- Email: [email protected]
RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-β1
Biochemical and Biophysical Research Communications xxx (2018) 1e8
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1 Dong-Ge Wang a, Tong-Min Li b, Xuan Liu c, * a
Department of Obstetrics and Gynecology, Jinhua Municipal Central Hospital, No. 365 People's East Road, Jinhua 321000, China The Maternal and Child Hospital of Dongchangfu District, Liaocheng 252000, China c Center of Department of Gynecology, Qingdao Women and Children's Hospital of Qingdao University, Qingdao 266000, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2018 Accepted 27 May 2018 Available online xxx
Cervical cancer is the second commonest cancer among women in the worldwide, and the majority cause of death in various countries, highlighting the importance of investigating new therapeutic targets. Rh family, C glycoprotein (RHCG) belongs to the Rhesus (Rh) family and was first identified as Rh blood group antigens. It has been confirmed to function in cancer progression, including prostate cancer and esophageal squamous cell carcinoma. However, its role in cervical cancer has never been explored. The present study indicated that RHCG was down-regulated in cervical cancers compared to that in normal cervical tissues, and further decreased in cervical cancer cell lines. Functionally, RHCG overexpression reduced cervical cancer cell proliferation and migration, as evidenced by the decreased transforming growth factor (TGF)-b1, matrix metalloproteinase (MMP)-2 and MMP-9 expressions in cancer cells; however, an opposite effect was observed when RHCG was knocked down. Further, increase of RHCG markedly induced apoptosis in cervical cancer cells by improving the cleavage of Caspase-3 and poly (ADP-Ribose) polymerase (PARP). And cells transfected with RHCG siRNA exhibited a notable reduction of cleaved Caspase-3 and PARP. Moreover, nucleus nuclear factor-kB (NF-kB) and whole cell xIPA expressions were markedly reduced by over-expressing RHCG. Conversely, suppressing RHCG elevated NF-kB activation and xIPA expression in cervical cancer cells. Notably, we found that TGF-b1 treatment could abolish the effects of RHCG over-expression on the reduction of cell migration and enhancement of apoptosis in cervical cancer cells. Over-expressing RHCG-reduced NF-kB activation and xIPA expression were also abrogated by TGF-b1 pre-treatment. Additionally, enhancing NF-kB activity could restore xIPA expressions and decrease apoptotic response in cervical cancer cells over-expressing RHCG. In vivo, we also found that RHCG over-expression reduced cervical tumor growth through the same signaling pathways as we found in vitro. Therefore, RHCG may be a potential prognostic biomarker and therapeutic target for human cervical cancer. © 2018 Published by Elsevier Inc.
Keywords: Cervical cancer RHCG Migration and apoptosis NF-kB TGF-b1
1. Introduction Cervical cancer remains one of the leading causes of tumorrelated deaths worldwide [1]. Although there is accumulating evidence that early detection of cervical cancer has decreased mortality, the prognosis of advanced or recurrent cervical cancer remains very poor [2]. Presently, therapeutic treatments for cervical cancer include surgery with chemotherapy, radiation therapy, and concurrent chemoradiation therapy [3]. Due to the poor prognosis of patients suffering from cervical cancer, targeted
* Corresponding author. E-mail address: [email protected] (X. Liu).
therapeutics attracted researchers attention to develop effective treatments of advanced, recurrent, or metastatic cervical cancer. Rh type C-glycoprotein (RHCG) functions as an electroneutral and bidirectional ammonium transporter [4]. RHCG belongs to Rhesus (Rh) family, which was first identified as Rh blood group antigens in human erythroid cells. Rh proteins are tightly associated with the family of ammonia transporter proteins, characterized by the presence of 12 transmembrane-spanning segments [5]. Hence, Rh family proteins can fall into two functionally distinct groups, including ammonia transporting Rh glycoproteins (RHAG, RHBG, and RHCG) and nontransporting Rh proteins (RHD and RHCE) [6,7]. The nonerythroid Rh glycoproteins, RHBG and RHCG, are ammonia transporters, which are widely expressed in various
https://doi.org/10.1016/j.bbrc.2018.05.183 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
2
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
tissues. And RHCG was highly expressed in human skeletal muscle, kidney and oral cavity; however, its physiological functions are not well explained [8,9]. As reported, RHCG was decreased in tongue squamous cell carcinoma and in esophageal squamous cell carcinoma [10,11]. However, the effects of RHGC decrease on tumor growth, including cervical cancer, are still not yet investigated. In the study, we calculated the expression of RHGC in human cervical cancer tissues and their corresponding non-tumor cervix uteri samples. Functional analysis of cervical cancer cells transfected with RHGC overexpression plasmid or RHGC siRNA were carried out to reveal the role of RHGC in cervical cancer progression. RHGC exhibited anti-tumor effects on cervical cancer, which might be a potential a potential therapy target for the treatment of cervical cancer. 2. Materials and methods 2.1. Patients and information A total of 51 patients with cervical cancer who received surgery at the Department of Obstetrics and Gynecology, Jinhua Municipal Central Hospital (Zhejiang, China) between January 2008 and December 2014 were included in the study. Their corresponding non-tumor cervix uteri samples were used as controls. Specimens were collected from patients for protein and RNA extraction. No patients received preoperative treatment. Informed consent was obtained from all patients before the collection of specimens. The study was reviewed and approved by the Institutional Review Board at Jinhua Municipal Central Hospital.
threshold cycle method and normalized to GAPDH. CelLytic™ NuCLEAR™ Extraction Kit (Sigma Aldrich, USA) was used for nuclear protein extraction. Cells or tumor tissue samples were lysed on ice using a cell lysis buffer (125 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.1%Triton X-100 and 5 mM EDTA) containing both 1% protease inhibitor and 1% phosphatase inhibitor cocktail (SigmaAldrich). The resulting lysates were separated on 10% SDS-PAGE, transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with 5% skim milk and incubated with primary and secondary antibodies (1:5000, Abcam, USA). The blots were visualized with ECL reagent (Millipore). The primary antibodies were shown as follows: antibody against RHCG (1:500, #sc-100287, Santa Cruz, USA), TGF-b1 (1:1000; #ab92486, Abcam), phosphorylated NF-kB (1:1000; #ab86299, Abcam), NF-kB (1:1000; #ab16502, Abcam), MMP-2 (1:500, #sc-13595, Santa Cruz), MMP-9 (1:500, #sc-13520, Santa Cruz), Caspase-3 (1:1000; #ab13847, Abcam), PARP (1:1000, #9532, Cell Signaling Technology, USA), xIAP (1:500, #sc-55551, Santa Cruz), Lamin B (1:500; #sc-374015, Santa Cruz), and GAPDH (1:1000, #ab8245, Abcam). 2.5. Flow cytometry analysis After double staining with 5 mL of APC-Annexin V, the cells were then incubated for 15 min at room temperature in the dark followed by the addition of 5 mL of propidium iodide (PI) at room temperature for 5 min in dark. Finally, cells were analyzed uisng a flow cytometer (FACScan; BD Biosciences, USA) equipped with Cellquest software (BD Biosciences). 2.6. Transwell analysis for migration
