Introduction

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Although interest in natural products as a source of innovation in drug discovery has decreased in the last few decades, therapeutic agents from microbial or plant origin account for more than 30% of the current worldwide human drug sales. For anticancer and anti-infective treatments, even 60% of the approved drugs and of the new drug candidates (excluding biologics) are of natural origin for the period 1989 to 1995 (Cragg et al., 1997).

Isostrychnopentamine (ISP) (Fig. 1) is an asymmetrical monoterpenoid bisindole alkaloid possessing an atypical structure with five nitrogen atoms. This alkaloid was isolated for the first time in our laboratory more than 20 years ago from the leaves of the plant Strychnos usambarensis Gilg (Angenot et al., 1978). S. usambarensis is a little tree or shrub distributed in almost all tropical Africa, from the Ivory Coast to South Africa. ISP is extracted [yield 0.2% (w/w)] from the leaves of the plant, which makes the supply of the raw material relatively easily obtained, if need be. This Strychnos was mainly known for its toxicity, due to quaternary curarizing alkaloids (Angenot, 1971; Angenot et al., 1975). Nevertheless, it is known that it contains also tertiary alkaloids, which possess cytotoxic activities against mammalian cells (Bassleer et al., 1982; Tits et al., 1984) and antiprotozoal activities (Wright et al., 1991; Frédérich et al., 1999).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Structure of ISP, an alkaloid from S. usambarensis.

Our interest in isostrychnopentamine stemmed from the previous studies conducted on its stereoisomer strychnopentamine. We have showed that strychnopentamine inhibits RNA synthesis and induces cytological modifications, such as lamellar bodies, vacuoles, and blebs in the cytoplasm of B16 mouse melanoma cells (Bonjean et al., 1996). Strychnopentamine, along with isostrychnopentamine and usambarensine (a closely related alkaloid from the roots of S. usambarensis) were then submitted to the National Cancer Institute for an in vitro screen of 60 cell lines (V. L. Narayanan, National Cancer Institute, unpublished data). Because ISP displayed an interesting activity pattern, we decided to investigate how ISP induced cell death in HCT-116 colon cancer cells.

In the present study, we report that ISP treatment of colon cancer cells induces G₂ cell cycle arrest and apoptosis through the intrinsic pathway. These findings provide for the first instance a role of ISP in cancer cell-induced apoptosis.

	Materials and Methods

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Drugs, Chemicals, and Plant Material. The leaves of S. usambarensis were collected by Professor F. Sandberg (University of Uppsala, Uppsala, Sweden) in Tanzania. Voucher specimens of the plant were deposited in the herbarium of the National Botanical Garden of Belgium (Meise, Belgium) and in the herbarium of the Pharmaceutical Institute (Liège, Belgium) (voucher no. Leeuwenberg 10826). ISP was isolated from S. usambarensis as described previously (Tavernier et al., 1987; Frédérich et al., 1999). Stock solutions (1 mM) were prepared with 5% dimethyl sulfoxide. Daunomycine-hydrochloride (Rhône-Poulenc-Rorer, Paris, France) and camptothecin and etoposide (Sigma Chemical, Bornem, Belgium) were stored in stock solutions, protected from light.

Cell Culture. Cell lines were obtained from American Type Culture Collection (Manassas, VA) and have been described previously (Hellin et al., 1998, 2000). HCT-116 and HCT-15 human colon carcinoma cells were grown in McCoy's 5A and RPMI 1640 medium supplemented with 1% L-glutamine (200 mM), 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (Invitrogen, Carlsbad, CA).

In Vitro Cytotoxicity Assay. This assay was performed as described previously (Bentires-Alj et al., 1999). Briefly, cells were seeded at the concentration of 7 × 10³ cells/well on 96-well flat-bottom microplates in 0.2 ml of medium. After 48 h of incubation with the drug, cell viability was measured by a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 (Roche Diagnostics, Basel, Switzerland) by mitochondrial dehydrogenases. Relative absorbencies were expressed as percentage of the respective controls (100% in ordinate). Means ± standard errors were calculated. IC₅₀ values were calculated from graphs.

DAPI/PI Staining. Cells were seeded at low density (4 × 10⁴ cells/well) on glass chamber slides (NUNC A/S, Roskilde, Denmark) and incubated for 24 h. The cells were then treated with or without 10 µM ISP. After 12 h, cells were washed twice with phosphate-buffered saline (PBS) and fixed with 3.7% paraformaldehyde for 15 min. Fixed cells were rinsed with PBS and then stained with DAPI/PI (1 and 0.6 µl/ml in PBS) for 20 min at room temperature in the dark. Finally, cells were washed three times with PBS and analyzed under a fluorescence microscope.

