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Cell cycle arrest and apoptosis of OVCAR-3 and MCF-7 cells induced by co-immobilized TNF- a plus IFN- g on polystyrene and the role of p53 activation
Yan-Qing Guan
a,1, Zhibin Li
a,1, Aini Yang
a, Zheng Huang
a, Zhe Zheng
a, Lin Zhang
a, Ling Li
a, Jun-Ming Liu
b,c,*aSchool of Life Science and Institute for Advanced Materials, South China Normal University, Guangzhou 510631, China
bLaboratory of Solid State Microstructures, Nanjing University, Nanjing 210093, China
cInstitute for Advanced Materials, South China Normal University, Guangzhou 510631, China
a r t i c l e i n f o
Article history:
Received 3 April 2012 Accepted 17 May 2012 Available online 8 June 2012
Keywords:
Co-immobilized TNF-aplus IFN-g OVCAR-3
MCF-7 p53 regulation
a b s t r a c t
The aim of this study is to reveal the biological mechanism for high anti-cancer efficiency of co- immobilized TNF-a plus IFN-gpolymeric drug (co-immobilized drug) in mediating two gynecologic cancer cell lines: MCF-7 and OVCAR-3. The co-immobilized drug is prepared by mixing 10 ng/ml TNF- aplus 10 ng/ml IFN-gwhich are then photo-immobilized onto cell culture polystyrene plates. The drug compositions and microstructures are characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. The MCF-7 and OVCAR-3 cell cycle arrest and programmed cell death are checked byflow cytometry, and the expression of p53 is probed by immunofluorescence staining. The phosphorylation sites of the p53 regulation and the apoptosis key protein expressions of caspase3, 8 and 9 are detected by western blot assay. Our data show that, in case of short treatment time (48 h) at low cytokine concentrations (20 ng/ml), the co-immobilized drug demonstrates visible effects in comparison with the treatment using TNF-aplus IFN-gfreely attached on the polymeric plate (free drug). It is revealed that the co-immobilized drug leads to significant cell arrest in the S phase or G1and G2phase and offer high efficiency in mediating a caspase-dependent apoptosis via p53 transcriptional regulation.
Moreover, upon the treatment by the co-immobilized drug, the two gynecologic cancer cell lines show different phosphorylation sites of p53 and then different caspase-dependent apoptosis pathways. The present work sheds deep insights into the p53 regulation mechanism responsible for the high anti- cancer efficiency of the co-immobilized TNF-aplus IFN-gpolymeric drug against MCF-7 and OVCAR-3.
Ó2012 Elsevier Ltd. All rights reserved.
1. Introduction
There are more than two hundred different kinds of cancers that we can find from human being. Among them, human ovarian carcinoma and human breast carcinoma are the most common ones that happen to female[1]. Conventional treatments include surgery, radiation, drugs and other therapies. However, the majority of cases, especially those advanced stage cancers, are still treated utilizing conventional chemotherapy. The drug resistance and toxicity remain to be major issues in chemotherapeutic
treatment, and therefore, a great need of developing novel thera- peutic drugs that will be more efficient or will synergize with the existing ones is appealed[2,3].
It is well known that cytokines are important for the developed biological treatment. Tumor necrosis factor-a(TNF-a) and inter- feron-g (IFN-g) are the two relatively affirmative anti-cancer cytokines which have remarkable synergistic effect [4,5]. Most recently, El-shazly et al reported that IFN-gand TNF-apotentiate prostaglandin D2-induced human eosinophil chemotaxis by up- regulating prostaglandin D2 (PGD2) receptor and demonstrated a novel synergism between IFN-gand TNF-a[6]. Lash et al also showed that TNF-a, in combination with IFN-g, inhibits extravillous trophoblast cells invasion via a mechanism associated with increased trophoblast apoptosis, decreased trophoblast prolifera- tion, and/or altered production of active proteases[7]. Our previous
* Corresponding author. Laboratory of Solid State Microstructures, Nanjing University, Nanjing 210093, China. Tel.:þ86 25 83596595; fax:þ86 25 83595535.
E-mail address:[email protected](J.-M. Liu).
