新的抗肿瘤药7_hydroxystaurosporine(UCN_01)抑制肿瘤浸润和转移
作者：孟庆慧 邵荣光 徐静文 樊赛军
单位：长岛Jewish医学中心分子肿瘤实验室，纽约美国
关键词：7_hydroxystaurosporine(UCN_01)前列腺肿瘤浸润转移
中国临床药理学与治疗学000401
目的和方法研究7_hydroxystaurosporine(UCN_01)对转移性前列腺肿瘤DU_145细胞浸润和转移的影响。用体外转移和创伤法检查细胞浸润和转移，用Westernblotting法检查蛋白质的表达。结果UCN_01在非毒性剂量下(100nmol·L-1)明显地抑制DU_145细胞浸润和转移。而且，UCN_01这种抗肿瘤浸润和转移功能与它增加细胞_细胞粘连分子E_cadherion的表达有关。结论这些实验首次证实UCN_01能抑制人前列腺肿瘤细胞的浸润和转移。UCN_01的临床应用可能更有效地控制前列腺肿瘤转移。
, 百拇医药
中图分类号R979.1
Suppression of invasion and migration by 7_hydroxystaurosporine(UCN_01),a new anti_tumor agent
MENG Qing_Hui FAN Sai_Jun
(Laboratory of Molecular Oncology, Department of Radiation Oncology, Long Island Jewish Medical Center,New York)
SHAO Rong_Guang
(Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing, China)
, 百拇医药
XU Jing_Wen
(Division of Rheumatology,Department of Medicine, The Long Island Campus for the Albert Einstein College of Medicine,New Hyde Park, New York, USA)
Aim and Methods To investigate the effect of UCN_01(7_hydroxystaurosporine) on cell migration and invasion ability of DU_145, an invasive human prostate cancer cell line.Results It was found that UCN_01 at non_cytotoxic doses (100 nmol· L- 1) significantly inhibited prostate cancer DU_145 cell invasion and migration behaviors.Moreover, this anti_invasion and migration activity of UCN_01 was associated with an up_regulation of cell adhesion molecule E_cadherin. Conclusion These results indicate for first time that UCN_01 inhibits the invasion and migration of human prostate cancer cells.Thus, clinical application of UCN_01 may contribute to the potential benefit for suppression of prostate cancer invasion and metastasis.
, 百拇医药
Key words 7_hydroxystaurosporine(UCN_01); prostate; tumor; invasion; migration
Protein kinase C(PKC) plays an important role in tumorigeneses,tumor cell invasion and metastasis[1,2],UCN_01(7_hydroxystaurosporine) is a selective PKC inhibitor derived from the non_selective protein kinase inhibitor staurosporine[3].Subsequently,a variety of studies from our and other laboratories have revealed that UCN_01 is a promising agent in the inhibition of tumor cell growth alone or in a combination with other chemotherapeutic agents,such as camptothecin,5_fluorouracil, cisplatin, mitomycin_C and ionizing radiation,in vitro and in vivo. The
, 百拇医药
anti_tumor activities of UCN_01 are associated with the arrest of cell cycle progression, including G1/S and/or G2/M, apoptosis induction and inhibition of DNA repair[4]. Therefore, these studies
indicate that UCN_01 is a profound anti_tumor agent in a larger number of tumors,including prostate cancer.Moreover,UCN_01 has been entered phase I trials in the United States and Japan as a single agent.In the present study, the effects of UCN_01 on the cell invasion and migration properties were investigated in DU_145, a human prostate cell line.Exposure to UCN_01 at non_ cytotoxic doses (100 nmol· L- 1) caused a significant repression of the cell invasion, speading and migration.Furthermore,UCN_01 caused an up_ regulation in the protein expression of cell adhesion molecule E_cadherin.
, 百拇医药
1 Materials and Methods
1. 1 Cell culture and UCN_01 The invasive human prostate cancer cell line DU_145 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained as monolayer cultures in D_MEM supplemented with 10% fetal calf serum (FCS), 2 mmol· L- 1 glutamine, 100 μ g· ml- 1 streptomycin and 100 unit/ml penicillin G (BioWhittaker, Walkersville, MD).UCN_01 was kindly obtained from the Laboratory of Molecular Pharmacology, Division of Basic Science, NCI, NIH (Bethesda, MD), stored at - 20 ℃ as a 10 mmol· L- 1 stock solution in 20% DMSO, and further diluted in medium prior to experiments.