Annexin V-FLUOS Test. HCT-116 cells (10⁶) treated or not with 15 µM ISP for 14 or 28 h were gently scraped and pooled with medium floating cells. Cells were individualized in 10 mM PBS-EDTA and stained as described by the manufacturer (Roche Diagnostics). Briefly, cell pellet was resuspended in 500 µl of 1/50 Annexin V-HEPES solution (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 5 mM CaCl₂) and incubated on ice for 30 min in the dark.

Cell Cycle Analysis. DNA content was measured as described by the manufacturer (Cycle Test Plus DNA reagent kit; BD Biosciences, San Jose, CA).

FACS Analysis. Cells (for Annexin V test and cell cycle analysis) were analyzed with a FACStar Plus (BD Biosciences) with a 100-mW air-cooled argon laser (Spinnaker 1161; Spectra Physics, Mountain View, CA) and the CellQuest software (Macintosh, Facstation; BD Biosciences).

Protein Extraction and Western Blotting. To obtain whole cell protein extract, control or ISP-treated cells were treated as described previously (Hellin et al., 1998). Then, 10 µg of total proteins was resolved on a 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Roche Diagnostics). The membrane was blocked overnight with Tris-buffered saline-Tween buffer plus 5% dry milk, incubated 1 h with the primary antibody, washed with Tris-buffered saline-Tween buffer, and then incubated with secondary antibody conjugated with peroxidase (1:10,000; DAKO, Glostrup, Denmark). The signal was detected using a chemiluminescent detection system (ECL kit; Amersham Biosciences AB, Uppsala, Sweden). The used antibodies were as follows: anti-PARP 85/116-kDa antibody (1:200; BD PharMingen, San Diego, CA); anti-p53 antibody (1:4000; Oncogene Research, San Diego, CA); anti-p21^WAF1 antibody (1:100, Oncogene Research), anti-cytochrome-c antibody (1:500; BD PharMingen); anti--actin antibody (1:500; Sigma Biosciences, Bornem, Belgium); anti-caspase-9 antibody (1:2000, BD PharMingen); and anti-caspase-8 antibody (1:600; BD PharMingen).

Cytoplasmic Nonmitochondrial Extracts. HCT-116 cells were harvested as described previously (Yang et al., 1997). Briefly, cells were centrifuged at 600g for 10 min at 4°C. The cell pellets were washed once with ice-cold PBS and resuspended in 5 volumes of buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCI, 1.5 mM MgCl₂, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride) containing 250 mM sucrose. The cells were homogenized with 12 strokes of a Teflon homogenizer, and the homogenates were centrifuged twice at 750g for 10 min at 4°C. The supernatants were centrifuged at 100,000g for 1 h at 4°C, and the resulting supernatants were divided into samples (eventually frozen at 80°C) and used for cytochrome c detection by Western blotting.

Caspase-3 Activity Assay. Caspase-3 activity was measured using the specific Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarine (Ac-DEVD) caspase-3 substrate from Calbiochem (San Diego, CA). Exponentially growing HCT-116 cells (2 × 10⁶) were incubated with ISP for various lengths of time. Extracts were prepared as described by the manufacturer. Proteins (50 µg) from this extract were then incubated with 150 µl of lysis buffer (Tris pH 8, 20 mM; NaCl, 137 mM; 10% glycerol, and 1% Nonidet P-40) with 20 mM caspase-3 substrate for 4 h at 37°C. The formation of a florigenic product was then measured at 460 nm (excitation wavelength, 355 nm) using SoftMAX Pro software and a SpectraMAX spectrofluorometer from Molecular Devices Corp. (Sunnyvale, CA).

DNA Laddering Assay. After treatment with ISP, HCT-116 cells were washed once in PBS, trypsinized, pooled with cells recovered from the supernatant, and centrifuged at 5000g for 2 min. Cells were washed again in PBS and treated with two cycles of lysis buffer (1% Nonidet P-40 in 20 mm EDTA, 50 mm Tris-HCI, pH 7.5, 10 p1/10⁶ cells, minimum 50 µl). After centrifugation for 5 min at 1600g, the supernatant was collected, treated with RNase A for 120 min at 56°C, brought to 1% (w/v) SDS, and finally digested with proteinase K (final concentration, 2.5 µg/µl) for at least 120 min at 37°C. DNA was then precipitated and analyzed by gel electrophoresis on 1% agarose gel.