1 These authors contributed equally to this work.
Contents lists available atSciVerse ScienceDirect
Biomaterials
j o u r n a l h o m e p a g e : w w w . e l s e v i e r. co m/ lo ca t e / b i o m a t e ri a l s
0142-9612/$esee front matterÓ2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2012.05.037
Biomaterials 33 (2012) 6162e6171
work revealed that TNF-aplus IFN-gco-immobilized on the poly- meric plate (hereafter abbreviated as co-immobilized polymeric drugs) shows much higher inhibition ratio, in terms of the syner- gistic effect, than that of using either TNF-a or IFN-g, allowing reduction of single cytokine dose[8]. Moreover, it was argued that the co-immobilized drugs exhibit a synergism mechanism which leads to the synergism of different programmed cell deaths (PCD) pathways[9,10].
In fact, a huge number of investigations indicate that biomate- rials seem to offer higher and different activities in comparison with free biological treatment[11e14]. Among the drugs to induce the PCD, polymeric drugs, with advantages of delayed release and long action, have been the trend of the development of novel drugs.
Along this line, Maysinger et al.[15]prepared the polymeric micelle drug carriers with intercellular pH-triggered drug release func- tionality, including the tumor permeability and high antitumor efficacyin vitroandvivo.More recently, Chen et al demonstrated the biological effects of commercial pristine graphene in murine RAW 264.7 macrophages, important effector cells of the innate immune system. They found that the pristine graphene can induce cytotoxicity through depleting the mitochondrial membrane potential (MMP) and increasing the intracellular reactive oxygen species (ROS), then triggering apoptosis by activating the mito- chondrial pathway[16]. Also, Liang et al synthesized a kind of novel Au nanoparticles (NPs). These NPs were functionalized with ther- apeutic peptide (PMI (p12)) and targeted peptide (CRGDK) in order to selectively bind to neuropilin-1(Nrp-1) receptors which over- expresses on the cancer cells. In particular, the enhanced tumor suppressor p53 expression allows the Au@p12 þCRGDK NPs to show highly effective cancer treatment, indicating the importance of tumor suppressor p53[17].
The tumor suppressor p53 in regulating the expression of genes that mediate cell cycle arrest and/or classical apoptosis in response to cellular stresses, including DNA damage, growth factor depri- vation, hypoxia, and oncogene activation, has been well recognized [18]. Classical apoptosis is one of the PCD pathways and charac- terized by chromatin condensation and DNA fragmentation. It can be mediated by cysteine protease family called caspases[19e21].
Actually, p53 can distinctly regulate all these different cellular pathways because it is the isoform of many proteins. Different p53 isoforms are expressed under specific cell conditions and have different roles in the cell[22]. The p53 protein senses and integrates these various stresses via a panoply of post-translational modifi- cations, including phosphorylation, acetylation, and ubiquitination.
p53 protein is expressed constitutively, but the levels are normally low due to its rapid ubiquitination by the HDM2 protein and rapid proteasomal degradation[23].
Following the environmental insults, the human p53 N- terminal region is phosphorylated on serines 6, 9, 15, 20, 33, 37 and threonine 18 by ATM, ATR, DNA-PK, p38 MAPK, Chk1, and Chk2. The phosphorylation of p53 disrupts its binding with HDM2, blocks the ubiquitination and proteolysis, and results in a rapid increase in p53 protein levels, allowing p53 to enter the nucleus, to bind to DNA, and to induce the expression of DNA repair and cell cycle inhibitor genes. Moreover, the activated p53 up-regulates the expression of downstream target genes, including Bax, the p53-upregulated modulator of classical apoptosis[24,25].
Although the regulation of the tumor suppressor p53 is of prime importance for cancer chemotherapeutic treatment (since the p53 tumor suppressor gene is mutated in half of all human cancers), the cancer cell signaling associated with phosphorylation of p53 is complex and largely unknown[22].
