Cyclooxygenase-2 Overexpression in Human Basal
Cell Carcinoma Cell Line Increases Antiapoptosis,
Angiogenesis, and Tumorigenesis
Jeng-Wei Tjiu
1,4, Yi-Hua Liao
1,4, Sung-Jan Lin
1, Yi-Ling Huang
1, Wei-Ling Tsai
1, Chia-Yu Chu
1,2,
Min-Liang Kuo
2and Shiou-Hwa Jee
1,3Cyclooxygenase-2 (COX-2) is critical for tumor formation, angiogenesis, metastasis, and prognosis. In this study, the role of COX-2 in antiapoptosis, tumorigenesis, and angiogenesis of human basal cell carcinoma (BCC) cells was investigated. Transfection of COX-2 constitutive expression vector into a BCC cell line yielded several overexpressing clones. All transfectants demonstrated remarkable resistance to ultraviolet B-induced apoptosis (confirmed by flow cytometry analysis, morphological change, and DNA fragmentation). Immunoblot analysis revealed marked increases in apoptosis-regulated genes Mcl-1 and Bcl-2. A 10-fold concentrated conditioned medium from COX-2-overexpressing BCC cells exhibited higher angiogenic activity in Matrigel plug and human umbilical vein endothelial cell tube formation assay. Cells exhibited increased levels of vascular endothelial growth factor-A (VEGF-A) mRNA and protein, and secreted VEGF-A and basic fibroblast growth factor (bFGF). COX-2-specific small interfering RNA markedly reduced the secreted species. After 7 weeks of inoculation, the tumor volume of COX-2-overexpressing cells in severe combined immunodeficient mice was significantly greater than that of vector control cells. Immunohistochemical analysis of CD31-positive vessels revealed a two-fold increase in microvessel density in COX-2 tumors, compared to control vector tumors. Our data indicate that Mcl-1 and Bcl-2, as well as VEGF-A and bFGF, are downstream effectors of COX-2-induced antiapoptosis and angiogenesis, respectively.
Journal of Investigative Dermatology (2006) 126, 1143–1151. doi:10.1038/sj.jid.5700191; published online 9 March 2006
INTRODUCTION
Cyclooxygenase (COX) is the key enzyme that mediates the production of prostaglandins from arachidonic acid. So far, two COX isoforms (cyclooxygenase-1 (COX-1) and cyclo-oxygenase-1 (COX-2)) have been identified. COX-1 is expressed constitutively, whereas COX-2 is induced by growth factors, tumor promoters, and cytokines (Taketo, 1998). To date, COX-2 has been determined to contribute to
tumorigenesis and the malignant phenotype of tumor cells via the inhibition of apoptosis, increased angiogenesis and invasiveness, and modulation of inflammation and immuno-suppression (Dempke et al., 2001).
Ultraviolet B (UVB, wavelength 290–320 nm) irradiation is a major etiologic factor in the pathogenesis of non-melanotic skin cancers, including squamous cell carcinoma and basal cell carcinoma (BCC) (de Gruijl et al., 2001). More than one million new non-melanotic skin cancers occur in the United States annually, with the majority of them comprising BCC (Miller and Weinstock, 1994). COX-2 and prostaglandin E2 (PGE2) expressions in human skin are increased after UVB
irradiation, especially in aged skin (Seo et al., 2003). Cultured human keratinocytes display increased COX-2 protein level and PGE2 excretion after UVB irradiation (Muller-Decker
et al., 1995; Buckman et al., 1998). Experimentally, increases in COX-2 expression and proliferation of basal layer keratinocytes have been demonstrated in Skh-1 hairless mice after UVB irradiation (Fischer et al., 1999). Reductions in UVB-induced skin papilloma or carcinoma occur upon feeding the mice with celecoxib (a selective COX-2 inhibitor) or indomethacin. Topical application of celecoxib also inhibits chronic inflammation and UVB-induced papilloma/ carcinoma in Skh/hr hairless mice (Wilgus et al., 2003). The ratio of squamous cell carcinoma to papillomas increases Received 16 November 2005; revised 16 November 2005; accepted 11
December 2005; published online 9 March 2006
1Department of Dermatology, National Taiwan University Hospital, National
Taiwan University College of Medicine, Taipei, Taiwan;2Laboratory of
Molecular and Cellular Toxicology, Institute of Toxicology, National Taiwan University College of Medicine, Taipei, Taiwan and3Department of
Dermatology, National Taiwan University College of Medicine, Taipei, Taiwan
Correspondence: Dr Shiou-Hwa Jee, Department of Dermatology, National Taiwan University Hospital and College of Medicine, National Taiwan University, 7, Chung-Shan South Road, Taipei, Taiwan.
E-mail: shiouhwa@ha.mc.ntu.edu.tw
4These authors contributed equally to this work
Abbreviations: BCC, basal cell carcinoma; bFGF, basic fibroblast growth factor; CM, conditioned medium; COX-2, cyclooxygenase-2; HUVEC, human umbilical vein endothelial cell; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; SCID, severe combined immunodeficiency; UVB, ultraviolet B; VEGF-A, vascular endothelial growth factor-A
markedly in the skin of COX-2-overexpressing transgenic mice treated with the known carcinogen 7,12-dimethyl-benz[a]anthracene (Muller-Decker et al., 2002).
