Saponins-Mediated Potentiation of Cisplatin Accumulation and Cytotoxicity in Human Colon Cancer Cells

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The triterpene saponins jenisseensosides A, B, C, D were found to increase the accumulation and cytotoxicity of the anticancer agent cisplatin in human colon tumor cells. These compounds are glycosides of quillaic acid whose fucose residue was acylated by a trans- or cis-p methoxycinnamic acid. In contrarst, other saponins derivatives without this acyl moiety were not found to potentiate the accumulation and cytotoxicity of cisplatin. These results suggested the importance of the acyl moiety for activity.

Cisplatin is an important anticancer drug for the treatment of many solid tumors but its efficiency is low in human colon cancer [1]. Decreased transport of cisplatin across the cell membrane plays an important role in the development of resistance to this agent. The use of drugs that influence membrane permeability and which influence transmembrane drug transport could be a useful method to circumvent the natural resistance of human colon cancer cells to cisplatin. Amphotericin B, a macrolide polyene antifungal antibiotic, has been shown to increase cisplatin accumulation and cytotoxicity on resistant HT29 human colon cancer cells [1]. It was thought to bind to membrane sterols and to form aqueous membrane pores that increase cell permeability to ions and to anticancer drugs. In this paper we describe the search of other compounds which could potentiate the cytotoxicity of cisplatin in colon cancer cells by interacting with the plasma membrane of cancer cells.[]

Saponins are triterpene or steroid glycosides, possessing various biological and pharmacological activities [2] [3] [4]. If we consider the mechanism of action, most of them are due to the interaction of the aglycone with the sterol of the plasma membrane followed by aggregation of the carbohydrate moieties inducing the rearrangement of the phospholipid bilayer and the pore formation [5]. This provokes a leak of electrolytes and metabolites and increases the permeability of the plasma cell membrane.

Quillaic-acid saponins of Quillaja saponaria and the steroid saponin digitonin of Digitalis purpurea were shown to promote the permeabilization of the plasma membrane in human leukemic H-L 60 cells [6] and in human ovarian carcinoma 2008 cells [7], respectively. In the course of our search for bioactive saponins from Caryophyllaceae, we describe here the effect of quillaic-acid saponins, jenisseensosides A, B, C and D, (1 - 4) (Chart 1) of two Silene species [8], [9] on the accumulation and cytotoxicity of cisplatin in human colon cancer cells.

The cytotoxic effect of the four saponins (1 - 4) and the induced potentiation of cisplatin were evaluated on HT 29 cells in serum free Ham’s F-10 medium using the Cristal Violet colorimetric assay (Fig. [1]). The saponins did not show any significant cytotoxic activity at concentrations up to 15 μg/ml (1, 2) and up to 25 μg/ml (3, 4). Saponin-induced potentiation of cisplatin was evaluated in the presence of 10 μg/ml of cisplatin, a concentration which was only slightly cytotoxic by itself (10 % cytotoxicity). Saponins 1, 2 (in a concentration range of 0 - 15 μg/ml) and to a lesser extent 3, 4 (in a concentration range of 0 - 15 μg/ml) displayed a clear potentiation of cisplatin cytotoxicity of 65 % and 60 % respectively, which was quite similar to that of amphotericin B (Fig. [1]). These results are corroborated by the fact that compounds 1, 2 and to a lessser extent 3, 4 increased the cellular platinum content in HT 29 colon cancer cells when incubation with 10 μg/ml of cisplatin was performed in protein-free medium (Fig. [2]). Furthermore, ethanol and DMSO used for dissolving saponins did not influence the platinum accumulation in HT-29 colon cancer cells (Fig. [2]).

Many other saponins isolated in our laboratory, mostly oleanane-type saponins without acyl substitutions such as 3-O-β-D-glucopyranosyl-oleanolic acid-28-O-β-D-glucopyranoside (5) previously isolated from Achyranthes bidentata [11] (Chart 1, Figs. 1, 2) did not show any accumulation and potentiation of cisplatin cytotoxicity in this asssay [10]. Taken together, these results suggest the importance of the p-methoxycinnamoyl groups in the potentiating effect of the saponins on the cytotoxicity of the antineoplastic drug cisplatin in HT 29 colon cancer cells. These results also corroborated the conclusion recently reported on acylated saponins [12] which suggested that the esterification might be a structural condition for the assumed pore-forming capacity [12]. Further investigations regarding the structure-activity relationship of other natural and hemisynthetic saponins as cisplatin cytotoxicity potentiating agents are currently in progress in order to confirm this hypothesis.