In order to gain further insight into the molecular basis by which the co-immobilized drugs induce PCD in other human
gynecologic cancer cells and also the role of the tumor suppressor p53, this study was designed not only to examine the cell morphology, cell mortalities, and cell cycle arrest, but also to investigate the inducing expression of p53 gene and phosphor- ylation sites of p53 regulation, the functional status of caspases, the apoptosis pathways of OVCAR-3 and MCF-7 cells, respec- tively, induced by the co-immobilized drugs. It is suggested that the co-immobilized drug strongly induce cell cycle arrest and apoptosis of OVCAR-3 and MCF-7 cells by activating p53 in different phosphorylation sites and generating different caspase- dependent cell death pathways. It is argued that the possible mechanism of p53 regulation may have a key role in modulating different cell cycle arrests and different apoptosis pathways of OVCAR-3 and MCF-7 cells. This possible mechanism thus provides more precise knowledge of potential therapeutic applications of the co-immobilized drug in treating human ovarian carcinoma and breast carcinoma.
2. Materials and methods
2.1. Drug synthesis and surface characterization
The preparation procedure of the co-immobilized drug, i.e. TNF-aplus IFN-g co-immobilized onto the polymer substrate (PSt), can be found in Supplementary Materials. As a reference, the TNF-aplus IFN-gfreely absorbed onto the identical PSt (hereafter abbreviated as free drug) was also prepared.
The raw PSt was chosen as control group drug for the present experiments. The
1.0 0.8 0.6 1.0 0.8 0.6 0.4 1.0 0.8 0.6 0.4 1.0 0.8 0.6
3500 3000 2500 2000 1500 1000
Wavenumber (cm-1)Transmittance (%)
PSt
2922-CH
3-N
AzPhTNF-a
AzPhIFN-γ
21063
-N
1390 C-N 21093
-N
21341652
O
C PSt-Co-immobilized TNF-α/IFN-γ
3
υ
A
B
C
D
Fig. 1.Measured Fourier transform infrared spectroscopy spectra of the polystyrene (A), AzPhTNF-a(B), AzPhIFN-g(C), and also the polystyrene co-immobilized with AzPhTNF-aplus AzPhIFN-g(D).
Fourier transform infrared spectroscopy (FTIR) (TENSOR27, Bruker, Germany) on these drugs was performed to probe the characteristic peaks associated with the chemical nature between TNF-a, IFN-g, and PSt, as shown inFig. 1.
Subsequently, the surface morphology and structure of the co-immobilized drug, free drug, and control group drug as one category, and the co-immobilized drug, free drug, and control group drug immersed in the identical cell culture medium, and then cultured with OVCAR-3 or MCF-7 cells for 48 h as the other group, were investigated by scanning electron microscopy (SEM, JSM-6330F, Japan). For the SEM characterization, these samples were desiccated and gold sprayed. Then the surface morphologies were observed, respectively (Fig. 2).
2.2. Cell culture
The OVCAR-3 and MCF-7 cells were obtained from Animal Center of Sun Yat-Sen University. These cells were grown in RPMI 1640 medium (Gibco BRL) with 10% fetal calf serum, supplemented with 0.03mg/ml penicillin and 0.05mg/ml streptomycin.
Then they were seeded at 1105cells/ml into 24-well polystyrene culture plate with serum-free medium, and induced by the co-immobilized and free drug up to final concentration of 20 ng/well for 48hrs.
2.3. Cell death and cell cycle arrest
After stimulated by the co-immobilized and free drug for 48 h, these cells were stained according to conventional Annexin V binding protocol provided by Onco- gene. Briefly, RPMI1640 medium containing floating cells was collected. The absorbed cells were washed with PBS without Ca2þand Mg2þand scraped after 10 min incubation with 2 mM EDTA in PBS without Ca2þand Mg2þat room temperature. The combined 0.5106cells were washed with Dulbecco PBS, re- suspended in binding buffer, and incubated with Annexin V- FITC for 15 min at room temperature in the dark. After centrifugation processing, the Annexin V- FITC was removed and the cells were stained with PI in binding buffer. Then, the cells were analyzed immediately byflow cytometry (Becton Dickinson, FACSCalibur, San Jose, CA) using Cell Quest Program (Fig. 3A).
In parallel, upon stimulated by the co-immobilized and free drug for 48 h, these cells were washed three times using phosphate-buffer saline (PBS), and then harvested andfixed in ice-cold 70% ethanol for 12 h. Subsequently, they were re-suspended in PBS containing propidium iodide and RNase A, and
analyzed by FCM. Data were analyzed using Cell Quest and Modfit software (Fig. 3B).
