Elsevier

Toxicology Letters

Volume 216, Issues 2–3, 4 February 2013, Pages 189-199
Toxicology Letters

The cucurbitacins E, D and I: Investigation of their cytotoxicity toward human chondrosarcoma SW 1353 cell line and their biotransformation in man liver

https://doi.org/10.1016/j.toxlet.2012.11.014Get rights and content

Abstract

Cucurbitacins are a class of natural compounds known for their numerous potential pharmacological effects. The purpose of this work was to compare the cytotoxicity of three cucurbitacins I, D, E on the chondrosarcoma SW 1353 cancer cell line and to investigate their biotransformation in man. Cucurbitacins I and D showed a very strong cytotoxicity, which was higher than that of cytochalasin D, used as a drug reference. Almost 100% of the cells were apoptotic as observed by DNA fragmentation (TUNEL assay) after 12 h with cucurbitacins I and D (1 μM) and cucurbitacin E (10 μM). In terms of IC50 values, cucurbitacins I and E presented a higher toxicity compared to that of cucurbitacin D (MTT assay). Cucurbitacin E was readily hydrolyzed by human hepatic microsomes, leading to cucurbitacin I (Km 22 μM, Vmax 571 nmol/mg proteins/min). On the other hand, the three cucurbitacins were hydroxylated at a very low extent, but they were sulfated and glucuronidated. In terms of Vmax/Km, the cucurbitacin E was the best substrate of UDP-glucuronosyltransferases. This study shows that cucurbitacins I, D and E present a potent cytotoxic activity toward the chondrosarcoma SW 1353 cell line and are metabolized as sulfate and glucuronide conjugates.

Highlights

Cucurbitacins I, D and E cytotoxicity toward chondrosarcoma was investigated. ► All cucurbitacins showed a very strong cytotoxicity and induced apoptosis. ► Cucurbitacins biotransformation in vitro in human liver was investigated. ► Cucurbitacin were glucuronidated by several UGTs isoforms at a very low extent. ► Cucurbitacin E was hydrolyzed by microsomal esterases leading to cucurbitacin I.

Introduction

Cucurbitacins are a family of highly oxygenated triterpenoids characterized by tetracyclic cucurbitane nucleus skeleton: the 19-(10  9β)-abeo-10α-lanost-5-ene (Chen et al., 2005, Seger et al., 2005). They are natural compounds found mainly in Cucurbitaceae and in large varieties of plants families (Chen et al., 2005, Dirr et al., 1986). Cucurbitacin synthesis in plants is initiated by the cucurbitadienol synthase (Shibuya et al., 2004). The primary cucurbitacins synthesized are cucurbitacins E and B that possess an acetyl function at C25 (Fig. 1) (Gry et al., 2006). The other cucurbitacins are produced due to several metabolic reactions as hydrogenation by cucurbitacin Δ23-reductase, deacetylation by cucurbitacin acetylesterases, hydroxylation, dehydrogenation and isomerization reactions (Dirr et al., 1986, Gry et al., 2006, Schabort and Teijema, 1968). Those reactions lead to more than 20 different species of cucurbitacins that differ from each other by their hydroxylation at C-2, -3, -19, -24, the presence of a double bond between C-1 and C-2 or between C-23 and C-24, the acetylation of the C-25 hydroxyl group and the presence of a ketone function at C-3 (Chen et al., 2005).

Cucurbitacins are of great interest for their pharmacological use because of their numerous biological activities. They show cytotoxicity toward a large number of human cancer cell lines such as human colon, breast, lung, central nervous system, prostate, nasopharynx (Balliano et al., 1983, Chen et al., 2005, Duncan et al., 1996, Jayaprakasam et al., 2003) by inhibiting the Jak/Stat3 pathway (Blaskovich et al., 2003, Sun et al., 2005) or by disrupting the actin and vimentin networks in cytoskeleton (Duncan et al., 1996). They are also known for their anti-inflammatory, anti-pyretic, analgesic, hepato-protective, anti-HIV and antioxidants activities (Chen et al., 2005, Escandell et al., 2007, Jayaprakasam et al., 2003, Peters et al., 1999, Siqueira et al., 2007).