Topoisomerase I and II DNA Cleavage Reaction. Native supercoiled pKMp27 DNA (0.5 µg) (lane DNA) was incubated for 30 min at 37°C with 4 units of human topoisomerase in the absence or presence of ISP at various concentrations (micromolar). Reaction was stopped with sodium dodecylsulfate and treatment with proteinase K. DNA samples were separated by electrophoresis on agarose gels containing ethidium bromide. After electrophoresis, the gels were washed with water and photographed under UV light. Etoposide and camptothecine were used as positive controls for topoisomerase I and II assays, respectively.

	Results

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Evaluation of ISP Cytotoxicity on Cancer Cells. Compared with other structurally related alkaloids such as strychnopentamine and usambarensine, ISP possessed a higher toxicity against colon and breast cancer cell lines (National Cancer Institute's in vitro 60-cell lines antitumor screen, unpublished data). We investigated then the effect of ISP on colon cancer HCT-116 and HCT-15 cells (human colon carcinoma cells containing a mutated p53 gene) by the WST-1 assay (Fig. 2). ISP promoted cell death in a dose-dependent manner in the two cell lines (IC₅₀ = 7 and 15 µM for HCT-116 and HCT-15 cell lines, respectively).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effect of ISP on HCT-116 and HCT-15 cell growth and viability. One day after seeding, cells were treated with increasing concentrations of ISP for 48 h. The number of cells was determined by WST-1 assay. Each value represents the mean of three measures ± S.D.

ISP Induces Apoptosis in HCT-116 Cells. Cells exposed to 10 µM ISP for 12 to 24 h shrank, rounded up, and detached from the dishes (data not shown), suggesting that they underwent apoptosis. To confirm this hypothesis, we used different methodological approaches. We analyzed the fluorescence of the nuclei of cells stained with the DNA-specific dye DAPI after 24 h of treatment. In untreated cells, we observed normal nuclei staining (Fig. 3B). In contrast, ISP-treated cells (10 µM) displayed typical condensed chromatin and fragmented nuclei (blebbing) (Fig. 3A). DNA fragmentation was further examined by the classical DNA laddering on agarose gel. Formation of internucleosomal fragments was observed after 24-h exposition of HCT-116 cells to 5 and 10 µM ISP (Fig. 4).

View larger version (87K): [in this window] [in a new window]   Fig. 3.   ISP induces condensation of nuclear chromatin and blebbing of the nuclei. One day after seeding, HCT-116 cells were treated with 0 or 10 µM ISP for 24 h, followed by fixation and staining with DAPI/PI as described under Materials and Methods. A, treated cells. B, control cells. The white column (B) represents 20 µM.

View larger version (49K): [in this window] [in a new window]   Fig. 4.   DNA fragmentation after exposure to ISP. HCT-116 cells were exposed to 5 or 10 µM ISP for 24 h. Genomic DNA was extracted as described under Materials and Methods, electrophoresed in a 1.5% agarose gel, and visualized with ethidium bromide staining.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Determination of necrotic versus apoptotic cells in HCT-116 ISP-treated cells. Cells were incubated with 0 or 15 µM ISP for various times and then stained with Annexin V-FLUOS and propidium iodide for FACS analysis (Annexin V-FLUOS in ordinate and PI in abscissa). Control (nontreated) cells (A) and cells incubated with 15 µM ISP for 14 h (B) or 28 h (C). Bottom left, living cells. Top left, apoptotic cells. Top right, necrotic cells. Bottom right, prenecrotic cells.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Caspase 3 activation. A, fluorometric assay. HCT-116 cells were incubated without or with 15 µM ISP for 6, 14, or 30 h before the addition of the caspase-3 substrate (Ac-DEVD), as described under Materials and Methods. The cell lysate was incubated for 4 h at 37°C and fluorescence was measured at 460 nm (excitation wavelength, 355 nm). B, Western blot was used to detect cleavage of full-length PARP (116-kDa band) into the 89-kDa fragment in untreated cells (control) and cells treated with ISP for 6, 12, or 24 h at 15 µM. Whole cell lysates where subjected to SDS-PAGE (as described under Materials and Methods) followed by blotting with an anti-PARP antibody (BD PharMingen).

