 h and 24 h post-USI treatment.
Finally, suppression of GRP78 also significantly increased the expression of caspase-12, CHOP and decreased GRP78 proteins in C33A and Siha cells compared to that in the untreated tumor cells at 6 h, 12 h and 24 h post-treatment (data not shown).
Ultrasound irradiation enhanced phosphorylation of JNK by downregulating GRP78 expression
The results showed that suppression of GRP78 enhanced the ratio of JNK p-JNK/JNK in Hela cells compared to that in the untreated tumor cells at 6 h, 12 h and 24 h post-treatment (Figure 8E, p < 0.05). Moreover, inhibition of GRP78 also significantly increased expression of p-JNK in C33A cells and Siha cells compared to that in untreated tumor cells (data not shown).
Cervical cancer is considered to be a leading cause of cancer-associated death worldwide for women.16 It is usually diagnosed clinically as advanced or moderate stages; however, the efficacy of traditional therapy is relatively poor.17 Therefore, there is a significant and urgent need to establish an effective and safe therapeutic strategy for cervical cancer. Although USI could induce apoptosis in both cancer cells and normal cells, the cancer cells are more sensitive to the treatment and more easily killed compared to normal cells.18, 19 Meanwhile, Lagneaux et al.20 reported that cancer cells are more susceptible to the USI due to higher cell apoptotic rates. Therefore, USI that can cause apoptosis might be of clinical importance as a novel therapeutic approach.
In this study, we first examined cell viability and apoptosis in both a normal cervical cell line (HaCaT cells) and cervical cancer cell lines (C33A, Hela and Siha). The MTT assay results showed that USI significantly reduced the cell viability of all cancer cells at 6 h, 12 h and 24 h post-treatment. The flow cytometry assay findings indicated that USI triggered significant amounts of apoptosis in all cervical cancer cells at 6 h, 12 h and 24 h post-treatment. However, USI demonstrated no effects on the cell viability and apoptosis of normal cervical cells. Therefore, we believed that USI treatment is safe for normal cervical cells, and only the cervical cancer cells were utilized in the following experiments for exploring mechanisms of cell viability and apoptosis.
Apoptosis, a process of programmed cell death, can maintain tissue homeostasis and eliminate dead cells from both normal and cancer tissues.21 Previous studies have reported that there are 2 main categories of apoptosis, including mitochondria-mediated22, 23 and ER stress-mediated apoptosis.14, 24 Ultrasound irradiation-induced apoptosis of cancer cells has previously been demonstrated to use the mitochondria-associated pathway25; therefore, this aspect is not discussed. In the present study, we investigated whether USI caused apoptosis by verifying the biomarker expression of ER stress-mediated apoptosis, including GRP78, CHOP and caspase-12.26 Our results showed that all of these ER stress biomarkers were significantly upregulated following USI in treated cervical cancer cells compared to untreated tumor cells. This suggests that USI induced cancer cell death (decreased cell viabilities) and apoptosis (formed apoptotic bodies) via the ER-mediated signaling pathway.
A previous study27 demonstrated that p-JNK is associated with cell apoptosis, and therefore we also evaluated the levels of p-JNK in USI-treated cervical cancer cells. Our results indicated that USI triggered a significant increase in p-JNK in both C33A cells and Siha cells. This result suggests that USI induced apoptosis in cervical cancer cells also via activating p-JNK, which is consistent with the previous study.28
The GRP78 protein plays critical roles in regulating ER-mediated apoptosis in cancer cells.29 When cancer cells are suffering from the stimuli, GRP78 is overexpressed to regulate any misfolded proteins, and in this way, silencing of the GRP78 gene might initiate ER stress.30 In this study, we designed the siRNA sequence of GRP78-homo-1548 to downregulate GRP78 expression and to clarify the role of this key molecule in USI-mediated apoptosis. The results indicated that silencing of GRP78 significantly upregulated caspase-12 and CHOP expression, which increases ER stress-mediated apoptosis of Hela cells. Meanwhile, p-JNK expression was also activated in Hela cells undergoing GRP78 silencing. These findings suggest that the GRP78 molecule might participate in apoptosis in USI-treated cervical cancer cells.
Although this study received a few interesting findings, there are also some limitations.
This study mainly focused on ER stress-induced apoptosis; however, we did not discuss the classical apoptosis pathway. Actually, some previous studies31, 32, 33, 34 have explored the classical pathways or molecules involved in USI-induced apoptosis of cancer cells. Xu et al.31 reported that ultrasound could induce apoptosis in cervical cancer cells via regulating Bcl-2 and Bax proteins. Xiao et al.32 found that ultrasound demonstrated a wider application in the clinical treatment of cervical cancer through modulating cleaved caspase-9. Chen et al.33 proved that ultrasound remarkably downregulated Bax and caspase-3 expression in cervical cancer cells. Finally, Tang et al.34 discovered that caspase-8 is also involved in the apoptosis in ultrasound-treated cancer cells. However, the intrinsic apoptosis pathway (involving tumor necrosis factor α (TNF-α), TRAIL and FAS-L molecule) and extrinsic apoptosis pathway (involving mitochondria-mediated molecules) have not been fully clarified herein, which is a limitation of our study.
The present findings indicated that USI could significantly inhibit the proliferation and induce apoptosis of cervical cancer cells. Ultrasound irradiation-induced cervical cell apoptosis was mediated by initiating the ER stress-associated signaling pathway and triggering the phosphorylation of JNK in cervical cancer cells. These results might provide the experimental insight for the therapeutic intervention of USI in cervical cancer therapy. In the future, we would investigate the effects of USI on tumor growth of xenografted mice. Moreover, we would also combine USI and ER stress inducers for enhancing the regression of tumors with the minimum off-target effects.