A previous immunohistochemical study described BCC to be COX-2 negative (Vogt et al., 2001). Several other published reports show BCC to be COX-2 positive in a low percentage of tested biopsies (Muller-Decker et al., 1999; Kagoura et al., 2001; Akita et al., 2004). However, a recent publication by O’Grady et al. (2004) demonstrated that COX-2 expression level correlated with angiogenesis in BCC specimens taken from renal transplant recipients and immunocompetent patients. Genetic and pharmacological evidence suggests that overexpression of COX-2 is critical for epithelial carcinogenesis. The purpose of the present study, therefore, was to investigate the effects of COX-2 over-expression on BCC cell apoptotic regulation, tumorigenesis, and angiogenesis. The cell regulators involved in apoptosis and angiogenesis were subsequently examined.
RESULTS
COX-2 overexpression induces resistance to apoptosis in human BCC cell line
The human COX-2 constitutive expression plasmid pCMV-COX-2 and the control vector pcDNA were transfected into human BCC cell line cells, which have a relatively low level of COX-2. After transfection, cells were cultured in a medium containing 100 mg/ml neomycin. Four stable clones (desig-nated as B4, C5, E4, and G7) were selected, based on their increased expression of COX-2 mRNA and protein compared with the vector control cells (Figure 1a and b). Elevated levels of PGE2production were found in clones B4 and G7, which
expressed the highest levels of COX-2 (Figure 1c).
Several apoptosis assays were performed to investigate whether COX-2 overexpression confers antiapoptotic ability to the human BCC cell line. A sensitive ELISA that measures cytoplasmic histone-associated DNA fragments further con-firmed that pcDNA clone (vector control) had a higher rate of apoptosis than COX-2-overexpressing clones (B4 and G7) at
12 hours after UVB irradiation. Enrichment factors were 12.2770.32, 4.6770.43, and 4.2070.73 (expressed as mean7SD) for pcDNA, B4, and G7, respectively (Po0.005 for B4 vs pcDNA and Po0.005 for G7 vs pcDNA) (Figure 2a). There was no significant variation in enrichment factors among pcDNA clone and COX-2-overexpressing clones (B4, G7) at 6 hours. Flow cytometric analysis also showed a significantly higher percentage of apoptotic cells in pcDNA than COX-2-overexpressing clones (pcDNA vs B4, Po0.001; pcDNA vs G7, Po0.05) (Figure 2b). Apoptotic cells were distinguished by staining with Hoechst 33258 fluorescent dye, which discerns chromatin condensation and nuclear segmentation, as an additional apoptosis assessment. As shown in Figure 2c, B4 and G7 cells had fewer apoptotic cells than the vector control cells when exposed to UVB irradiation.
The expression levels of the Bcl-2 family proteins were examined to identify possible downstream genes regulated by COX-2. Figure 3 reveals that the level of Mcl-1 protein was increased three- to four-fold in COX-2-overexpressing stable clones as compared with the vector control clone, and the level of Bcl-2 protein increased by 1.8- to 2.6-fold in
pcDNA 1.0 2.5 3.1 2.7 1.8 1.0 1.0 1.0 1.0 1.0 1.0 1.0 3.9 2.5 2.6 3.8 1.0 1.0 1.0 1.0 COX-2 -Actin COX-2 -Tubulin B4 C5 E4 G7 pcDNA B4 C5 E4 G7 pcDNA BCC 0 50 100 150 PGE 2 (pg/ml) 200 P<0.005 P<0.005 P<0.005 P<0.005 250 300 B4 G7 a c b
Figure 1. Determination of COX-2 mRNA, protein, and secreted PGE2levels
in COX-2-transfected BCC cells.(a) The mRNA of various cell lines as
indicated was extracted and semiquantified by reverse transcriptase-PCR of COX-2. (b) The protein extracted from cell lysates was quantified by Western blotting for COX-2. (a, b) The number below each lane indicates the relative densitometric intensity to control (defined as 1.0). This represents one of three experiments. (c) The secretion of PGE2in culture supernatant was measured
by ELISA. Concentration of PGE2was expressed as mean7SD in five
experiments. Student’s t-test was used for comparison. pcDNA: vector control; B4, C5, E4, and G7: clones of COX-2 transfectants.
pcDNA pcDNA B4 G7 0 2 4 6 8
Enrichment factor (EF)
10 12 14 a c b P<0.005 P<0.05 P<0.001 P<0.005 B4 G7 pcDNA 0 5 10 15 % of apoptotic cells 20 25 B4 G7
Figure 2. Expression of COX-2 protects BCC cells against apoptosis.(a) The
cell apoptosis was measured by cell death ELISA. Enrichment factor (EF) was defined as mU of sample (12 hours after UVB irradiation)/mU of the negative control (without UVB irradiation) (mU ¼ absorbance (103)). (b) Flow
cytometric analysis with propidium iodide staining. (c) Nuclear fragmentation and chromatin condensation (arrow) determined by staining with Hoechst 33258 fluorescent dye. Each cell line was treated with 25 mJ/cm2UVB, and
harvested at 12 hours (a) and at 24 hours (b, c). Data are expressed as mean7SD in (a, b). (c) One of three reproducible experiments. Student’s t-test was used for comparison (n ¼ 3). pcDNA: vector control; B4 and G7: COX-2-overexpressing BCC clones. Bar ¼ 10 mm.
