Notoginsenoside R1 (NGR1) induce DNA damage to inhibit cervical cancer cells proliferation by inhibiting PHF6 activity

Notoginsenoside R1 (NGR1) is isolated from the panax notoginseng which is a kind of Traditional Chinese Medicine and edible plant with good healthful effect that using range is very wide for medical treatment and health care. It had been demonstrated to inhibit various tumors proliferation, but whether it inhibited cervical cancer cells proliferation and its mechanism was not unclear. In this study, we showed that NGR1 could inhibit cervical cancer cells to proliferate with a time and dose dependent manner, induce cervical cancer cells apoptosis and arrest cervical cancer cells in G1/S-phase . We also found that NGR1 could make H2AX phosphorylation and inhibit PHF6 expression with a time and dose dependent manner. Furthermore, when over expression PHF6 gene, the γH2AX haven't any change , but silenced PHF6 gene with siRNA, the γH2AX increased significantly. That mean PHF6 has negative correlation with γH2AX. Subsequently , we added NGR1 to intervene, something interesting happened that PHF6 protein fell even more, but the γH2AX more up regulation in the siPHF6 and NGR1 group. In the PEGFP-C1-PHF6 plasmid vector and NGR1 group, inversely , the PHF6 protein declined, and the γH2AX still up regulation . All those results indicate d that NGR1 caused DNA injury by inhibiting PHF6 activity pathway and arrested cervical cancer cells in G1/S-phase.

Introduction genes assembling, which is involved in embryonic development, neurogenesis, hematopoiesis and tumorigenesis [19,20,[23][24][25]. Recent studies have shown that deletion of the PHF6 gene can lead to instability increased of the rDNA genome and block the cells in G2/M to repair DNA [26].
In currently, chemotherapy also is an important method for therapy cervical cancer, especially suit for the patients with terminal cancers or metastatic cancers. But, chemotherapy is easy to cause different degrees of drugs tolerance, furthermore, most chemotherapy drugs are highly toxic and prone to many adverse reactions [27,28]. So, it lead to a series of serious impacts on the quality of life of cancer patients. Therefore, it metastasis of lung cancer [37]. But, Whether NGR1 has an effect on cervical cancer is unknown, and its mechanism is also indistinct. So, in this paper, firstly, we demonstrated that NGR1 has an inhibitory effect on cervical cancer. Secondly, we investigated the molecular mechanisms that NGR1 induce DNA damage to inhibit cervical cancer cells proliferation with down-regulation the expression of PHF6 protein.

NGR1 inhibits cervical cancer cells proliferation
At first, we demonstrated whether the NGR1 could inhibit the cervical cancer cells growth and proliferation. The NGR1 structure was showed in Fig. 1A. HeLa cells were treated with difference concentration of NGR1(0mM, 1mM, 2mM, 4mM, 8mM), by the CCK8 assay, we found that NGR1 could inhibit the cervical cancer cells growth and proliferation compared with non-treated cells, and the inhibition ratio is also depend on concentration gradient and time duration (Fig. 1B). Soft agar cloning formation experiment is an important symbolic of malignant cancer cells that connected with tumorigenesis, invasiveness and metastasis. Fig. 1C showed that the colony formation ability of HeLa cells were also obviously inhibited by NGR1 with a dose-dependent manner. Compared with the control group, the number of cells colony of the treated groups significantly decreased. Along with increasing the concentration, the cells colony formation was decreased. That mean the NGR1 could effectively inhibit the cervical cancer cells growth and proliferation.

NGR1 induces cervical cancer cells apoptosis
In this step, we observed whether NGR1 induces cervical cancer cell apoptosis. After Hela cells were treated with NGR1 for 24h, follow on, stained with DAPI. In the treatment group(8mM, NGR1), the results showed that the cell nucleus became irregular and small, even had some typical apoptotic bodies after NGR1 treated. However, in the control group, the cell nucleus morphology were roundish and they nucleus color were homogeneous ( Fig. 2A). Thereafter, HeLa cells were treated with a series concentration of NGR1 (0mM, 1mM, 2mM, 4mM, 8mM) for 12h. The cells were stained with AnnexinV-FITC/PI, then using flow cytometry to analyse apoptosis rate of the cells. The funny fact was that the cells apoptosis rate had a significant increase with a dose-dependent manner (Fig. 2B).
Caspase3 play a key role in the process of cell apoptosis. So we detected the caspase3 of Hela cells after NGR1 treatment for 24h. The results showed that caspase3 was activated after NGR1 treatment, and caspase3 up-regulated after NGR1-treated cells compared to the control cells (Fig. 2C).

