Cancer chemotherapy combination treatments with etoposide.
Abstract
The pharmaceutically important anticancer drugs etoposide and teniposide are derived from podophyllotoxin, a natural product isolated from roots of Podophyllum hexandrum growing in the wild. The overexploitation of this endangered plant has led to the search for alternative sources. Metabolic engineering aimed at constructing the pathway in another host cell is very appealing, but for that approach, an in-depth knowledge of the pathway toward podophyllotoxin is necessary. In this chapter, we give an overview of the lignan pathway leading to podophyllotoxin. Subsequently, we will discuss the engineering possibilities to produce podophyllotoxin in a heterologous host. This will require detailed knowledge on the cellular localization of the enzymes of the lignan biosynthesis pathway. Due to the high number of enzymes involved and the scarce information on compartmentalization, the heterologous production of podophyllotoxin still remains a tremendous challenge. At the moment, research is focusing on the last step(s) in the conversion of deoxypodophyllotoxin to (epi)podophyllotoxin and 4′-demethyldesoxypodophyllotoxin by plant cytochromes.
Keywords
- etoposide
- podophyllotoxin
- Podophyllum hexandrum
- Anthriscus sylvestris
- metabolic engineering
1. Introduction
The high demand of podophyllotoxin derivatives for chemotherapy gives a severe pressure on the natural sources, such as
2. Lignans and their biological activities
In 1936, Haworth was the first to describe a group of phenylpropanoid dimers (C6C3) linked by the central carbon (C8) as lignans [8]. The Haworth’s definition of lignan has been adopted by the IUPAC nomenclature recommendations in 2000 [9]. According to this nomenclature, lignans can be divided into eight subgroups based on the oxygen incorporation into the skeleton and the cyclization pattern [10]. In the lignan pathway toward podophyllotoxin, six subgroups of lignans can be defined in the order of occurrence: furofuran, furan, dibenzylbutane, dibenzylbutyrolactol, dibenzylbutyrolactone, and aryltetralin (Figure 1). The other two subgroups are arylnaphthalene and dibenzocyclooctadiene. Dibenzylbutanes are only linked by the 8,8′ bond. An additional oxygen bridge is found in furofurans, furans, dibenzylbutyrolactols, and dibenzylbutyrolactones. A second carbon-carbon link is found in aryltetralins, arylnaphthalenes, and dibenzocyclooctadienes [10, 11]. The majority of the lignans has oxygen at the C9 (C9′) carbon; however, some lignans in the dibenzylbutanes, furans, and dibenzocyclooctadiene subgroups are missing this oxygen [10]. Humans metabolize the furofurans pinoresinol and sesamin, the furan lariciresinol, the dibenzylbutane secoisolariciresinol, and the dibenzylbutyrolactone matairesinol. These lignans are phytoestrogens, which can be converted into enterolactone or enterodiol by intestinal bacteria [12, 13]. Enterolactone and enterodiol have antioxidant, estrogenic, and anti-estrogenic activities in humans; furthermore, they may protect against certain chronic diseases [14]. Several lignans have been described to have antiviral properties; however, therapeutic applications are limited due to the toxicity [15]. The extract, podophyllin, of
3. Importance of podophyllotoxin and derivatives for chemotherapy
Podophyllotoxin is a tubulin-interacting agent that inhibits mitotic spindle formation [21]. As podophyllotoxin is severely toxic if applied systemic, a number of less toxic derivatives have been generated and these are now widely used in cancer chemotherapy. Interestingly, the derivatives currently used in the clinic, etoposide, and teniposide, have a different mode of action than podophyllotoxin. They inhibit topoisomerase II by stabilizing its binding to DNA, which results in double-stranded breaks in the DNA and arrest of the cell cycle in the G2 phase [21]. Etoposide (VP-16, VePesid®) was synthesized in 1966 by Sandoz and was further developed by Bristol-Meyers from 1978 onwards. In 1983, it was approved by the FDA for the treatment of testicular cancer [22]. As etoposide is poorly soluble in water, the etoposide prodrug etoposide