COX-2-expressing clones. In contrast, the levels of the other Bcl-2 family members phospho-BAD and Bcl-XL were not significantly affected in COX-2 stable clones.
COX-2 overexpression increases angiogenesis both in vitro and in vivo
The effect of COX-2 on angiogenesis in vitro was initially examined by tube formation of human umbilical vein endothelial cells (HUVECs) cultured with conditioned med-ium (CM). Formation of the network-like tubular HUVEC structure involves the migration, invasion, and differentiation of endothelial cells. The CM obtained from the COX-2-overexpressing clones B4 and G7 showed increased tube formation per microscopic field as compared to controls (10.471.78, Po0.0005 for B4 and 10.271.78, Po0.0005 for G7 clone vs 0 for the vector control) (Figure 4a and b).
The effect of COX-2 on the induction of angiogenesis was assessed in vivo using the Matrigel plug assay. Upon visual examination, substantial vasculature was evident in plugs embedded with CM from COX-2-overexpressing BCC cells, even with hemorrhaging in the plugs, as compared to vector controls (Figure 4c). The B4 and G7 clones showed significant increases in hemoglobin relative to the control (4.5571.7 mg hemoglobin/mouse for B4, 4.3272.5 mg hemoglobin/mouse for G7 vs 0.2270.16 hemoglobin mg/ mouse for control) (both Po0.05) (Figure 4d).
The observation that COX-2 induced angiogenesis in mice prompted the study of COX-2-transfected BCC cells to determine their expression of angiogenic factors. Reverse transcriptase-PCR and immunoblotting were employed to analyze the expression of angiogenic factors including IL-8, vascular endothelial growth factor-A (VEGF-A), basic fibro-blast growth factor (bFGF), and platelet-derived growth factor. As shown in Figure 5a, the expression of VEGF-A mRNA in COX-2-transfected BCC cells was higher than in vector control cells. The expression of IL-8 mRNA was also slightly increased in COX-2-overexpressing clones (varying from 1.1- to 1.6-fold). Immunoblotting analysis showed that COX-2-overexpressing BCC cells expressed more abundant
VEGF-A than vector controls (Figure 5b). Secreted levels of VEGF-A and bFGF were also increased significantly in B4 and G7 clones in comparison with vector control or parent BCC cells (Po0.0001) (Figure 5c).
A loss-of-function assay was performed by blocking COX-2 function using COX-2-specific siRNA. Protein levels of COX-2 reduced by 51.7% and secreted PGE2decreased by
57.38 % in COX-2-overexpressing clones transiently trans-fected with COX-2-specific siRNA, which confirmed the knockdown effect of RNA interference (data not shown). The B4 and G7 clones showed statistically significant decreases in both secreted VEGF-A (54 and 56.8%, respectively) and bFGF (74 and 47.5%, respectively) after being transiently transfected with COX-2-specific siRNA (Figure 5d).
pcDNA 1.0 1.8 1.9 2.6 2.0 1.0 3.6 3.8 4.0 3.5 1.0 0.9 1.1 1.0 1.1 1.0 0.9 1.0 1.0 1.0 1.0 0.5 0.9 1.1 1.1 Bcl-2 Bcl-XL Phospho-BAD Mcl-1 -Tubulin B4 C5 E4 G7
Figure 3. Immunoblot analysis of Mcl-1, Bcl-2, Bcl-XL, and phosphor-BAD
proteins in different COX-2-expressing cell lines.Cell lysates were prepared
and immunoblotting was performed as described in Materials and Methods. Equal aliquots of protein extracted from pcDNA, B4, C5, E4, and G7 clones were electrophoresed, and the proteins were transferred to a nitrocellulose support. The nitrocellulose was probed with specific antibodies as indicated. The number below each line indicates the relative densitometric density to control (defined as 1.0). This represents one of three reproducible experi-ments. pcDNA: vector control; B4, C5, E4, and G7: COX-2-overexpressing BCC clones. pcDNA BCC 0 2 4 6 8 Number of tubes 0 2 1 4 3 6 5 8 7 Hemoglobulin (mg/dl) 10 12 14 P<0.05 P<0.05 P<0.001 P<0.001 P<0.001 pcDNA B4 G7 B4 G7 pcDNA B4 G7 pcDNA BCC B4 G7 a b d c P<0.001
Figure 4. COX-2 overexpression increases angiogenesis both in vivo and in
vitro. (a) Ten times concentrated culture supernatants (CM) collected from
COX-2 transfectants and vector control incubated with HUVECs as depicted. (b) The number of tube-like structures was expressed as the mean7SD following a random count of five 9.7 mm2microscopic fields. (c) CM from the
COX-2-overexpressing clones and vector control were used for Matrigel blood plug assay in C57BL6j mice (five per group). The mice were killed 7 days after implantation. Gross appearance of the plugs was photographed. (d) The blood plugs were extracted and quantitation of hemoglobin was performed using the Drabkin method. The mean amount of hemoglobin was calculated. Student’s t-test was used for comparison. Bar ¼ 100 mm.