DNA damaged and PHF6 protein activated caused by NGR1
DNA damage is an early event, it may immediately trigger DNA repair response. When damaged DNA can not be repaired, cells are likely to activate the apoptotic system to apoptosis. In the present study, HeLa cells were treated with different hours (0, 3, 6, 12, 24,48h) by NGR1(4mM), cells harvest for protein assay by western blot, we found that NGR1 could damage DNA and activate PHF6 protein with time-dependent manner (Fig. 3A).
In concretely describe, compare to the control group, DNA was damaged by NGR1 in early stage, because γH2AX increased from 3h to 12h, but it gradually returned to normal levels as time extension(from 24h to 48h). This mean NGR1 may sustained to destroy the DNA double stranded structure with time during until the DNA double stranded were completely destroyed. Meanwhile, we founded that PHF6 protein decreased by NGR1 treatment with the stress time going on. In addition, we also observed the relationship of γH2AX and PHF6 protein with the concentration of NGR1. HeLa cells were treated with difference concentration of NGR1(0, 1, 2, 4, 8mM) for 12h, we found that γH2AX increased and PHF6 protein decreased with dose-dependent manner (Fig. 3B).

NGR1 induce DNA damage by inhibiting PHF6 activity
PHF6 protein is a nucleolus protein which contains two zinc finger domains (ZaP1: aa14-134;ZaP2: aa 209-332). So we observed whether PHF6 protein locate in nucleolus though immunofluorescence assay. First, compare with DAPI group, we found PHF6 protein location in cell nucleus (Fig. 4A). Further investigation, compare with DAPI and nucleolin(C23, a nucleolus indicator protein) group , we found PHF6 protein location in nucleolus (Fig. 4B). Nucleolus is not only a site for storage rRNA, synthesis and assembly of ribosome subunits, but it is also involved in a number of important biological processes.
Recently study have shown that PHF6 may play an important role in nucleolar transcriptional regulation and/or chromatin remodeling [21]. So, we investigated the relation of DNA damage and PHF6 protein. When the PHF6 gene was independently over expressed by PEGFP-C1-PHF6 plasmid vector, we found the γH2AX haven't any change, but PHF6 gene silenced solely by siRNA, the γH2AX increased significantly. That mean PHF6 has negative correlation with γH2AX. It indicate that PHF6 deletion may have triggered a series of stress responses in the nucleus to repair the rDNA genome. Subsequently, we added NGR1 to intervene. We found, compared to the siPHF6 or control group, PHF6 protein fall even more, but the γH2AX more up regulation in the siPHF6 and NGR1 group.
In the PEGFP-C1-PHF6 plasmid vector and NGR1 group, inversely, the PHF6 protein declined, and the γH2AX still up regulation (Fig. 4C). The results indicate that NGR1 induce DNA damage by inhibiting PHF6 activity.

