Inhibitory Effect of Centipede Scolopendra Extract on Gallbladder Carcinoma via Up-regulation of PUMA

Background: To observe the effect of centipede scolopendra extract on gallbladder carcinoma (GBC) and further investigate its underlying mechanism. Methods: The GBC cell line GBC-SD was purchased and cultured. Small interfering RNA (siRNA) was constructed. The mRNA expression levels of PUMA were measured by reverse transcription-quantitative PCR (RT-qPCR) and protein expression levels of p53, PUMA, Bax and Bcl-2 were measured by western blotting respectively. Viability and proliferation were detected using MTT and colony formation assays respectively. The apoptosis rate was determined by Annexin-V-FITC/PI double staining. Results: The GBC-SD cell line was successfully obtained and cultured. MTT and colony formation assays demonstrated that the viability and proliferation of GBC-SD cells could be markedly inhibited by centipede scolopendra extract in a concentration-dependent manner in the limited concentration range. Following pretreatment with centipede scolopendra extract, RT-qPCR demonstrated that the expression levels of PUMA were markedly increased in the GBC-SD cells, and western blotting showed that the high expression of PUMA was accompanied by upregulation of Bax and downregulation of p53 and Bcl-2 in the GBC-SD cells. However, this effect has proved hard to reproduce after PUMA-siRNA. Flow cytometry revealed that the apoptosis rate was 9.8±2.2%, 25.3±3.6%, 10.6±2.0%, and 13.5±2.4% in control group, centipede scolopendra extract group, PUMA-siRNA group, and centipede scolopendra extract combined with PUMA-siRNA group respectively. Conclusions: Centipede scolopendra extract could induce the apoptosis of GBC-SD cells by promoting the PUMA-Bax signaling pathway. It could serve for GBC p53, PUMA, Bax and Bcl-2 were detected by western blotting. The results revealed that centipede scolopendra extract could markedly up-regulate the expressions of PUMA and Bax, while it markedly down-regulate the expressions of p53 and Bcl-2 in a concentration-dependent manner in the limited concentration range. This indicated that inducing apoptotic effect of centipede scolopendra extract was closely associated with the PUMA-Bax signaling pathway, which was consistent with the results of ow cytometry. However, further research are still required to clarify the other potential mechanisms of the regulatory process.

thousands of years. In recent years, a study has reported that centipede scolopendra extract has extensive biological activities, such as anti-in ammatory, anti-oxidation, anti-allergic, and anti-convulsant etc [10] . Besides, several studies have reported anti-tumor effect of centipede scolopendra extract including against epidermoid carcinoma and human embryonic kidney 293 cells [11] , human breast tumor [12] , hepatocarcinoma [13] and pancreatic cancer [14) . However, the effect of centipede scolopendra extract on gallbladder carcinoma have not been elucidated.
PUMA gene is located on chromosome 19q, spans a 119-kb length, encodes a 193-amino-acid protein, and contains Bcl-2 homology domain 3 (BH3) which presents in many apoptotic factors of Bcl-2 family.
As a strong inducer of apoptosis, the upregulation of PUMA could activate Bax by translocating Bax into the mitochondrial. Besides, it also could indirectly activate Bax by displacing Bax and binding the antiapoptotic protein Bcl-2/Bcl-xL to relieve the inhibition of Bcl-2/Bcl-xL on Bax. These could markedly promote the apoptosis of tumor cells [15] .
Taking advantage of the traditional medicine and exploring effective anti-tumor components may improve the e cacy of chemotherapy in GBC. In the present study, the effects of centipede scolopendra extract on GBC were preliminarily investigated, and the potential underlying mechanism was further elucidated.