2.2. Culture and transfection of cells Human cervical cancer cell lines, including Siha, Caski, Hela, C41, and C-33a, and normal endocervical cell line (End1/E6E7) were obtained from American Type Culture Collection (ATCC, USA). Human cervical cancer cell line of TC-1 was purchased from the Tumor Center of Chinese Academy of Medical Sciences. All cells were routinely maintained in RIP1640 or DMEM medium (Invitrogen, USA) supplemented with 10% FBS (Gibco, USA) at 37 C in a humidified air atmosphere containing 5% CO2. For RHGC overexpression plasmid, vector pcDNA3.1 plasmid was obtained from KpnI and EcoRV (TransGen Biotech, china). The code sequence of RHGC amplified using PCR with Quick-Fusion Cloning Kit (Bimake, Houston, USA). The siRNA against RHGC and negative control siRNA were obtained from Generay Co., Ltd (Shanghai, China). Cells were transfected with plasmids with DNA Transfection Reagent (Bimake) or with RHGC siRNA using Lipofectamine™-2000 (Invitrogen) when they grew to 70% confluence following the manufacturer's protocols. 2.3. Cell viability assay Cell proliferation analysis was performed using the MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's protocol. The results were measured by absorbance at 450 nm. 2.4. Quantitative real time-PCR (RTePCR) and western blot analysis Total RNA was isolated using TRIZOL (Invitrogen) and reverse transcribed into cDNA using Prime Script™ RT Master Mix (Takara, Japan). Gene transcripts were quantified using CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with SYBR Premix Ex Taq (Takara) and normalized with GAPDH. All primers are listed in Supplementary Table 1. Ct values were analyzed using comparative
An 8-mm pore size transwell chamber (Corning, USA) was used for transwell migration assay. A total of 1 105 cells in 100 mL medium in the absence of FBS were plated in the upper chamber and 500 mL medium containing 10% FBS was covered on the bottom chambers as chemoattractant. After incubation for 24 h, nonmigratory cells on the upper membrane surface were removed, and the cells on the bottom surface were fixed with 4% polyoxymethylene and then stained with 0.1% crystal violet for 10 min. Cancer cells were counted through photographing 5 random fields using a light microscope. 2.7. Immunofluorescent (IF) analysis Cells were fixed with 4% paraformaldehyde phosphate buffer solution and incubated with antibody against RHCG (1:100, #sc100287, Santa Cruz), and antibody against NF-kB (1:100, #ab86299, Abcam, USA) at 4 C overnight. Alexa Fluor 594-conjugated secondary antibodies (Abcam) were incubated at room temperature for 30 min. The nuclei were stained with DAPI (KeyGen Biotech). Then, the images were taken with a fluorescent microscope. 2.8. Cervical tumor xenograft model Animal experiments were performed according to the guideline for the Regulations for Animal Experiments and Related Activities at Jinhua Municipal Central Hospital. 6-week-old female Balb/c nude mice were purchased from Nanjing Peng Sheng Biological Technology Co Ltd (Nanjing, China). 1.0 107 Siha cells stably transfected with OE-Con, OE-RHCG, si-Con or si-RHCG were subcutaneously injected into the flank region of nude mice to establish cervical cancer xenograft model (n ¼ 6/group). Tumor volume was monitored through measuring the longest dimension (length) and shortest dimension (width) using dial calipers at 3-day intervals. Tumor volume was evaluated using the following formula: tumor
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
diameter ¼ 0.5 (length þ width). 2.9. Statistical analysis All data were expressed as mean ± SEM. Statistical analyses were performed using Graphpad Prism (version 6.0, USA). Differences among groups were analyzed by one-way ANOVA followed by Tukey's test. P < 0.05 was considered statistically significant. 3. Results 3.1. Expression of RHCG is associated with human cervical cancer development We calculated the relationship between RHCG expression in cervical cancer tissue and different clinicopathological characteristics (Table 1). High RHCG expression was associated with larger tumor size, lymph node metastasis and higher FIGO stage, indicating that RHCG expression was associated with aggressive oncogenic behavior of cervical cancer. To evaluate the expression and significance of RHCG in cervical cancer, RHCG mRNA expression levels were measured. As shown in Fig. 1AeC, RHCG expression was clearly decreased in cervical cancer tissues compared to non-tumor cervical tissues. Western blot analysis further confirmed the down-regulation of RHCG in human cervical cancer tissue samples (Fig. 1D). Kaplan-Meier survival analysis indicated that patients with high RHCG expression had better overall survival rate than those with low RHCG expression (Fig. 1E). A significant decrease of RHCG was observed in human cervical cancer cell lines using RT-qPCR, western blot and IF analysis (Fig. 1FeH). Overexpression and suppression was achieved by transfection of RHCG plasmid or siRNAs into Siha and Caski cell lines, as measured by RT-qPCR and western blot assays (Fig. 1I and J). 3.2. RHCG regulates migration and apoptosis in human cervical cancer cell lines As shown in Fig. 2A, the cell viability was significantly reduced after transfection of RHCG plasmid and increased after transfection with RHCG siRNA. The results also showed that the number of Siha and Caski cells for migrating in RHCG plasmid transfection group was markedly down-regulated in comparison to that in the control group, while suppression of RHCG by siRNA transfection markedly
Table 1 Relationship between RHCG expression in cervical cancer patients and clinicopathologic characteristics. Characteristics
No
RHCG expression High
Age (years) 49 <49 Tumor size (cm) 4.0 <4.0 Lymph node metastasis Yes NO FIGO stage IA IB II Parametrial invasion Yes NO a
p value
3
elevated the migration ability (Fig. 2B). It was also found that the protein levels of TGF-b1, MMP-2 and MMP-9 were significantly decreased, when RHCG was over-expressed in both cervical cancer cells. Inversely, cells transfected with RHCG siRNA showed a remarkable increase of TGF-b1, MMP-2 and MMP-9 in cervical cancer cells (Fig. 2C). RT-qPCR analysis indicated that the expression of Snail, TWIST, EGFR, and Vimentin obviously down-regulated in cells transfected with OE-RHCG plasmid compared with those infected with OE-Con plasmid. On the contrary, inhibition of RHCG by siRNAs clearly enhanced the expression of these signals. And a reverse result was observed in the change of E-cadherin in cancer cells after transfection with OE-RHCG plasmid or RHCG siRNA (Fig. 2D). RHCG obviously elevated the apoptosis in cervical cancer cells. Conversely, suppressing RHCG clearly reduced apoptosis (Fig. 2E). Western blot analysis indicated that RHCG overexpression markedly increased the cleavage of Caspase-3 and PARP. However, suppressing RHCG significantly decreased the cleaved Caspase-3 and PARP expressions (Fig. 2F). The number of pNF-kB-positive cells was evidently decreased as compared with that in the OE-Con group; however, a notable increase in p-NF-kBpositive cells was observed in cancer cells transfected with RHCG siRNA (Fig. 2G). Further, western blot analysis showed that nucleus NF-kB and cellular xIPA expressions were markedly downregulated in cells transfected with OE-RHCG. When transfected with RHCG siRNA, a significant increase of nucleus NF-kB and cellular xIPA levels was observed (Fig. 2H and I). 3.3. RHCG modulates TGF-b1 expressions to reduce migration and induce apoptosis in human cervical cancer cell lines In this section, we first found that OE-RHCG plasmid-induced reduction of Snail, TWIST, EGFR and Vimentin, and elevation of Ecadherin was abolished by TGF-b1 pre-treatment (Fig. 3A). The evident increase of apoptosis in cells transfected with RHCG plasmid was reversed by the pre-treatment of TGF-b1 (Fig. 3B). Consistently, pre-treatment of TGF-b1 clearly reduced the cleavage of Caspase-3 and PARP in RHCG over-expressed cells (Fig. 3C). In addition, the number of cells expressing p-NF-kB reduced by RHCG over-expression was markedly restored by TGF-b1 pre-treatment (Fig. 3D). As shown in Fig. 3E and F, the reduction of nucleus NFkB and cellular xIPA caused by RHCG over-expression was also eliminated by TGF-b1 pre-treatment. Following, NF-kB was activated by LPS and TNF-a addition (Fig. 3G). Both the expressions in nucleus NF-kB and cellular xIPA decreased by RHCG overexpression were recovered in LPS- and TNF-a-treated cells (Fig. 3H and I). Inversely, OE-RHCG-induced apoptosis was abrogated by LPS or TNF-a pre-treatment (Fig. 3J). Also, PS or TNF-a pretreatment led to a remarkable decrease of cleaved Caspase-3 and PARP in cancer cells transfected with OE-RHCG (Fig. 3K).
Low
3.4. RHCG reduces tumor growth in vivo 23 28
13 15
10 13
0.453
a
26 25
5 9
21 16
0.032
17 34
4 15
13 19
0.025a
3 38 10
1 12 2
2 26 8
0.022a
7 44
3 19
4 25
0.418
Statistically significant (p < 0.05).
