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ORIGINAL ARTICLE
Year : 2015  |  Volume : 11  |  Issue : 8  |  Page : 239-243

Effect of capilliposide for induction apoptosis in human nasopharyngeal cancer CNE-2 cells through up-regulating PUMA expression


Department of Radiation Oncology, Zhejiang Cancer Hospital, Hangzhou 310022, China

Date of Web Publication26-Nov-2015

Correspondence Address:
Hu Qiaoying
Department of Radiation Oncology, Zhejiang Cancer Hospital, Hangzhou 310022
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.170529

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 > Abstract 

Objective: To observe the apoptosis of capilliposide against human nasopharyngeal cancer CNE-2 cells and to study its primary mechanisms.
Materials and Methods: Vectors pSilencer-PUMA-small interfering RNA (siRNA) were constructed to transcribe functional siRNA specially targeting PUMA. The interfering plasmids were used to transfect CNE-2 cells with lipofectamine 2000 transfection reagent. PUMA messenger RNA (mRNA) expression levels were analyzed by polymerase chain reaction. The proliferation of CNE-2 cells was detected using MTT colorimetry. Annexin V/propidium iodide double staining was applied to detect the apoptosis rate of CNE-2 cells. The protein levels of p53, PUMA, and Bax were detected using Western blot analysis.
Results: Recombinant siRNA expression vector targeting PUMA was constructed. MTT assays showed capilliposide inhibited the proliferation of CNE-2 cells in a concentration-dependent manner. The inhibition was strengthened along with increased concentrations. Apoptosis detected by flow cytometry in control group, drug group, siRNA group, and drug combined siRNA group was 9.3 ± 2.3%, 31.4 ± 5.6%, 12.3 ± 4.1%, and 13.2 ± 3.7%, respectively. After pretreated by capilliposide, PUMA protein was upregulated, and BAX was distributed to mitochondria in CNE-2 cells using Western blot analysis, but this effect can be interrupted by PUMA-siRNA.
Conclusions: Capilliposide could induce the apoptosis of CNE-2 cells, which might be related with the increasing in PUMA-Bax pathway.

Keywords: Apoptosis, capilliposide, PUMA, small interfering RNA


How to cite this article:
Yonghong H, Qiaoying H, Yongfeng P, Qiu T, Jiangguo F. Effect of capilliposide for induction apoptosis in human nasopharyngeal cancer CNE-2 cells through up-regulating PUMA expression. J Can Res Ther 2015;11, Suppl S4:239-43

How to cite this URL:
Yonghong H, Qiaoying H, Yongfeng P, Qiu T, Jiangguo F. Effect of capilliposide for induction apoptosis in human nasopharyngeal cancer CNE-2 cells through up-regulating PUMA expression. J Can Res Ther [serial online] 2015 [cited 2020 Sep 20];11:239-43. Available from: http://www.cancerjournal.net/text.asp?2015/11/8/239/170529


 > Introduction Top


Nasopharyngeal carcinoma is one of the most common malignant tumors in China, and the incidence rate ranks sixth in malignant tumor. Radiotherapy is the primary treatment for nasopharyngeal carcinoma, but more than 70% of locally advanced nasopharyngeal carcinoma must receive comprehensive treatment.[1] The current comprehensive treatment strategy is combined with radiotherapy, chemotherapy, and targeted therapy–Radiotherapy a regional treatment, chemotherapy and targeted therapy a system of treatment. In recent years, significant improvement was achieved and the overall 5 years survival rate was about 70%, but more than 40% of the patients eventually faced the treatment failure.[2] Taking advantage of the traditional medicine, exploring effective anti-cancer components, and participating in different period of the treatment of nasopharyngeal carcinoma may improve the therapeutic efficacy.

By analysis of ancient prescription, our study group found the capilliposide has antitumor activity by inducing the apoptosis of human nasopharyngeal carcinoma CNE-2 cells. To further understanding the antitumor effect of capilliposide, in this study, we analyze the function of P53 and PUMA in the apoptosis signal pathway using loss of function strategy.


 > Materials and Methods Top


Cells and cell culture

Human nasopharyngeal carcinoma CNE-2 cell line was supplied by the Zhejiang Provincial Cancer Hospital Research Institute. The cells were grown in RPMI-1640 medium (Hangzhou Sijiqing Biotechnology Limited) and supplemented with 10% fetal calf serum (Sigma Company) in 5% CO2 and saturated humidity at 37°C. Cells in the logarithmic growth phase were digested routinely for experimental use.