Materials and Methods

The trans- and cis-p-methoxycinnamoyl triterpene-saponins were isolated as inseparable mixtures from the roots of Silene jenisseensis (1, 2) and Silene fortunei (3, 4) and characterized by spectral analyses (IR, FABMS, 2D-NMR such as COSY, HMBC, HMQC) in previous papers [8], [9]. 3-O-β-D-Glucopyranosyl-oleanolic acid-28-O-β-D-glucopyranoside (5) was isolated and characterized from the roots of Achyranthes bidentata as previously described [11]. Each saponin was dissolved in DMSO and then in culture medium and tested at different concentrations. Cisplatin (cis-diammine-dichloroplatinum II, CDDP) was purchased from Roger Bellon Laboratories (Neuilly, France). Amphotericin B (Am B) was purchased from Sigma Aldrich (Ulrich, France). The human colon cancer cell line HT 29 was obtained from the American Tissue Culture Collection (ATCC, Rockville, USA). Cells were grown as monolayers in a controlled atmosphere (37 °C, 5 % CO₂) in Ham’s F-10 medium (Bio Whittaker, Ververs, Belgium) supplemented with 10 % fetal calf serum (Boerhinger Ingelheim, Gagny, France). Culture medium was changed 3 times a week.

Cytotoxicity assay: HT-29 cells (2 × 10⁴ per well) were seeded in 96-well culture plates and cultured for two days before treatment. Saponins were dissolved immediately before use in a mixture of DMSO and absolute ethyl alcohol (1 : 1, V/V), then diluted in serum-free Ham’s F-10 medium. Final concentrations of DMSO and EtOH did not exceed 0.5 % and did not affect cell survival. Cells were treated for 3 h with saponins alone or together with 10 μg/ml cisplatin. After treatment, cells were washed twice with Ham’s F-10 and cultured again for 7 days in drug-free culture medium. Cell survival was measured by the the Cristal Violet colorimetric assay. In brief, cells were rinsed by phosphate buffered saline (PSB), then surviving adherent cells were fixed for 5 minutes by pure ethanol. After drying, cells were stained by Cristal Violet (5 g/l in distilled water). Dye in excess was flushed by tap water. Cell fixed dye was eluted by 33 % acetic acid. Optical density (OD) was measured in each well on automatic spectrophotometer (Anthos Labtec Instruments, Salzburg, Austria). Cell survival was expressed as

OD in 4 treated wells___________________ × 100

OD in 4 control wells

Results were expressed as the percentage of surviving cells compared to control untreated cells. Each point was the mean of four determinations.

Platinum accumulation in cancer cells: HT 29 cells (10⁶ per well) were grown in 6- well culture plates for 72 h and then incubated for 3 h with 10 μg/ml cisplatin and saponins at 10 μg/ml concentration. Cells were washed 2- fold with Ham’s F-10 medium, then detached by a trypsin solution (0.25 μg/ml) in Hank’s Balance Salt solution. After counting and centrifugation, cell pellets were lyophilized and kept at room temperature until assay. After dilution in ultra-pure water, the platinum concentration of samples was measured using a Zeeman atomic absorption spectrometer (SpectrAA-300Z; VARIAN, Les Ulis, France). Values are expressed as mean ± SD of triplicate experiments.

Acknowledgements

This work was supported by the ”Ligue Bourguignonne Contre le Cancer”.

References

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  Search in Google Scholar
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  Search in Google Scholar
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 10 Lacaille-Dubois M A, Wagner H, Correia M, Chauffert B. Biological Screening of Plant Saponins on the Potentiation of the Cisplatin Cytotoxicity in Colon Cancer Cells. International Symposium Antitumour Products from Higher Plants Paris; 8 - 10 January 1998: 124
  Search in Google Scholar
• 11 Marouf A, Desbene S, Khanh T C, Wagner H, Correia M, Chauffert B, Lacaille-Dubois M A. Triterpene saponins from the roots of Achyranthes bidentata .  Pharmaceutical Biology,. 2001;  39 263-7
  CrossrefPubMedSearch in Google Scholar
• 12 Melzig M F, Bader G, Loose R. Investigations of the mechanism of membrane activity of selected triterpenoid saponins.  Planta Medica,. 2001;  67 43-8
  Thieme ConnectPubMedSearch in Google Scholar

