Experimental immunology
The influence of Rhodiola rosea, Rhodiola kirilowii and Rhodiola quadrifida extracts on cutaneous angiogenesis induced in mice after grafting of human kidney cancer tissue

Introduction
Dr Judah Folkman was the first who recognized that the growth of solid tumor and its metastasis is strongly dependent on the recruitment and ingrowth of new blood vessels from nearby normal tissues. Now, it is general agreement, that inhibition of angiogenesis may become a promising strategy in cancer therapy [1]. We previously reported, that aqueous and hydro-alcoholic extracts of 3 Rhodiola species – R. rosea, R. kirilowii and R. quadrifida suppressed angiogenic reaction induced in mice skin by grafting of syngeneic L-1 sarcoma cells [2-4]. The question arise whether this suppression was dependent on inhibition of angiogenic factors production by transplanted cells, or was the result of attenuation of angiogenic signaling for endothelial cells. For this purpose we decided to repeat our previous experiments using sonicated homogenate of human kidney cancer tissue instead of living murine sarcoma cells, and comparing the results to the results of simultaneously performed experiments with L-1 sarcoma living cells. In both type of experiments we used 0.4 mg daily dose of Rhodiola extracts. From our earlier studies we have known that tissue homogenates and cell suspensions prepared from various types of human cancers (kidney, ovary, lung, urinary bladder), as well as human sera and human recombinant angiogenic factors, when injected intradermally to recipient mice induce strong neovascular reaction, which may be suppressed or attenuated by various substances of synthetic and natural origin [5-13].

Materials and Methods
R. rosea and R. kirilowii plants originated from many years’ cultivation of the Research Institute of Medicinal Plants (RIMP) in Poznań, thanks to prof. P. M. Mrozikiewicz and dr W. Buchwald. Rhizomes and roots of R. quadrifida were collected in Altai mountain in Mongolia, thanks to dr H. Wiedenfeld and prof. M. Furmanowa. Samples extractions were prepared by the methods as described [14, 15]. Briefly, aqueous extracts: finely powdered roots were extracted with water two times in the temperature 40-45°C. The supernatants were combined and after centrifugation at 3000 rpm for 15 min, were lyophylized. Hydro alcoholic extracts: finely powdered roots were extracted with ethanol/water solution (1/1, V/V) by the percolation method. The percolates were lyophylized which was preceded by the distilling off the ethanol in the temperature 40-45°C.

Biological studies
The study was performed on 7-8 weeks old inbred Balb/ mice, weighing about 20 g, females, delivered from the Polish Academy of Sciences breeding colony. Rhodiola extracts were administered to mice per os in daily doses 0.4 mg. These doses corresponded to 200 mg given to 70 kg person (applying the counter 7 for differences between mouse and human in relation of the surface to body mass). Mice received drugs by Eppendorff pipette, in 40 µl of 10 % ethyl alcohol, for 3 days after tumor homogenate or cells grafting. Control animals received 10% alcohol.
Tumour tissue was obtained surgically from patient with kidney clear cell carcinoma. 2.5 g of tissue were suspended in 5 ml of phosphate-buffered saline(PBS), homogenized with an ultrasonic disrupter VirSonic (Virtis) for 2 minutes, at frequency 22.5 KHz and stored at -70°C, in 1 ml aliquots.
Sarcoma cells were delivered from Warsaw’s Cancer Centre collection, thanks to dr Henryk Skurzak, and then passaged through several generations of Balb/c mice, according to the method described [16]. Briefly, sarcoma cells were grafted (106/0.1 ml) subcutaneously into subscapular region. After 14 days the tumours were excised, cut to smaller pieces, rubbed through sieve and suspended in 5 ml of PBS. The suspension was left for 10 min at room temperature. After sedimentation the supernatant was collected and centrifuged for 10 min at 300 × g. Obtained sarcoma cells were washed once with PBS for 10 min, then centrifuged at 300 × g, and resuspended in Parker medium in concentration of 4 × 106/ml.
