Adhesion to type V collagen enhances staurosporine-induced apoptosis of adrenocortical cancer cells
Tiziana Nardo · Geraldina Micalizzi · Roberto Vicinanza · Francesca De Iuliis ·
Ludovica Taglieri · Susanna Scarpa
Received: 7 April 2014 / Accepted: 24 June 2014/Published online: 10 July 2014 C International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Adrenocortical carcinoma (ACC) is a rare and ag- gressive tumor characterized by poor prognosis and resistance to conventional chemotherapy. Many chemotherapy agents act determining apoptosis, therefore, studying the responsive- ness of ACC to apoptosis inducing molecules, can help to identify possible conditions to promote cancer cell death. Tumor progression is strictly related to the interaction between cancer cells and stroma; yet, extracellular matrix remodeling regulates tumor cell proliferation and apoptosis. At this pur- pose, we have studied staurosporine-induced apoptosis of ACC cell line H295R adherent to different extracellular matrix molecules. H295R cells grown on plastic showed a low re- sponsiveness to staurosporine, with an apoptotic rate of 24 %, as compared to breast cancer MCF7 cells, with an apoptotic rate of 60 %. The adhesion of H295R cells to type V collagen induced a significant increase of apoptosis up to 52 %; this effect was inhibited by anti-integrin alpha2 antibody. At the same time, the adhesion of H295R cells on polylysine, matrigel, lamimin, fibronectin, and type I-III collagens didn’t modify staurosporine-induced apoptosis. Staurosporine- treated H295R cells showed an increase of PARP cleavage and of annexin-V expression, when adherent to type V colla- gen. Yet, staurosporine induced Akt and Erk activation on H295R cells: the adhesion on type V collagen didn’t modify Akt activation, while determined a dramatic inhibition of Erk activation. The described data demonstrate that the adhesion to type V collagen specifically increases the responsiveness of ACC cells to staurosporine-induced apoptosis and that this is probably obtained through the inhibition of Erk activation.
Keywords Adrenocortical cancer . Type V collagen . Apoptosis · Staurosporine
Introduction
Adrenocortical carcinoma is a rare tumor that is characterized by an aggressive malignancy and a resistance to conventional therapy; about 50 % of cancer patients don’t survive 2 years from the diagnosis. Surgery is still the unique valid therapy for this tumor and it can be beneficial only when performed on patients without advanced tumors; approximately 40 % of all patients who undergo a radical resection of this cancer will be live 5 years later [1].
Chemotherapy with the drug mitotane has a little to add in the treatment of adrenocortical carcinoma; prognosis and life expectancy of this tumor haven’t improved in the last 30 years [2]. Actually, some biological therapies have been utilized in clinical trials, such as the use of anti-angiogenic molecules, among these the drug bevacizumab, that is a monoclonal antibody inhibiting vascular endothelial growth factor A (VEGF-A) [3, 4]. A recent research performed on H295R adrenal cancer cell line has demonstrated an anti- proliferative activity of resorcylic acid lactone, a potent and specific inhibitor of MEK (mitogen activated extracellular Kinase) [5, 6]. MEK regulation can be mediated by the interaction between extracellular matrix and integrins, which activates intracellular signal transduction pathways leading to the control of either cell survival, growth, or death [7]: several extracellular matrix components, such as laminin, fibronectin, and collagens, can be involved in this cross talk with cellular integrins. For example, alpha5beta1 interacts with fibronectin to activate intracellular pathway of Erk which induces cell proliferation [8]; at the same time, alpha5beta1 integrin can support cell survival through the activation of three different pathways, FAK, PI3K/Akt, and Calmodulin-dependent
T. Nardo · G. Micalizzi · R. Vicinanza · F. De Iuliis · L. Taglieri · S. Scarpa ☒
Experimental Medicine Department, Sapienza University of Rome, 00161 Rome, Italy
e-mail: susanna.scarpa@uniroma1.it
protein kinase [9]. Anchorage dependence has long been recognized as a requirement for cell viability and cells that denied adhesion undergo apoptosis called anoikis [10]. Usu- ally, integrin-mediated signaling negatively regulates the in- trinsic apoptotic cascade; however, integrins can also promote the extrinsic apoptotic cascade by positively regulating a form of apoptosis called integrin-mediated death (IMD) [11]. Thus, different ligands, or different forms of particular ligands, can transmit through integrin distinct signals of the survival or death of the cell. Furthermore, emerging evidence is growing towards an important role played by extracellular matrix dur- ing tumor progression and response to therapy [12].