2.4. Immunofluorescence analysis
Similarly, after stimulated by the co-immobilized and free drugs for 48 h, the OVCAR-3 and MCF-7 cells were washed using phosphate-buffer saline (PBS), and then incubated with appropriately diluted antibodies to p53 (Beijing Zhongshan Golden Biotechnology Co; China) overnight, washed and incubated with corre- sponding secondary antibodies labeled with IgG-Cy3(568e574 nm, red, Wuhan Boster Biological technology, China) for 1 h. At last, they were washed with PBS, stained with DAPI (0.5mg/ml) and viewed under thefluorescence microscopy, as shown inFig. 4. The control group drug was comprised of cells processed without primary antibody.
2.5. Western blot analysis
After stimulated by the co-immobilized and free drug for 48 h, the cells were lysed in extraction buffer (125 mM Tris.cl pH 6.8, 2%SDS, 4 M urea, 20% glycerol, 5%b- ME). The protein samples were separated by SDS-PAGE (12%) and electro-transferred onto NC (nitrocellulose) membrane (Boster Biotechnology Co., Ltd., China). The expression of proteins was obtained using antibodies to p53, p53ser-6, p53ser-37, p53ser-315 (Signalway Antibody Co., Ltd., USA) and caspase-8, caspase-9, caspase-3 (Boster Biotechnology Co., Ltd., China).
The blots were incubated with appropriate secondary antibodies conjugated to toalkaline phosphatase (AP) peroxidase (Boster Biological Technology Co., Ltd., China) and developed using ALP reagent (Beyotime Institute of Biotech- nology, China). The protein levels were normalized by reprobing the blots with antibody tob-actin (Boster Biological Technology Co., Ltd., China) (Fig. 5,Fig. 6 and Fig. 7). The protein expression was determined using the BandScan4.3 software.
2.6. Statistical analysis
Analysis on the significance of results was evaluated by two sample Student’st- test using the statistical software SPSS. Data are presented as meanSD. Differences are considered to be statistically significant atP<0.05.
Fig. 2.Scanning electron microscopy (SEM) of three polystyrene surfaces (A, D, G), three polystyrene surfaces cultured with OVCAR-3 cells (B, E, H), and three polystyrene surfaces cultured with MCF-7 cells (C, F, I) for 48 h.
Y.-Q. Guan et al. / Biomaterials 33 (2012) 6162e6171 6164
3. Results
3.1. Spectroscopy identification and microscopic surface analysis
A schematic diagram of the preparation and molecular structures of photoactive TNF-a or IFN-g (AzPhTNF-a or AzPhIFN-g) is shown in Supplementary Materials Figure.S1.
Since the spectroscopy evidences in our recent paper show clearly the effective reaction of TNF-a or IFN-g with N-(4-
azidobenzoyloxy) succinimide, i.e. formation of AzPhTNF-a or AzPhIFN-g[21,22], it is critical to check the attachment of the photoactive TNF-aor IFN-g onto the PSt plate after the photo- immobilization procedure.
The measured FTIR data on the raw PSt, AzPhTNF-a, AzPhIFN-g, and as-prepared co-immobilized drugs, are shown in Fig. 1.
