Cytotoxicity of Two Triterpenoids from Nigella Glandulifera

During an investigation of antitumor substances from Nigella glandulifera Freyn et Sint. (Ranunculaceae) the cytotoxicity of two oleanane triterpene saponins isolated from the seeds of this species, kalopanaxsaponins A and I, was evaluated against HepG2, drug resistant HepG2 (R-HepG2) (two hepatocyte cell lines) and primary cultured normal mouse hepatocytes. Evident cytotoxic activities were observed. Morphological observations and cell cycle analysis suggest that these compounds inhibit the proliferation of hepatoma by inducing apoptosis and consequently kalopanaxsaponins A and I may be potential therapeutic agents for the treatment of parental and drug resistant hepatoma.


Introduction
The genus Nigella (Ranunculaceae) consists of about 20 species, three of which -Nigella glandulifera Freyn et Sint, Nigella sativa and Nigella damascena L. are used in traditional medicine.Their seeds, commonly added in many food preparations of the Uyghur ethnic group, possess diuretic, analgesic, spasmolytic, galactagogue and bronchodilator effects and have been used for a long time in the treatment of oedema, urinary calculus and bronchial asthma.Flavone glycosides, triterpenoids and alkaloids are the main constituents in the Nigella genus [1][2][3][4][5].Nigella glandulifera Freyn et Sint. is an erect annual herbaceous plant widely distributed in the west of China.The whole herb has been used as a folk remedy for treatment of colds, cough and insomnia.Previous phytochemical study on the seeds of this plant revealed the presence of several compounds, including a new alkaloid, nigeglanine [6], which is only the third reported naturally occurring product with an indazole ring after nigelicine and nigellidine [7,8].1) are important oleanane triterpene saponins previously found in Kalopanax pictus [9,10].Their isolation for the first time from the seeds of Nigella glandulifera has been described recently [11].Compounds 1 and 2 have been reported to display cytotoxicity in J82, T24, Colon26 and 3LL cancer cells, with IC 50 values ranging from 1.1-16.5 µM [12].In addition, kalopanaxsaponin A significantly reduced the LL/2 tumor weight of mice at a i.p dose of 5-10 mg and apparently prolonged the life span of mice bearing Colon 26 and 3LL Lewis lung carcinomas at an i.p dose of 15 mg [2,12].However, there are no reports on their activities towards drug resistant tumor and normal cells and the underlying mechanisms of cytotoxic activity of these two saponins remains unclear.
In the present study, the cytotoxicity of kalopanaxsaponins A and I was evaluated on HepG2, drug resistant HepG2 (R-HepG2) (two hepatocyte cell lines) and primary cultured normal mouse hepatocytes, in order to explore their potential in parental and drug resistant hepatoma therapy.

Results and Discussion
Multi-drug resistance is one of the most serious limitations in chemotherapy and always leads to cancer treatment failure.Meanwhile, lack of selectivity of the active ingredient(s) usually contributes to the many observed side effects.Consequently, there is an urgent need to find new chemical agents that can differentiate between normal and cancerous cells, in order to selectively kill the cancerous cells and drug resistant tumors with reduced toxicity [13].Therefore, two hepatocyte cell lines (HepG2, R-HepG2) and the primary cultured normal mouse hepatocytes were used to test the cytotoxicicity, drug resistance and selectivity of kalopanaxsaponins A and I. Betulinic acid, a selective cytotoxic triterpenoid acid, was used as positive control [14].Based on our results, kalopanaxsaponins A and I showed significant cytotoxic activities towards all two cell lines and primary cultured normal mouse hepatocytes in a concentration-dependent manner and the IC 50 values were derived from the dose effect curves (Table 1).The cytotoxicities of kalopanaxsaponins A and I on R-HepG2 were as effective as on HepG2, but at the same time, the IC 50 values towards normal mouse hepatocytes were very low.This indicated that kalopanaxsaponins A and I were effective against the drug resistant HepG2 cell line, but had no selective cytotoxicity towards cancer cells.Cell death can occur by either of two distinct mechanisms: necrosis ("accidental" cell death) and apoptosis ("programmed" cell death) [15,16].Necrosis causes severe inflammation, while apoptosis does not.Most of the cytotoxic antitumor drugs in current use have been shown to induce apoptosis in susceptible cells.Harmlessly disposing of cancer cells is one of the main considerations in chemotherapy [17] and consequently induction of apoptosis is an important strategy in anticancer drug research and development.In our study, treatment with kalopanaxsaponins A and I at concentrations of one third of their IC 50 (6 and 3 µM, respectively) for 24 h resulted in significant morphological changes in HepG2 cells (Figure 2), and the changes induced by kalopanaxsaponins A and I were very similar.After treatment with kalopanaxsaponins A and I, chromatin aggregation, nuclear and cytoplasmic condensation and partition of cytoplasm and nucleus into membrane bound-vesicles (apoptotic bodies) occurred in the cells.
Many anticancer agents and DNA-damaging agents arrest the cell cycle at the G 0 /G 1 , S, or G 2 /M phase [18,19].After exposure to the tested compound at concentration of one third of their IC 50 (6 and 3 µM) respectively for 24 h, the distinct sub-G 1 peak which is recognized as apoptotic fraction was observed on the HepG2 cells treated with kalopanaxsaponins A and I, but no significant change of cell cycle progression was detected (Figure 3).The apoptosis fraction induced by kalopanaxsaponins A and I was 13.61% and 20.81% respectively.While, the apoptosis triggered by positive control betulinic acid was 9.12%.This result confirmed the apoptosis observed in morphological observation.Experimental General