, 百拇医药
1.2 In vitro invasion assay In vitro invasion assay was carried out using a modified Boyden chamber[5]. Briefly, the surfaces of filter (0.8 μ m pore size) were coated with a uniform thickness of 25 μ g of Matrigel for 1 h at room temperature.Uniformity of the coating was checked by Coomassie blue staining and low_ power microscope observation.The lower chamber was filled with 10% FCS medium containing fibronectin (16 μ g/chamber) as the chemoattractant.Cells (1× 105 cells/ml) resuspended in the medium containing 2.5 % FCS and 100 nmol· L- 1 UCN_01 were carefully transferred onto the upper surface of filters hold in the chamber.After 24 h incubation, the filter was gently removed from the chamber, the cells on the upper surface were removed by wiping with a cotton swab, the cells that invaded through the Matrigel and attached to the lower surface of the filter were fixed, stained with H＆ E, and counted in 15 randomly selected microscopic fields(× 400) per filter. Experiments were independently performed at least three times.
, 百拇医药
1.3 Scratch wound assay The spreading and migration capabilities of DU_145 cells were assessed using a scratch wound assay[5],which measures expansion of a cell population on surfaces. The cells were seeded into six_well tissue culture dishes at a concentration of 2.5× 105 cells and cultured in medium containing 10% FCS to nearly confluent cell monolayers, which were then carefully wounded using 1 ml sterile pipette tips and any cellular debris was removed by washing with PBS.The wounded monolayers were then incubated in 10% FCS D_MEM containing different doses of UCN_01 for 48 h and photographed under a light microscope(× 200).The experiments were repeated in quadruplicate wells at least three times.
, 百拇医药
1.4 Immunoblot assay Protein expression was assayed using an immunoblot assay as described previously[5].UCN_01_untreated and treated cells were lysed in a mini_modified ice_cold RIPA lysis buffer containing protease inhibitors (Calbiochem, San Diego, CA) and 100 mmol· L- 1 sodium orthovanadate.100 μ g of total protein lysates was electrophoresed on 10% SDS_ polyacrylamide gels and transferred to membranes by electroblotting. The membranes were incubated with a monoclonal E_cadherin antibody (Trans_ duction Laboratories, Lexington, KY) and then incubated with secondary antibodies after being extensively washed.Antibody reaction was revealed using an enhanced chemiluminescence detection system (Amersham Life Science, Arlington Heights, Illinois) as instructed by the manufacturer.Equal protein loading and the protein transfer were confirmed by immunoblotting for determination of α _actin protein using a polyclonal α _actin antibody(I_19, Santa Cruz, Hercule, CA) on the same Western blots.A colored marker (Bio_Rad Laboratories, Hercules, CA) was used as a molecular size standard.
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2 Results
Using a MTT assay, the cytotoxicity of UCN_01 was determined in DU_145 human prostate cancer cells after treatment with UCN_ 01.Consistent wit previous studies[6],>100 nmol · L- 1 doses of UCN_01 (48 h exposure) caused a significant cytotoxicity(data not shown). Therefore, the effects of UCN_01 on DU_145 cell invasion and migration ability were carried out using < 100 nmol· L- 1 doses, this will exclude any possible effects from the cytotoxicity of UCN_01 on anti_invasion and migration activities of UCN_ 01.Tumor cell invasion is a crucial aspect of the processes of tumor metastasis. For the evaluation of cellular invasiveness, a modified Boyden chamber assay was carried out to determine the ability of DU_145 glioma cells to invade through biological matrices in vitro.The relevance of this assay for other invasion assays and for in vivo malignancy has been documented extensively[7], based on the percentage of cells penetrating the reconstituted basement membrane_coated filters and attached on the lower surface of filter. As shown in Figure 1, the invasion capacity of DU_145 cells was markedly inhibited when UCN_01 was present in the upper chamber, a reduction of about 71% (14% from 42% ) of the invasiveness of DU_145 cells were observed for 100 nmol· L- 1 UCN_01 after 24 h treatment.An inhibition was also observed following 48 h exposure to UCN_ 01, a reduction of approximately 58% (28% from 67% ).