The COX-2-specific inhibitor NS-398 was also used to demonstrate specificity of the COX-2 transfection effects, and the secreted levels of PGE2were decreased by 90.3% in B4
clone and by 91.1% in G7 clone after 25 mM NS-398
treatment for 24 hours (data not shown). The B4 and G7 clones showed significant decreases in the secreted levels of VEGF-A (59.0 and 68.%, respectively) and bFGF (86.3 and 81.8%, respectively) after treatment with 25 mMNS-398 for
24 hours (Figure 5e).
Increased tumor growth and tumor angiogenesis in BCC cells with COX-2 overexpression in SCID mice
To compare the cell transformation ability of COX-2 transfectants and vector control cells, we employed a soft agar growth assay. A total of 1 103cells from pcDNA, B4, and G7 clones were seeded into soft agar plate and colony numbers were counted 28 days after seeding. The results showed 9.272.0, 24.773.7, and 25.875.4 colonies in a high-power field ( 200) for pcDNA, B4, and G7 respec-tively (B4 vs pcDNA, Po0.001; G7 vs pcDNA, Po0.001)
(Figure 6a). Microscopic examination also revealed that both B4 and G7 clones showed colonies much larger than those of the pcNDA clone (Figure 6b).
Tumorigenicity of COX-2-overexpressing clones and vector control cells in severe combined immunodeficient (SCID) mice model was next explored. Cells from pcDNA, B4, and G7 (5 106each) were subcutaneously injected into
5-week-old SCID mice. The mean tumor size in G7-transplanted SCID mice was approximately 6,833 mm3 on day 47. The B4 xenograft reached a mean of 4,377 mm3on day 47. In contrast, the mean tumor volume in SCID mice injected with pcDNA cells reached only a mean of 1,821 mm3by the same day (P ¼ 0.0097) (Figure 7a and b).
The xenograft volume reached a statistically significant difference 22 days after implantation (P ¼ 0.0231). Immuno-histochemical analysis of xenograft resection specimens confirmed that COX-2 staining was higher in COX-2-overexpressing clones (data not shown).
The number of blood vessels in COX-2-overexpressing xenograft tumors was quantified. A correlation was evident
pcDNA 1.0 2.2 1.4 1.9 1.8 1.0 1.0 1.0 1.0 0.9 1.0 1.0 0.9 1.0 1.0 1.0 0.9 1.0 1.0 1.0 1.0 3.3 3.5 3.9 4.3 1.0 1.2 1.2 0.4 1.1 1.0 1.1 1.3 1.5 1.6 -Actin VEGF-A VEGF-A VEGF-A bFGF bFGF PDGF IL-8 -Tubulin B4 C5 E4 G7 pcDNA B4 C5 E4 G7 pcDNA 0 0 20 40 60 80 100 120 500 1,000 1,500 2,000 2,500 3,000 pg/ml pg/ml 3,500 4,000 4,500 5,000 BCC P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 P<0.001 B4 G7 BCC pcDNA B4 G7 a b c VEGF-A bFGF VEGF-A bFGF 20 50 100 150 200 40 60 80 pg/ml 20 10 0 0 200 400 600 800 1,000 1,200 1,400 1,600 40 30 50 60 pg/ml pg/ml pg/ml P<0.005 P<0.005 P<0.005 P<0.01 P<0.05 P<0.05 P<0.001 P<0.001 B4 B4 +siRNA G7 +siRNA G7 B4 B4+ NS-398 25 M G7+ NS-398 25 M G7 B4 B4+ NS-398 25 M G7+ NS-398 25 M G7 B4 B4 +siRNA G7 +siRNA G7 d e
Figure 5. Expression of angiogenic factors in COX-2-transfected BCC cells. (a) Reverse transcriptase-PCR analysis of the level of VEGF-A, bFGF,
platelet-derived growth factor, and IL-8 mRNA in COX-2 transfectants (B4, C5, E4, and G7) and vector control (pcDNA) cells. (b) Western blot analysis of VEGF-A protein in COX-2-overexpressing and vector control BCC cells. (c–e) Changes of secreted VEGF-A and bFGF were measured by ELISA. Concentrations of VEGF-A and bFGF were expressed as the mean7SD of three independent experiments. Student’s t-test was used for statistical analysis (c–e). Different culture conditions were used. (c) A total of 2 105cells of BCC, pcDNA, B4, and G7 clones were seeded in 1.6 cm diameter culture plates, and the supernatants were collected at
48 hours. (d) A total of 3 105cells of B4 and G7 clones were seeded in 10 cm diameter culture dishes and transiently transfected with COX-2-specific siRNA.
Culture supernatants were collected at 48 hours for ELISA after siRNA transfection. (e) A total of 2 105cells of B4 and G7 were seeded in 1.6 cm diameter
between the level of expression of COX-2 and the density of tumor microvessels (8.9672.26 vessels/microscopic field for B4 cells, n ¼ 5, Po0.05, 8.2271.25 vessels/micro-scopic field for G7 cells, Po0.05, n ¼ 5 vs 4.4670.77
vessels/microscopic field for pcDNA cells, n ¼ 5) (Figure 8a and b).