Considered that adrenocortical carcinoma patients often evolve in resistance to chemotherapy, and that one of the mechanisms involved in developing tumor cell chemoresistance is the escape from apoptotic response, we choose the adrenocortical carcinoma cell line H295R to eval- uate its sensitivity to staurosporine, a potent apoptosis inducer. Staurosporine is a protein kinase A (PKA) inhibitor that determines apoptosis through the activation of caspase 3 [13]. We initially found a significant low responsiveness of H295R cells to staurosporine-induced apoptosis, as compared to other cancer cells; therefore, the aim of our research was to understand whether the adhesion to specific components of the extracellular matrix could modify the described low re- sponsiveness of this adrenocortical carcinoma cell line to staurosporine-induced apoptosis.
Materials and methods
Cell cultures and substrates
H295R adrenocortical carcinoma cell line [14] was supplied from ATCC (Rockville, MD. USA), and it was grown in DMEM/HAM’S F-12 Medium supplemented with penicillin-streptomycin 50 U/ml and with 10 % fetal calf serum, and enriched with a mixture of insulin, transferrin, and selenium ITS (Sigma). MCF7 was grown in RPMI-1640 Medium supplemented with penicillin-streptomycin 50 U/ml and with 10 % fetal calf serum.
Staurosporine (Sigma) was dissolved in DMSO (Sigma) in stock solution of 1 mM and conserved frozen, the following dilutions were made in culture medium.
The substrates were all purchased from Sigma-Aldrich and diluted in PBS: 25 µg/ml for fibronectin and laminin, 10 µg/ml for polylysine, and 40 µg/ml for Matrigel; type I- III-V collagens were diluted in 0.5 M acetic acid at the concentration of 25 µg/ml. The plates were covered with each substrate at the final concentration of 2.5 µg/cm2, and the plates were exhaustively washed with PBS before use.
The neutralization of integrin was obtained by incubating the cells after trypsinization in 100 ul medium with 1 ul of
mouse antibody anti-integrin «2 (Millipore) for 30 min at room temperature (RT), the cells were then normally plated.
Western blot
Cell lysates were obtained scraping the cells in lysis buffer 1 % Triton, 0.1 % SDS, 150 mM NaCl, 50 mM TrisHCl pH 7.4, and 2 mM EDTA plus protease inhibitor cocktail tablet (Roche Applied Sciences) for 30 min at 4 ℃, lysates were then centrifuged at 12,000 rpm for 15 min at 4 ℃. Protein concen- tration was evaluated by Bio-Rad Protein Concentration Assay.
Fifty micrograms of lysate were separated by molecular weight on 8 or 10 % SDS-PAGE and then transferred onto a nitrocellulose membrane. Blots were blocked for 1 h at RT in 5 % non-fat dry milk and then incubated with primary anti- body, washed in Tris-buffered saline with 0.1 % Tween-20 and then incubated with horseradish peroxidase conjugated anti-mouse or anti-rabbit antibodies (1:5,000 diluted) (Sigma- Aldrich). The filters were then developed by enhanced chemi- luminescence (Super Signal West Pico Chemiluminescent Substrate, Thermo Scientific) using Kodak X-Omat films. The used of primary antibodies were mouse anti-cleaved caspase-3 (1:500 diluted) (Cell Signaling), mouse anti-Parp- 1 (1:500 diluted) (Santa Cruz Biotechnology), rabbit anti- AKT (1:2,000 diluted) (Abcam, Cambridge, UK), rabbit anti-phospho S473 AKT (1:2,000 diluted) (Abcam, Cam- bridge, UK), rabbit anti-ERK (1:1,000 diluted) (Santa Cruz Biotechnology), mouse anti-phospho S7383 ERK (1:1,000 diluted) (Santa Cruz Biotechnology), and mouse anti-actin (1:1,000 diluted) (Sigma Aldrich).
Proliferation test
To determine cell density, sulforhodamine B colorimetric assay was performed [15]: 1.5x104 cells were plated on 96 wells plate and grown for 24 h untreated or 14 h staurosporine treated. Cells were then fixed with 50 % trichloroacetic acid for 1 h at 4 ℃ and stained for 30 min at RT with 0.4 % sulforhodamine B in 1 % acetic acid. The excess dye was removed by washing four times with 1 % acetic acid. The protein-bound dye was dissolved in 10 mM TRIS pH 10, and OD was determined at 510 nm using a microplate reader.