Extract of Centipede Scolopendra
The centipede Scolopendra specimens were purchased from Changsha Jiuzhitang Pharmaceutical Co., Ltd in Hunan province of China. The specimens were identi ed as authentic by the Department of traditional Chinese medicine of our hospital where a voucher specimen was deposited. The centipede scolopendra was crushed into ne powder. First, 50 g specimens were decocted in 1500 ml alcohol solution (3/2 by v/v; alcohol/water) for 1 h, followed by ltering and collection of the ltrate concentrate.
Second, 750 ml alcohol solution (3/2 by v/v; alcohol/water) was added into the lter residue, and the aforementioned procedures were repeated. Third, the two ltrate concentrates were combined, ltered once again and then evaporated to 25 ml. Finally, the stock solution (2 g/ml, 25 ml) was bathed in water at 80 ℃ for 30 min with intermittent sterilization for 3 consecutive days. Subsequently, the samples were centrifuged at 1000 R/min for 10 min and stored at -20 ℃. When in use, the sterile normal saline was added to 400 mg/ml and stored at 4 ℃ and the bacteria were removed by suction. The rotary evaporator was used to concentrate the extract [16] .

Cell culture
The GBC-SD cell line was supplied by the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modi ed Eagle's medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/ml penicillin (Invitrogen, Carlsbad, CA, USA) and 100 U/ml streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were cultured with 5% CO 2 at saturated humidity at 37°C.
MTT assay GBC-SD cells in the logarithmic growth phase were homogeneously transferred to a 96-well plate (2×10 4 cells/well). After 24 h of culture in complete medium, the cells were treated with centipede scolopendra extract at concentrations of 3.125 mg/ml, 6.250 mg/ml, 12.500 mg/ml, 25.000 mg/ml and 50 mg/ml respectively. After  The A 490 were measured in the experimental group and control group, and the cell inhibition rate was calculated using the formula as follows: the inhibition rate (%) = [1 -(A 490 mean value of experimental group/A 490 mean value of control group)] × 100%. According to the changes of cell inhibition in the different concentration of centipede scolopendra extract and treatment duration, the optimum concentration and treatment duration of centipede scolopendra extract were con rmed. The experiment was repeated 3 times and the mean value was used to draw the cell growth curve

Colony formation assay
The cells were treated with centipede scolopendra extract as aforementioned. After 48 h of treatment, the cells were harvested and counted after trypsinization. A total of 500 cells/well were placed in a 6-well plate and cultured at 37˚C. The medium was refreshed once every 3 days. After 14 days, the cells were washed with 1×PBS, xed with 3.7% methanol, and stained with 0.1% crystal violet at 37˚C for 10 min (Sigma-Aldrich, St. Louis, MO, USA). Visible colonies were manually counted using an optical microscope (Olympus Corporation) (each colony contains at least 50 cells).

PUMA-siRNA synthesis
According to the GenBank database, the sequences of primers were designed in Table 1. Three interference siRNAs and one negative control siRNA were synthesized and determined no homology with other nonrelated genes by Genechem Co., Ltd ( Table 1). The cells were inoculated in a 6-well plate (5×10 5 cells/well), and the lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) was used for transfection with the siRNAs at a concentration of 120 nM according to the manufacturer's instructions. The cells were divided into ve groups as follows: PUMA-siRNA-1 group, PUMA-siRNA-2 group, PUMA-siRNA-3 group, PUMA-siRNA-NC group, and normal group. After 24 h, total RNA was extracted and ampli ed with RT-qPCR, then the more e cient PUMA-siRNA was selected to perform the subsequent experiments. The stability of selected PUMA-siRNA in the presence of centipede scolopendra extract was detected. The cells were divided into four groups as follows: PUMA-siRNA group, PUMA-siRNA combined with centipede scolopendra extract group, centipede scolopendra extract group and normal control group. After 24 h, total RNA was extracted and ampli ed with RT-qPCR.