Because of the presence of azido-phenyl group, a shift of the characteristic peaks associated with this group (2106 cm1, 2109 cm1, 2134 cm1) is demonstrated in the AzPhTNF-a(B),
Control Free Co-immobilized Cell apoptosis
MCF-7OVCAR-3 101
10
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210
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410
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4102 103 104 105 100 101 102 103 104
10
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4 100 101 102 103 10410
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4 100 101 102 103 1040.1% 2.4%
9.5%
88%
1.0% 2.4%
22.6% 74%
0.9% 13.6%
13.3% 72.2%
1.8% 25.2%
65.6%
7.4%
0.8%
0.7%
71.2%
27.3%
1.7% 1.4%
74.2% 22.7%
PI
PI
100 80 60 40 20 0
100 80 60 40 20 0
CK F C
CK F C
% Apoptosis ratio% Apoptosis ratio
**
**
ANNEXIN ANNEXIN ANNEXIN
Control Free Co-immobilized Cell arrest
OVCAR-3MCF-7 Cell NumberCell Number
500 400 300 200 100 0
0 40 80 120 160 240 500 400 300 200 100 0
0 40 80 120 160 240 500 400 300 200 100 0
0 40 80 120 160 240 500
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0 40 80 120 160 240 500
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0 40 80 120 160 240 500
400 300 200 100 0
0 40 80 120 160 240
DNA Content DNA Content DNA Content
%G1=63.0
%G2=0.4
%S=36.6
%G1=58.9
%G2=1.2
%S=40.0
%G1=40.4
%G2=0.2
%S=59.4
%G1=73.9
%G2=25.5
%S=0.7
%G1=46.2
%G2=6.8
%S=47.0
%G1=30.3
%G2=1.4
%S=68.3
% Cell Ratio
80 60 40 20 0
% Cell Ratio
80 60 40 20 0
G1 S G2
G1 S G2
Control Free Co-immobilized
Control Free Co-immobilized
** **
** **
A
B
a b c d
e f g h
a b c d
e f g h
Fig. 3.Measured cell mortalities (A) and cell cycle (B) data byflow cytometry for OVCAR-3 and MCF-7 cells were treated with co-immobilized and free drugs for 48 h. The upper and lower panel were OVCAR-3 cells and MCF-7 cells group (a, e: control; b, f: free; c, g: immobilized; d, h: cell mortalities or cell ratio), respectively. Evaluated cell mortalities or cell ratio for the corresponding treatments, with the significancep<0.05 labeled by symbol * andp<0.01 labeled by symbol **, co-immobilized and free drugs in comparison with each other. The bars stand for the standard deviations (n¼3).
AzPhIFN-g(C), and the co-immobilized drug(D). Besides, since the nitrogen in the azido group captured the hydrogen from carbon on the surface of PSt, the bond of -CH3(methyl) featured by the characteristic peak (2922 cm1) in the PSt is not detected in the co-immobilized drugs, demonstrating the specific binding of AzPhIFN-gor AzPhTNF-amolecules onto the polymeric substrates (The azido groups from the AzPhIFN-gor AzPhTNF-a molecules will change to nitrogen radicals and capture hydrogen from any carbon to form the C-N covalent bonds).
Furthermore, Fig. 2 presents three SEM images for the PSt surface (A, D, G), the PSt surface plus OVCAR-3 cells (B, E, H), and the PSt surface plus MCF-7 cells (C, F, I), respectively. In comparison with the PSt surface (A) and (D), the PSt surface (G) shows nano- sized dot structure. Also, normal cell morphology on the PSt surface plus OVCAR-3 cells (B) and MCF-7 cells(C) in control groups can be seen, but the PSt surface plus OVCAR-3 cells (E, H) and the PSt surface plus MCF-7 cells (F, I) show clear cell morphology of apoptosis, which may be due to the effect induced by the free and co-immobilized drugs. In this case, our results demonstrate that both the OVCAR-3 and MCF-7 cell death may involve an apoptosis pathway.
3.2. Programmed cell death and cell cycle arrest in OVCAR-3 or MCF-7 cells
On the other hand, the treatments by the free and co-immobilized drugs lead to the ovarian carcinoma cell line OVCAR-3 mortality rate as high as 76.4% and 85.8%, respectively, while upon the treatment by the free and co-immobilized drugs to the breast carcinoma cell line MCF-7, the cell mortality rate was 72.0% and 90.8%, respectively, as shown inFig. 3A.
Moreover, the DNA content histograms of the ovarian carcinoma cell line OVCAR-3 and the breast carcinoma cell line MCF-7 induced
by the co-immobilized drug after 48hrs are presented respectively inFig. 3B. Our results suggest that the cell cycle arrests of OVCAR-3 and MCF-7 induced by the co-immobilized drug are different from the cell cycle arrests of OVCAR-3 and MCF-7 induced by the free drug. In addition,Fig. 3B reveals that the OVCAR-3 and MCF-7 cells are arrested in the S phase and in G1/G2 phase, respectively. It seems that in this case, the OVCAR-3 and MCF-7 cells induced by the co-immobilized drug may not take the identical cell cycle arrest mechanisms.
