INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY

Vol. 26, no. 4, 977-982 (2013)

LETTER TO THE EDITOR

TANSHINONE VI INHIBITS THE EXPRESSION OF INTERCELLULAR ADHESION MOLECULE-l AND VASCULAR CELL ADHESION MOLECULE-l V. NICOLINI, F. BOSSP, A. VIGGIAN0 2, R. VALENTINP and S.L. NORP I

Clinical Department ofMedical, Surgical and Health Science, Trieste, Italy; 'Department of Medicine and Surgery University ofSalerno, Baronissi, Salerno, Italy Received March 26, 2013 - Accepted October 16, 2013

This study investigated the possible antitumor mechanisms of action of Tanshinone VI, one of the components of Salvia miltiorrhiza Bunge, which is used in traditional Chinese herbal medicine. To this end, the expression of intercellular adhesion molecule-l (ICAM-l) and vascular cell adhesion molecule-l (VCAM-l), were evaluated in-vitro in tumor necrosis factor-a (TNF-a)-stimulated endothelial cells, with, or without the addition of Tanshinone VI (10, 20, 30, or 40 mM) in the culture medium; the effects of Tanshinone VI on angiogenesis was also evaluated with an epithelial cell tube formation assay and its toxicity was evaluated with a colorimetric (MTT) cell viability assay. The results showed that the upregulation of ICAM-l and VCAM-l induced by TNF-a was dose-dependently inhibited by Tanshinone VI, with restoration of control levels at the dose of 40 mM; Tanshinone VI also had a remarkable antiangiogenesis effect, already at the dose of 10 mM, while none of the doses tested had significant effects on cell viability. These results indicate that the antitumor properties of Tanshinone VI can be ascribed to the inhibition of cell adhesion, due to blockage of the up-regulation of cell adhesion molecules, with the consequent inhibition of metastases formation and/or angiogenesis. The lack of toxic effects at the dosage used makes Tanshinone VI a good candidate for its therapeutic use in humans. Cancer progression is a multi-step process in which some adhesion molecules play a pivotal role in the development of recurrent, invasive and distant metastases. Multiple and diverse cell adhesion molecules take part in the inter-cellular and cell-extracellular matrix interactions of cancer; cell adhesion molecules (CAMs) are expressed on a variety of cells, including vascular endothelial cells (ECs), lymphocytes, fibroblasts, hematopoietic cells and tumor cells. Some CAMs, such as the vascular cell adhesion molecule-l (VCAM-l) and the intercellular adhesion molecule-l (ICAM-l) are expressed on ECs that have been activated by cytokines such as interleukin-l a, interleukin-6 or

tumor necrosis factor-a (TNF-a) which are typically produced in inflammatory and tumor processes (13). Thus, the negative prognostic meaning of high levels of those cytokines in cancer disease, is likely due to the induction of the cited CAMs which permit the growth of a tumor mass and the implantation of metastases; accordingly, if a drug is able to inhibit that cytokine-mediated induction of the expression of CAMs, that drug is expected to have a beneficial effect for the treatment of cancer disease. Danshen (Salvia miltiorrhiza Bunge) is a herb that has been widely used in traditional Chinese medicine for treating coronary heart diseases, such as angina pectoris and myocardial infarction (4, 5). Along

Key words: Tanshinone VI, angiogenesis, adhesion molecules, vascular endothelial growth factor (VEGF) Mailingaddress: Dr S.L. Nori, Department of Medicine and Surgery, University of Salerno, Baronissi Campus, via S. Allende, Baronissi, 84081Salerno, Italy Tel.: +39089965078 Fax: +39089968794 e-mail: [email protected]

0394-6320 (2013)

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DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF INTEREST RELEVANT TO THIS ARTICLE.