Flow cytometry
The cells were inoculated in a 6-well plate (1×10 5 cells/well) and divided into four groups: PUMA-siRNA group, PUMA-siRNA combined with centipede scolopendra extract group, centipede scolopendra extract group, and control group. After treatment with different experimental materials according to the grouping, the cells were harvested, washed twice, resuspended in binding buffer, and stained with the Annexin V and PI solution at room temperature for 30 min. The Annexin V-FITC apoptosis detection kit (Beckman Coulter, Brea, CA, USA) was used to detect the apoptosis of cells by Annexin V-FITC and PI double staining according to the manufacturer's instructions. The analysis was performed using a BD FACSAria™ II ow cytometer (Becton, Dickinson and Company), and the data were analyzed using CellQuest Pro software (version 5.1; Becton, Dickinson and Company).

RT-qPCR
Following treatment with centipede scolopendra extract and/or PUMA-siRNA, total RNA was exacted from GBC-SD cell lines using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), which were then treated with DNase I (Invitrogen, Carlsbad, CA, USA) for puri cation according to the manufacturer's instructions.
Reverse transcription was performed at 42˚C for 60 min using miRcute miRNA First-Strand cDNA Synthesis Kit (TaKaRa biotechnology, Dalian, China) according to the manufacturer's instructions. qPCR was performed using the SYBR® Green PCR detection kit (TaKaRa biotechnology, Dalian, China) in a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scienti c, Inc.) according to the manufacturer's instructions. The sequences of the primers were summarized in Table 1. The 2 −ΔΔCt method was used to calculate the relative expression level of target gene [17] . The GAPDH was used as an internal control.
Western blot analysis for the protein expression of p53, PUMA, Bax and Bcl-2 Following treatment with centipede scolopendra extract and/or PUMA-siRNA, total protein was extracted using RIPA lysis buffer (50 mM Tris pH 8.0, 120 mM Nacl, 0.5% sodium deoxycholate, 0.5% NP-40, 0.1% SDS, 1 mM EDTA, 50 mM NaF, 1 mM Na2VO4, 1 mM PMSF, and 2 ug/ml aprotinin) on ice for 30 min. The protein concentration was determined by BCA assay, and 50 µg protein/lane was separated using the 10% sodium dodecyl sulfate-poly-acrylamide gel electrophoresis at 120 V for 60 min, which were then transferred onto polyvinylidene di uoride (PVDF) membranes. The membranes were blocked with the 5% non-fat milk in TBST (10 mM  Statistical analysis SPSS v17.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analyses. All data were expressed as the mean ± standard deviation. The differences were analyzed using T test for two groups and One-way Analysis of Variance (ANOVA) with the Bonferroni correction for multiple groups. P < 0.05 was considered statistically signi cant. All experiments were repeated at least 3 times.

Results
Optimum concentration and treatment duration of centipede scolopendra extract The GBC-SD cells were treated with centipede scolopendra extract at concentrations of 3.125 mg/ml, 6.250 mg/ml, 12.500 mg/ml, 25.000 mg/ml and 50.000 mg/ml for 24 h, 48 h and 72 h respectively. The results shown in Fig. 1 revealed that there was no signi cant difference among groups treated with different concentrations of centipede scolopendra extract in the rst 24 h. At 48 h after treatment with centipede scolopendra extract at the concentration of 12.500 mg/ml, the inhibition rate markedly increased to 59.68 ± 4.85% and continued to slowly increase with the growing concentration, however, excessive toxicity was observed at the concentration of 50.000 mg/ml and all cells were dead. The toxicity was observed as early as at the concentration of 12.500 mg/ml after treatment for 72 h, and when the concentration reached 25.000 mg/ml, all cells died. In this study, the concentration of 12.500 mg/ml and treatment duration of 48 h were selected for the subsequent experiments.
Designation of PUMA-siRNA The cells were transfected with PUMA-siRNA-1, PUMA-siRNA-2, PUMA-siRNA-3, and PUMA-siRNA-NC respectively using lipofectamine 2000 for 24 h, and then total RNA was extracted, ampli ed and measured. The results shown in Fig. 2A demonstrated that PUMA-siRNA-2 was more e cient, and the silencing e ciency could reach 71.85 ± 6.68% in the PUMA-siRNA-2 group. Therefore, the PUMA-siRNA-2 was selected to use in the next experiment.
The stability of PUMA-siRNA-2 in the presence of centipede scolopendra extract was proved in Fig. 2B.
The silencing e ciency of PUMA-siRNA-2 group was almost equal to that of PUMA-siRNA-2 combined with centipede scolopendra extract group.