Cucurbitacins E, D and I are the most studied cucurbitacins as they are among the most abundant in plants. Cucurbitacin E is the acetylated form of cucurbitacin I (Fig. 1) (Chen et al., 2005). So far, two studies have been reported, only focused on the metabolism of cucurbitacins E and I. The first study reported that cucurbitacins E and I were rapidly metabolized in S9-supernatants of rat liver, but without attempting to identify the metabolites formed (Lorenz et al., 2005). The second reported the in vitro hydrolysis of cucurbitacin E into cucurbitacin I in human plasma by paraoxonase (Saade et al., 2009). The metabolic pathway of cucurbitacins is still unclear. Therefore, it is necessary to bring additional information on the biotransformation of cucurbitacins in man. Particularly, the presence of several hydroxyl groups on the molecules suggests that they are potential substrates of UDP-glucuronosyltransferases and sulfotransferases.

In this work, a systematic comparative study of the cytotoxicity against the human bone chondrosarcoma cell lines, SW 1353 has been measured. Chondrosarcomas are rare malignant cartilage tumors divided into subgroups based on their matrix composition and cellular behavior. These tumors exhibit degradation of cartilage matrix composition and organization by displaying high water content and peripheral enchondral ossification (Murphey et al., 2003). They still have no cures, and surgery remains the main form of treatment (Herget et al., 2011). With the aim to develop new therapeutic drugs against this cancer with poor diagnosis, the cytotoxicity potency of cucurbitacins on the chondrosarcoma cell line SW 1353 has been investigated.

Section snippets

Chemicals

Cucurbitacins I, D and E were purchased from Extrasynthese (Genay Cedex, France). UDP-[U-14C]-glucuronic acid (specific activity 285 mCi/mmol) and [35S]3′-phosphoadenosine-5′-phosphosulfate (0.289 Ci/mmol) were purchased from Perkin Elmer (Courtaboeuf, France). UDP-glucuronic acid, 1-naphthol, nifedipine, 4-nitrophenol, 4-aminobiphenyl, eugenol, RS-ketoprofen, hyodeoxycholic acid, octylgallate, nicotinamide adenine dinucleotide phosphate (NADPH), cytochalasin D, phenylmethionyl sulfonylfluoride

MTT assay

The viability of the chondrosarcoma SW 1353 cell line was investigated by MTT assay after addition in the culture medium of 0, 0.1, 1, 10 and 100 μM cucurbitacins I (Fig. 2a), D (Fig. 2b) and E (Fig. 2c) for 6, 12, 24, 46 and 70 h. It was compared with that measured after cytochalasin D addition (Fig. 2d). The presence of dimethyl sulfoxide (0.1%, v/v) in the culture medium led to a 2–2.5-fold increase in cell viable number, when compared to that observed in the culture medium in which the

Discussion

Cucurbitacins are natural compounds known for their numerous potent pharmaceutical effects. They have shown cytotoxic effects on several cancer cell lines (Nakashima et al., 2010) and anti-inflammatory activities on rheumatoid arthritis (Escandell et al., 2007). These effects are mainly due to their strong inhibition of STAT3 (Signal Transducers and Activators of Transcription-3) and of IKK/NF-κB pathways leading to a decrease in the activity of key inflammatory enzymes, such as

Conclusion

In conclusion, our results show that cucurbitacins D, E and I present a potent cytotoxicity toward the chondrosarcoma SW 1353 cancer cell line and they are metabolized by conjugation reactions. More work is needed to determine the fate of these compounds in man before to consider their utilization on a therapeutic basis.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by a CEDRE grant no. 75/2010, by Lebanese National Council for Scientific Research (Ref. 02-04-09) and by the Ligue Régionale contre le Cancer.

References (40)

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