DISCUSSION
Accumulated evidence suggests that cancer cells expressing higher levels of COX-2 may obtain a survival advantage that eventually facilitates tumor development and progression (Dannenberg et al., 2005). We have shown that IL-6 overexpression in BCC cells increases expression of COX-2 and is accompanied by high vascularization (Jee et al., 2001). Blockage of COX-2 function by siRNA abrogates IL-6-mediated angiogenic activities (Jee et al., 2004) in a manner consistent with a downstream COX-2 effect of IL-6-induced angiogenesis in BCC cells. Several reports described that BCC had a low positive rate for COX-2 immunohistochemical staining in tested biopsies (Muller-Decker et al., 1999; Kagoura et al., 2001; Vogt et al., 2001; Akita et al., 2004). But recently, O’Grady et al. (2004) showed a positive staining of COX-2 in BCC, and demonstrated that COX-2 expression was correlated with microvessel densities. We found that higher levels of COX-2 expression not only associated with neovascularization but also correlated with tumor invasion depth in human BCC specimens (manuscript in submission).
5 10 15 20 25 Colony count 30 35 0 P<0.001 P<0.001 pcDNA B4 G7 pcDNA B4 G7 a b
Figure 6. Anchorage-independent growth of COX-2-overexpressing BCC
clones. (a) Anchorage-independent growth of COX-2-transfected BCC cells.
A total of 1 103cells were mixed in 0.35% soft agar and plated in a 3.5 cm
diameter plate. Twenty eight days after seeding, the number of colonies was determined. Results shown are the mean7SD of three experiments. (b) Colony morphology of COX-2 overexpression clones (B4 and G7) and vector control (pcDNA) cells. Bar ¼ 0.2 mm.
pcDNA 10,000 pcDNA B4 G7 8,000 6,000 4,000 2,000 0 0 10 20 30 Day Volume (mm 3) 40 50 B4 G7 a b
Figure 7. Tumorigenic effect of COX-2-overexpressing BCC cells in SCID
mice.The vector control and COX-2-overexpressing clones (B4 and G7) were
injected into SCID mice by subcutaneous inoculation of 5 106cells (four
mice per group). (a) The gross appearance of the xenograft following the killing of SCID mice performed 7 weeks after injection of BCC cells (a) and tumor size were assessed as described in Materials and Methods (b). Four independent experiments were performed. Significance was assessed using the Kruskal–Wallis equality of populations rank test.
P<0.05 P<0.05 pcDNA 0 2 4 6 Microvessel density (number/HPF) 8 10 12 B4 B4 pcDNA G7 G7 a b
Figure 8. Microvessel density was increased in COX-2-overexpressing
xenografts.(a) Immunohistochemical staining of sections of xenografts using
anti-CD31 antibody. (b) Quantitative analysis of CD31-positive vessels (mean7SD) by the standard method of microvessel density determination. pcDNA: vector control; B4 and G7: COX-2-overexpressing BCC clones. Bar ¼ 50 mm.
In the present study, we demonstrate the function of COX-2 in tumorigenesis and angiogenesis by both molecular dissection and in vivo inspection. COX-2 is an enzyme that mediates many physiological functions, such as inhibition of cell apoptosis, augmentation of angiogenesis, and increased cell motility. The COX-2-mediated functions are influenced in part by various genes such as VEGF-A (Ma et al., 2002), CD44, and matrix metalloproteinases (Dohadwala et al., 2002). High COX-2 levels in the human colon (Tsujii et al., 1998) and gastric cancers (Jones et al., 1999) greatly promote tumor growth by increasing microvessel density (ie angiogen-esis). VEGF-A is a major downstream effecter gene of COX-2-induced angiogenesis in colon cancer (Seno et al., 2002). Recently, it was shown in Apc/COX-2 double knockout mice that stromal expression of COX-2 is required for the induction of VEGF and subsequent tumor angiogenesis (Seno et al., 2002). Tumors in COX-2 knockout mice showed decreased expression of VEGF mRNA and a 30% decrease in vascular density compared with wild type (Williams et al., 2000). Loss of wild-type p53, a tumor suppressor gene, is associated with increased expression of COX-2 mRNA and increased release of bFGF (Galy et al., 2001) and VEGF (Yuan et al., 2002). The product of COX-2 enzymatic function, PGE2, stimulates
VEGF production by promoting the translocation of hypoxia-inducible factor-a from cytosol to nucleus (Liu et al., 2002). PGE2was reported to induce the accumulation of IL-8 mRNA
and protein production in human colonic epithelial cells (Yu and Chadee, 1998). PGE2induces IL-8 mRNA transcription
through PGE2receptor 1- and 4-dependent signal
transduc-tion pathway in human T cells (Caristi et al., 2005). IL-8 is well known for its angiogenesis capability in many human cancers. Our study revealed that there are slight increases in IL-8 mRNA levels in COX-2-overexpressing clones (varying from 1.1- to 1.6-fold), whereas VEGF-A showed a more prominent increase in mRNA (1.8- to 2.4-fold). Secreted bFGF was also increased markedly in COX-2-overexpressing clones, by ELISA. Therefore, in addition to VEGF-A and bFGF, the role of IL-8 in mediating angiogenesis in COX-2-overexpressing BCC is worthy of further investigation.