NGR1 arrest cervical cancer cells in G1/S phase
The previous results showed that NGR1 inhibited cervical cancer cells proliferation, So we continue to verify whether NGR1 affected the cells cycle progression. HeLa cells were treated with NGR1 at 2, 4, 8mM for 24h, respectively.
To collect and analyze the cells by flow cytometry assay. The specific procedures were similar to the apoptosis rate experiment. Compared with the control group (25.90%), both NGR1 at 2 mM, 4 mM and 8mM groups had more percentage in G1/S-phase, 34.13%, 44.32% and 58.63% respectively (Fig. 5). All those results clearly indicate that NGR1 arrest cervical cancer cells in G1/S phase and had a significant increase with a dose-dependent manner. Discussion NGR1 is isolated from panax notoginseng, a valuable Chinese herbal medicine and edible plants with good healthful effect for human. Several studies have found it has anti-tumor activity[37-39]. However, there are few reports that NGR1 has an anti-cervical cancer activity and its mechanism. In this study, we showed that NGR1 could effectively inhibit cervical cancer cells proliferation with time and dose dependent manner. The inhibition rate of cells were increased with extension the time or increasing the concentration of NGR1 (Fig. 1). It indicate that NGR1 has some symptomatic therapeutic effect on cervical cancer.
Apoptosis is a programmed cell death, a common phenomenon at the special developing stages in the prokaryotic and eukaryotic cells, which is an active mode of death and is controlled by certain genes [40][41][42]. It is very important for reproductive development, organogenesis and keeping the organism to maintain dynamic homeostasis [43,44] Recently, increasing NGR1 could induce SW480 human colorectal cancer cells apoptosis and arrest them in G1/S phase [36]. We also demonstrated that NGR1 induced cervical cancer cells to apoptotic and arrest them in G1/S phase( Fig. 2 and Fig. 5). The result is supported by nuclear morphology and cells cycle has change in NGR1 treated group.
Furthermore, the apoptosis rate of cells were increased with dose-dependent manner of NGR1 (Fig. 2B), the augment of G1/S ratio in the proliferation cycle of cells presents a dose-dependent manner (Fig. 5). As we known, Caspase3 activation plays an important role in the development of cell apoptosis [45][46][47]. In the NGR1 treated cells groups, we observed that caspase3 was increased relay on increasing the concentration of NGR1 (Fig.  2C). The results suggested that NGR1 activated caspase3 to apoptotic with dose dependent manner.
DNA damage is an early event, it may immediately trigger DNA repair response [48,49].
When damaged DNA can not be repaired, cells are likely to activate the apoptotic system to apoptosis. DNA damage response (DDR) is a series of regulatory events which including DNA damage, cell cycle arrest, and repair the damaged DNA to ensure the maintenance of genomic stability and cell viability [50]. In order to clarify its initial mechanism, we found that NGR1 indeed induce cervical cancer apoptosis, when we increased the NGR1 concentration to treat HeLa cells, An interesting phenomenon happened that NGR1 could make H2AX phosphorylation with a time and dose dependent manner (Fig. 3). Above all, our data suggest that DNA damage is an early event after NGR1 treatment and may trigger DNA repair processes, and that the failure of repair of DNA damage may trigger apoptosis program.
PHF6 deletion may have triggered a series of stress responses in the nucleus to repair the rDNA genome, blocking the progression of the cell cycle and inhibiting the proliferation of cancer cells, and delay the damaged genome to repair [26,51]. In this study, we found that NGR1 inhibited PHF6 expression with a time and dose dependent manner and PHF6 has negative correlation with γH2AX. Furthermore, when over expression PHF6 gene, the γH2AX haven't any change, but silenced PHF6 gene with siRNA, the γH2AX increased significantly (Fig. 3). That mean PHF6 has negative correlation with γH2AX. Subsequently, we added NGR1 to intervene, more interestingly were that PHF6 protein fall even more, but the γH2AX more up regulation in the siPHF6 and NGR1 group. In the PEGFP-C1-PHF6 plasmid vector and NGR1 group, inversely, the PHF6 protein declined, and the γH2AX still up regulation (Fig. 4).. All those results indicate that NGR1 caused DNA injury by inhibiting PHF6 activity.
In summary, our data showed NGR1 can effectively inhibit cervical cancer proliferation with a time and dose dependent manner, due to it induced cervical cancer cells apoptosis and arrests cells at G1/S-phase. In addition, our study also suggested that NGR1 induce DNA damage by activation the nucleolus protein of PHF6. It will provide a new target and therapeutic strategy for cancers from the molecular mechanism researches. Finally, It is worth noting that DNA repair function defects can cause tumors, but in cancer cells it can rapid and complete to repair DNA damage to resist chemotherapy drugs. This is the reason why most anticancer drugs do not work. But in this research we found that NGR1 could induce early DNA damage and lead to apoptotic. Its mean cervical cancer cells could not resist NGR1 to work. Furthermore, NGR1 has a function to enhance the organic immunity[52]. So, we believe that NGR1 may be extensively applied in clinical to treat various cancers in the future, due to it can improve patients' immunity to reduce drug resistance of cancers.