In order to further evaluate the role of RHCG in regulating cervical cancer progression, the in vivo study was performed. As shown in Fig. 4AeC, RHCG over-expression markedly reduced the tumor size, tumor volume and tumor weight compared to the OECon group. In contrary, a significant increase of tumor size, volume and weight was observed in the group of si-RHCG than that in the si-Con. Western blot analysis indicated that RHCG over-expression led to a significant reduction of TGF-b1, MMP-2 and MMP-9, as well as p-NF-kB and xIPA in tumor tissue samples; however, a remarkable increase of these proteins was observed in si-RHCG group. Moreover, RHCG over-expression markedly increased the cleaved Caspase-3 and PARP expressions. And in si-RHCG group, we observed an evident decrease of Caspase-3 and PARP cleavage
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
4
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
Fig. 1. Down-regulation of RHCG in human cervical cancer tissues and cell lines. (AeC) RT-qPCR analysis of RHCG in 51 pairs of human cervical cancer tissues and their corresponding non-tumor cervix uteri samples. (D) Western blot analysis of RHCG in 24 pairs of human cervical cancer tissues and their corresponding normal cervix uteri samples. (E) Kaplan-Meier analysis indicated patients with high expression of RHCG showing better overall survival rate. (F) RT-qPCR, (G) western blot and (H) IF staining analysis of RHCG in cervical cancer cell lines and normal cervix uteri cells. (I,J) Confirmation of RHCG over-expression or inhibition after transfection with OE-RHCG plasmid or siRNAs in Siha and Caski cells by RT-qPCR and western blot assays. Data are presented as mean ± SEM of at least three independent experiments. n ¼ 6/group. ***p < 0.001.
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
5
Fig. 2. RHCG regulates migration and apoptosis in human cervical cancer cell lines. Siha and Caski cells were transfected with RHCG overexpression plasmid or siRNAs for 24 h (A) MTT analysis of Siha and Caski cells. (B) Transwell analysis for migration. (C) Western blot analysis of TGF-b1, MMP-2 and MMP-9. (D) RT-qPCR analysis of EMT-markers (Snail, TWIST, EGFR, Vimentin and E-cadherin). (E) Flow cytomery analysis of cells for apoptosis measurements. (F) Western blot analysis of cleaved Caspase-3 (C-Casp3) and PARP (C-PARP). (G) IF staining of p-NF-kB in cells. (H) Western blot analysis of NF-kB in nuclear of cells. (I) Western blot analysis of xIPA in whole cells. Data are presented as mean ± SEM of at least three independent experiments. n ¼ 6/group. *p < 0.05, **p < 0.01 and ***p < 0.001.
(Fig. 4DeF). Thus, RHGC showed anti-tumor role in human cervical cancer. 4. Discussion RHCG, an integral membrane protein, belongs to Rh family and forms homotrimer in plasma membrane. Furthermore, RHCG is highly expressed in human cervix, squamous epithelia of esophagus and oral cavity [4e7,9,10]. Nevertheless, the biological function of RHGC in these tissues or organs has not been investigated. RHGC was reported to show anti-tumor role in squamous cell carcinoma from esophagus and tongue [10,11]. Presently, the association between RHGC and human cervical cancer is little to be explored. In the study, we firstly discovered that RHGC was decreased in cervical cancers in comparison to that in the normal cervical tissue samples. Functionally, RHGC over-expression markedly reduced cervical cancer cell proliferation and migration, whereas RHGC knockdown generated an opposite result. Further, enhancing RHGC expression reduced TGF-b1 expressions, and migration-associated markers. Moreover, RHGC over-expression induced apoptosis in cervical cancer cells compared to the control group, while in the RHGC-silenced group, a remarkable decrease of apoptosis was observed through reducing cleaved Caspase-3 and PARP expressions. In addition, nuclear NF-kB and cellular xIAP expressions were found to be decreased in RHGC-overexpressing cells, while an opposite effect was observed in RHGC-knockdown group. Importantly, the in vitro results suggested that RHGCsuppressed cervical cancer progression was at least partly dependent on TGF-b1 inhibition to reduce cell migration and to induce apoptosis. In vivo study confirmed the results that RHGC over-
expression reduced the tumor growth via the same signaling pathways as we discovered in vitro. All these findings demonstrated that RHGC might be a tumor suppressor, playing an essential role in cervical cancer inhibition. It was validated that epithelial-mesenchymal transition (EMT) elevated cell migration and invasion, resulting in tumor cell dissemination, and RHGC was discovered to exert a suppressive effect on the migration of cervical cancer cells [12,13]. Overexpressing RHGC reduced EMT-markers of Snail, TWIST, EGFR, and Vimentin [12,14], while enhanced E-cadherin, which were abolished by RHGC suppression. MMPs were suggested to be involved in tumor metastasis through enhancing cell motility and degrading the extracellular matrix. MMP2 and MMP9 accelerated cell proliferation and migration, therefore promoting tumor development [15]. Our findings indicated that RHGC might impede MMP-2 and MMP-9 expressions to suppress cervical cancer cell migration. Therefore, in addition to the suppression of MMP-1 in esophageal squamous cell carcinoma, here we further found that RHGC could reduce MMP-2 and -9 to suppress tumor growth [9]. TGF-b1 is known to play an essential role in tumor growth. TGF-b1 could facilitate tumor invasion and metastasis, induce cells undergoing EMT, and modulate tumor microenvironment remodeling, which are in favor of tumor development and progression [16,17]. In our study, we also found that over-expressing RHGC significantly reduced TGF-b1 expressions in cancer cells, while an opposite result was observed in RHGC-knockdown cells. More importantly, TGF-b1 treatment could diminish RHGC-reduced migration, as evidenced by the restored expression of Snail, TWIST, EGFR, and Vimentin, and the down-regulated E-cadherin in cervical cancer cells. Therefore, we hypothesized that RHGC could
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
6
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
Fig. 3. RHCG modulates TGF-b1 expressions to reduce migration and induce apoptosis in human cervical cancer cell lines. Siha and Caski cells were pre-treated with TGF-b1 (10 ng/ml) for 24 h, followed by transfection with RHCG overexpression plasmid for another 24 h (A) RT-qPCR analysis of EMT-markers. (B) Flow cytomery analysis of cells. (C) Western blot analysis of C-Casp3 and C-PARP. (D) IF staining of p-NF-kB in cells. (E) Western blot analysis of NF-kB in nuclear of cells. (F) Western blot analysis of xIPA in whole cells. (G) Western blot analysis of p-NF-kB and xIAP in whole cells after treated with LPS (100 ng/ml) or TNF-a (100 ng/ml) for 24 h. Siha and Caski cells were pre-treated with LPS (100 ng/ ml) or TNF-a (100 ng/ml) for 24 h, followed by transfection with RHCG overexpression plasmid for another 24 h (H,I) Western blot analysis of nuclear NF-kB and cellular xIPA. (J) Flow cytomery analysis of cells. (K) Western blot analysis of C-Casp3 and C-PARP. Data are presented as mean ± SEM of at least three independent experiments. n ¼ 6/group. * p < 0.05, **p < 0.01 and ***p < 0.001.
suppress TGF-b1 expression to inhibit migration of cervical cancer (Fig. 4G). Apoptosis is an orchestrated event that cells are programmed to die after receiving specific stimuli, and thus is an essential component of cell growth control [18]. Apoptosis is characterized by morphologic alterations, including chromatin condensation, reduction of cell volume and nuclear fragmentation. Biochemical changes, such as Caspase activation, DNA and protein breakdown and membrane surface changes, are also critical characteristics of apoptosis [19,20]. When disturbed, an imbalance between life and
death of cells could result in tumor progression [21]. Here, for the first time, we found that RHGC over-expression could clearly induce apoptosis in cervical cancer cells; however, knocking down RHGC expressions resulted in a significant decrease in apoptosis, which was supported by the down-regulated activity of Caspase-3 and PARP. NF-kB is a transcription factor, which has been suggested to be significantly up-regulated in various tumors [22]. NF-kB is shown to inhibit apoptotic response to promote tumor growth [23]. Hence, inactivation of the NF-kB pathway could be served as a therapeutic target for preventing tumor growth, including cervical
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
7
Fig. 4. RHCG reduces tumor growth in vivo. (A) Representative images of tumor tissue samples isolated from each group. (B) Tumor volume was recorded every 2 days. (C) Tumor weight was measured. Western blot analysis of (D) TGF-b1, MMP-2 and MMP-9, (E) C-Casp3 and C-PARP, as well as (F) p-NF-kB and xIAP in tumor tissue sample. (G) Hypothesis of RHCG axis suppressing tumor growth by migration inhibition and apoptosis induction via TGF-b1. Data are presented as mean ± SEM of at least three independent experiments. n ¼ 6/group. *p < 0.05, **p < 0.01 and ***p < 0.001.