3.3. Localization of p53 protein in OVCAR-3 or MCF-7 cells
In this experiment, the OVCAR-3 and MCF-7 cells were treated with the free and co-immobilized drugs for 48 h so that the cell morphology and protein expression of p53 can be observed by fluorescence microscopy, as shown inFig. 4. It seems that the co- immobilized drug is better than the free drug in terms of the apoptosis promotion. The cells become smaller and some cell chromosomes are stained into dark-blue or blue-black color. Some apoptosis featured by the cell shrinkage, fragmentation, and enhanced staining, can be identified.
In addition, we address the effect of the co-immobilized drug on the protein expression of p53 in OVCAR-3 and MCF-7 cells by immunofluorescence staining for 48 h, as shown inFig. 4. The difference in the amount of p53 protein can be detected upon the treatment by the free and co-immobilized drugs, respectively. In this case, we argue that both the OVCAR-3 and MCF-7 cell death may involve p53-dependent apoptosis pathway.
3.4. Phosphorylation sites of p53 protein in OVCAR-3 or MCF-7 cells
Moreover, we analysized p53 total protein level and the three phosphorylation sites for p53 using antibodies to p53ser-6, Fig. 4.Representative photomicrographs and by measured protein expression data of p53 immunofluorescence staining for OVCAR-3 and MCF-7 cells treated with co-immobilized and free drugs for 48 h. The middle and lower panel were free and co-immobilized group (AeC andDeF: control; G-I and JeL: free; MeO and PeR: immobilized), respectively. The white arrows mean that the cells are in the stage of the classical programmed cell death, and the yellow arrows mean that the protein expression of p53. Only one of three representative experiments is shown here. white bars, 10mm. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)
Y.-Q. Guan et al. / Biomaterials 33 (2012) 6162e6171 6166
p53ser-37, p53ser-315, and the data are presented inFig. 5. The blots show no apparent difference in p53ser-6 for both the OVCAR-3 cells and MCF-7 ones, although obvious up-regulation of the expression of p53ser-6 upon the treatment by the co- immobilized drugs at 48 h is identified. The co-immobilized drug induces remarkable up-regulation of the levels of p53 total protein, p53ser-6, p53ser-37, and p53ser-315 expression at 48 h in the MCF-7 cells, as shown in Fig. 5. In this case, we confirm that the apoptosis of OVCAR-3 and MCF-7 cells induced
by the co-immobilized drug may involve different active sites of p53 phosphorylation.
3.5. Caspase activity in OVCAR-3 or MCF-7 cells
Now we come to investigate the caspase activity in response to the two different cases and two cancer cell lines. To proceed, the OVCAR-3 and MCF-7 cells were induced by the free and co- immobilized drugs for 48 h, respectively. The antibody
OVCAR-3 MCF-7
CK F C CK F C
p53 total(53KD)→
p53ser6(53KD)→
p53ser37(53KD)→
p53ser315(53KD)→
β
-actin(43KD)→
p53 total
p53 phosphor-ser37 p53 phosphor-ser315 p53 phosphor-ser6 0.8
0.6 0.4 0.2
0.0
Expression (%)
OVCAR-3 MCF-7
0.8 0.6
0.4 0.2
0.0
Expression (%)
OVCAR-3 MCF-7
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Expression (%)
OVCAR-3 MCF-7
0.8
0.6 0.4 0.2 0.0
Expression (%)
OVCAR-3 MCF-7
Control Free
Co-immobilized
Control Free
Co-immobilized
Control Free
Co-immobilized Control
Free
Co-immobilized
**
**
** **
*
Fig. 5.Measured protein expression data of p53, p53ser-6, p53ser-37, p53ser-315 by western blotting for OVCAR-3 and MCF-7 cells. Cells were cultured in serum free medium and treated with control (CK), free TNF-aplus IFN-g(F) and co-immobilized TNF-aplus IFN-g(C) for 48 h, respectively. The protein expression was determined using BandScan4.3 software. The relative levels are plotted with the significancep<0.05 labeled by symbol * andp<0.01 labeled by symbol **, co-immobilized and free drugs in comparison with each other. The bars stand for the standard deviations (n¼3).
identifies three caspase-9 bands with molecular weights of 47 kDa, 37 kDa, and 35 kDa, and three caspase-3 bands with molecular weights of 32 kDa, 17 kDa, and 12 kDa, respectively, as shown inFigs. 6and7.