Centipede scolopendra extract inhibits viability and proliferation of GBC-SD cells
To test the effect of centipede scolopendra extract on cell growth, the GBC-SD cells were treated with extract at concentrations of 3.125 mg/ml, 6.250 mg/ml 12.500 mg/ml and PUMA-siRNA-NC combined with centipede scolopendra extract at concentrations of 12.500 mg/ml for 12 h, 24 h and 48 h. The results shown in Fig. 3A demonstrated that the growth of GBC-SD cells were markedly inhibited by treatment with centipede scolopendra extract in a concentration-dependent manner in the limited concentration range according to the MTT assay.
Colony formation assay was performed to verify the effect of centipede scolopendra extract on proliferation ability of GBC-SD cells. As shown in Fig. 3B, the proliferation ability of GBC-SD cell was markedly suppressed by treatment with centipede scolopendra extract in a concentration-dependent manner in the limited concentration range.
Centipede scolopendra extract promotes cell apoptosis Following treatment with centipede scolopendra extract and/or PUMA-siRNA, the apoptosis of GBC-SD cells was detected by ow cytometry. As shown in Fig. 4, the GBC-SD cell apoptosis rate was 9.8 ± 2.2% in the control group, but this sharply increased to 25.3 ± 3.6% in the centipede scolopendra extract group (P < 0.01). Nevertheless, when PUMA-siRNA-2 was used to block PUMA expression, the GBC-SD cell apoptosis rate was 10.6 ± 2.0% in the PUMA-siRNA group compared with 13.5 ± 2.4% in the PUMA-siRNA combined with centipede scolopendra extract group (P > 0.05).
Underlying mechanism of Centipede scolopendra extract on GBC-SD To further uncover the potential mechanism of the inhibitory effect of centipede scolopendra extract on GBC-SD cells, RT-qPCR was performed to detect the expression of PUMA. As shown in Fig. 5, compared with the control group, the PUMA expression was markedly increased in the experiment groups (P < 0.05).
Western blotting was performed to detect the expressions of several key regulators. As shown in Fig. 6, compared with the control group, the PUMA and Bax expression were markedly increased while the p53 and Bcl-2 expression were markedly decreased following treatment with centipede scolopendra extract in a concentration-dependent manner in the limited concentration range. These results demonstrated that the PUMA-Bax signaling pathway may contribute to the inhibitory effect of centipede scolopendra extract on the GBC-SD cells.