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with 20 phenolic acids, 30 diterpene compounds, including the relatively abundant tanshinones, tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone, have been isolated from Danshen (6, 7). These abundant tanshinones are the major diterpenes isolated from Danshen and show cytotoxic effects on cell lines derived from various human carcinomas of the colon, breast, prostate, ovary, lung and mouth (8, 9). Recent studies suggest that tanshinones can suppress angiogenesis by inhibiting endothelial proliferation and angiogenic differentiation; this effect is probably due to a modulation of angiogenic regulators such as VEGF, HIF-Ia, c-Myc, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). In 2005 tanshinones were reported to inhibit the proliferation of fetal bovine serum (FBS)-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) in a concentration-dependent manner, along with inactivation of ERK 1/2, increase of P21 and down-regulation of cyclin D I (10). Tsai et al. (II) used human umbilical vein endothelial cells (HUVECs) as a model and found that Tanshinone IIA inhibited FBS-induced migration, TNF-ainduced invasion, and extracellular matrix-induced tube formation. Thus, the aim of the present study was to investigate the effect of another tanshinone, Tanshinone VI (Fig. I), in a model similar to that of Tsai et al. (II). In particular, the following were evaluated: i) the expression of ICAM-I and VCAM-I in TNF-a-stimulated HUVECs with or without the addition ofTanshinone VI; ii) the ability of Tanshinone VI to inhibit cell tube formation of HUVECs, that is an in-vitro model of angiogenesis; iii) the cell viability after the addition ofTanshinone VI, as an index of possible toxicity. MATERIALS AND METHODS Reagents Analytical grade solvents were obtained from Carlo Erba, Milano, Italy. HPLC grade acetonitrile (CH3N), methanol (MeOH), and formic acid were purchased from J.T. Baker (Baker Mallinckrodt, Phillipsburg, NJ, USA). HPLC-grade water (18 mV) was prepared using a Millipore (Bedford, MA, USA) Milli-Q purification system. Recombinant PIGF-I protein (aminoacids 19149) produced in E.coli in our laboratory was used in all the experiments. The recombinant growth factors,

receptors and antibodies used were from R&D Systems. Human recombinant TNF-u was from PeproTech (Rocky Hill, NJ, USA) and was used at a concentration of 10 ng/ mL. Primary and peroxidase-conjugated antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA).

Tanshinone VI The crude Tanshinone VI used in the present investigation was obtained by extraction with ethanol-nhexane (I: I, v/v) from S. milthiorriza Bunge. Preparative HSCCC with two phase solvent systems composed of n-hexane-ethanol-water (10:7:3 v/v) was successfully performed in a stepwise elution yielding six relatively pure diterpenoids from 300 mg of the crude extract in a single run. The Tanshinone VI structure was demonstrated by ID and 20 NMR spectroscopy as well as ESI mass spectrometry and compared with published data. Cells Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase treatment and cultured in medium 199 supplemented with 20% newborn calf serum (Invitrogen, Carlsbad, CA, USA), 50 ug/ml, endothelial growth supplement, 50 ug/ml, heparin, 100 U/mL penicillin, and 100 ug/rnl, streptomycin (Sigma-Aldrich) and grown in tissue culture plates (Costar, Cambridge, MA, USA) coated with 2% endotoxin-free gelatin. ELISA The expression of adhesion molecules on HUVEC surface was assessed on cells grown to confluence in 96-well tissue culture plates (Costar, Cambridge, MA) divided into 12 groups: with the sole culture medium (groups I, 2); with the addition of TNF-u 10 ng/ml (groups 3, 4); with the addition of TNF-u 10 ng/ml and Tanshinone VI 10 11M (groups 5, 6); with the addition of TNF-u 10 ng/ml and Tanshinone VI 20 11M (groups 7, 8); with the addition of TNF-u 10 ng/ml and Tanshinone VI 30 11M (groups 9, 10); and with the addition ofTNF-u 10 ng/ml and Tanshinone VI 40 11M (groups II, 12). The cells were incubated with 100 III anti-VCAM mAb clone IAC3 (odd groups), or anti-ICAM-I mAb clone 8AA6 (even groups) for I h at room temperature followed by antimouse peroxidase-conjugated antibodies. The enzymatic reaction was developed with p-nitrophenyl phosphate (PNPP, I mg/ml) as substrate and read kinetically at 405 nm using a Titertek Multiskan ELISA reader (Flow Labs, Milano, Italy).