Immunofluorescence
The cells were grown directly on Labteck chamber slides (Nunc) for at least 24 h, the cells were then washed with PBS with Ca/Mg, and fixed with 4 % buffered paraformalde- hyde (Sigma) for 20 min at 4 ℃. The cells were incubated with 3 % bovine serum albumin for 1 h at RT and then with rabbit anti-cleaved caspase-3 antibody (1:200 diluted) (Cell Signalling) for 1 h at RT, then washed twice with PBS with Ca/Mg and then incubated with the secondary anti-rabbit
120
a
b
100
*
% viable cells
Staurosporine 5 µ M
80
*
Control
*
60
*
*
*
40
Caspase-3
- 32 kDa
20
ß-actin
- 45 kDa
0
CT
1μ.Μ
CT
1 μΜ
2 μ.Μ
3 μ.Μ
4 μ.Μ
5 μ.Μ
MCF-7
H295R
C
Control
ST 5μ.Μ
antibody Alexa Fluor 594 conjugated (1:400 diluted) for 1 h at RT. The cells were finally washed twice with PBS with Ca/ Mg, nuclear staining was performed with 0.05 % Hoechst for 5 min, mounted with Prolong Antifade reagent (Life Technol- ogies), and analyzed by a fluorescence microscope (Olympus BX52); image acquisition and processing were conducted by IAS 2000 software.
Annexin V staining was performed using the annexin V- FITC Detection Kit (PromoKine): untreated and 2 h 1 uM staurosporine-treated cells were washed using PBS with Ca/ Mg and incubated with ready to use anti-annexin V-FITC conjugated antibody for 15 min in the dark; in parallel, the cells were also stained with ready to use propidium iodide, the cells were then fixed with 2 % buffered paraformaldehyde
140
*
*
*
*
120
*
*
*
ns
ns
ns
100
ns
ns
% viable cells
ns
*
80
60
40
20
0
9
ST
₡2
C
ST
₡2
CT
₡2
CT
@2
Q2
ST
CX
ST
C7
ST
EST 2
PL
ECM
LM
FN
C-I
C-III
C-V
2 Springer
(Sigma) for 10 min, washed, nuclear staining was performed, and finally mounted and analyzed at fluorescence microscope. Annexin V positive and negative cells were counted in differ- ent fields for a total of 1,000 cells for each sample.
Statistical analysis and graphic programs
All results were analyzed by ANOVA, and the significance was evaluated by the Tukey HSD post hoc test (Honestly Significant Difference). All figures were elaborated by Adobe Photoshop CS5. All graphs were elaborated by GraphPad Prism 5.0.
Results
We first investigated the responsiveness of adrenocortical cancer cells H295R to staurosporine that is one of the most
used inducers of apoptosis [16]: H295R cells were treated for 14 h with 1, 2, 3, 4, and 5 uM staurosporine, utilizing breast adenocarcinoma MCF7 cells as positive control; cell replication was then evaluated by sulforhodamine B stain- ing. H295R cells showed responsiveness to staurosporine much lower than MCF7 cells: at standard concentration of 1 µM, MCF7 had a cell loss of 61 %, while H295R had only 24 %; in fact, it was necessary the concentration of 5 uM to reach a cell death of 50 % for H295R (Fig.1a). In order to confirm that staurosporine treatment induced apo- ptosis in H295R cells, a Western blot was performed for cleaved caspase-3, which resulted positive on 5 uM staurosporine-treated cells and negative in DMSO solvent-treated H295R (Fig. 1b). In accordance, the immu- nofluorescent staining of activated caspase-3 resulted neg- ative in H295R cells treated only with DMSO solvent and became positive in 5 uM staurosporine-treated cells (Fig.1c).
PL Control
PL Staurosporine
Poli Control
Poli Staurosporine
Col V Control
Col V Staurosporine
a
MW
PARP
116 kDa
Cleaved PARP
89 kDa
ß-Actin
45 kDa
b
10
9
p<0.01
Densitometry (Cleaved PARP/PARP) / ß-Actin
8
7
6
5
4
3
ns
2
1
0
PL Control
PL Staurosporine
Poli Control
Poli Staurosporine
Col V Control
Col V Staurosporine
Once observed the low responsiveness of H295R to staurosporine-induced apoptosis, we investigated whether the adhesion to different extracellular matrix components could modify this resistance of H295R. Therefore, H295R cells were plated on polylysine, fibronectin, laminin, type I, III, V collagens, and Matrigel for 24 h and then treated with 1 µM staurosporine for 14 h and then sulforhodamine B staining was performed. The adhesion to the utilized sub- strates didn’t determined significant modifications on H295R cell apoptosis rate, except for type V collagen adhe- sion, that increased significantly cell apoptosis rate, from 24 % in the cells grown on plastic to 50 % in the cells plated on type V collagen, with an increase in apoptosis rate of 26 % (Fig. 2). In order to demonstrate the specificity of the described effect, we pretreated the cells with anti-integrin alpha2 antibody, after immunofluorescent control of the positive expression of integrin alpha2 subunit by H295R (data not shown). Cell pretreatment with anti-integrin alpha2 totally abolished the increase of apoptosis observed on staurosporine-treated cells adherent on type V collagen (Fig. 2).