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Fig1 Effect of UCN_01 on in vitro invasion of DU_145 cells
Subconfluent DU_145 cells were trypsized and counted, resuspended in 2.5% serum medium containing UCN_01(50 or100nmol· L- 1) and transferred into the upper compartment of the modified Boyden chambers (1× 105 cells/chamber). Fibronectin (16 μ g/chamber) as the chemoattracter is added to the lower compartment. At 24 or 48 h later, invaded cells that attached to the lower surface of the filter were counted in 15 randomly selected microscopic fields (× 400) per filter. Data (± s) are expressed as the percentage of the control response from two independent experiment assays that yielded similar results
, 百拇医药
Fig 2 Effect of UCN_01 on in vitro invasion of DU_145 cells
Confluent DU_145 cells cultured in six_well dishes were carefully wounded using a pipette tip and then re_cultured in the medium with or without UCN_01 (100 nmol· L- 1). The cells were photographed under a phase contrast microscope at 48 h after wounded
To examine whether the UCN_01 anti_ invasion potential was associated with its suppression on the cell spreading and migration, the effect of UCN_01 on the motility of DU_145 cells was also analyzed using the scratch wound assay. Confluent monolayers of DU_145 cells cultured in six_well tissue culture dishes were scratch wounded with sterile pipette tips, post_incubated for further 48 h in the absence or the presence of 100 nmol· L- 1 UCN_01 and photographed.As
, 百拇医药
representative fields shown in Fig2, 100 nmol· L- 1 UCN_01 markedly inhibited the flattening and spread of both cell lines along the edges of the wound compared to the untreated control cells.
To ascertain whether the suppression of DU_145 cell invasiveness and migration by UCN_01 was accompanied with an alteration of cell adhesion molecule E_cadherin, the expression of E_cadherin protein was analyzed by the immunoblot assay.As illustrated in Fig 3 a, DU_145 cells contained a low base level of endogenous E_cadherin protein,however,48 h exposure to UCN_01 resulted in a significant dose_dependent increase.The bands of E_cadherin protein were quantitated by densitometry and presented as a percentage of the untreated control relevant to the corresponding α _actin binds in Fig 3b. 100 nmol· L- 1 UCN_01 caused a ?_fold increase of E_cadherin protein.
, 百拇医药
Fig 3 Effect of UCN_01 on expression of E_cadherin protein
a:Subconfluent growing DU_145 cells ware treated with UCN_01 at the indicated doses for 24 h and then harvested for Western blotting.50 μ l of total protein lysates was loaded each lane.
b:Protein bands were quantitated by densitometry and expressed relative to the α _actin bands and untreated control bands
3 Discussion
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It has been known that activation of PKC promotes prostate cancer cell metastatic ability[8,9], whereas inactivation of PKC activity reduces in vitro invasion and migration behaviors of tumor cells by utility of selective PKC inhibitors, such as staurosporine[10],sphingosine[11],cal_ phostin C[12], tamoxifen[13] and hypericin[14]. Therefore, PKC appears to be a new target for inhibition of tumor cell invasion and migration, and thus metastasis[1,2].In the present study, we found that UCN_01,at a non_cytotoxic dose (100 nmol· L- 1), significantly reduces or suppresses the cell invasion and migration capabilities in human prostate cancer DU_145 cells.These findings further support the previous observations that the activity of PKC plays an important role in the invasion of human prostate cancer[8,9].Therefore, the promising inhibition of tumor cell invasion and migration is a novel feature of this compound, which is added to the anti_tumor activities of UCN_01. Further studies in our laboratory are in progress to determine the potential effects of UCN_01 on metastasis and angiogenesis of prostate cancer in vivo.
, 百拇医药
The mechanisms for PKC in the inhibition of tumor cell invasion and migration may be complicated[1,2].Although the mechanisms by which suppression of cell invasion and migration by UCN_01 occurs remain to be elucidated, an important finding in our present study is that a concurrent up_regulated expression of E_cadherin was observed accompanying with the inhibition of cell invasion and migration after treatment with UCN_01.E_cadherin,a transmembrane glycoprotein, is a key mediator of cell_cell adhesion via forming a complex with three major cytoplasmic catenins (α , β and γ ) and has been reported to play a key role in the control of the invasive and metastatic progression in a variety of human carcinoma cells, including prostate cancer[15,16]. Disruption of the E_cadherin/catenin complex, due primarily to loss or decreased expression of E_cadherin, results in reduced cellular adhesiveness and is shown to be correlated with progression of tumors by increasing cell proliferation, motility and invasiveness [16]. Moreover, it is al
, 百拇医药
so found that tamoxifen, one of PKC inhibitors, blocks the invasion and migration of tumor cells by increasing the expression and functions of E_cadherin/catenin complexes [17].Therefore, it is believed that E_cadherin may be an important mediator in anti_invasion and migration of UCN_01, although further experiments need to clarify the relationship between E_cadherin expression and PCK activity in anti_tumor invasion and migration of UCN_01.