Cutaneous angiogenesis assay was performed according to [17] with own modifications [18]. Briefly, multiple 0.05 ml samples of homogenate or cell suspension were injected intradermally into partly shaved, narcotised Balb/c mice (3-4 mice per group, 4-6 injections per mouse). In order to facilitate the localisation of injection sites later on, the suspension was coloured with 0.1% of trypan blue. On the day of grafting and on the following two days mice were fed tested substances, or 10% ethyl alcohol as a control. After 72 hours mice were sacrificed with lethal dose of Morbital. All newly formed blood vessels were identified and counted in dissection microscope, on the inner skin surface, at magnification of 6×, in 1/3 central area of microscopic field. Identification was based on the fact that new blood vessels, directed to the point of cells injection, differ from the background vasculature in their tortuosity and divarications. All experiments were performed in anaesthesia (3.6% chloral hydrate, 0.1 ml per 10 g of body mass).
For all experiments animals were handled according to the Polish law on the protection of animals and NIH standards. All experiments were accepted by the local Ethical Committee.
Statistical evaluation of the results was performed by one-way ANOVA and the significance of differences between the groups was verified with a Dunnett’s Multiple Comparison Test (GraphPadPrism software package).

Results
The results of Rhodiolas extracts administration to mice injected with human kidney tumor homogenates are presented on the Fig. 1. R. rosea and R. kirilowii extracts significantly diminished neovascular reaction, R. quadrifida extracts, however, were ineffective. Water extract of R. rosea (RRW) presented lower activity than aqueous (RKW) and hydro-alcoholic (RKA) Rhodiola kirilowii extracts, as well as Rhodiola rosea hydro-alcoholic(RRA) one.
The main differences between the results obtained for human kidney cancer homogenate and living L-1 sarcoma cells (Fig. 2) was: firstly, lack of the effect of both R. quadrifida extracts in the former and significant inhibition in the latter type of experiment, secondly, lack of the effect of R. kirilowii water extract on neovascular response induced by L-1 sarcoma cells, and its full activity in the experiment with human cancer homogenate.
R. kirilowii hydro-alcoholic and R. rosea extracts were effective in both types of experiment.

Discussion
Present experiments show, that attenuation of neovascular reaction, observed by us in mice grafted with L-1 sarcoma living cells and fed Rhodiola quadrifida extracts, is probably dependent on the suppressing effect of these herbal preparations on the production, (or/and on the release), of pro-angiogenic factors by transplanted cells. In situation when cocktail of various angiogenic cytokines present in the homogenate of human kidney cancer was introduced to mice skin, R. quadrifida extracts were ineffective, not influencing the efferent arc of cutaneous angiogenic response. Other extracts (except aqueous extract of R. kirilowii) probably influenced both arcs of neovascular reaction.
The differences may be connected with different chemical composition of our various Rhodiola extracts [14, 19, 20]. We have found angioinhibitory effect of rosavin and salidroside in L-1 sarcoma model [2, 3]. Rosavin is characteristic for R. rosea extracts, salidroside is a common compound for all of them. We also observed in other model (serum-induced angiogenesis) inhibitory activity of epigallocatechin gallate (EGCG), one of the compounds present in R. kirilowii extracts [10, 21]. Many questions remain unanswered and a lot of experimental studies should yet be done before recommending selected Rhodiola extracts or some of their active compounds as safe and effective antiangiogenic agents.

References
1. Folkman J (2002): Role of angiogenesis in tumor growth and metastasis. Seminars in Oncology 29 (S16): 15-18.
2. Skopińska-Różewska E, Malinowski M, Wasiutyński A et al. (2008): The influence of Rhodiola quadrifida 50% hydro-alcoholic extract and salidroside on tumor-induced angiogenesis in mice. Pol J Vet Sci 11: 97-104.
3. Skopińska-Różewska E, Hartwich M, Siwicki A et al. (2008): The influence of Rhodiola rosea extracts and rosavin on cutaneous angiogenesis induced in mice after grafting of syngeneic tumor cells. Centr Eur J Immunol 33: 102-107.
4. Skopińska-Różewska E, Mścisz A, Sommer E et al. (2005):The influence of different Rhodiola extracts on murine tumor cells angiogenic activity. Herba Polonica 51 (Suppl 1): 172.
5. Skopińska-Różewska E, Krotkiewski M, Sommer E et al. (1999): Inhibitory effect of shark liver oil on cutaneous angiogenesis induced in Balb/c mice by syngeneic sarcoma L-1, human urinary bladder and human kidney tumor cells. Oncol Rep 6: 1341-1344.