To further confirm staurosporine-dependent apoptosis on H295R cells adherent to type V collagen, the cleavage of poly ADP-ribose polymerase (PARP) upon staurosporine treatment was evidenced by Western blot: H295R untreated cells showed uncleaved PARP band migrating at 116 kDa, and after 1 µM staurosporine treatment for 14 h a double band of cleaved PARP was visible, migrating at 116 and 89 kDa; when the cells were grown on type V collagen, the band of cleaved PARP at 89 kDa had an higher density, as compared to the cells grown on plastic and on polylysine (Fig. 3).
In order to better quantify the apoptosis rate and to evaluate whether the described increase of apoptosis induced by type V collagen was specific to H295R, the expression of annexin V bound to phosphatidylserine was analysed by immunofluo- rescent staining on H295R and MCF7 cells plated on plastic, polylysine, and type V collagen and then treated with 1 uM staurosporine for 2 h. Both untreated H295R and MCF7 cells were totally negative for annexin V; once treated with staurosporine, both cells became positive (Fig. 4). Annexin V positivity rate of treated cells was 6 % for H295R and 20 % for MCF7 when grown on plastic, 8 % for H295R, and 21 % for MCF7 when adherent on polylysine and 18 % for H295R and 21 % for MCF7 when adherent on type V collagen (Fig. 4). When H295R cells grown on type V collagen were pretreated with anti-integrin alpha2 and then treated with staurosporine, the percentage of positive cells to annexin V was 5 % (Fig. 4). These data confirmed the increased apopto- sis rate induced by adhesion to type V collagen upon staurosporine treatment and demonstrated the specificity of this effect to H295R cells, as compared to MCF7 cells.
It was finally investigated whether staurosporine treatment on ACC cells adherent to type V collagen could induce any modification on signaling pathways of Akt and Erk that are
a
d
b
e
C
f
% annexin-V positive cells
25
20
T
15
10
5
T
0
Q
PL
c-V
c-V + @2
H295R
MCF7
important regulators of cancer cell survival and resistance to treatment. Western blot analysis demonstrated that 1 µM staurosporine activated Akt, as evidenced by phosphory- lated Akt band expressed only by H295R-treated cells and not by untreated cells; however, the level of phospho-Akt in the cells adherent to type V collagen remained similar to that in the control cells, indicating
that the adhesion to type V collagen had no effect on Akt pathway (Fig. 5). Also, Erk was activated by staurosporine, since the band of phosphorylated Erk ap- peared visible only on treated cells; yet, staurosporine- dependent Erk activation was partially inhibited by cell adhesion to type V collagen, and the density of phospho- Erk band in staurosporine-treated cells was much lower in the cells adherent to type V collagen than in the cells grown on plastic (Fig. 5).