For the OVCAR-3 cells, the results show no essential up- regulating in the level of caspase-8 expression (57 kDa, 45 kDa, and 18 kDa) upon the treatment by the co-immobilized drug (Fig. 7A), while the expression of caspase-9 at 47 kDa, 37 kDa, and 35 kDa shows obvious up-regulating upon the treatment by the co- immobilized drug at 48 h (Fig. 7C). For the MCF-7 cells, the levels of caspase-9 (17 kDa and 12 kDa) for both the free and co-immobilized drug have no much difference (Fig. 7D).
However, the co-immobilized drug induces respectively the up- regulating of the level of caspase-3 expression (37 kDa and 17 kDa) and caspase-8 expression (57 kDa, 45 kDa, and 18 kDa) at 48 h, as shown inFig. 7B and F.
On the other hand, we found both the high expression of the cleaved forms of caspases and procaspases in the co- immobilized TNF-a plus IFN-g case. We argue that it might be due to the long time stimulation of the co-immobilized TNF-aplus IFN-g.
4. Discussion
Motivated by the advantages of immobilized cytokines[26e29]
and based on our earlier works[8e10], in the present study, we explore whether the co-immobilized drug treatment can effectively induce the programmed cell death on the other gynecologic cancer cell lines (OVCAR-3 or MCF-7) and what is the potential molecular basis for OVCAR-3 or MCF-7 cells.
Generally, it is of great importance to understand the mech- anisms of PCD in cancer cells, because PCD is believed to be one of the major consequences of anti-cancer drug treatment against malignancies. Classical apoptosis is a physiological mechanism for eliminating malignant cells or cancer cells without eliciting damage to normal cells or surrounding tissues. Thus, induction of apoptosis in target cells is a key mechanism by which anti- cancer therapy works[30]. Apoptotic cells are characterized by detachment, shrinkage and rounding, plasma membrane
blebbing, nuclear collapse and chromatin condensation[31]. In our work, the results clearly show the classical apoptotic cells morphology of OVCAR-3 and MCF-7 cells lines, as shown in Figs. 2,3 and 4respectively. Some of the cells present fragmented DNA, while at the same time almost all of the cells in the culture were rounded up or detached from the surface of the PSt co- immobilized with IFN-gplus TNF-a.
On the other hand, cell cycle arrest is well known as an early stage sign for programmed cell death[32]. More recently, the cell cycle alterations and cellular mechanisms induced by C-reactive protein (CRP) in H9c2 cardiac myocytes were revealed. It was shown that the CRP-treated H9c2 cells display cell cycle arrest in the G0/G1phase (the G1/S checkpoint) and CRP causes an increase in the p53 accumulation and its phosphorylation on Ser15, leading to the p21 up-regulation[33]. Moreover, p53 is also involved in inhibiting cell cycle progression from the G2phase to mitosis (the G2/M checkpoint). Bourougaa et al reported that in addition to previously reported induction of apoptosis, ER stress also leads to a p53-dependent G2arrest[34]. Hiraoka et al also reported that purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G1phase to the S phase as well as at the G2/M phase at higher concentrations, inducing significant apoptosis and cell death in J774 cells[35]. This p53-mediated arrest should allow repair of DNA damage or the elimination of highly damaged cells by apoptosis, in order to prevent the survival of genetically modified cells. However, in our study, the mechanisms by which p53 could monitor cell cycle progression seem to be diverse. The co-immobilized drug leads to the cell cycle arrest in the S phase (DNA replication (the intra-S checkpoint)) of OVCAR-3 and the G1plus G2phase of MCF-7 cells, compared with the case of the free drug at 48 h.