Tube/ormation assay Twenty-four-well culture plates (Nalge Nunc International, Naperville, IL), divided into 6 groups, were coated with 50 ml low-growth-factor synthetic

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matrix (Matrigel, BD Biosciences, San Jose, CA, USA) and incubated for 30 min at 37°C. HUVECs, that were serum starved for 4 h, were treated with trypsinethylenediaminetetra-acetic acid and suspended in a cond itioned medium for 24 h at 37°C with the addit ion of: nothing else (group I); VEGF (group 2); VEGF plus Tanshinone VI 10 11M (group 3); VEGF plus Tanshinone VI 20 11M (group 4); VEGF plus Tanshinone VI 30 11M (group 5); VEGF plus Tanshinone VI 40 11M (group 6). HUVECs were seeded at a density of Ix I04 cells on the synthet ic matrix and then incubated for 17 h at 37°C. Microg raphs of the 96-well plates were taken by phase contra st microscopy at a magnification of 100x. Images of tube formation were selected randomly in three fields.

was used for pair-wise compari sons.

RESULTS The ELISA test demonstrated that TNF-a, at the concentration of 10 ng/ml, was effecti ve in inducing the expression of both ICAM-I and VCAM-l, while the treatment with Tanshinone VI exerted a dose-dependent inhibition of such induction, nearly restoring the values to that of untreated cells at the

Cell viability The effect of Tanshinone VI on cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphe nyltetrazolium bromide (MIT) assay. The cells were firstly grown to confluence in 96-well culture plates (Costar, Cambridge, MA) divided into 5 group s; the cultures were then treated with Tanshinone VI 0-10-2030-40 11M (groups I, 2, 3, 4, 5 respe ctively) for 24h at 37°C, 5% C02. After further incubation at 37°C for 12 h, the number of viable cells was estimated using the MTT assay. Statistical analysis Data are presented as means±S.E. One- way analysis of variance (ANOVA) was used to test significant difference between the groups and the Newmann-K euls post-hoc test

1.8 1.6

O ICA M-1 - VCA M-1

*

*

Fig. l. 2D chemical structure of Tanshinon e VI estract from Danshen.

*

*

1.4

**

1.2 c

~

ci

o

*

1.0 0.2 0.6 0.4 0.2 0

c-

TNF

TS 10

TS 20

TS 30

TS 40

Fig. 2. Expression ofadhesion molecules after the administration ofTanshinone VI at different concentrations. -C, without either TNF-a or Tanshinone VI; TNF, TNF-a 10 ng/ml; TSIO-TS40, TNF-a 10 ng/ml plus Tanshinone VI (It), 20. 30, 40 JIM respectively). Different asterisks indicate statistical significant difference (P < 0.05) compared to other groups for the same antigen (ICAM- l or VCA M-l) . Compared to the TNF group, Tanshinone VI significantly reduced the expressio n of ICAM-l at the dose of40 JlM and that of VCAM-l at doses above 20 JlM

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Fig. 3. Effects ofTanshinone VI on H U VECs tube fo rmation. as an in vitro model of angi ogenesis. A ) Ne ither VEGF nor Tanshinone were added to the culture medium; no tube struc tures are present. B) The addition of VEGF to the culture medium induced the formation of tub e structures. C-D) The addition of VEGF plus Tansh inon e VI (10 flM in C, 40 flM in D) in the culture medium resu lted in the prevention of tube f ormation.

cell v iab ility (MTT assay)

1.4.--- - - - - - - - - - - - - - - - - - - - - - - - ............., 1.2 1.0

5 0 .8 o 00.6 0 .4 0 .2

o

Fig. 4. Effects of Tanshinone VI on HU VECs viability evaluated by MTT assay. The groups treated with Tanshinone VI (TS 10-TS40; 10, 20, 30. 40 p M, respectively) did not differ f rom the control group (ctrl), demonstratin g lack ofsignificant toxicity in this model.

concentration of 40 IlM. The ANOVA showed a significant difference between groups (P