In conclusion, we have first found that UCN_01, a selective PKC inhibitor, suppresses the cell migration and invasion in invasive human prostate cancer DU_145 cells.Moreover, this inhibition of prostate cell invasion and migration is characterized by an enhanced expression of cell adhesion molecule E_cadherin.Therefore, UCN_ 01 is a potent anti_tumor agent in therapy of human prostate cancer not only via the suppression of proliferation of prostate cancer at higher doses, but also via the inhibition of cell invasion and metastasis at lower doses. In addition, our results indicate that the PKC signal transduction pathway may play an important role in prostate cancer cell invasion and metastasis, the suppression of this pathway may limit the malignant progression of human prostate cancer.
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To whom correspondence should be sent:Saijun Fan, MD, Ph.D, Laboratory of Molecular Oncology,Department of Radiation Oncology,Long Island Jewish Medical Center(The Long Island Campus for the Albert Einstein College of Medicine)270_05,76th Avenue, New Hyde Park, New York 11040
References
1，Caponigro F, French RC, Kaye SB. Protein kinase C:a worthwhile target for anticancer drugs? Anti_cancer Drugs,1997;8:26
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2，Gomez DE, Skilton G, Alonso DF,et al.The role of protein kinase C and novel phorbol ester receptors in tumor cell invasion and metastasis. Oncol Rep,1999; 6:1363
3，Takahashi I, Kobayashi E, Asano K,et al. UCN_01, a selective inhibitor of protein kinase C from Strep tomyces.J Antibiot(Tokyo),1987;40:1782
4，Fan S, Meng Q. UCN_01 (7_hydroxystaurosporine): A profound anti_cancer agent. Chin J Clin Phar macolTherap(in press)
, 百拇医药
5，Meng Q,Qi M,Chen DZ, et al. Suppression of breast cancer invasion and migration by indole_3_carbinol:associated with up_regulation of BRCA1 and E_cad_ herin/catenin complexes. J Mol Med, 2000;78:155
6，Kruger EA, Blagosklonny MV, Dixon SC,et al. UCN_01,a protein kinase C inhibitor, inhibits en_dothelial cell proliferation and angiogenic hypoxic re_sponse. Invasion Metastasis,1998;18:209
7，Hendrix MJ, Seftor EA, Seftor RE,et al.A simple quantitative assay for studying the invasive potential of high and low human metastatic variants. Cancer Lett,1987;38:137
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8，Liu B, Maher RJ, Hannun YA,et al.12(S)_HETE enhancement of prostate tumor cell invasion: selective role of PKC alpha. J Natl Cancer Inst,1994;86: 1145
9，Timar J, Raso E, Fazakas ZS,et al. Multiple use of a signal transduction pathway in tumor cell invasion. Anticancer Res,1996;16:3299
10，Szaniawska B, Gawrychowski K, Janik P. The effect of protein kinase C inhibitors on invasion of human ovary cancer cells. Neoplasma,1998;45:7
, http://www.100md.com
11，Stam JC, Michiels F, Van der Kammen RA,et al. Invasion of T_lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling. Embo J, 1998;17:4066
12，Shimao Y, Nabeshima K, Inoue T, et al.TPA_ en_hanced motility and invasion in a highly metastatic variant (L_10) of human rectal adenocarcinoma cell line RCM_1: selective role of PKC_alpha and its inhi_bition by a combination of PDBu_induced PKC down_regulation and antisense oligonucleotides treatment. Clin Exp Metastasis, 1999;17:351
, http://www.100md.com
13，Hoelting T, Duh QY, Clark OH,et al.Tamoxifen antagonizes proliferation and invasion of estrogen re_ceptor_negative metastatic follicular thyroid cancer cells via protein kinase C. Cancer Lett,1996;100:89
14，Zhang W, Law RE, Hinton DR,et al. Inhibition of human malignant glioma cell motility and invasion in vitro by hypericin, a potent protein kinase C in_ hibitor. Cancer Lett, 1997;120:31
15，Bracke ME, Van Roy FM, Mareel MM. The E_cad_ herin/catenin complex in invasion and metastasis.Curr Top Microbiol Immunol,1996;213:123
, http://www.100md.com
16，schiesche W,Schonborn I,Behrens J,et al. Expres_ sion of E_cadherin and catenins in invasive mammary carcinomas.Anticancer Res,1997;17:561