Several apoptosis regulators were examined in our study. Mcl-1 and Bcl-2, both members of Bcl-2 family, were significantly upregulated in COX-2-overexpressing BCC cells, whereas other Bcl-2 members were not. This implicates Mcl-1 and Bcl-2 in COX-2-mediated antiapoptotic effects. Over-expression of COX-2 on rat intestinal epithelial cells results in inhibition of apoptosis and overexpression of Bcl-2 (Tsujii and DuBois, 1995). Exposure of a variety of cancer cells to a selective COX-2 inhibitor induces apoptosis (Arico et al., 2002; Elder et al., 2002). One potential mechanism for the proapoptotic activity of COX-2 inhibitors is the downregula-tion of Bcl-2 (Sheng et al., 1998). Tumor tissue expressed reduced levels of proapoptotic proteins Bax and Bcl-XL, and increased levels of the antiapoptotic protein Bcl-2 in COX-2 transgenic mice model. Decreased apoptosis of mammary epithelial cells contributes to tumorigenesis in COX-2-over-expressing transgenic mice (Liu et al., 2001).
The Mcl-1 gene was originally identified as an early gene that is induced during differentiation of ML-1 myeloid
leukemia cells (Kozopas et al., 1993). Overexpression of Mcl-1 delays apoptosis via a broad array of agents, such as c-Myc overexpression, growth factor withdrawal, and other cytotoxic agents (Ando et al., 1998; Chao et al., 1998; Wang et al., 1999). The intracellular distribution of Mcl-1 protein overlaps with that of Bcl-2 protein, with prominent mito-chondrial membrane localization (Yang et al., 1995). Previously, we demonstrated that Mcl-1 upregulation is involved in IL-6-mediated antiapoptotic effects. Recently, Mcl-1 expression has been shown to be upregulated by COX-2/PGE2 in lung adenocarcinoma cell line (Lin et al., 2001).
Increased levels of Mcl-1 induced by the overexpression of COX-2 were reported in cholangiocarcinoma and lympho-mas (Nzeako et al., 2002; Wun et al., 2004).
COX-2 is present in normal skin, benign epidermal proliferation, and malignant skin neoplasm (Buckman et al., 1998; Kagoura et al., 2001). Sun exposure is the essential causal factor of BCC. Acute exposure of human keratinocytes to UVB irradiation results in increased production of PGE2
and increased expression of COX-2 (Buckman et al., 1998). COX-2 protein expression is upregulated in the human epidermis after exposure to UVB (Buckman et al., 1998). Chronic inflammation upregulates mediators of inflammatory response, such as COX-2, leading to the production of inflammatory cytokines and prostaglandins, which may suppress cell-mediated immune response and promote angiogenesis (O’Byrne and Dalgleish, 2001). Topical appli-cation of celecoxib also inhibits chronic inflammation and UVB-induced papilloma/carcinoma in Skh/hr mice (Wilgus et al., 2003). Recently, COX-2 was reported as a target of treatment in conjunction with photodynamic therapy in many skin cancers (Akita et al., 2004). Combination of 5-fluoro-uracil and celecoxib also displayed synergistic regression of UVB-induced skin tumors (Wilgus et al., 2004). Our data showed that tumor aggressiveness, defined as a higher microvessel density and deeper local invasion depth, was associated with levels of COX-2 expression (manuscript in preparation). COX-2 thus might serve as a chemopreventive and therapeutic target in human skin cancers.
Finally, the present study reveals that COX-2 induces antiapoptotic activity and angiogenesis in BCC by upregula-tion of Bcl-2 and Mcl-1, as well as secreted VEGF-A and bFGF. The exact molecular mechanism and downstream effectors that mediate angiogenesis and tumorigenesis in COX-2-overexpressing cell line remain to be elucidated by a further genome-wide analysis. Our findings suggest that blocking COX-2 function may increase susceptibility to apoptosis and inhibit angiogenesis in BCC, thus providing a feasible scheme for treatment and prevention of human BCC. Further clinical trials may be needed to test the effectiveness of COX-2-specific inhibitor combined with chemotherapeu-tic agents or photodynamic therapy in the treatment of human BCC. COX-2 inhibition might have beneficial effects, especially for BCC with a higher level of COX-2 expression or aggressive phenotypes. We hope to find more specific therapeutic targets that can be useful in treatment of human BCC in the near future.
MATERIALS AND METHODS
The medical ethical committee of the National Taiwan University College of Medicine approved all described studies. The study was conducted according to the Declaration of Helsinki Principles.
Cell origin, cell culture, and establishment of COX-2 transfectants
The human BCC cell line (parental BCC cells, originally designated BCC-KMC-1) originated from an undifferentiated BCC derived from a trauma scar. The 130–135th passages of this cell line were used in this study. The pCMV-COX-2, a constitutive expression vector, carries full-length human COX-2 cDNA under the control of the cytomegalovirus promoter/enhancer sequence and with a neomycin selection marker. The BCC cells were transfected with pCMV-COX-2 or control pcDNA3 vector (GIBCO Invitrogen Co., Grand Island, NY), containing a cytomegalovirus promoter and a neomycin selection marker, using the TransFastTM transfection reagent (Pro-mega Co., Madison, WI). After 24 hours, cells were replanted in RPMI 1640 medium (GibcoBRL, Rockville, MD) with 10% fetal calf serum and 500 ng/ml G418 (Sigma Chemical Co., St Louis, MO). The G418-resistant clones were selected and expanded. For this study, we used high-expression transfectants (B4, C5, E4, and G7), the parental BCC cells and BCC cells transfected with control vector (pcDNA) served as controls. All of these cells were grown at 371C and 5% CO2in RPMI 1640 medium supplemented with 10% (vol/ vol) fetal bovine serum.