phosphate (Etopophos®) was designed by Bristol-Meyers Squibb, which was approved by the FDA in 1996 [23]. The prodrug is converted to etoposide within 30 min presumably by alkaline phosphatases. Furthermore, the pharmacokinetics and toxicity of etoposide phosphate are equal to etoposide [24, 25]. According to the National Cancer Institute and the Dutch government etoposide, phosphate should be used in combination therapy for various cancers (Table 1) [26–28]. Teniposide (VM-26, Vumon®) was synthesized in 1967 by Sandoz and was further developed by Bristol-Meyers from 1978 onwards [22]. It is used in the treatment of acute myeloid leukemia and myelodysplastic syndromes in children and in acute lymphocytic leukemia [29, 30]. Toxicity problems are still an issue with etoposide; therefore, novel derivatives were designed and evaluated in preclinical and clinical studies [31]. The derivatives NK611, Gl-311, and TOP-53 were discontinued after phase I or II studies [22, 32, 33]. NK611, which is more water soluble than etoposide, shows similar toxic effects in humans as etoposide. However, only few patients showed efficacy in phase I studies [34–36]. No data of the phase I or II studies were found for GL-311 and TOP-53. Four newer derivatives are tafluposide, F14512, Adva-27a, and QS-ZYX-1-61 [31, 32]. Tafluposide (F-11782), a pentafluorinated epipodophylloid, inhibits topoisomerase I and II activity [37, 38]. In phase I study, stable disease was observed in 7 out of 21 patients with advanced solid tumors, such as choroid and skin melanoma [39]. Increasing the selectivity of anticancer agents is of great interest. As the polyamine transport system is upregulated in cancer cells, F14512 was designed to target the transport system by linking the epipodophyllotoxin core to a spermine chain [40]. Phase I study in adult patients with acute meloid leukemia showed clinical activity in relapsed patients, but limited activity in refractory patients [41]. F14512 will be tested in combination with cytarabine in a phase II study [41]. The minimal therapeutic effect of etoposide on dogs with relapsing lymphomas has resulted in a phase I study of F14512, which showed a strong therapeutic efficacy [42]. The derivative adva-27a, a GEM-difluorinated C-glycoside derivate of podophyllotoxin, is effective against multidrug resistant cancer cells [43]. Preparations are being made for a phase I study in pancreatic and breast cancer patients in Canada [44]. The derivative QS-ZYX-1-61 induces apoptosis by inhibition of topoisomerase II in human non–small-cell lung cancer [45]. Further investigations are necessary for this compound.
Cancer | Combination of drugs |
---|---|
Vincristine sulfate, etoposide phosphate, prednisone, doxorubicin hydrochloride | |
Doxorubicin hydrochloride, bleomycin, vincristine sulfate, etoposide phosphate | |
Doxorubicin hydrochloride, bleomycin, vincristine sulfate, etoposide phosphate, prednisone, cyclophosphamide | |
- All | Rituximab, ifosfamide, carboplatin, etoposide phosphate |
Etoposide phosphate, ifosfamide, methotrexate | |
Lomustine, etoposide phosphate, chlorambucil, prednisolone | |
- B-cell | Rituximab, etoposide phosphate, prednisone, vincristine sulfate, cyclophosphamide, doxorubicin hydrochloride |
- Nonbrain | Cisplatin, etoposide phosphate, bleomycin |
- Ovarian/testicular | Bleomycin, etoposide phosphate, cisplatin |
- Advanced testicular | Etoposide phosphate, ifosfamide, cisplatin |
- Children | Cytarabine, daunorubicin hydrochloride, etoposide phosphate |
- Phase II | Cytarabine and amsacrine, etoposide or mitoxantrone |
Carboplatin, etoposide phosphate, vincristine sulfate | |
Etoposide with cisplatin or carboplatin | |
Cisplatin, cyclophosphamide, doxorubicin, vincristine, methotrexate | |
Ifosfamide, carboplatin, and etoposide |
4. Overview of the lignan biosynthetic pathway
Podophyllotoxin is produced in the lignan pathway, which we will discuss in more detail in this section (Figure 1). Lignins and lignans are the major metabolic products of the phenylpropanoid pathway in vascular plants. Lignins are derived from coumaryl, coniferyl, and sinapyl alcohol, whereas lignans are derived from coniferyl alcohol [46].