Detection of Caspase-8, Caspase-9, and Cytochrome c. To further characterize the apoptotic pathway activated by ISP, extracts from HCT-116 cells were examined by Western blotting using caspase-8- and caspase-9-specific antibodies (Fig. 7). No change of the caspase-8 content was detected in this cell line. In contrast, the amount of caspase-9 protein (48 kDa) decreased after 6 h of exposition to 15 µM ISP, whereas the cleaved active (37-kDa) form of caspase-9 increased significantly. We then examined whether a cytoplasmic release of cytochrome c occurred. Figure 7 showed that cytochrome c was detected in the cytosol after 6- to 12-h exposition to 15 µM ISP. Cytochrome c release was maximum at 12 h and decreased strongly 24 h after ISP treatment. Altogether, the data strongly supported the conclusion that the mitochondrial pathway is used to trigger apoptosis after an ISP exposure.

View larger version (53K): [in this window] [in a new window]   Fig. 7.   Western blot analysis of caspase-8, caspase-9, and cytochrome c proteins in cytoplasmic nonmitochondrial or total extracts from untreated cells (control) and cells treated with 15 µM ISP for 6 to 24 h. The protocol was described under Materials and Methods. Actin blotting is shown to assess that equivalent amounts of proteins were loaded in each line.

Changes in Cell Cycle after Exposition to ISP. HCT-116 cell cycle was examined 3 and 12 h after addition of 15 µM ISP to the culture medium (Fig. 8). Without treatment, most cells (83%) were in G₁ and S phases, due to the high proliferative state of this cell line. After 12 h of exposition to 15 µM ISP, we observed a marked increase in the percentage of cells in the G₂-M phase of the cell cycle. This increase was accompanied by a decrease in the percentage of cells in G₁ phase (from 49 to 32%), which indicated a cell cycle arrest in G₂-M phase.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effect of ISP on HCT-116 cell cycle. One day after seeding, cells were untreated (A) or treated with 15 µM ISP for 3 h (B) or 15 µM ISP for 12 h (C). Distribution of the cells in G0-G1, S, and G2-M phases was analyzed by flow cytometry as described under Materials and Methods.

Role of p53 and p21 Proteins in Cell Cycle Arrest and Apoptosis. Extracts from HCT-116 cells after ISP treatment at various concentrations were examined by Western blotting using p53- and p21-specific antibodies. p53 expression was unchanged in the variously ISP-treated HCT-116 cells (Fig. 9A). ISP until 50 µM did not induce any p53 recruitment (data not shown). In contrast, an increase in the p21 protein content occurred after treatment with 10 or 15 µM ISP (Fig. 9A). To confirm that ISP can induce p21 in a p53-independent manner, we examined p21 protein content in HCT-15 cells after treatment by ISP. HCT-15 cells are human colon carcinoma cells where p53 gene is deleted. Figure 9B show that p21 protein content was raised in HCT-15 cells after treatment with 10 to 15 µM ISP for 12 to 24 h. Apoptosis was also confirmed in these HCT-15 cells by observation of PARP cleavage after treatment with 10 to 15 µM ISP during 24 h (data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 9.   A, Western blot analysis of p53 and p21-WAF1 proteins in HCT-116 cells, untreated (control) and treated with ISP for 12 or 24 h at 10, 15, or 20 µM. Daunomycine treatment at 1 µM (for 24 h) was used as positive control. Whole cell lysates where subjected to SDS-PAGE (as described under Materials and Methods) followed by blotting with an anti-p53, anti-actin, or anti-p21 antibody. Actin blotting is shown to assess that equivalent amounts of proteins were loaded in each line. B, Western blot analysis of p21-WAF1 proteins in HCT-15 cells, untreated (control) and treated with ISP for 12 or 24 h at 10 or 15 µM. Whole cell lysates where subjected to SDS-PAGE (as described under Materials and Methods) followed by blotting with an anti-p21 or anti-actin antibody. Actin blotting is shown to assess that equivalent amounts of proteins were loaded in each line.

ISP Treatment Does Not Inhibit Topoisomerases I and II. The effect of ISP on the catalytic activity of human topoisomerases I and II was investigated using a conventional plasmid DNA relaxation assay (Bailly, 2001). ISP did not affect the electrophoretic mobility of the DNA in the presence of either topoisomerase I or II, even at a high concentration of 100 µM. The antitumor drugs camptothecin and etoposide, used as positive controls in the same experiment, strongly promoted permanent single- and double-stranded DNA cleavages due to topoisomerases I and II inhibition (data not shown). ISP is therefore not a topoisomerase poison.