Antibodies and reagents
Anti-Mcl-1 (BD Pharmingen, San Diego, CA), anti-Bcl-2 (BD Pharmingen), anti-Bcl-XL (Santa Cruz Biotechnology, Santa Cruz, CA), anti-BAD (Santa Cruz Biotechnology), anti-VEGF-A (R&D Systems, Minneapolis, MN), anti-COX-2 (Cayman Chemical, Ann Arbor, MI), and anti-a-tubulin antibody (NeoMarkers, Freemont, CA) were used in immunoblotting analysis. Neutralizing antibody (goat polyclonal anti-bFGF antibody) was obtained from R&D Systems. The NS-398, a COX-2-specific inhibitor, was obtained from Cayman Chemical (Ann Arbor, MI).
Apoptosis ELISA assay
A sensitive ELISA that detects cytoplasmic histone-associated DNA fragments was performed, according to the manufacturer’s protocol (Cell Death Detection ELISAPLUS; Roche Diagnostic GmbH, Mannheim, Germany). Briefly, human BCC cell lines were cultured in 6 cm Petri dishes (106cells/dish) overnight, then treated with UVB 25 mJ/cm2and harvested at 6 and 12 hours. An amount of 1 104 cells was then centrifuged at 200 g. The cells were lysed with lysis buffer for 30 minutes, followed by centrifugation at 200 g for 10 minutes at room temperature. The supernatant was collected and incubated with immunoreagent for 2 hours. After washing, a substrate solution was added into each well and kept in the dark until the color developed. The reaction was determined in a spectrophotometry at 405 nm, with 490 nm as the reference wavelength. The result was presented as enrichment factor (EF). The EF is defined as mU of sample/mU of the corresponding negative control (mU ¼ absorbance (103)).
Flow cytometry analysis
The BCC/COX-2 cells and vector controls (1 106) were treated with UVB 25 mJ/cm2. At an indicated time, cells were harvested and fixed
in 75% methanol at 201C for at least 30 minutes. After centrifuga-tion (100 g) for 5 minutes at 41C, the cell pellets were resuspended in 1 ml of phosphate-buffered saline (PBS) containing 2 mg/ml RNase A and incubated for 30 minutes. Finally, propidium iodide solution was added to a final concentration of 40 mg/ml. Fluorescence emitted from the PI-DNA complex was quantified after laser excitation of the fluorescent dye using FACS flow cytometry (Becton-Dickinson, Labware, Bedford, MA).
Preparation of CM
Parental BCC, pcDNA, B4, or G7 cells were plated in 1 ml of culture medium at 2 105cells/well in 24-well 18 mm culture dishes. After serum starvation for 1 day, the culture supernatants were collected 24 hours later and centrifuged sequentially at 12,500 g with Microcon YM-3 centrifugal filter devices (Millipore Co., Bedford, MA) (with the cutoff of molecules smaller than 3,000 Da) for 30 minutes to obtain a 10-fold concentrate culture supernatant.
Matrigel blood plug in vivo assay
Matrigel blood plug assay was performed as described previously (Plunkett and Hailey, 1990; O’Reilly et al., 1997). Briefly, C57BL6j mice (five per group) were each injected subcutaneously with 50 ml of CM from various cell lines mixed with 450 ml Matrigel (growth factor reduced) (Becton-Dickinson, Labware, Bedford, MA) and 40 U heparin/ml at 41C. Mice were killed 6 days later; gels were recovered and processed for further studies. Neovessels was quantified by Drabkin method, measuring hemoglobin of the plug with the Drabkin reagent kit 525 (Sigma, St Louis, MI).
In vitro capillary tube formation on Matrigel
Human umbilical vein was collected with consent. The HUVEC capillary tube formation was evaluated as follows. Twenty-four-well 18 mm tissue culture dishes were coated with Matrigel basement membrane matrix (300 ml/well) (Becton-Dickinson) at 41C and allowed to polymerize at 371C for at least 30 minutes. The HUVECs (5 104cells/well, in 24-well 18 mm tissue culture dishes) were grown in a final volume of 0.4 ml culture medium containing 150 ml M199 (GibcoBRL, Rockville, MD) and 250 ml CM. After 6 hours incubation, tube formation was observed through an inverted, phase-contrast photomicroscope, then photographed and counted. The number of tube formations was measured by counting the number of tube-like structures formed by connected endothelial cells in five randomly selected 9.7 mm2microscopic fields. The assay was performed in triplicate.
COX-2-specific RNA interference
The target sequence for the COX-2 siRNA was bases 291–313 of NM000963.1 (50-aactgctcaacaccggaattttt-30) following the descrip-tion by Denkert et al. (2003). The pRNA-U6/neo (GenScript Corp., Piscataway, NJ) is an siRNA expression vector with neomycin as the selection marker. We constructed pRNA-U6/COX-2, a constitutively expressed vector, which carries the COX-2 siRNA sequence under the control of U6 promoter. The BCC/COX-2 cells were transiently transfected with pRNA-U6/COX-2 overnight. Inhibition of COX-2 protein was confirmed by Western blotting and function inhibition was measured by the PGE2EIA kit.