Reagents and antibodies
PEGFP-C1-PHF6 plasmid was gift from Ottawa Hospital Research Institute(professor Picketts DJ). NGR1 was obtain from Chen du Pure Chem-Standard Co.,Ltd(Chen du, China).
Its chemical structure is shown at Fig. 1A, and the purity is no less than 98%. NGR1 was dissolved in dimethyl sulphoxide (DMSO) which as 100 mM stock solution stored at -20°C, the stock solution was diluted using cell culture medium for the indicated concentration.

Cell viablility assays by CCK8 assay
HeLa cells were digested by trypsin and made the cells suspension concentration as 1×10 5 /ml. Inoculated 100μl the cells suspension into a 96-well plate to make per well with 1×10 4 cells. Cultured them incubator at 37℃ for 4h. After cells adherence, used different concentrations of NGR1 treatment them with 1, 2, 4, 8,and 16mM for 24h and 48h, at the same time each concentration were set 6 replicates. Added 100μl of DMEM medium with 10% CCK8 to each well as a liquid to change. Be carefully, do not has any bubble in the wells to avoid affecting OD value. After incubated its at 37℃ for 4h, the OD value was measured using a spectrophotometer at 450nm. Finally, the cells viability rate were calculated according to the formula: Cell viability rate = [(As -Ab)/(Ac -Ab)] ×100% As: absorbance of experimental wells (culture medium containing cells, CCK-8 and NGR1); Ac: absorbance of control wells (culture medium containing cells and CCK-8, but no NGR1); Ab: absorbance of blank wells (culture medium without cells, CCK-8 and NGR1). the operating instructions, Briefly, the cells (1×10 6 ) were collected using trypsinization to digest , washed twice with cold PBS. Cell pellets were fixed in 70% ethanol, washed the cells in cold PBS, resuspended the cells in 0.535 mL of dye buffer containing 10μl RNase(50×) and 25μl PI(25×), incubated the samples in the dark for 30 min at room temperature, and analyzed with a BD Accuri C6 flow cytometer. The siRNA or PEGFP-C1-PHF6 plasmid and Lipofectamine™2000 with Opti-MEM culture medium which without FBS were mixed gently, then incubated them on ice for 20min. The complexes was added to each cell well. After 6h, the culture medium was exchanged that instead of complete medium to keep cultivating.

Immunofluorescence staining
Immunofluorescence staining analyses was performed as previously described [53]. Briefly, HeLa cells were fixed with 4% paraformaldehyde for 20min, and then added 0.1% TritonX-100 incubation for 5min. The cells were blocked with 3% bovine serum albumin(BSA) for 1h, and then incubated with anti-PHF6 over nigh at 4°C. After washing, fluoreseinconjugated secondary antibody were incubated to conjugate the anti-PHF6. DAPI was use to label the cell nuclear. The all samples in the same experiments were observed under Olympus Confocal laser scanning microscope.

Western-blotting analysis
Western-blotting analysis was performed aspreviously described [53]. Briefly, cells were lysed in RIPA buffer containing 25mM Tris (pH7.6), 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1mM PMSF (Sigma), and 1% protease inhibitors cocktail(Thermo Fisher Scientific). The protein in lysate was measured with BCA Protein Assay kit. Equal amounts of protein extracts were loaded on 10% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred them to a PVDF membranes. The membranes were blocked with 5% skim milk for 1h at room temperature and then incubated them with the desired primary antibodies overnight at 4°C. After washing, its were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Detection was performed by using a chemiluminescent ECL advance western blotting detection kit. The primary antibodies used were anti-PHF6, anti-γH2AX and anti-caspase3 antibody. GAPDH served as control. Western-blotting radioactive signal was quantified using Image J.

statistical analysis
All experiments were repeated at least three times and the data were presented as the mean ± standard deviation (SD). Student's t-test was used to compare the differences in all the measurable variables in this study. P values less than 0.05 considered statistical significance.

Conflicts of interest
The authors declare that they have no competing interests in regards to this work.