cancer [24]. The inhibitor of apoptosis (IAPs) protein family presents another negative modulator of apoptotic pathway. IAPs, mainly xIAP, directly suppress caspases through several mechanisms [25,26]. In the study, we found that RHGC over-expression markedly reduced NF-kB activation, which was in line with previous studies [11]. Also, xIAP expression was decreased in cells transfected with OE-RHGC plasmid. We further found that promoting NF-kB activation using LPS or TNF-a could elevate xIAP expressions, and reduce apoptotic response in cervical cancer cells over-expressing RHGC. TGF-b1 is reported to modulate NF-kB activity in various diseases, including cancer [27,28]. As expected, here we found that TGF-b1 treatment recovered NF-kB activation, and xIAP expressions in cervical cancer cells with RHGC overexpressions, accompanied with reduced apoptosis. Therefore, we concluded that RHGC inhibited human cervical cancer progression by inducing apoptosis and inactivating NF-kB pathway dependent on TGF-b1 suppression (Fig. 4G). In summary, our study indicated that RHGC was decreased in cervical cancers in comparison to that in normal cervical tissues.
Additionally, RHGC functioned as a tumor suppressor by decreasing the migration and inducing apoptosis in cervical cancer cells through down-regulating TGF-b1 expressions. These findings might provide useful clues for developing effective diagnostic and therapeutic strategies for cervical cancer. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.05.183. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.05.183 References [1] K.S. Tewari, M.W. Sill, H.J. Long III, et al., Improved survival with bevacizumab in advanced cervical cancer, N. Engl. J. Med. 370 (8) (2014) 734e743.
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
8
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
[2] S. Cao, W. Liu, F. Li, et al., Decreased expression of lncRNA GAS5 predicts a poor prognosis in cervical cancer, Int. J. Clin. Exp. Pathol. 7 (10) (2014) 6776. [3] Z.Z. Fu, Y. Peng, L.Y. Cao, et al., Value of apparent diffusion coefficient (ADC) in assessing radiotherapy and chemotherapy success in cervical cancer, MRI (Magn. Reson. Imaging) 33 (5) (2015) 516e524. [4] I.D. Weiner, J.W. Verlander, Renal and hepatic expression of the ammonium transporter proteins, Rh B glycoprotein and Rh C glycoprotein, Acta Physiologica 179 (4) (2003) 331e338. [5] C.H. Huang, J. Peng, Evolutionary conservation and diversification of Rh family genes and proteins, Proc. Natl. Acad. Sci. Unit. States Am. 102 (43) (2005) 15512e15517. [6] F. Quentin, D. Eladari, L. Cheval, et al., RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron, J. Am. Soc. Nephrol. 14 (3) (2003) 545e554. [7] D.O.D. Mak, B. Dang, I.D. Weiner, et al., Characterization of ammonia transport by the kidney Rh glycoproteins RhBG and RhCG, Am. J. Physiol. Ren. Physiol. 290 (2) (2006) F297eF305. [8] J.W. Verlander, R.T. Miller, A.E. Frank, et al., Localization of the ammonium transporter proteins RhBG and RhCG in mouse kidney, Am. J. Physiol. Ren. Physiol. 284 (2) (2003) F323eF337. [9] K. Takeda, T. Takemasa, Expression of ammonia transporters Rhbg and Rhcg in mouse skeletal muscle and the effect of 6-week training on these proteins, Physiological reports (10) (2015) 3. [10] B.S. Chen, Z.X. Xu, X. Xu, et al., RhCG is downregulated in oesophageal squamous cell carcinomas, but expressed in multiple squamous epithelia, EJC (Eur. J. Cancer) 38 (14) (2002) 1927e1936. [11] X.Y. Ming, X. Zhang, T.T. Cao, et al., RHCG suppresses tumorigenicity and metastasis in esophageal squamous cell carcinoma via inhibiting NF-kB signaling and MMP1 expression, Theranostics 8 (1) (2018) 185. [12] E.W. Thompson, D.F. Newgreen, Carcinoma invasion and metastasis: a role for epithelial-mesenchymal transition? Canc. Res. 65 (14) (2005) 5991e5995. [13] R. Kalluri, R.A. Weinberg, The basics of epithelial-mesenchymal transition, JCI (J. Clin. Investig.) 119 (6) (2009) 1420e1428. [14] S. Maseki, K. Ijichi, H. Tanaka, et al., Acquisition of EMT phenotype in the gefitinib-resistant cells of a head and neck squamous cell carcinoma cell line through Akt/GSK-3b/snail signalling pathway, BJC (Br. J. Cancer) 106 (6) (2012) 1196. €kela €, M. Kylma €niemi, et al., Expression of matrix [15] T. Salo, M. Ma
[16]
[17]
[18] [19]
[20] [21]
[22] [23] [24]
[25]
[26]
[27]
[28]
metalloproteinase-2 and-9 during early human wound healing, Lab. Invest. J. Tech. Methods. Pathol. 70 (2) (1994) 176e182. L.R. Gomes, L.F. Terra, R.A.M. Wailemann, et al., TGF-b1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells, BMC Cancer 12 (1) (2012) 26. H.R. Kim, J.S. Jo, K.E. Hwang, et al., Salinomycin Suppresses TGF-b1-induced EMT by Down-regulating MMP-2, 9 via AMPK-sirt1 Pathway in a Non-small Cell Lung Cancer, 2017. J.L. Koff, S. Ramachandiran, L. Bernal-Mizrachi, A time to kill: targeting apoptosis in cancer, Int. J. Mol. Sci. 16 (2) (2015) 2942e2955. G. Kiraly, A.S. Simonyi, M. Turani, et al., Micronucleus formation during chromatin condensation and under apoptotic conditions, Apoptosis 22 (2) (2017) 207e219. Y. Kiraz, A. Adan, M.K. Yandim, et al., Major apoptotic mechanisms and genes involved in apoptosis, Tumor Biol. 37 (7) (2016) 8471e8486. M. Redza-Dutordoir, D.A. Averill-Bates, Activation of apoptosis signalling pathways by reactive oxygen species, Biochim. Biophys. Acta Mol. Cell Res. 1863 (12) (2016) 2977e2992. M.H. Park, J.T. Hong, Roles of NF-kB in cancer and inflammatory diseases and their therapeutic approaches, Cells 5 (2) (2016) 15. A.L. Rinkenbaugh, A.S. Baldwin, The NF-kB pathway and cancer stem cells, Cells 5 (2) (2016) 16. H.H. Chen, S.P. Chen, Q.L. Zheng, et al., Genistein promotes proliferation of human cervical cancer cells through estrogen receptor-mediated PI3K/AktNF-kB pathway, J. Canc. 9 (2) (2018) 288. C.Y. Wang, M.W. Mayo, R.G. Korneluk, et al., NF-kB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation, Science 281 (5383) (1998) 1680e1683. E. Strekalova, D. Malin, H. Rajanala, et al., Metformin sensitizes triple-negative breast cancer to proapoptotic TRAIL receptor agonists by suppressing XIAP expression, Breast Cancer Research and Treatment 163 (3) (2017) 435e447. M. Arsura, G.R. Panta, J.D. Bilyeu, et al., Transient activation of NF-kB through a TAK1/IKK kinase pathway by TGF-b1 inhibits AP-1/SMAD signaling and apoptosis: implications in liver tumor formation, Oncogene 22 (3) (2003) 412. C. Freudlsperger, Y. Bian, S.C. Wise, et al., TGF-b and NF-kB signal pathway cross-talk is mediated through TAK1 and SMAD7 in a subset of head and neck cancers, Oncogene 32 (12) (2013) 1549.