Reverse transcriptase-PCR
The RNAs from BCC/COX-2 and vector control were isolated using Trizol followed by chloroform. The mixture was then centrifuged, which yielded an upper aqueous phase containing total RNA. After precipitation with isopropanol, the RNA pellet was redissolved in denaturing solution, reprecipitated with isopropanol, and washed with 75% ethanol. An amount of 5 mg of total RNA was subjected to first-strand synthesis using Random Hexamer (Promega, Madison, WI) and M-MLV Reverse Transcriptase (RNase H Minus) (Promega, Madison, WI) at 371C for 3 hours. The cDNA was then diluted to a final volume of 20 ml and quantified. Each 1 ml PCR reaction contained Taq polymerase (0.5 U/l; Protech Technology, Taipei, Taiwan) and 25 pmol each of the sense and the antisense primers in PCR buffer (10 mM Tris pH 9, 50 mM KCl, 6 mM MgCl2, 0.01% gelatin, and 0.1% Triton X-100). Primer sequences are as follows: IL-8: sense 50-CTC TCT TGG CAG CCT TCC TGA TT-30and antisense 50-AAC TTC TCC ACA ACC CTC TGC AC-30; VEGF-A: sense 50-AGC TAC TGC CAT CCA ATC GC-30 and antisense 50-GGG CGA ATC CAA TTC CAA GAG-30; platelet-derived growth factor: sense 50 -AAG CAT GTG CCG GAG -AAG CG-30 and antisense 50-TCC TCT AAC CTC ACC TGG AC-30; bFGF: sense 50-AGT CTC CTG GAG AAA GCT-30and antisense 50-CCC TTG ATG GAC ACA ACT-30; and b-actin: sense 50-CGT CTG GAC CTG GCT GGC CGG GAC C-30 and antisense 50-CTA GAA GCA TTT GCG GTG GAC GAT G-30. The reaction mixture was incubated for 5 minutes at 941C and then amplified by 25 PCR cycles (denaturing for 1 minute at 941C, annealing for 1 minute at 561C, and extending for 1 minute at 721C). Each PCR product was then analyzed on a 2% agarose gel stained with 1 mg/ml ethidium bromide and photographed (Syngene; Syngene Bio Imaging System). Intensity of bands on the photographs was quantified by scanning laser densitometry.
Enzyme immunoassay (ELISA)
The PGE2EIA kit (Cayman Chemical, Ann Arbor, MI) and bFGF and VEGF ELISA kits (R&D Systems) were used to determine the respective levels of PGE2, bFGF, and VEGF in cell culture supernatants. Each measurement was carried out according to the manufacturer’s instructions, and each experiment was repeated in triplicate.
Western blot analysis
Cell lysates were prepared as described previously (Kuo et al., 1998). A 50 mg sample of each lysate was subjected to electrophoresis on a 10% SDS-polyacrylamide gel for detection of Mcl-1, 2, Bax, Bcl-x/L, VEGF-A, and COX-2. The samples were then electroblotted onto nitrocellulose paper. After blocking, blots were incubated with anti-Mcl-1, anti-Bcl-2, anti-Bax, anti-Bcl-x/L, anti-VEGF, and anti-Cox-2 antibodies in PBS containing Triton X-100 (PBST) for 1 hour followed by two washes (15 minutes each) in PBST. The samples were then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG for 30 minutes. After washing, blots were incubated for 1 minute with the Western blotting reagent ECL (Amersham Biosciences UK Limited, Buckinghamshire, England) and chemiluminescence was detected by exposing the filters to Kodak-X-Omat films for 30 seconds to 3 minutes.
Colony growth in soft agar
Growth in soft agar was determined in six-well 35 mm diameter dishes prepared with a lower layer of 0.7% agar solution in RPMI
with 10% fetal bovine serum, then overlaid with a 0.35% agar solution, also in growth medium, in which 1 106 cells were resuspended. Colonies were scored 28 days after preparation. (The colonies larger than 0.1 mm in diameter were scored as positive.) The pcDNA (vector control), B4, and G7 (COX-2-overexpressing clones) were subjected for colony formation assays.
Tumorigenicity assay in SCID mice.The SCID mice purchased from Tzu-Chi University Animal Center in Taiwan were housed in a dedicated SCID mouse facility with microisolator caging. The mice were handled under a unidirectional laminar airflow hood. Control-transfected BCC cells (pcDNA) and COX-2-Control-transfected BCC cells (B4 and G7 clones) were trypsinized, washed with PBS, resuspended in PBS, and adjusted to a concentration of 5 106cells/100 ml in PBS. The cell suspensions were then mixed with an equal volume of Matrigel before subcutaneous injection into SCID mice. Tumor development was followed in individual animals (four per group) by weekly sequential caliper measurements of length (L), width (W), and depth (H). Tumor volume was calculated by the formula L W H 0.5236. After 7 weeks, the mice were killed; the tumors were removed and weighed. A segment of the tumor was excised and fixed either in OCT embedding medium (Sakura Finetek USA Inc., Torrance, CA) or 10% neutral buffered formalin.
CONFLICT OF INTEREST
The authors state no conflict of interest.
ACKNOWLEDGMENTS
This work was supported by grants from National Science Council, Taiwan (NSC92-2314-B-002-156, NSC-93-2314-B-002-072, and NSC-93-3112-B-002-034) and a private donation from Mr Chao-Chai Lin.
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