Discussion
Adrenocortical carcinoma is a tumor usually characterized by a poor response to conventional chemotherapy in vivo; in accordance with this tumor feature, our data demonstrate that the studied adrenocortical carcinoma cell line has a low re- sponsiveness to staurosporine-induced apoptosis. Our data further evidence that the adhesion of these cells to type V collagen increases of 26 % the low apoptosis rate induced by staurosporine. Interestingly, we also demonstrate that this effect is specific for H295R cells, as compared to staurosporine responsive MCF7 cells, which don’t show any increase of apoptosis on type V collagen, probably because their apoptosis rate is already high when adherent on plastic. Luparello has recently reported that the adhesion to type V collagen is able to impair the survival of several breast carci- noma cells in vitro by promoting caspase-dependent apoptosis [17]. Another study performed on a cohort of non-small lung cancer patients has demonstrated that the group of tumors characterized by an extracellular matrix rich of type V colla- gen fibers has a significant increase in tumor apoptosis rate and better values of patient survival times [18]. Type V collagen is a minor component localized in the pericellular matrices of several normal and tumor cells types, and may be specific for extra-membranous structures which are closely associated with basal laminae [19]; its biological role remains still poorly understood, since the cellular responses that in- duces can be multiple and sometimes opposite, depending on
the cell histotype. This collagen sometimes represents a good substrate for attachment of normal and cancer cells [20]; on the opposite, it can behave also as an anti-adhesive and anti- proliferative molecule for tumor cells, determining the block of cell proliferation and impairing cell invasive activity [21-23]. Our data correlate with these researches and demon- strate that the specific and unique adhesion to type V collagen of these adrenocortical carcinoma cells significantly enhances their rate of responsiveness to staurosporine-induced apopto- sis. The described capacity to positively regulate tumor cell apoptosis is not a unique feature of type V collagens among the extracellular matrix proteins; in fact, our group previously demonstrated that the adhesion of MCF7 breast cancer cells on fibronectin and laminin determined a significant increase of staurosporine-induced apoptosis through BCL2 down- regulation and increase of PARP cleavage [24]. Other authors stated that extracellular matrix does not always represent a pro-survival stimulus, and several of its components have been described to impair cell viability and to promote cell death, as is reported for thrombospondin, EMILIN2, or pro- tein CCN1 that induces apoptosis trough integrin x6ß1 [25]. In fact it has been suggested that tumor microenvironment can actively contribute to tumor initiation, progression, and me- tastasis [26], this is reached through the binding of extracel- lular matrix proteins to integrins. This interaction regulates several intracellular signal transduction cascades, among which the pathway of Ras-Raf-MEK-ERK is perhaps the best characterized; yet, curiously, Erk activation has been associ- ated with either stimulation or inhibition of cell proliferation [7]. When we investigated the cellular pathway that can be targeted by staurosporine in these cells, we found that Akt and Erk were both activated in H295R staurosporine-treated cells. The G1 arrest induced by staurosporine has been shown to correlate with activation of phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway [27]; our data suggest that also in H295R cells the activation of Akt signaling pathway prob- ably contributes to staurosporine-induced apoptosis. One of the strategies utilized by tumor cells to reduce apoptosis is to enhance endogenous survival pathways, one of these may be
ERK tot.
AKT tot.
ß-actin
PL
C-V
CT
ST
CT
ST
PL
C-V
CT
ST
CT
ST
p-ERK
-43 kDa
p-AKT
-60 kDa
ß-actin
-45 kDa
the mitogen activated protein kinase/extracellular signal reg- ulated kinase (MAPK/Erk), which supports cell survival and can be controlled during staurosporine-mediated cell death [16]. It has in fact been described that in staurosporine- treated neuroblastoma cells enhanced ERK dependent CREB activation reduces apoptosis [28]. Actually, we observed in H295R cells a rate of apoptosis much lower than in other tumor cells, and in parallel we found Erk activation: therefore, it is possible that this can be the mechanism by which these cells escape apoptosis. Interestingly, H295R cells adhesion to type V collagen drastically decreases the rate of staurosporine- induced Erk activation and, at the same time, the apoptosis rate elicited by staurosporine increases significantly.
Taken together, these data suggest that the stromal compo- sition of adrenocortical cancer can drive the response to che- motherapy by regulating the apoptotic elimination of tumor cells. This information can be important in identifying pos- sible microenvironment conditions capable to promote adre- nocortical carcinoma cell death.
References
1. Allolio M, Fassnacht M. Clinical review: Adrenocortical carcinoma clinical update. J Clin Endocrinol Metab. 2006;91(6):2027-37.
2. Kirschner LS. The next generation of therapies for adrenocortical cancers. Trends Endocrinol Metab. 2012;23(7):343-50.
3. Jubb AM, Harris AL. Biomarkers to predict the clinical efficacy of bevacizumab in cancer. Lancet Oncol. 2010;11(12):1172-83.
4. Wu JM, Staton CA. Anti angiogenic drug discovery lessons from the past and thoughts for the future. Expert Opin Drug Discov. 2012;7(8):723-43.
5. Lawnicka H, Kowalewicz-Kulbat M, Sicinska P, Altmann KH, Hofmann T, Stepien H. Resorcylic acid lactone L-783, 277 inhibits the growth of the human adrenal cancer cell line H295R in vitro. Int J Immunopathol Pharmacol. 2009;22(4):889-95.