	Discussion

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Many naturally occurring indolomonoterpenic alkaloids (camptothecin, ellipticine, vincristine) are or were used as anticancer agents. Their chemotherapeutic effect is mediated by different biochemical modes of action, such as inhibition of topoisomerase I and II and inhibition of microtubules formation. Herein, we investigated the mechanisms responsible for the cell death-promoting activity of isostrychnopentamine, a Strychnos alkaloid whose derivatives usambarensine and strychnopentamine were known for their cytotoxicity (Bonjean et al., 1996). Our results clearly showed for the first time that ISP (15 µM) induced cell cycle arrest and subsequent apoptotic death in the human colon cancer cell line HCT-116. Necrotic or lytic effects were excluded by the lack of plasma membrane permeability as assessed by the absence of PI uptake.

We clearly observed many of the typical structural and ultrastructural modifications that happen during the apoptotic process, including translocation of phosphatidylserine to the outer layer of the plasma membrane, condensation of nuclear chromatin, internucleosomal DNA fragmentation, caspase-3 activation, and PARP cleavage. Further investigations of the apoptotic process showed that the effector caspases were activated by the mitochondrial pathway, caspase-9 activation, and cytochrome c release from mitochondria.

The weak difference of ISP cytoxicity on HCT-15 and HCT-116 cells could be linked to the mutation of p53 in HCT-15 cells. However, we showed that triggering of apoptosis in HCT-116 cells did not require p53. Multidrug resistance in HCT-15 cells (M. Bentires-Alj, unpublished data) could be an alternate explanation. Further experiment should be done to address whether the multidrug resistance expression reduces ISP cytotoxic effect.

Camptothecin (and its hemisynthetic analogs topotecan and irinotecan), ellipticine, vincristine, and vinblastine are antitumor agents that are ISP biosynthetically related alkaloids. However, in contrast to the indolomonoterpenic alkaloids camptothecin and ellipticin, ISP inhibited neither topoisomerases I or II. On the other hand, disruption of microtublule structure by vincristine or vinblastine results in induction of tumor suppressor gene p53 and in phosphorylation of Bcl-2, leading to apoptosis (Wang et al., 1999). Here, we clearly showed that ISP-induced HCT-116 cell death was not associated with a change in p53 expression. ISP cytotoxicity is thus probably mediated by a different biochemical mechanism.

The protein p21 is an inducer of cell cycle arrest by inhibition of cyclin-dependent kinases, which regulate transitions between different phases of the cell cycle. The expression of p21 is increased in response to a variety of stressfull stimuli, including DNA-damaging drugs (such as daunomycin), ionizing radiation, and agents affecting mitosis (such as paclitaxel or vincristine). The response to these factors are mediated primarily through transcriptional activation of the p21-WAF1 gene by p53 (for review, see Roninson, 2002). Nevertheless, p21 gene expression can uncommonly be regulated by p53-independent mechanisms in response to ultraviolet c (Haapajarvi et al., 1999), reactive oxygen species (Qiu et al., 1996), growth factors (Michieli et al., 1994; Datto et al., 1995), and alkylphospholipids (Patel et al., 2002), or by some natural drugs such as the isoflavone genistein (Choi et al., 2000) or indole-3-carbinol (Chinni et al., 2001). p53-independent p21 induction is probably regulated in part at the level of transcription, through various cis-regulatory sites in the p21 promoter, and in part at the level of mRNA and protein stability (Gartel and Tyner, 1999). Here, we have demonstrated that the ISP induction of p21 was independent from p53, both in wild-type p53 HCT-116 and in p53-deleted HCT-15 colon cancer cells. ISP might be used as a tool to help to understand the mechanisms regulating p53-independent inductions of p21.

The resistance of cancer cells to classical chemotherapy may be due in part to the high frequency of mutation in p53 that impairs p53-dependent apoptosis. Most of the common anticancer agents such as doxorubicine, etoposide, paclitaxel, vincristine, or 5-fluorouracil induce apoptosis via a p53-dependent pathway (Lowe et al., 1993; Mesner et al., 1997). The requirement of p53 tumor suppressor gene for activation of apoptosis by these agents provides an attractive explanation for their poor efficacy on p53 mutant tumors. Thus, chemotherapeutic agents such as ISP, which induce p21-dependent cell cycle arrest and apoptosis independently from the p53 apoptotic pathway are of a major interest. It is now important to investigate the molecular targets and effectors, which are affected by ISP to trigger programmed cell death.