Marie Aleth Lacaille-Dubois

Laboratoire de Pharmacognosie

Faculté de Pharmacie

Université de Bourgogne

7, Bd. Jeanne d’ Arc

BP 87900

21079 Dijon Cedex

France

Phone: +33-3-80393229

Fax: +33-3-80393300

Email: malacd@u-bourgogne.fr

References

• 1 Assem M, Bonvalot S, Beltramo J L, Garrido C, Dimanche Boitrel M T, Genne P, Rebibou J M, Caillot D, Chauffert B. Deleterious effect of serum proteins on amphotericin B-induced potentiation of cisplatin in human colon cancer cells.  British Journal of Cancer. 1994;  70 631-5
  PubMedSearch in Google Scholar
• 2 Lacaille-Dubois M A, Wagner H. A review of the biological and pharmacological activities of saponins.  Phytomedicine. 1996;  2 363-86
  PubMedSearch in Google Scholar
• 3 Lacaille-Dubois M A, Wagner H. Bioactive saponins from plants: an update. In: Atta-Ur-Rahman, editor Studies in Natural Products Chemistry, Bioactive Natural Products (Part B). Amsterdam; Elsevier Vol. 21 2000: 633-87
  Search in Google Scholar
• 4 Lacaille-Dubois M A. Biologically and pharmacologically active saponins from plants: recent advances. In: Oleszek W, Marston, A, editors Saponins in Food, Feedstuffs and Medicinal Plants. Dordrecht, Boston, London; Kluwer Academic Publishers 2000: 205-18
  Search in Google Scholar
• 5 Armah C N, Mackie A R, Roy C PK, Osbourn A E, Bowyer P, Ladha S. The membrane permeabilizating effect of avenacin A-1 involves the reorganization of bilayer cholesterol.  Biophysical Journal. 1999;  76 281-90
  PubMedSearch in Google Scholar
• 6 Leng Y M, Ou Y J, Kwan C Y, Loh T T. Specific interaction between tetrandrine and Quillaja saponins in promoting permeabilisation of plasma membrane in human leukemic HL-60 cells.  Biochimica et Biophysica Acta. 1997;  1325 318-28
  PubMedSearch in Google Scholar
• 7 Jekunen A P, Shalinsky D R, Hom D K, Albright K D, Heath D, Howell S. Modulation of cisplatin cytotoxicity by permeabilization of the plasma membrane by digitonin in vitro .  Biochemical Pharmacology. 1993;  45 2079-85
  CrossrefPubMedSearch in Google Scholar
• 8 Lacaille-Dubois M A, Hanquet B, Cui Z H, Lou Z C, Wagner H. Acylated triterpene saponins from Silene jenisseensis .  Phytochemistry. 1995;  40 509-14
  CrossrefPubMedSearch in Google Scholar
• 9 Lacaille-Dubois M A, Hanquet B, Cui Z H, Lou Z C, Wagner H. Jenisseensosides C and D, biologically active acylated triterpene saponins from Silene jenisseensis .  Phytochemistry. 1997;  45 985-90
  CrossrefPubMedSearch in Google Scholar
• 10 Lacaille-Dubois M A, Wagner H, Correia M, Chauffert B. Biological Screening of Plant Saponins on the Potentiation of the Cisplatin Cytotoxicity in Colon Cancer Cells. International Symposium Antitumour Products from Higher Plants Paris; 8 - 10 January 1998: 124
  Search in Google Scholar
• 11 Marouf A, Desbene S, Khanh T C, Wagner H, Correia M, Chauffert B, Lacaille-Dubois M A. Triterpene saponins from the roots of Achyranthes bidentata .  Pharmaceutical Biology,. 2001;  39 263-7
  CrossrefPubMedSearch in Google Scholar
• 12 Melzig M F, Bader G, Loose R. Investigations of the mechanism of membrane activity of selected triterpenoid saponins.  Planta Medica,. 2001;  67 43-8
  Thieme ConnectPubMedSearch in Google Scholar

Marie Aleth Lacaille-Dubois

Laboratoire de Pharmacognosie

Faculté de Pharmacie

Université de Bourgogne

7, Bd. Jeanne d’ Arc

BP 87900

21079 Dijon Cedex

France

Phone: +33-3-80393229

Fax: +33-3-80393300

Email: malacd@u-bourgogne.fr