17，Bracke ME, Charlier C, Bruyneel EA,et al. Tamox_ ifen restores the E_cadherin function in human breast cancer MCF_7/6 cells and suppresses their invasive phenotype. Cancer Res,1994;54:4607
2000-08-01 Received， 2000-10-20 Accepted, http://www.100md.com
单位：长岛Jewish医学中心分子肿瘤实验室，纽约美国
关键词：7_hydroxystaurosporine(UCN_01)前列腺肿瘤浸润转移
中国临床药理学与治疗学000401
目的和方法研究7_hydroxystaurosporine(UCN_01)对转移性前列腺肿瘤DU_145细胞浸润和转移的影响。用体外转移和创伤法检查细胞浸润和转移，用Westernblotting法检查蛋白质的表达。结果UCN_01在非毒性剂量下(100nmol·L-1)明显地抑制DU_145细胞浸润和转移。而且，UCN_01这种抗肿瘤浸润和转移功能与它增加细胞_细胞粘连分子E_cadherion的表达有关。结论这些实验首次证实UCN_01能抑制人前列腺肿瘤细胞的浸润和转移。UCN_01的临床应用可能更有效地控制前列腺肿瘤转移。
, 百拇医药
中图分类号R979.1
Suppression of invasion and migration by 7_hydroxystaurosporine(UCN_01),a new anti_tumor agent
MENG Qing_Hui FAN Sai_Jun
(Laboratory of Molecular Oncology, Department of Radiation Oncology, Long Island Jewish Medical Center,New York)
SHAO Rong_Guang
(Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing, China)
, 百拇医药
XU Jing_Wen
(Division of Rheumatology,Department of Medicine, The Long Island Campus for the Albert Einstein College of Medicine,New Hyde Park, New York, USA)
Aim and Methods To investigate the effect of UCN_01(7_hydroxystaurosporine) on cell migration and invasion ability of DU_145, an invasive human prostate cancer cell line.Results It was found that UCN_01 at non_cytotoxic doses (100 nmol· L- 1) significantly inhibited prostate cancer DU_145 cell invasion and migration behaviors.Moreover, this anti_invasion and migration activity of UCN_01 was associated with an up_regulation of cell adhesion molecule E_cadherin. Conclusion These results indicate for first time that UCN_01 inhibits the invasion and migration of human prostate cancer cells.Thus, clinical application of UCN_01 may contribute to the potential benefit for suppression of prostate cancer invasion and metastasis.
, 百拇医药
Key words 7_hydroxystaurosporine(UCN_01); prostate; tumor; invasion; migration
Protein kinase C(PKC) plays an important role in tumorigeneses,tumor cell invasion and metastasis[1,2],UCN_01(7_hydroxystaurosporine) is a selective PKC inhibitor derived from the non_selective protein kinase inhibitor staurosporine[3].Subsequently,a variety of studies from our and other laboratories have revealed that UCN_01 is a promising agent in the inhibition of tumor cell growth alone or in a combination with other chemotherapeutic agents,such as camptothecin,5_fluorouracil, cisplatin, mitomycin_C and ionizing radiation,in vitro and in vivo. The
, 百拇医药
anti_tumor activities of UCN_01 are associated with the arrest of cell cycle progression, including G1/S and/or G2/M, apoptosis induction and inhibition of DNA repair[4]. Therefore, these studies
indicate that UCN_01 is a profound anti_tumor agent in a larger number of tumors,including prostate cancer.Moreover,UCN_01 has been entered phase I trials in the United States and Japan as a single agent.In the present study, the effects of UCN_01 on the cell invasion and migration properties were investigated in DU_145, a human prostate cell line.Exposure to UCN_01 at non_ cytotoxic doses (100 nmol· L- 1) caused a significant repression of the cell invasion, speading and migration.Furthermore,UCN_01 caused an up_ regulation in the protein expression of cell adhesion molecule E_cadherin.
, 百拇医药
1 Materials and Methods
1. 1 Cell culture and UCN_01 The invasive human prostate cancer cell line DU_145 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained as monolayer cultures in D_MEM supplemented with 10% fetal calf serum (FCS), 2 mmol· L- 1 glutamine, 100 μ g· ml- 1 streptomycin and 100 unit/ml penicillin G (BioWhittaker, Walkersville, MD).UCN_01 was kindly obtained from the Laboratory of Molecular Pharmacology, Division of Basic Science, NCI, NIH (Bethesda, MD), stored at - 20 ℃ as a 10 mmol· L- 1 stock solution in 20% DMSO, and further diluted in medium prior to experiments.