MTT cell viability assay

CNE-2 cells in the logarithmic growth phase were digested routinely with 0.25% trypsin (Gino Biomedical Technology Co., Ltd., Hangzhou, China) and then centrifuged to remove residual culture medium and trypsin. Subsequent to counting, the cells were homogeneously transferred to a 96-well plate (1 × 104 cells/well). After 12 h culture in serum-free medium, cells treated with 2, 4, 8, 20, and 40 μg/mL capilliposide (supplied by Professor Tian Bingkui Laboratory, Zhejiang University, China). After 0 h, 24 h, 48 h, and 72 h, each hole added 10 uL MTT (Sigma Company), culturing for 4 h, discarding the supernatant, adding 150 uL DMSO (Sigma Company) per hole, shaking for 10 min, and then 120 uL cell suspensions was transferred to another 96 well plate, and 120 uL DMSO solution was taken as a blank. Optical density was measured with enzyme mark instrument at 570 nm wavelength. The experiment was repeated 3 times. Cell growth curve was drawn with mean value.

Synthesis and transfection of small interfering RNA

According to the complementary DNA sequence of PUMA and GAPDH in GenBank, primer sequences were designed with PRIMER 5 software (PRIMER Biosoft, USA) as upstream of puma 5 '-CCCTGGAGGGTCCTGTACAA, downstream 5'-CTCTGTGGCCCCTGGGTAA, fragment length 61 bp, upstream of GAPDH 5 '-GAAGGTGAAGGTCGGAGTC, downstream 5'-GAAGATGGTGATGGGATTTC, and fragment length 225 bp. We designed 3 interference sequences and 1 negative control (NC) sequence and determined no homology with other nonrelated genes. Small interfering RNA (SiRNA) sequence was synthesized by Baiao MEIKO Biological Technology Co., Ltd., [Table 1]. CNE-2 cells were inoculated in the 60 mm culture dish (1 × 105 cells/dish), using lipofectamine 2000 reagent (Invitrogen Company) for transfection, dividing into five group: hs-puma-si-1, hs-puma-si-2, hs-puma-si-3, siRNA in NC group, and normal group (program according to the lipofectamine 2000 specification). Forty-eight hours after transfection, total RNA was extracted, amplified with a two-step Q-polymerase chain reaction, and then the siRNA of high-efficiency silence was selected for downstream experiment.
Table 1: Puma siRNA sequence

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Flow cytometry for the detection of apoptosis of CNE-2 cells treated with capilliposide

CNE-2 cells were inoculated in 6-well plate (2 mL, 1.5 × 105 cells/hole) and divided into the following four groups as siRNA, siRNA combined with capilliposide, capilliposide, and control group. Of the two groups with siRNA, 4–6 h after transfection, one of the groups was incubated with capilliposide for 48 h, and then apoptosis was detected. Cell density was adjusted to 1 × 106/mL with 100 uL. Binding buffer, washing, and suspending with phosphate-buffered saline (PBS) for 2 times, adding 5 uL Annexin V-FITC markers (Beyotime Company) and 5 uL PI solutions, then mixing gently. The apoptosis of cells were detected with a flow cytometer (Beckman Coulter, Miami, FL, USA) with 1 h after staining for 15 min (room temperature, avoiding light) and suspension in 400 uL binding buffer.

Western blot analysis for the protein expression of PUMA, BAX, and P53

Total protein and mitochondrial protein were extracted from CNE-2 cells in each group, and protein concentrations were determined by protein quantification kit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel was prepared. SDS-PAGE gel was prepared. Protein samples were subjected to electrophoretic separation by SDS-PAGE for 90–120 min. The damp-dry protein samples were transferred to polyvinylidene difluoride membranes (PVDF, PALL Company) and rinsed in Tris-buffered saline with TBST solution for 3 times, 5 min each time. Western blocking buffer was added for 60 min at room temperature. The blocked membranes were rinsed with PBS with Tween 20 solution. The membranes were blocked for 1 h with 5% skim milk powder under room temperature, and then treated with primary antibody at 4°C overnight. On the 2nd day, the membranes were thrice rinsed with TBST solution. The membranes were treated with secondary antibody at 37°C for 1 h, and then rinsed in TBST solution for 3 times, 5 min each time. The membranes were colored in the chemiluminescence analyzer (Getein biotech Inc, Jiangsu, China), and gray values were calculated using grayscale scanner. The ratio of the inner reference βeferen expression bands represented the target protein expression level.

Statistical analysis

All experiments were repeated 3 times. All data are presented as the mean ± standard error of the mean and analyzed using SPSS 12.0 software (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was performed for comparisons between groups. P <0.05 was considered statistically significant.