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1 Dong-Ge Wang a, Tong-Min Li b, Xuan Liu c, * a
Department of Obstetrics and Gynecology, Jinhua Municipal Central Hospital, No. 365 People's East Road, Jinhua 321000, China The Maternal and Child Hospital of Dongchangfu District, Liaocheng 252000, China c Center of Department of Gynecology, Qingdao Women and Children's Hospital of Qingdao University, Qingdao 266000, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2018 Accepted 27 May 2018 Available online xxx
Cervical cancer is the second commonest cancer among women in the worldwide, and the majority cause of death in various countries, highlighting the importance of investigating new therapeutic targets. Rh family, C glycoprotein (RHCG) belongs to the Rhesus (Rh) family and was first identified as Rh blood group antigens. It has been confirmed to function in cancer progression, including prostate cancer and esophageal squamous cell carcinoma. However, its role in cervical cancer has never been explored. The present study indicated that RHCG was down-regulated in cervical cancers compared to that in normal cervical tissues, and further decreased in cervical cancer cell lines. Functionally, RHCG overexpression reduced cervical cancer cell proliferation and migration, as evidenced by the decreased transforming growth factor (TGF)-b1, matrix metalloproteinase (MMP)-2 and MMP-9 expressions in cancer cells; however, an opposite effect was observed when RHCG was knocked down. Further, increase of RHCG markedly induced apoptosis in cervical cancer cells by improving the cleavage of Caspase-3 and poly (ADP-Ribose) polymerase (PARP). And cells transfected with RHCG siRNA exhibited a notable reduction of cleaved Caspase-3 and PARP. Moreover, nucleus nuclear factor-kB (NF-kB) and whole cell xIPA expressions were markedly reduced by over-expressing RHCG. Conversely, suppressing RHCG elevated NF-kB activation and xIPA expression in cervical cancer cells. Notably, we found that TGF-b1 treatment could abolish the effects of RHCG over-expression on the reduction of cell migration and enhancement of apoptosis in cervical cancer cells. Over-expressing RHCG-reduced NF-kB activation and xIPA expression were also abrogated by TGF-b1 pre-treatment. Additionally, enhancing NF-kB activity could restore xIPA expressions and decrease apoptotic response in cervical cancer cells over-expressing RHCG. In vivo, we also found that RHCG over-expression reduced cervical tumor growth through the same signaling pathways as we found in vitro. Therefore, RHCG may be a potential prognostic biomarker and therapeutic target for human cervical cancer. © 2018 Published by Elsevier Inc.
Keywords: Cervical cancer RHCG Migration and apoptosis NF-kB TGF-b1
1. Introduction Cervical cancer remains one of the leading causes of tumorrelated deaths worldwide [1]. Although there is accumulating evidence that early detection of cervical cancer has decreased mortality, the prognosis of advanced or recurrent cervical cancer remains very poor [2]. Presently, therapeutic treatments for cervical cancer include surgery with chemotherapy, radiation therapy, and concurrent chemoradiation therapy [3]. Due to the poor prognosis of patients suffering from cervical cancer, targeted
* Corresponding author. E-mail address: [email protected] (X. Liu).
therapeutics attracted researchers attention to develop effective treatments of advanced, recurrent, or metastatic cervical cancer. Rh type C-glycoprotein (RHCG) functions as an electroneutral and bidirectional ammonium transporter [4]. RHCG belongs to Rhesus (Rh) family, which was first identified as Rh blood group antigens in human erythroid cells. Rh proteins are tightly associated with the family of ammonia transporter proteins, characterized by the presence of 12 transmembrane-spanning segments [5]. Hence, Rh family proteins can fall into two functionally distinct groups, including ammonia transporting Rh glycoproteins (RHAG, RHBG, and RHCG) and nontransporting Rh proteins (RHD and RHCE) [6,7]. The nonerythroid Rh glycoproteins, RHBG and RHCG, are ammonia transporters, which are widely expressed in various
https://doi.org/10.1016/j.bbrc.2018.05.183 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
2
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
tissues. And RHCG was highly expressed in human skeletal muscle, kidney and oral cavity; however, its physiological functions are not well explained [8,9]. As reported, RHCG was decreased in tongue squamous cell carcinoma and in esophageal squamous cell carcinoma [10,11]. However, the effects of RHGC decrease on tumor growth, including cervical cancer, are still not yet investigated. In the study, we calculated the expression of RHGC in human cervical cancer tissues and their corresponding non-tumor cervix uteri samples. Functional analysis of cervical cancer cells transfected with RHGC overexpression plasmid or RHGC siRNA were carried out to reveal the role of RHGC in cervical cancer progression. RHGC exhibited anti-tumor effects on cervical cancer, which might be a potential a potential therapy target for the treatment of cervical cancer. 2. Materials and methods 2.1. Patients and information A total of 51 patients with cervical cancer who received surgery at the Department of Obstetrics and Gynecology, Jinhua Municipal Central Hospital (Zhejiang, China) between January 2008 and December 2014 were included in the study. Their corresponding non-tumor cervix uteri samples were used as controls. Specimens were collected from patients for protein and RNA extraction. No patients received preoperative treatment. Informed consent was obtained from all patients before the collection of specimens. The study was reviewed and approved by the Institutional Review Board at Jinhua Municipal Central Hospital.
threshold cycle method and normalized to GAPDH. CelLytic™ NuCLEAR™ Extraction Kit (Sigma Aldrich, USA) was used for nuclear protein extraction. Cells or tumor tissue samples were lysed on ice using a cell lysis buffer (125 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.1%Triton X-100 and 5 mM EDTA) containing both 1% protease inhibitor and 1% phosphatase inhibitor cocktail (SigmaAldrich). The resulting lysates were separated on 10% SDS-PAGE, transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with 5% skim milk and incubated with primary and secondary antibodies (1:5000, Abcam, USA). The blots were visualized with ECL reagent (Millipore). The primary antibodies were shown as follows: antibody against RHCG (1:500, #sc-100287, Santa Cruz, USA), TGF-b1 (1:1000; #ab92486, Abcam), phosphorylated NF-kB (1:1000; #ab86299, Abcam), NF-kB (1:1000; #ab16502, Abcam), MMP-2 (1:500, #sc-13595, Santa Cruz), MMP-9 (1:500, #sc-13520, Santa Cruz), Caspase-3 (1:1000; #ab13847, Abcam), PARP (1:1000, #9532, Cell Signaling Technology, USA), xIAP (1:500, #sc-55551, Santa Cruz), Lamin B (1:500; #sc-374015, Santa Cruz), and GAPDH (1:1000, #ab8245, Abcam). 2.5. Flow cytometry analysis After double staining with 5 mL of APC-Annexin V, the cells were then incubated for 15 min at room temperature in the dark followed by the addition of 5 mL of propidium iodide (PI) at room temperature for 5 min in dark. Finally, cells were analyzed uisng a flow cytometer (FACScan; BD Biosciences, USA) equipped with Cellquest software (BD Biosciences). 2.6. Transwell analysis for migration