6. Skopińska-Różewska E, Piazza GA, Sommer E et al. (1998): Inhibition of angiogenesis by Sulindac and its sulfone metabolite (FGN-1): A potential mechanism for their antineoplastic properties. Int J Tissue Reactions 20: 85-89.
7. Rogala E, Skopińska-Różewska E, Wasiutyński A et al. (2008): Echinacea purpurea diminishes neovascular reaction induced in mice skin by human cancer cells and stimulates non-specific cellular immunity in humans. Centr Eur J Immunol 33: 127-130.
8. Skopińska-Różewska E, Janik P, Przybyszewska M et al. (1998): Inhibitory effect of theobromine on induction of angiogenesis and VEGF mRNA expression in v-raf transfectants of human urothelial cells HCV-29. Int J Mol Med 2: 649-652.
9. Bałan BJ, Skopińska-Różewska E, Barcz E et al. (1999): The effect of selected phenolic acids on angiogenic activity of ovarian cancer cells-preliminary report. Onkol Pol 2: 203-208.
10. Skopiński P, Szaflik J, Duda-Król B et al. (2004): Suppression of angiogenic activity of sera from diabetic patients with non-proliferative retinopathy by compounds of herbal origin and sulindac sulfone. Int J Mol Med 14: 707-711.
11. Skopiński P, Rogala E, Duda-król B et al. (2005): Increased IL-18 content and angiogenic activity of sera from diabetic (type 2) patients with background retinopathy. J Diabetes Complications 19: 335-338.
12. Skopiński P, Skopińska-Różewska E, Sommer E et al. (2005): The effect of some diet-derived angiogenesis inhibitors and sulindac sulfone on the ability of VEGF, bFGF and IL-18 to induce cutaneous neo-vascular response in mice. Pol J Environ Studies 14 (SII, partI): 325-329.
13. Jung L, Chorostowska-Wynimko J, Skopińska-Różewska E et al. (2008): The effect of a single dose of enoxaparine on the angiogenic potential of human serum and plasma. Centr Eur J Immunol 33: 50-53.
14. Mielcarek S, Mścisz A, Buchwald W et al. (2005): Phytochemical investigation of Rhodiola sp.root extracts.Herba Polonica 51(Suppl 1): 159.
15. Siwicki AK, Skopińska-Różewska E, Hartwich M et al. (2007): The influence of Rhodiola rosea extracts on non-specific and specific cellular immunity in pigs, rats and mice. Centr Eur J Immunol 32: 84-91.
16. Skopińska-Różewska E, Skurzak H, Wasiutyński A (2007): Sarcoma L-1 in mice as a model for the study of experimental angiogenesis. Centr Eur J Immunol 32: 77-83.
17. Auerbach R, Kubai L, Sidky Y (1976): Angiogenesis induction by tumors, embryonic tissues and lymphocytes. Cancer Res 36: 3435-3440.
18. Skopińska-Różewska E, Sommer E, Demkow U et al. (1997): Screening of angiogenesis inhibitors by modified tumor-induced angiogenesis (TIA) test in lung cancer. Ann Acad Med Bialostocensis 42: 287-296.
19. Wiedenfeld H, Dumaa M, Malinowski M et al. (2007): Phytochemical and analytical studies of extracts from Rhodiola rosea and Rhodiola quadrifida. Die Pharmazie 4: 308-311.
20. Wiedenfeld H, Zych M, Buchwald W, Furmanowa M (2007): New compounds from Rhodiola kirilowii. Scientia Pharmaceutica 75: 29-34.
21. Skopiński P, Skopińska-Różewska E, Sommer E et al. ((2004): Inhibitory effect of complement inactivation and epigallocatechin gallate on the human serum ability to induce cutaneous neovascular reaction in mice. Bull Vet Inst Pulawy 48: 485-487.
22. Folkman J (2002): Role of angiogenesis in tumor growth and metastasis. Seminars in Oncology 29 (S16): 15-18.
23. Skopińska-Różewska E, Malinowski M, Wasiutyński A et al. (2008): The influence of Rhodiola quadrifida 50% hydro-alcoholic extract and salidroside on tumor-induced angiogenesis in mice. Pol J Vet Sci 11: 97-104.
24. Skopińska-Różewska E, Hartwich M, Siwicki A et al. (2008): The influence of Rhodiola rosea extracts and rosavin on cutaneous angiogenesis induced in mice after grafting of syngeneic tumor cells. Centr Eur J Immunol 33: 102-107.