 > Results Top


Effective concentration of capilliposide

Capilliposide has inhibitory effect on the proliferation of nasopharyngeal carcinoma CNE-2 cells. On the base of the results of MTT, different concentrations of capilliposide did not show significant difference between the inhibition rates in the first 24 h but after 48 h, the inhibition rate significantly increased to 55% at the concentration of 8 ug/mL and continued to increase with the growing concentrations [Figure 1]. When the concentration reached 40 ug/mL, capilliposide exhibited excessive toxicity to CNE-2 cells, and all cells were dead after treated for 48 h. On base of data and the results of the loss of function strategy, in this study, we adopted the concentration of 15 ug/mL and action time of 48 h for the further research.
Figure 1: Inhibition rates at different concentration of capilliposide

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Synthesis and designation of PUMA-small interfering RNA

Hs-puma-si-1, hs-puma-si-2, hs-puma-si-3, and siRNA NC were respectively transfected into CNE-2 cells using lipofectamine 2000, and then RNA was extracted and amplified. Transcription levels of PUMA-siRNA mRNA were quantitated. Compared to the normal group, silence efficiency can reach 67% in hs-puma-si-1 transfection group. On base of the results, hs-puma-si-1 was selected for next step experiment [Figure 2].
Figure 2: Relative messenger RNA level of puma in different small interfering RNA groups

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Cell apoptosis

CNE-2 cell apoptosis was detected using flow cytometry. Compared with the control group, CNE-2 cell apoptosis rate increased from 9.3 ± 2.3% to 31.4 ± 5.6% (P = 0.01) after treated with capilliposide. However, when blocked PUMA expression with siRNA, the apoptosis rate in capilliposide group was 13.2 ± 3.7%, correspondingly, 12.3 ± 4.1% in PUMA-siRNA group (P = 0.13). From the results, the effect of apoptosis induced by capilliposide can be inhibited when PUMA expression was blocked, and this suggested capilliposide induced the apoptosis through the pathway of upregulating the expression of PUMA [Figure 3].
Figure 3: Apoptosis of CNE-2 cell in different groups: (a) Control group, (b) capilliposide group, (c) small interfering RNA combined with capilliposide group, and (d) small interfering RNA group

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Protein expression of p53, PUMA, and bax

CNE-2 cells were treated with capilliposide, PUMA-siRNA, or a combination of capilliposide and PUMA-siRNA, respectively. Compared with the control group, PUMA expression was upregulated in capilliposide group, while low expression in PUMA-siRNA group or in combination with capilliposide group. Whole cell lysates were isolated, and Bax expression, a downstream molecule of PUMA, was detected with Western blot. Bax protein increased and distributed in mitochondrial in capilliposide group, but these phenomena were not observed in PUMA-siRNA group or in combination with capilliposide group [Figure 4].
Figure 4: CNE-2 cells were treated with capilliposide or small interfering RNA; whole cell lysates were isolated and analyzed by Western blot with respective antibody

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Control group siRNA combined with capilliposide group capilliposide group siRNA group.


 > Discussion Top


Lysimachiacapillipes Hemsl, also known as Lysimachia or Anisochilus carnosua (L.) Wall, a kind of plant of Primulaceae, mainly distributed in Jiangxi, Fujian, Guangdong and Guizhou, etc., L.capillipes Hemsl tastes sweet and the whole plant, can be made into medicine. It can be used to treat cold, cough, asthma, rheumatic pain, irregular menstruation, neurasthenia, and tumor.[3] By exploring the ancient prescriptions, our study group found L.capillipes Hemsl has antitumor activity, and Lysimachia capilliposide plays the major role. In our study, apoptosis of nasopharyngeal carcinoma cell line CNE-2 can be induced by capilliposide proved by flow cytometry technology, and p53-PUMA pathway plays an important part in the process proved by loss of function strategy.

We explore the effective concentration and time of anti CNE-2 cell by detecting the proliferation through the method of MTT. The results of the study show that capilliposide has significant growth inhibition on CNE-2 cells, and the effect has the time-concentration dependence. According to the cell proliferation curve, the cell proliferation inhibition was not obvious before 24 h, but significant after 48 h. On the base of the results, we choose 48 h as treated time and the corresponding IC50 is 7.4 ug/mL. Considering the follow research of the loss of function strategy, we increase concentration of capilliposide to 15 ug/mL. By this optimization, in the case of blocking the expression of PUMA, apoptosis rate of CNE-2 cells will not be too low to influence our observations.

Apoptosis plays a very important role in the occurrence and development of tumor, inducing apoptosis of tumor cells is one of the common ways of various anti-cancer drugs.[4] After determining the action concentration and the time of capilliposide, we explore the apoptosis by flow cytometry. The results confirmed capilliposide can induce apoptosis of CNE-2 cells, but when using capilliposide, at the same time, inhibiting PUMA expression by siRNA, apoptosis of CNE-2 cells was significantly inhibited. On the base of results, we analyzed that capilliposide may induce apoptosis of CNE-2 cells through PUMA pathway.