, 百拇医药
1.2 In vitro invasion assay In vitro invasion assay was carried out using a modified Boyden chamber[5]. Briefly, the surfaces of filter (0.8 μ m pore size) were coated with a uniform thickness of 25 μ g of Matrigel for 1 h at room temperature.Uniformity of the coating was checked by Coomassie blue staining and low_ power microscope observation.The lower chamber was filled with 10% FCS medium containing fibronectin (16 μ g/chamber) as the chemoattractant.Cells (1× 105 cells/ml) resuspended in the medium containing 2.5 % FCS and 100 nmol· L- 1 UCN_01 were carefully transferred onto the upper surface of filters hold in the chamber.After 24 h incubation, the filter was gently removed from the chamber, the cells on the upper surface were removed by wiping with a cotton swab, the cells that invaded through the Matrigel and attached to the lower surface of the filter were fixed, stained with H＆ E, and counted in 15 randomly selected microscopic fields(× 400) per filter. Experiments were independently performed at least three times.
, 百拇医药
1.3 Scratch wound assay The spreading and migration capabilities of DU_145 cells were assessed using a scratch wound assay[5],which measures expansion of a cell population on surfaces. The cells were seeded into six_well tissue culture dishes at a concentration of 2.5× 105 cells and cultured in medium containing 10% FCS to nearly confluent cell monolayers, which were then carefully wounded using 1 ml sterile pipette tips and any cellular debris was removed by washing with PBS.The wounded monolayers were then incubated in 10% FCS D_MEM containing different doses of UCN_01 for 48 h and photographed under a light microscope(× 200).The experiments were repeated in quadruplicate wells at least three times.
, 百拇医药
1.4 Immunoblot assay Protein expression was assayed using an immunoblot assay as described previously[5].UCN_01_untreated and treated cells were lysed in a mini_modified ice_cold RIPA lysis buffer containing protease inhibitors (Calbiochem, San Diego, CA) and 100 mmol· L- 1 sodium orthovanadate.100 μ g of total protein lysates was electrophoresed on 10% SDS_ polyacrylamide gels and transferred to membranes by electroblotting. The membranes were incubated with a monoclonal E_cadherin antibody (Trans_ duction Laboratories, Lexington, KY) and then incubated with secondary antibodies after being extensively washed.Antibody reaction was revealed using an enhanced chemiluminescence detection system (Amersham Life Science, Arlington Heights, Illinois) as instructed by the manufacturer.Equal protein loading and the protein transfer were confirmed by immunoblotting for determination of α _actin protein using a polyclonal α _actin antibody(I_19, Santa Cruz, Hercule, CA) on the same Western blots.A colored marker (Bio_Rad Laboratories, Hercules, CA) was used as a molecular size standard.
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2 Results
Using a MTT assay, the cytotoxicity of UCN_01 was determined in DU_145 human prostate cancer cells after treatment with UCN_ 01.Consistent wit previous studies[6],>100 nmol · L- 1 doses of UCN_01 (48 h exposure) caused a significant cytotoxicity(data not shown). Therefore, the effects of UCN_01 on DU_145 cell invasion and migration ability were carried out using < 100 nmol· L- 1 doses, this will exclude any possible effects from the cytotoxicity of UCN_01 on anti_invasion and migration activities of UCN_ 01.Tumor cell invasion is a crucial aspect of the processes of tumor metastasis. For the evaluation of cellular invasiveness, a modified Boyden chamber assay was carried out to determine the ability of DU_145 glioma cells to invade through biological matrices in vitro.The relevance of this assay for other invasion assays and for in vivo malignancy has been documented extensively[7], based on the percentage of cells penetrating the reconstituted basement membrane_coated filters and attached on the lower surface of filter. As shown in Figure 1, the invasion capacity of DU_145 cells was markedly inhibited when UCN_01 was present in the upper chamber, a reduction of about 71% (14% from 42% ) of the invasiveness of DU_145 cells were observed for 100 nmol· L- 1 UCN_01 after 24 h treatment.An inhibition was also observed following 48 h exposure to UCN_ 01, a reduction of approximately 58% (28% from 67% ).