Discussion
As a common type of cancer with an annually increasing occurrence rate, GBC has unsatisfactory outcomes of current therapies, which mainly included surgery, chemotherapy, radiotherapy, and immunotherapy and an extremely poor prognosis [7][8] . This is not only due to its high malignancy and a lack of early speci c diagnostic methods which lead to most patients at an advanced stage when diagnosed, but GBC also usually present low sensitivity to chemotherapy and radiation [9] . Additonally, the vast majority of patients cannot afford the expensive cost of immunotherapy drugs which are generally not covered by medical insurance in China. All these factors lead to the poor outcome of patients with GBC in China. It is imperative to identify novel and effective treatments for patients with GBC.
As a traditional Chinese medicine, centipede scolopendra has been used to treat a series of conditions such as childhood convulsion, diphtheria, poisonous nodules, spasm, and seizure for thousands of years in China [18][19] . There are venom glands in the rst two limbs of centipede scolopendra, which contain many species of chemical constituents such as histamine, sero-tonin, polysaccharids, polypeptides and lipids [20][21] . Previous studies have demonstrated that they present different biochemical and pharmacological properties [10] . Among these, the effect to inhibit tumor growth has been widely concerned [11][12][13][14]22] .
In the present study, the authors have demonstrated that centipede scolopendra extract served a notable role in inducing cell apoptosis to inhibit growth of GBC-SD cells. The results revealed that viability and proliferation of GBC-SD cells were markedly inhibited by centipede scolopendra extract in a concentration-dependent manner in the limited concentration range. This suggested that centipede scolopendra extract could serve as a novel treatment for patients with GBC in clinical practice.
Cell apoptosis plays a critical role in oncogenesis and progression and inducing apoptosis is a very common way of anti-tumor treatment [23] . Tamm has reported several mechanisms of apoptosis contributing to oncotherapy as follows: i) promoting growth-factor-independent cell death; ii) interfering with bypassing of cell-cycle checkpoints; and iii) enhancing sensitivity of tumor cells to immune-based cytotoxicity [24] . Therefore, the Annexin V-FITC and PI double staining assay was used to detect the apoptotic effect of centipede scolopendra extract on GBC-SD cells. The results demonstrated that centipede scolopendra extract could markedly induce apoptosis of GBC-SD cells. However, the effect of inducing apoptosis could be markedly inhibited by PUMA-siRNA, which suggested that centipede scolopendra extract may induce apoptosis of GBC-SD cells via the PUMA signaling pathway.
The biological functions of the PUMA gene has been introduced in detail in the previous article [11] . The p53-dependent and non-dependent pathways play key roles in PUMA inducing cell apoptosis. Previous studies have reported that the mechanism of PUMA-induced apoptosis may include the followings: i) PUMA and BCL-2/Bcl2xl combine to produce an inhibitory effect on Bax/Bak in the normal or tumor cells; ii) PUMA combines with p53/Bcl2xl so that p53 could be set free, which further activates Bax to induce cell apoptosis; and iii) PUMA directly binds to Bax/Bak to change its conformation, and then Bax is activated to induce cell apoptosis [25][26][27][28] .
In order to gain further insights into the mechanism of centipede scolopendra extract-induced GBC-SD cell apoptosis, further experiments were performed. Since the Bcl-2 family plays a vital role in the process of cell apoptosis [29][30][31] , the expressions of p53, PUMA, Bax and Bcl-2 were detected by western blotting. The results revealed that centipede scolopendra extract could markedly up-regulate the expressions of PUMA and Bax, while it markedly down-regulate the expressions of p53 and Bcl-2 in a concentrationdependent manner in the limited concentration range. This indicated that inducing apoptotic effect of centipede scolopendra extract was closely associated with the PUMA-Bax signaling pathway, which was consistent with the results of ow cytometry. However, further research are still required to clarify the other potential mechanisms of the regulatory process.
In this study, the inhibitory effect of centipede scolopendra extract on GBC and its underlying mechanism were only investigated by cell experiments in vitro. More studies will be carried out to con rm the results by animal experiments in vivo in the following research.

Conclusion
In summary, the present study demonstrated that centipede scolopendra extract could induce GBC-SD cell apoptosis via up-regulation of PUMA and Bax expression, correlating with down-regulation of p53 and Bcl-2 expression. This contributed to the inhibition by centipede scolopendra extract on GBC-SD cell growth. The present study offered effective data for centipede scolopendra extract as a potential novel strategy against GBC in clinical practice.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
WC conceived and designed the study. CY, YX and JJ obtained the cell lines and were the major contributors in acquiring the data. CY and YX performed the cell experiments and drafted the manuscript. ZL, WZ and ZC analyzed and interpreted the data. QL and WC revised the manuscript. CY, YX and WC con rm the authenticity of all the raw data. All authors read and approved the nal manuscript.