2.2. Culture and transfection of cells Human cervical cancer cell lines, including Siha, Caski, Hela, C41, and C-33a, and normal endocervical cell line (End1/E6E7) were obtained from American Type Culture Collection (ATCC, USA). Human cervical cancer cell line of TC-1 was purchased from the Tumor Center of Chinese Academy of Medical Sciences. All cells were routinely maintained in RIP1640 or DMEM medium (Invitrogen, USA) supplemented with 10% FBS (Gibco, USA) at 37 C in a humidified air atmosphere containing 5% CO2. For RHGC overexpression plasmid, vector pcDNA3.1 plasmid was obtained from KpnI and EcoRV (TransGen Biotech, china). The code sequence of RHGC amplified using PCR with Quick-Fusion Cloning Kit (Bimake, Houston, USA). The siRNA against RHGC and negative control siRNA were obtained from Generay Co., Ltd (Shanghai, China). Cells were transfected with plasmids with DNA Transfection Reagent (Bimake) or with RHGC siRNA using Lipofectamine™-2000 (Invitrogen) when they grew to 70% confluence following the manufacturer's protocols. 2.3. Cell viability assay Cell proliferation analysis was performed using the MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's protocol. The results were measured by absorbance at 450 nm. 2.4. Quantitative real time-PCR (RTePCR) and western blot analysis Total RNA was isolated using TRIZOL (Invitrogen) and reverse transcribed into cDNA using Prime Script™ RT Master Mix (Takara, Japan). Gene transcripts were quantified using CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with SYBR Premix Ex Taq (Takara) and normalized with GAPDH. All primers are listed in Supplementary Table 1. Ct values were analyzed using comparative
An 8-mm pore size transwell chamber (Corning, USA) was used for transwell migration assay. A total of 1 105 cells in 100 mL medium in the absence of FBS were plated in the upper chamber and 500 mL medium containing 10% FBS was covered on the bottom chambers as chemoattractant. After incubation for 24 h, nonmigratory cells on the upper membrane surface were removed, and the cells on the bottom surface were fixed with 4% polyoxymethylene and then stained with 0.1% crystal violet for 10 min. Cancer cells were counted through photographing 5 random fields using a light microscope. 2.7. Immunofluorescent (IF) analysis Cells were fixed with 4% paraformaldehyde phosphate buffer solution and incubated with antibody against RHCG (1:100, #sc100287, Santa Cruz), and antibody against NF-kB (1:100, #ab86299, Abcam, USA) at 4 C overnight. Alexa Fluor 594-conjugated secondary antibodies (Abcam) were incubated at room temperature for 30 min. The nuclei were stained with DAPI (KeyGen Biotech). Then, the images were taken with a fluorescent microscope. 2.8. Cervical tumor xenograft model Animal experiments were performed according to the guideline for the Regulations for Animal Experiments and Related Activities at Jinhua Municipal Central Hospital. 6-week-old female Balb/c nude mice were purchased from Nanjing Peng Sheng Biological Technology Co Ltd (Nanjing, China). 1.0 107 Siha cells stably transfected with OE-Con, OE-RHCG, si-Con or si-RHCG were subcutaneously injected into the flank region of nude mice to establish cervical cancer xenograft model (n ¼ 6/group). Tumor volume was monitored through measuring the longest dimension (length) and shortest dimension (width) using dial calipers at 3-day intervals. Tumor volume was evaluated using the following formula: tumor
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
diameter ¼ 0.5 (length þ width). 2.9. Statistical analysis All data were expressed as mean ± SEM. Statistical analyses were performed using Graphpad Prism (version 6.0, USA). Differences among groups were analyzed by one-way ANOVA followed by Tukey's test. P < 0.05 was considered statistically significant. 3. Results 3.1. Expression of RHCG is associated with human cervical cancer development We calculated the relationship between RHCG expression in cervical cancer tissue and different clinicopathological characteristics (Table 1). High RHCG expression was associated with larger tumor size, lymph node metastasis and higher FIGO stage, indicating that RHCG expression was associated with aggressive oncogenic behavior of cervical cancer. To evaluate the expression and significance of RHCG in cervical cancer, RHCG mRNA expression levels were measured. As shown in Fig. 1AeC, RHCG expression was clearly decreased in cervical cancer tissues compared to non-tumor cervical tissues. Western blot analysis further confirmed the down-regulation of RHCG in human cervical cancer tissue samples (Fig. 1D). Kaplan-Meier survival analysis indicated that patients with high RHCG expression had better overall survival rate than those with low RHCG expression (Fig. 1E). A significant decrease of RHCG was observed in human cervical cancer cell lines using RT-qPCR, western blot and IF analysis (Fig. 1FeH). Overexpression and suppression was achieved by transfection of RHCG plasmid or siRNAs into Siha and Caski cell lines, as measured by RT-qPCR and western blot assays (Fig. 1I and J). 3.2. RHCG regulates migration and apoptosis in human cervical cancer cell lines As shown in Fig. 2A, the cell viability was significantly reduced after transfection of RHCG plasmid and increased after transfection with RHCG siRNA. The results also showed that the number of Siha and Caski cells for migrating in RHCG plasmid transfection group was markedly down-regulated in comparison to that in the control group, while suppression of RHCG by siRNA transfection markedly
Table 1 Relationship between RHCG expression in cervical cancer patients and clinicopathologic characteristics. Characteristics
No
RHCG expression High
Age (years) 49 <49 Tumor size (cm) 4.0 <4.0 Lymph node metastasis Yes NO FIGO stage IA IB II Parametrial invasion Yes NO a
p value
3
elevated the migration ability (Fig. 2B). It was also found that the protein levels of TGF-b1, MMP-2 and MMP-9 were significantly decreased, when RHCG was over-expressed in both cervical cancer cells. Inversely, cells transfected with RHCG siRNA showed a remarkable increase of TGF-b1, MMP-2 and MMP-9 in cervical cancer cells (Fig. 2C). RT-qPCR analysis indicated that the expression of Snail, TWIST, EGFR, and Vimentin obviously down-regulated in cells transfected with OE-RHCG plasmid compared with those infected with OE-Con plasmid. On the contrary, inhibition of RHCG by siRNAs clearly enhanced the expression of these signals. And a reverse result was observed in the change of E-cadherin in cancer cells after transfection with OE-RHCG plasmid or RHCG siRNA (Fig. 2D). RHCG obviously elevated the apoptosis in cervical cancer cells. Conversely, suppressing RHCG clearly reduced apoptosis (Fig. 2E). Western blot analysis indicated that RHCG overexpression markedly increased the cleavage of Caspase-3 and PARP. However, suppressing RHCG significantly decreased the cleaved Caspase-3 and PARP expressions (Fig. 2F). The number of pNF-kB-positive cells was evidently decreased as compared with that in the OE-Con group; however, a notable increase in p-NF-kBpositive cells was observed in cancer cells transfected with RHCG siRNA (Fig. 2G). Further, western blot analysis showed that nucleus NF-kB and cellular xIPA expressions were markedly downregulated in cells transfected with OE-RHCG. When transfected with RHCG siRNA, a significant increase of nucleus NF-kB and cellular xIPA levels was observed (Fig. 2H and I). 3.3. RHCG modulates TGF-b1 expressions to reduce migration and induce apoptosis in human cervical cancer cell lines In this section, we first found that OE-RHCG plasmid-induced reduction of Snail, TWIST, EGFR and Vimentin, and elevation of Ecadherin was abolished by TGF-b1 pre-treatment (Fig. 3A). The evident increase of apoptosis in cells transfected with RHCG plasmid was reversed by the pre-treatment of TGF-b1 (Fig. 3B). Consistently, pre-treatment of TGF-b1 clearly reduced the cleavage of Caspase-3 and PARP in RHCG over-expressed cells (Fig. 3C). In addition, the number of cells expressing p-NF-kB reduced by RHCG over-expression was markedly restored by TGF-b1 pre-treatment (Fig. 3D). As shown in Fig. 3E and F, the reduction of nucleus NFkB and cellular xIPA caused by RHCG over-expression was also eliminated by TGF-b1 pre-treatment. Following, NF-kB was activated by LPS and TNF-a addition (Fig. 3G). Both the expressions in nucleus NF-kB and cellular xIPA decreased by RHCG overexpression were recovered in LPS- and TNF-a-treated cells (Fig. 3H and I). Inversely, OE-RHCG-induced apoptosis was abrogated by LPS or TNF-a pre-treatment (Fig. 3J). Also, PS or TNF-a pretreatment led to a remarkable decrease of cleaved Caspase-3 and PARP in cancer cells transfected with OE-RHCG (Fig. 3K).
Low
3.4. RHCG reduces tumor growth in vivo 23 28
13 15
10 13
0.453
a
26 25
5 9
21 16
0.032
17 34
4 15
13 19
0.025a
3 38 10
1 12 2
2 26 8
0.022a
7 44
3 19
4 25
0.418
Statistically significant (p < 0.05).