25. Skopińska-Różewska E, Mścisz A, Sommer E et al. (2005): The influence of different Rhodiola extracts on murine tumor cells angiogenic activity. Herba Polonica 51 (Suppl 1): 172.
26. Skopińska-Różewska E, Krotkiewski M, Sommer E et al. (1999): Inhibitory effect of shark liver oil on cutaneous angiogenesis induced in Balb/c mice by syngeneic sarcoma L-1, human urinary bladder and human kidney tumor cells. Oncol Rep 6: 1341-1344.
27. Skopińska-Różewska E, Piazza GA, Sommer E et al. (1998): Inhibition of angiogenesis by Sulindac and its sulfone metabolite (FGN-1): A potential mechanism for their antineoplastic properties. Int J Tissue Reactions 20: 85-89.
28. Rogala E, Skopińska-Różewska E, Wasiutyński A et al. (2008):Echinacea purpurea diminishes neovascular reaction induced in mice skin by human cancer cells and stimulates non-specific cellular immunity in humans. Centr Eur J Immunol 33: 127-130.
29. Skopińska-Różewska E, Janik P, Przybyszewska M et al. (1998): Inhibitory effect of theobromine on induction of angiogenesis and VEGF mRNA expression in v-raf transfectants of human urothelial cells HCV-29. Int J Mol Med 2: 649-652.
30. Bałan BJ, Skopińska-Różewska E, Barcz E et al. (1999): The effect of selected phenolic acids on angiogenic activity of ovarian cancer cells – preliminary report. Onkol Pol 2: 203-208.
31. Skopiński P, Szaflik J, Duda-Krół B et al. (2004): Suppression of angiogenic activity of sera from diabetic patients with non-proliferative retinopathy by compounds of herbal origin and sulindac sulfone. Int J Mol Med 14: 707-711.
32. Skopiński P, Rogala E, Duda-król B et al. (2005): Increased IL-18 content and angiogenic activity of sera from diabetic (type 2) patients with background retinopathy. J Diabetes Complications 19: 335-338.
33. Skopiński P, Skopińska-Różewska E, Sommer E et al. (2005): The effect of some diet-derived angiogenesis inhibitors and sulindac sulfone on the ability of VEGF, bFGF and IL-18 to induce cutaneous neo-vascular response in mice. Pol J Environ Studies 14 (SII, part I): 325-329.
34. Jung L, Chorostowska-Wynimko J, Skopińska-Różewska E et al. (2008): The effect of a single dose of enoxaparine on the angiogenic potential of human serum and plasma. Centr Eur J Immunol 33: 50-53.
35. Mielcarek S, Mścisz A, Buchwald W et al. (2005): Phytochemical investigation of Rhodiola sp.root extracts. Herba Polonica 51 (Suppl 1): 159.
36. Siwicki AK, Skopińska-Różewska E, Hartwich M et al. (2007): The influence of Rhodiola rosea extracts on non-specific and specific cellular immunity in pigs, rats and mice. Centr Eur J Immunol 32: 84-91.
37. Skopińska-Różewska E, Skurzak H, Wasiutyński A (2007): Sarcoma L-1 in mice as a model for the study of experimental angiogenesis. Centr Eur J Immunol 32: 77-83.
38. Auerbach R, Kubai L, Sidky Y (1976): Angiogenesis induction by tumors, embryonic tissues and lymphocytes. Cancer Res 36: 3435-3440.
39. Skopińska-Różewska E, Sommer E, Demkow U et al. (1997): Screening of angiogenesis inhibitors by modified tumor-induced angiogenesis (TIA) test in lung cancer. Ann Acad Med Bialostocensis 42: 287-296.
40. Wiedenfeld H, Dumaa M, Malinowski M et al. (2007): Phytochemical and analytical studies of extracts from Rhodiola rosea and Rhodiola quadrifida. Die Pharmazie 4: 308-311.
41. Wiedenfeld H, Zych M, Buchwald W, Furmanowa M (2007): New compounds from Rhodiola kirilowii. Scientia Pharmaceutica 75: 29-34.
42. Skopiński P, Skopińska-Różewska E, Sommer E et al. (2004): Inhibitory effect of complement inactivation and epigallocatechin gallate on the human serum ability to induce cutaneous neovascular reaction in mice. Bull Vet Inst Pulawy 48: 485-487.