6. Lawnicka H, Kowalewicz-Kulbat M, Sicinska P, Kazimierczuk Z, Grieb P, Stepien H. Anti-neoplastic effect of protein kinase CK2 inhibitor, DMAT, on growth and hormonal activity of human adre- nocortical carcinoma cell line (H295R) in vitro. Cell Tissue Res. 2010;340(2):371-9.
7. Roovers K, Assoian RK. Integrating the MAP kinase signal into the G1 phase of cell cycle machinery. Bioessays. 2000;22(9):818-26.
8. Aguirre G. Genes and diseases in man and models. Prog Brain Res. 2001;131(1):663-78.
9. Lee BH, Ruoslahti E. Alpha5beta1 integrin stimulates Bcl2 expres- sion and cell survival through Akt, focal adhesion kinase and Ca2+/ calmodulin dependent protein kinase IV. J Cell Biochem. 2005;95(6): 1214-23.
10. Frisch SM, Screaton RA. Anoikis mechanism. Curr Opin Cell Biol. 2001;13(5):555-62.
11. Stupack DG, Puente XS, Boutsaboualoy S, Storgard CM, Cheresh DA. Apoptosis of adherent cells by recruitement of caspase-8 unligated integrins. J Cell Biol. 2001;155(3):459- 70.
12. Denys H, Braems G, Lambein K, Pauwels P, Hendrix A, De Boeck A, et al. The extracellular matrix regulates cancer progression and therapy response: implications for prognosis and treatment. Curr Pharm Des. 2009;15(12):1373-84.
13. Tang D, Lahti JM, Kidd VJ. Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis. J Biol Chem. 2000;275(13):9303-7.
14. Rainey WE, Bird IM, Sawetawan C, Hanley NA, McCarthy JL, McGee EA, et al. Regulation of human adrenal carcinoma cell (NCIH295) production of C19 steroids. J Clin Endocrinol Metab. 1993;77(3):731-7.
15. Vichaj V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc. 2006;1(3):1112-6.
16. Antonsson A, Persson JL. Induction of apoptosis by staurosporine involves the inhibition of expression of major cell cycle proteins at the G2/M checkpoint accompanied by alterations in Erk and Akt kinase activities. Anticancer Res. 2009;29(8):2893-8.
17. Luparello C, Sirchia R, Longo A. Type V collagen and protein kinase Cn down-regulation in 8701-BC breast cancer cells. Mol Carcinog. 2013;52(5):348-58.
18. Souza P, Rizzardi F, Noleto G, Atanazio M, Bianchi O, Parra ER, et al. Refractory remodeling of the microenvironment by abnormal type V collagen, apoptosis, and immune response in non-small cell lung cancer. Hum Pathol. 2010;41(2):239-48.
19. Sage H. Collagens of basement membranes. J Invest Dermatol. 1982; 79 (1): 51 s-59s
20. Ruggiero F, Champliaud MF, Garrone R, Aumailley M. Interactions between cells and collagen V molecules or single chains involve distinct mechanisms. Exp Cell Res. 1994;210(2): 215-23.
21. Luparello C, David F, Campisi G, Sirchia R. T47-D cells and type V collagen: a model for the study of apoptotic gene expression by breast cancer cells. Biol Chem. 2003;384(6):965-75.
22. Luparello C, Sirchia R. Type V collagen regulates the expression of apoptotic and stress response genes by breast cancer cells. J Cell Physiol. 2005;202(2):411-21.
23. Sirchia R, Ciacciofera V, Luparello C. Tumor cell collagen interac- tions: Identification and semi-quantitative evaluation of selectively expressed genes by combination of differential display and multiplex PCR. Biol Proced Online. 2003;5(4):222-7.
24. Vasaturo F, Malacrino C, Sallusti E, Coppotelli G, Birarelli P, Giuffrida A, et al. Role of extracellular matrix in regulation of staurosporine-induced apoptosis in breast cancer cells. Oncol Rep. 2005;13(4):745-50.
25. Marastoni S, Ligresti G, Lorenzon E, Colombatti A, Mongiat M. Extracellular matrix: a matter of life and death. Connect Tissue Res. 2008;49(3):203-6.
26. Hu M, Polyak K. Microenvironmental regulation of cancer develop- ment. Curr Opin Genet Dev. 2008;18(1):27-34.
27. Testa JR, Bellacosa A. AKT plays a central role in tumorigenesis. Proc Natl Acad Sci U S A. 2001;98(20):10983-5.
28. Park EM, Cho S. Enhanced ERK dependent CREB activation re- duces apoptosis in staurosporine treated neuroblastoma SK-N-BE2C cells. Neurosci Lett. 2006;402(13):190-4.