, 百拇医药
Fig1 Effect of UCN_01 on in vitro invasion of DU_145 cells
Subconfluent DU_145 cells were trypsized and counted, resuspended in 2.5% serum medium containing UCN_01(50 or100nmol· L- 1) and transferred into the upper compartment of the modified Boyden chambers (1× 105 cells/chamber). Fibronectin (16 μ g/chamber) as the chemoattracter is added to the lower compartment. At 24 or 48 h later, invaded cells that attached to the lower surface of the filter were counted in 15 randomly selected microscopic fields (× 400) per filter. Data (± s) are expressed as the percentage of the control response from two independent experiment assays that yielded similar results
, 百拇医药
Fig 2 Effect of UCN_01 on in vitro invasion of DU_145 cells
Confluent DU_145 cells cultured in six_well dishes were carefully wounded using a pipette tip and then re_cultured in the medium with or without UCN_01 (100 nmol· L- 1). The cells were photographed under a phase contrast microscope at 48 h after wounded
To examine whether the UCN_01 anti_ invasion potential was associated with its suppression on the cell spreading and migration, the effect of UCN_01 on the motility of DU_145 cells was also analyzed using the scratch wound assay. Confluent monolayers of DU_145 cells cultured in six_well tissue culture dishes were scratch wounded with sterile pipette tips, post_incubated for further 48 h in the absence or the presence of 100 nmol· L- 1 UCN_01 and photographed.As
, 百拇医药
representative fields shown in Fig2, 100 nmol· L- 1 UCN_01 markedly inhibited the flattening and spread of both cell lines along the edges of the wound compared to the untreated control cells.
To ascertain whether the suppression of DU_145 cell invasiveness and migration by UCN_01 was accompanied with an alteration of cell adhesion molecule E_cadherin, the expression of E_cadherin protein was analyzed by the immunoblot assay.As illustrated in Fig 3 a, DU_145 cells contained a low base level of endogenous E_cadherin protein,however,48 h exposure to UCN_01 resulted in a significant dose_dependent increase.The bands of E_cadherin protein were quantitated by densitometry and presented as a percentage of the untreated control relevant to the corresponding α _actin binds in Fig 3b. 100 nmol· L- 1 UCN_01 caused a ?_fold increase of E_cadherin protein.
, 百拇医药
Fig 3 Effect of UCN_01 on expression of E_cadherin protein
a:Subconfluent growing DU_145 cells ware treated with UCN_01 at the indicated doses for 24 h and then harvested for Western blotting.50 μ l of total protein lysates was loaded each lane.
b:Protein bands were quantitated by densitometry and expressed relative to the α _actin bands and untreated control bands
3 Discussion
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It has been known that activation of PKC promotes prostate cancer cell metastatic ability[8,9], whereas inactivation of PKC activity reduces in vitro invasion and migration behaviors of tumor cells by utility of selective PKC inhibitors, such as staurosporine[10],sphingosine[11],cal_ phostin C[12], tamoxifen[13] and hypericin[14]. Therefore, PKC appears to be a new target for inhibition of tumor cell invasion and migration, and thus metastasis[1,2].In the present study, we found that UCN_01,at a non_cytotoxic dose (100 nmol· L- 1), significantly reduces or suppresses the cell invasion and migration capabilities in human prostate cancer DU_145 cells.These findings further support the previous observations that the activity of PKC plays an important role in the invasion of human prostate cancer[8,9].Therefore, the promising inhibition of tumor cell invasion and migration is a novel feature of this compound, which is added to the anti_tumor activities of UCN_01. Further studies in our laboratory are in progress to determine the potential effects of UCN_01 on metastasis and angiogenesis of prostate cancer in vivo.
, 百拇医药
The mechanisms for PKC in the inhibition of tumor cell invasion and migration may be complicated[1,2].Although the mechanisms by which suppression of cell invasion and migration by UCN_01 occurs remain to be elucidated, an important finding in our present study is that a concurrent up_regulated expression of E_cadherin was observed accompanying with the inhibition of cell invasion and migration after treatment with UCN_01.E_cadherin,a transmembrane glycoprotein, is a key mediator of cell_cell adhesion via forming a complex with three major cytoplasmic catenins (α , β and γ ) and has been reported to play a key role in the control of the invasive and metastatic progression in a variety of human carcinoma cells, including prostate cancer[15,16]. Disruption of the E_cadherin/catenin complex, due primarily to loss or decreased expression of E_cadherin, results in reduced cellular adhesiveness and is shown to be correlated with progression of tumors by increasing cell proliferation, motility and invasiveness [16]. Moreover, it is al
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so found that tamoxifen, one of PKC inhibitors, blocks the invasion and migration of tumor cells by increasing the expression and functions of E_cadherin/catenin complexes [17].Therefore, it is believed that E_cadherin may be an important mediator in anti_invasion and migration of UCN_01, although further experiments need to clarify the relationship between E_cadherin expression and PCK activity in anti_tumor invasion and migration of UCN_01.