PUMA, its gene located on chromosome 19q31, was primarily found by three independent research teams by Yu et al.,[5] Nakano et al.,[6] and Han et al.[7] The promoter region was rich in GC sequence, and this may be associated with the low expression in normal cells.[1],[8] The promoter region had the binding site of transcription factor c-Myc and P53, which was related to the PUMA transcription activation in the condition of stress. As a strong inducer of apoptosis, the apoptotic effect of PUMA has two important features–one is that PUMA almost mediated all the P53 dependent apoptosis signal,[9],[10] for the animals or cells of PUMA gene deletion, P53 cannot induce apoptosis; the other is that PUMA also mediated P53 independent apoptosis signal.[7] In the downstream pathways, PUMA can bind directly to Bax and promote Bax translocation from the cytosol into the mitochondrial; on the other hand, PUMA can indirectly activate Bax by displacing Bax and binding the anti-apoptotic protein Bcl-2/Bcl-xL located in the mitochondrial membrane, relieving the inhibition of Bcl-2/Bcl-xL on Bax. Therefore, in this research, we explored the apoptotic mechanism by detecting P53 and PUMA expression after treated with capilliposide using Western blot technology, as long as detecting the distribution of PUMA downstream molecule Bax from the cell total protein and mitochondrial protein.

After treated with capilliposide, Western blot examination found that both P53 and PUMA were upregulated in CNE-2 cells. This suggested that capilliposide could induce PUMA expression by P53 dependent pathway. When blockaded PUMA expression with capilliposide and siRNA in CNE-2 cells, the expression of P53 remained elevated, but PUMA expression was inhibited, this phenomenon was consistent with the apoptosis inhibition examined by flow cytometry.


 > Conclusion Top


Our study found that capilliposide can induce CNE-2 cell apoptosis by up-regulating p53 and PUMA expression, and promoting Bax to the mitochondrial distribution. Though more in vitro and in vivo studies are needed to establish the anti-tumor activity and mechanisms of capilliposide, findings presented herein collectively provide fundamental insight of the usefulness of capilliposide in human nasopharyngeal cancer therapy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

1.
Pignon JP, Bourhis J, Domenge C, Designé L. Chemotherapy added to locoregional treatment for head and neck squamous-cell carcinoma: three meta-analyses of updated individual data. MACH-NC Collaborative Group. Meta-Analysis of Chemotherapy on Head and Neck Cancer. Lancet 2000;355:949-55.  Back to cited text no. 1
    
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Peng G, Wang T, Yang KY, Zhang S, Zhang T, Li Q, et al. A prospective, randomized study comparing outcomes and toxicities of intensity-modulated radiotherapy vs. conventional two-dimensional radiotherapy for the treatment of nasopharyngeal carcinoma. Radiother Oncol 2012;104:286-93.  Back to cited text no. 2
    
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Shi KH, Xie WM. Cultivation techniques of Lysimachia capillipes Hemsl. MinXi Tech 1997;2:37-8.  Back to cited text no. 3
    
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Indran IR, Tufo G, Pervaiz S, Brenner C. Recent advances in apoptosis, mitochondria and drug resistance in cancer cells. Biochim Biophys Acta 2011;1807:735-45.  Back to cited text no. 4
    
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Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell 2001;7:673-82.  Back to cited text no. 5
    
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Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 2001;7:683-94.  Back to cited text no. 6
    
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Han J, Flemington C, Houghton AB, Gu Z, Zambetti GP, Lutz RJ, et al. Expression of bbc3, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals. Proc Natl Acad Sci U S A 2001;98:11318-23.  Back to cited text no. 7
    
8.
Jeffers JR, Parganas E, Lee Y, Yang C, Wang J, Brennan J, et al. Puma is an essential mediator of p53-dependent and -independent apoptotic pathways. Cancer Cell 2003;4:321-8.  Back to cited text no. 8
    
9.
Li Y, Feng H, Gu H, Lewis DW, Yuan Y, Zhang L, et al. The p53-PUMA axis suppresses iPSC generation. Nat Commun 2013;4:2174-93.  Back to cited text no. 9
    
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Uo T, Veenstra TD, Morrison RS. Histone deacetylase inhibitors prevent p53-dependent and p53-independent Bax-mediated neuronal apoptosis through two distinct mechanisms. J Neurosci 2009;29:2824-32.  Back to cited text no. 10
    


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