In order to further evaluate the role of RHCG in regulating cervical cancer progression, the in vivo study was performed. As shown in Fig. 4AeC, RHCG over-expression markedly reduced the tumor size, tumor volume and tumor weight compared to the OECon group. In contrary, a significant increase of tumor size, volume and weight was observed in the group of si-RHCG than that in the si-Con. Western blot analysis indicated that RHCG over-expression led to a significant reduction of TGF-b1, MMP-2 and MMP-9, as well as p-NF-kB and xIPA in tumor tissue samples; however, a remarkable increase of these proteins was observed in si-RHCG group. Moreover, RHCG over-expression markedly increased the cleaved Caspase-3 and PARP expressions. And in si-RHCG group, we observed an evident decrease of Caspase-3 and PARP cleavage
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
4
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
Fig. 1. Down-regulation of RHCG in human cervical cancer tissues and cell lines. (AeC) RT-qPCR analysis of RHCG in 51 pairs of human cervical cancer tissues and their corresponding non-tumor cervix uteri samples. (D) Western blot analysis of RHCG in 24 pairs of human cervical cancer tissues and their corresponding normal cervix uteri samples. (E) Kaplan-Meier analysis indicated patients with high expression of RHCG showing better overall survival rate. (F) RT-qPCR, (G) western blot and (H) IF staining analysis of RHCG in cervical cancer cell lines and normal cervix uteri cells. (I,J) Confirmation of RHCG over-expression or inhibition after transfection with OE-RHCG plasmid or siRNAs in Siha and Caski cells by RT-qPCR and western blot assays. Data are presented as mean ± SEM of at least three independent experiments. n ¼ 6/group. ***p < 0.001.
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
5
Fig. 2. RHCG regulates migration and apoptosis in human cervical cancer cell lines. Siha and Caski cells were transfected with RHCG overexpression plasmid or siRNAs for 24 h (A) MTT analysis of Siha and Caski cells. (B) Transwell analysis for migration. (C) Western blot analysis of TGF-b1, MMP-2 and MMP-9. (D) RT-qPCR analysis of EMT-markers (Snail, TWIST, EGFR, Vimentin and E-cadherin). (E) Flow cytomery analysis of cells for apoptosis measurements. (F) Western blot analysis of cleaved Caspase-3 (C-Casp3) and PARP (C-PARP). (G) IF staining of p-NF-kB in cells. (H) Western blot analysis of NF-kB in nuclear of cells. (I) Western blot analysis of xIPA in whole cells. Data are presented as mean ± SEM of at least three independent experiments. n ¼ 6/group. *p < 0.05, **p < 0.01 and ***p < 0.001.
(Fig. 4DeF). Thus, RHGC showed anti-tumor role in human cervical cancer. 4. Discussion RHCG, an integral membrane protein, belongs to Rh family and forms homotrimer in plasma membrane. Furthermore, RHCG is highly expressed in human cervix, squamous epithelia of esophagus and oral cavity [4e7,9,10]. Nevertheless, the biological function of RHGC in these tissues or organs has not been investigated. RHGC was reported to show anti-tumor role in squamous cell carcinoma from esophagus and tongue [10,11]. Presently, the association between RHGC and human cervical cancer is little to be explored. In the study, we firstly discovered that RHGC was decreased in cervical cancers in comparison to that in the normal cervical tissue samples. Functionally, RHGC over-expression markedly reduced cervical cancer cell proliferation and migration, whereas RHGC knockdown generated an opposite result. Further, enhancing RHGC expression reduced TGF-b1 expressions, and migration-associated markers. Moreover, RHGC over-expression induced apoptosis in cervical cancer cells compared to the control group, while in the RHGC-silenced group, a remarkable decrease of apoptosis was observed through reducing cleaved Caspase-3 and PARP expressions. In addition, nuclear NF-kB and cellular xIAP expressions were found to be decreased in RHGC-overexpressing cells, while an opposite effect was observed in RHGC-knockdown group. Importantly, the in vitro results suggested that RHGCsuppressed cervical cancer progression was at least partly dependent on TGF-b1 inhibition to reduce cell migration and to induce apoptosis. In vivo study confirmed the results that RHGC over-
expression reduced the tumor growth via the same signaling pathways as we discovered in vitro. All these findings demonstrated that RHGC might be a tumor suppressor, playing an essential role in cervical cancer inhibition. It was validated that epithelial-mesenchymal transition (EMT) elevated cell migration and invasion, resulting in tumor cell dissemination, and RHGC was discovered to exert a suppressive effect on the migration of cervical cancer cells [12,13]. Overexpressing RHGC reduced EMT-markers of Snail, TWIST, EGFR, and Vimentin [12,14], while enhanced E-cadherin, which were abolished by RHGC suppression. MMPs were suggested to be involved in tumor metastasis through enhancing cell motility and degrading the extracellular matrix. MMP2 and MMP9 accelerated cell proliferation and migration, therefore promoting tumor development [15]. Our findings indicated that RHGC might impede MMP-2 and MMP-9 expressions to suppress cervical cancer cell migration. Therefore, in addition to the suppression of MMP-1 in esophageal squamous cell carcinoma, here we further found that RHGC could reduce MMP-2 and -9 to suppress tumor growth [9]. TGF-b1 is known to play an essential role in tumor growth. TGF-b1 could facilitate tumor invasion and metastasis, induce cells undergoing EMT, and modulate tumor microenvironment remodeling, which are in favor of tumor development and progression [16,17]. In our study, we also found that over-expressing RHGC significantly reduced TGF-b1 expressions in cancer cells, while an opposite result was observed in RHGC-knockdown cells. More importantly, TGF-b1 treatment could diminish RHGC-reduced migration, as evidenced by the restored expression of Snail, TWIST, EGFR, and Vimentin, and the down-regulated E-cadherin in cervical cancer cells. Therefore, we hypothesized that RHGC could
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
6
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
Fig. 3. RHCG modulates TGF-b1 expressions to reduce migration and induce apoptosis in human cervical cancer cell lines. Siha and Caski cells were pre-treated with TGF-b1 (10 ng/ml) for 24 h, followed by transfection with RHCG overexpression plasmid for another 24 h (A) RT-qPCR analysis of EMT-markers. (B) Flow cytomery analysis of cells. (C) Western blot analysis of C-Casp3 and C-PARP. (D) IF staining of p-NF-kB in cells. (E) Western blot analysis of NF-kB in nuclear of cells. (F) Western blot analysis of xIPA in whole cells. (G) Western blot analysis of p-NF-kB and xIAP in whole cells after treated with LPS (100 ng/ml) or TNF-a (100 ng/ml) for 24 h. Siha and Caski cells were pre-treated with LPS (100 ng/ ml) or TNF-a (100 ng/ml) for 24 h, followed by transfection with RHCG overexpression plasmid for another 24 h (H,I) Western blot analysis of nuclear NF-kB and cellular xIPA. (J) Flow cytomery analysis of cells. (K) Western blot analysis of C-Casp3 and C-PARP. Data are presented as mean ± SEM of at least three independent experiments. n ¼ 6/group. * p < 0.05, **p < 0.01 and ***p < 0.001.
suppress TGF-b1 expression to inhibit migration of cervical cancer (Fig. 4G). Apoptosis is an orchestrated event that cells are programmed to die after receiving specific stimuli, and thus is an essential component of cell growth control [18]. Apoptosis is characterized by morphologic alterations, including chromatin condensation, reduction of cell volume and nuclear fragmentation. Biochemical changes, such as Caspase activation, DNA and protein breakdown and membrane surface changes, are also critical characteristics of apoptosis [19,20]. When disturbed, an imbalance between life and
death of cells could result in tumor progression [21]. Here, for the first time, we found that RHGC over-expression could clearly induce apoptosis in cervical cancer cells; however, knocking down RHGC expressions resulted in a significant decrease in apoptosis, which was supported by the down-regulated activity of Caspase-3 and PARP. NF-kB is a transcription factor, which has been suggested to be significantly up-regulated in various tumors [22]. NF-kB is shown to inhibit apoptotic response to promote tumor growth [23]. Hence, inactivation of the NF-kB pathway could be served as a therapeutic target for preventing tumor growth, including cervical
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
7
Fig. 4. RHCG reduces tumor growth in vivo. (A) Representative images of tumor tissue samples isolated from each group. (B) Tumor volume was recorded every 2 days. (C) Tumor weight was measured. Western blot analysis of (D) TGF-b1, MMP-2 and MMP-9, (E) C-Casp3 and C-PARP, as well as (F) p-NF-kB and xIAP in tumor tissue sample. (G) Hypothesis of RHCG axis suppressing tumor growth by migration inhibition and apoptosis induction via TGF-b1. Data are presented as mean ± SEM of at least three independent experiments. n ¼ 6/group. *p < 0.05, **p < 0.01 and ***p < 0.001.