In conclusion, we have first found that UCN_01, a selective PKC inhibitor, suppresses the cell migration and invasion in invasive human prostate cancer DU_145 cells.Moreover, this inhibition of prostate cell invasion and migration is characterized by an enhanced expression of cell adhesion molecule E_cadherin.Therefore, UCN_ 01 is a potent anti_tumor agent in therapy of human prostate cancer not only via the suppression of proliferation of prostate cancer at higher doses, but also via the inhibition of cell invasion and metastasis at lower doses. In addition, our results indicate that the PKC signal transduction pathway may play an important role in prostate cancer cell invasion and metastasis, the suppression of this pathway may limit the malignant progression of human prostate cancer.
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To whom correspondence should be sent:Saijun Fan, MD, Ph.D, Laboratory of Molecular Oncology,Department of Radiation Oncology,Long Island Jewish Medical Center(The Long Island Campus for the Albert Einstein College of Medicine)270_05,76th Avenue, New Hyde Park, New York 11040
References
1，Caponigro F, French RC, Kaye SB. Protein kinase C:a worthwhile target for anticancer drugs? Anti_cancer Drugs,1997;8:26
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2，Gomez DE, Skilton G, Alonso DF,et al.The role of protein kinase C and novel phorbol ester receptors in tumor cell invasion and metastasis. Oncol Rep,1999; 6:1363
3，Takahashi I, Kobayashi E, Asano K,et al. UCN_01, a selective inhibitor of protein kinase C from Strep tomyces.J Antibiot(Tokyo),1987;40:1782
4，Fan S, Meng Q. UCN_01 (7_hydroxystaurosporine): A profound anti_cancer agent. Chin J Clin Phar macolTherap(in press)
, 百拇医药
5，Meng Q,Qi M,Chen DZ, et al. Suppression of breast cancer invasion and migration by indole_3_carbinol:associated with up_regulation of BRCA1 and E_cad_ herin/catenin complexes. J Mol Med, 2000;78:155
6，Kruger EA, Blagosklonny MV, Dixon SC,et al. UCN_01,a protein kinase C inhibitor, inhibits en_dothelial cell proliferation and angiogenic hypoxic re_sponse. Invasion Metastasis,1998;18:209
7，Hendrix MJ, Seftor EA, Seftor RE,et al.A simple quantitative assay for studying the invasive potential of high and low human metastatic variants. Cancer Lett,1987;38:137
, 百拇医药
8，Liu B, Maher RJ, Hannun YA,et al.12(S)_HETE enhancement of prostate tumor cell invasion: selective role of PKC alpha. J Natl Cancer Inst,1994;86: 1145
9，Timar J, Raso E, Fazakas ZS,et al. Multiple use of a signal transduction pathway in tumor cell invasion. Anticancer Res,1996;16:3299
10，Szaniawska B, Gawrychowski K, Janik P. The effect of protein kinase C inhibitors on invasion of human ovary cancer cells. Neoplasma,1998;45:7
, http://www.100md.com
11，Stam JC, Michiels F, Van der Kammen RA,et al. Invasion of T_lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling. Embo J, 1998;17:4066
12，Shimao Y, Nabeshima K, Inoue T, et al.TPA_ en_hanced motility and invasion in a highly metastatic variant (L_10) of human rectal adenocarcinoma cell line RCM_1: selective role of PKC_alpha and its inhi_bition by a combination of PDBu_induced PKC down_regulation and antisense oligonucleotides treatment. Clin Exp Metastasis, 1999;17:351
, http://www.100md.com
13，Hoelting T, Duh QY, Clark OH,et al.Tamoxifen antagonizes proliferation and invasion of estrogen re_ceptor_negative metastatic follicular thyroid cancer cells via protein kinase C. Cancer Lett,1996;100:89
14，Zhang W, Law RE, Hinton DR,et al. Inhibition of human malignant glioma cell motility and invasion in vitro by hypericin, a potent protein kinase C in_ hibitor. Cancer Lett, 1997;120:31
15，Bracke ME, Van Roy FM, Mareel MM. The E_cad_ herin/catenin complex in invasion and metastasis.Curr Top Microbiol Immunol,1996;213:123
, http://www.100md.com
16，schiesche W,Schonborn I,Behrens J,et al. Expres_ sion of E_cadherin and catenins in invasive mammary carcinomas.Anticancer Res,1997;17:561
17，Bracke ME, Charlier C, Bruyneel EA,et al. Tamox_ ifen restores the E_cadherin function in human breast cancer MCF_7/6 cells and suppresses their invasive phenotype. Cancer Res,1994;54:4607
2000-08-01 Received， 2000-10-20 Accepted, http://www.100md.com