Cell culture and drug treatment
HepG2 (ATCC) cells were maintained in RPMI 1640 (Gibco) containing 10% FBS (Gibco), sodium bicarbonate (2 mg/mL,) penicillin sodium salt (100 µg/mL) and streptomycin sulfate (100 µg/mL).R-HepG2 (City University of Hong Kong) cells were maintained in the presence of 1.2 µM doxorubicin (Sigma).Cells were grown to 60% confluence, trypsinized with 0.25% trypsin/2 mM EDTA, and plated for experimental use.In all experiments, cells were grown in RPMI-1640 medium with 10% FBS for 24 h prior to treatment.Mouse hepatocytes were isolated from normal Kunming mice (Experimental Animal Center of Zhongshan Medical University) by the enzymatic perfusion technique as we have described previously [20].The viability of mouse hepatocytes was about 80% tested with Trypan blue.Compounds were dissolved in DMSO at a concentration of 100mM, and diluted in tissue culture medium before use.

Cytotoxicity assay
Cells were seeded in 96 well tissue culture plates and treated with the saponins or vehicle (0.1% DMSO) at various concentrations and incubated for 48 h followed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay [21].Betulinic acid was used as positive control.Briefly, IC 50 values for the two saponins and betulinic acid on different cell lines and the primary cultured normal mouse hepatocytes were obtained from the concentration-effect curves.

Morphological observations
HepG2 cells were cultured in 3.5 cm dishes.The tested compounds were added to the medium at a concentration of one third of their IC 50 values (6 and 3 µM, respectively).A vehicle treated sample and betulinic acid were used as negative and positive controls separately.After treatment, all the cultures were incubated at 37 o C, 5% CO 2 for 24 h.Photographs were taken under an inverted Leica fluorescence 40×10 microscope after AO (acridine orange)/EB (ethidium bromide) staining [22].

Flow cytometry analysis
Flow cytometry was used to elucidate the cytotoxicity of the two saponins.HepG2 cells were treated with the compounds at concentration of one third of their IC 50 values (6 and 3 µM, respectively) for 24 h.A vehicle (0.1% DMSO) treated sample and betulinic acid were used as negative and positive controls respectively.Cells were collected and fixed in 70% cold (-20°C) ethanol overnight.After washing twice with PBS, cells were resuspended in PBS containing 1% FCS (vertex).RNA in the fixed cells was digested using RNase A (0.5 mg/mL) at 37 o C for 1 h.Finally, the cells were stained by propidium iodide (PI, 2.5 µg/mL).The DNA content of cells was then analyzed with a FACSCalibur instrument (Becton-Dickinson).

Table 1 .
IC 50 values of kalopanaxsaponins A and I on different cell lines and normal hepatocytes a Data shown represent the mean±SD of three independent experiments.