Since the tumor suppressor p53 in regulating the expression of genes that mediate cell cycle arrest and then classical apoptosis has been well recognized[18], ourfindings (the clas- sical apoptotic cells morphology of OVCAR-3 and MCF-7 cells lines and complicated cell cycle phase distribution) allow us to examine p53 activation in response to the co-immobilized TNF-aplus IFN-g.
Then, our results demonstrated that the cell death of both OVCAR-3 and MCF-7 may involve p53-dependent pathway. We found that the co-immobilized drug induces the remarkable up- regulating of the level of p53ser-37 and p53ser-315 expression only in the MCF-7 cells, which means that the cell death of OVCAR-3 and MCF-7 cells may involve different phosphorylation sites for the activation of p53, induced by co-immobilized drug. We thus argue that there might be increased transcriptional regulation of p53 that induces the different cell cycle arrests and classical apoptotic pathway.
Furthermore, the key proteins called caspases appear to be involved in regulating the activation of apoptotic signal trans- mission[36e40]. In our experiments, caspase-9 (both OVCAR-3 and MCF-7 cells), caspase-8 (MCF-7 cells), and caspase-3 (both OVCAR-3 and MCF-7 cells) were activated in the PCD process induced by the co-immobilized drug, as shown inFigs. 6and7. We think that the PCD of MCF-7 cells is stronger than the OVCAR-3 cells due to the stronger activities of caspases. Also, the stronger activities of cas- pases may be induced by the increased transcriptional regulation of p53 in MCF-7 cells.
We thus present our mechanistic model for the OVCAR-3 and MCF-7 apoptosis induced by the co-immobilized polymeric drug, as shown inFig. 8. Given the higher inhibiting activities for OVCAR-3 and MCF-7 cells, it is reasonable to conclude that the increased p53 transcriptional regulation and a caspase-dependent death pathway are the main reasons for the significant antiproliferative effect induced by the PSt co-immobilized with TNF-aplus IFN-g. OVCAR-3 MCF-7
CK F C CK F C
Pro-caspase-8(57KD)→
Pro-caspase-9(47KD)→
Pro-caspase-3(37KD)→
β
-actin(43KD)→
Cleaved-caspase-8(45KD)→
Cleaved-caspase-9(37KD)→
Cleaved-caspase-3(17KD)→
Cleaved-caspase-9(35KD)→
Cleaved-caspase-8(18KD)→
Fig. 6.Measured protein expression data of caspase-8, caspase-9, and caspase-3 by western blotting for OVCAR-3 and MCF-7 cells. Cells were cultured in serum free medium and treated with control (CK), free TNF-aplus IFN-g(F) and co-immobilized TNF-aplus IFN-g(C) for 48 h, respectively.
Y.-Q. Guan et al. / Biomaterials 33 (2012) 6162e6171 6168
Fig. 7.Measured protein expression data of caspase-8, caspase-9, and caspase-3 by western blotting for OVCAR-3 and MCF-7 cells. The protein expression was determined using BandScan software. The relative levels are plotted with the significancep<0.05 labeled by symbol * andp<0.01 labeled by symbol **, co-immobilized and free drugs in comparison with each other. The bars stand for the standard deviations (n¼3).
5. Conclusion
In summary, we have characterized the surface morphology of the co-immobilized TNF-aplus IFN-gpolymeric drug and discussed its high activity on inducing the programmed cell death for the other gynecologic cancer OVCAR-3 and MCF-7 cells. It has been revealed that the induced cell death mechanism of the co- immobilized TNF-aplus IFN-g is p53-regulated apoptosis. It has been suggested that co-immobilized TNF-aplus IFN-ghas potential therapeutic applications in treatment of human ovarian carcinoma and breast carcinoma.
Acknowledgments
The expenses of this work were supported by the Natural Science Foundation of China (30970731 and 31170919), the Natural Science Foundation of Guangdong Province (9151063101000015, S2011020003276), and the Priority Academic Program Develop- ment of Jiangsu Higher Education Institutions, China.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, atdoi:10.1016/j.biomaterials.2012.05.037.
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PSt matrix
TNF-αα IFN-γ
FADD TRADD
Pro-caspase8
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Pro-caspase3
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