4.1. Coniferyl alcohol toward matairesinol
The pathway toward podophyllotoxin starts with pinoresinol, lariciresinol, secoisolariciresinol, and matairesinol. Pinoresinol and lariciresinol are found in most vascular plants, such as
4.1.1. Dirigent protein
In 1997, Davin and coworker showed that the dirigent protein (DIR) from
4.1.2. Pinoresinol-lariciresinol reductase
In 1996, Dinkova-Kostova and coworkers found the pinoresinol-lariciresinol reductase (PLR) in
4.1.3. Secoisolariciresinol dehydrogenase
Secoisolariciresinol dehydrogenase (SDH) from
4.2. Matairesinol toward deoxypodophyllotoxin
Plant feeding experiments performed by various groups have revealed the metabolites intermediate between matairesinol and podophyllotoxin, such as yatein and deoxypodophyllotoxin in
4.3. Conversion of deoxypodophyllotoxin into demethyldesoxypodophyllotoxin
The
5. Engineering approaches
In this part, we will focus on genetic engineering approach`es to produce podophyllotoxin in a heterologous system. In order to produce podophyllotoxin in
5.1. Production of coniferyl alcohol in E. coli and S. cerevisiae
Coniferyl alcohol can be produced in
5.2. Cellular localization of enzymes from the lignan pathway
In order to engineer the lignan pathway for podophyllotoxin production in a heterologous cell, knowledge about the localization of lignans and their corresponding enzymes is necessary. Localization to the wrong organelle might abolish or lower production, as was shown for penicillin production [77]. The monolignol coniferyl alcohol is synthesized in the cytosol and transported over the plasma membrane for incorporation into lignin or lignan by an ABC membrane transporter, whereas the glucosylated form (coniferin) for storage could only be transported over the vacuolar membrane possibly by another ABC membrane transporter or proton-coupled antiporter [78, 79]. Analyses of transmembrane helices by the TMHMM predictor [80] indicated that DIR has one transmembrane helix. Furthermore, the DIR protein is a glycoprotein with a secretory signal peptide [50]. This indicates that the DIR protein is membrane attached, which is consistent with the findings in
5.3. Conversion of deoxypodophyllotoxin to (epi)podophyllotoxin by engineering
In 2006, Vasilev and coworkers showed that the human liver cytochrome P450 3A4 (CYP3A4) together with human NADPH P450 reductase can convert deoxypodophyllotoxin stereoselectivity into epipodophyllotoxin [93]. The disadvantage of this system is the usage of frozen cells and therefore the need to supply a regenerative system, such as glucose-6-phosphate dehydrogenase and NADP. Changing the system to a resting cell assay or cell-free assay with the usage of a cheaper cofactor and increasing the electron transfer between cytochrome and reductase would greatly increase the usability of this system. As CYP3A4 is quite unspecific, an approach to find a dedicated cytochrome converting deoxypodophyllotoxin into podophyllotoxin could be provided by the systematic analysis of cytochrome encoding genes found by Kumari and coworkers, who analyzed the transcriptome of
5.4. Production of etoposide
Industrially, podophyllotoxin is chemically converted to etoposide (Figure 3). Podophyllotoxin is converted to 4′-demethyl-epipodophyllotoxin by demethylation and epimerization in two steps with a yield of 52% followed by the protection of the phenolic group by conversion to 4′-O-carbobenzoxy-epipodophyllotoxin in one step with 89% yield [97]. 4′-O-carbobenzoxy-epipodophyllotoxin is then glycosylated to the esterification of
6. Future perspectives
Recent insights in the lignan biosynthetic pathway by Lau and Sattely [69] have progressed the research in the lignan pathway enormously. Engineering of the lignan pathway in a heterologous host will become feasible, if the localization of the enzymes in the pathway has been determined. Depending on this localization, either
Acknowledgments
This work was supported by EU regional funding. The PhytoSana project in the INTERREG IV A Deutschland-Nederland program: 34- INTERREG IV A I-1-01=193.
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