cancer [24]. The inhibitor of apoptosis (IAPs) protein family presents another negative modulator of apoptotic pathway. IAPs, mainly xIAP, directly suppress caspases through several mechanisms [25,26]. In the study, we found that RHGC over-expression markedly reduced NF-kB activation, which was in line with previous studies [11]. Also, xIAP expression was decreased in cells transfected with OE-RHGC plasmid. We further found that promoting NF-kB activation using LPS or TNF-a could elevate xIAP expressions, and reduce apoptotic response in cervical cancer cells over-expressing RHGC. TGF-b1 is reported to modulate NF-kB activity in various diseases, including cancer [27,28]. As expected, here we found that TGF-b1 treatment recovered NF-kB activation, and xIAP expressions in cervical cancer cells with RHGC overexpressions, accompanied with reduced apoptosis. Therefore, we concluded that RHGC inhibited human cervical cancer progression by inducing apoptosis and inactivating NF-kB pathway dependent on TGF-b1 suppression (Fig. 4G). In summary, our study indicated that RHGC was decreased in cervical cancers in comparison to that in normal cervical tissues.
Additionally, RHGC functioned as a tumor suppressor by decreasing the migration and inducing apoptosis in cervical cancer cells through down-regulating TGF-b1 expressions. These findings might provide useful clues for developing effective diagnostic and therapeutic strategies for cervical cancer. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.05.183. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.05.183 References [1] K.S. Tewari, M.W. Sill, H.J. Long III, et al., Improved survival with bevacizumab in advanced cervical cancer, N. Engl. J. Med. 370 (8) (2014) 734e743.
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183
8
D.-G. Wang et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
[2] S. Cao, W. Liu, F. Li, et al., Decreased expression of lncRNA GAS5 predicts a poor prognosis in cervical cancer, Int. J. Clin. Exp. Pathol. 7 (10) (2014) 6776. [3] Z.Z. Fu, Y. Peng, L.Y. Cao, et al., Value of apparent diffusion coefficient (ADC) in assessing radiotherapy and chemotherapy success in cervical cancer, MRI (Magn. Reson. Imaging) 33 (5) (2015) 516e524. [4] I.D. Weiner, J.W. Verlander, Renal and hepatic expression of the ammonium transporter proteins, Rh B glycoprotein and Rh C glycoprotein, Acta Physiologica 179 (4) (2003) 331e338. [5] C.H. Huang, J. Peng, Evolutionary conservation and diversification of Rh family genes and proteins, Proc. Natl. Acad. Sci. Unit. States Am. 102 (43) (2005) 15512e15517. [6] F. Quentin, D. Eladari, L. Cheval, et al., RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron, J. Am. Soc. Nephrol. 14 (3) (2003) 545e554. [7] D.O.D. Mak, B. Dang, I.D. Weiner, et al., Characterization of ammonia transport by the kidney Rh glycoproteins RhBG and RhCG, Am. J. Physiol. Ren. Physiol. 290 (2) (2006) F297eF305. [8] J.W. Verlander, R.T. Miller, A.E. Frank, et al., Localization of the ammonium transporter proteins RhBG and RhCG in mouse kidney, Am. J. Physiol. Ren. Physiol. 284 (2) (2003) F323eF337. [9] K. Takeda, T. Takemasa, Expression of ammonia transporters Rhbg and Rhcg in mouse skeletal muscle and the effect of 6-week training on these proteins, Physiological reports (10) (2015) 3. [10] B.S. Chen, Z.X. Xu, X. Xu, et al., RhCG is downregulated in oesophageal squamous cell carcinomas, but expressed in multiple squamous epithelia, EJC (Eur. J. Cancer) 38 (14) (2002) 1927e1936. [11] X.Y. Ming, X. Zhang, T.T. Cao, et al., RHCG suppresses tumorigenicity and metastasis in esophageal squamous cell carcinoma via inhibiting NF-kB signaling and MMP1 expression, Theranostics 8 (1) (2018) 185. [12] E.W. Thompson, D.F. Newgreen, Carcinoma invasion and metastasis: a role for epithelial-mesenchymal transition? Canc. Res. 65 (14) (2005) 5991e5995. [13] R. Kalluri, R.A. Weinberg, The basics of epithelial-mesenchymal transition, JCI (J. Clin. Investig.) 119 (6) (2009) 1420e1428. [14] S. Maseki, K. Ijichi, H. Tanaka, et al., Acquisition of EMT phenotype in the gefitinib-resistant cells of a head and neck squamous cell carcinoma cell line through Akt/GSK-3b/snail signalling pathway, BJC (Br. J. Cancer) 106 (6) (2012) 1196. €kela €, M. Kylma €niemi, et al., Expression of matrix [15] T. Salo, M. Ma
[16]
[17]
[18] [19]
[20] [21]
[22] [23] [24]
[25]
[26]
[27]
[28]
metalloproteinase-2 and-9 during early human wound healing, Lab. Invest. J. Tech. Methods. Pathol. 70 (2) (1994) 176e182. L.R. Gomes, L.F. Terra, R.A.M. Wailemann, et al., TGF-b1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells, BMC Cancer 12 (1) (2012) 26. H.R. Kim, J.S. Jo, K.E. Hwang, et al., Salinomycin Suppresses TGF-b1-induced EMT by Down-regulating MMP-2, 9 via AMPK-sirt1 Pathway in a Non-small Cell Lung Cancer, 2017. J.L. Koff, S. Ramachandiran, L. Bernal-Mizrachi, A time to kill: targeting apoptosis in cancer, Int. J. Mol. Sci. 16 (2) (2015) 2942e2955. G. Kiraly, A.S. Simonyi, M. Turani, et al., Micronucleus formation during chromatin condensation and under apoptotic conditions, Apoptosis 22 (2) (2017) 207e219. Y. Kiraz, A. Adan, M.K. Yandim, et al., Major apoptotic mechanisms and genes involved in apoptosis, Tumor Biol. 37 (7) (2016) 8471e8486. M. Redza-Dutordoir, D.A. Averill-Bates, Activation of apoptosis signalling pathways by reactive oxygen species, Biochim. Biophys. Acta Mol. Cell Res. 1863 (12) (2016) 2977e2992. M.H. Park, J.T. Hong, Roles of NF-kB in cancer and inflammatory diseases and their therapeutic approaches, Cells 5 (2) (2016) 15. A.L. Rinkenbaugh, A.S. Baldwin, The NF-kB pathway and cancer stem cells, Cells 5 (2) (2016) 16. H.H. Chen, S.P. Chen, Q.L. Zheng, et al., Genistein promotes proliferation of human cervical cancer cells through estrogen receptor-mediated PI3K/AktNF-kB pathway, J. Canc. 9 (2) (2018) 288. C.Y. Wang, M.W. Mayo, R.G. Korneluk, et al., NF-kB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation, Science 281 (5383) (1998) 1680e1683. E. Strekalova, D. Malin, H. Rajanala, et al., Metformin sensitizes triple-negative breast cancer to proapoptotic TRAIL receptor agonists by suppressing XIAP expression, Breast Cancer Research and Treatment 163 (3) (2017) 435e447. M. Arsura, G.R. Panta, J.D. Bilyeu, et al., Transient activation of NF-kB through a TAK1/IKK kinase pathway by TGF-b1 inhibits AP-1/SMAD signaling and apoptosis: implications in liver tumor formation, Oncogene 22 (3) (2003) 412. C. Freudlsperger, Y. Bian, S.C. Wise, et al., TGF-b and NF-kB signal pathway cross-talk is mediated through TAK1 and SMAD7 in a subset of head and neck cancers, Oncogene 32 (12) (2013) 1549.
Please cite this article in press as: D.-G. Wang, et al., RHCG suppresses cervical cancer progression through inhibiting migration and inducing apoptosis regulated by TGF-b1, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.05.183