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“Formula” for creating a powerful antitumour agent from an endangered plant

Podophyllum hexandrum is an endangered plant found in the Himalayas and on the American continent. In the past, the native people of these regions knew the purgative effects of this species, but were unaware that its metabolism hid a much more important formula.

Today, this plant is the main source of podophyllotoxin, a compound which other agents, such as etoposide, come from. Its powerful antitumour action against refractory testicular cancer, non-lymphocytic leukaemia, non-Hodgkin lymphoma and lung cancer makes this compound a very important drug for medicine. As with many plants, Podophyllum hexandrum has become a real “natural source” of medicine. However, it has one key characteristic: it is an endangered species. Harvesting this plant has been prohibited in the United States since 1985, and exporting it in India has also been forbidden since 1994.

In order to conserve it and maintain biodiversity, using “omics” technologies in R&D is essential while cancer-fighting drugs are researched and developed. In this sense, work carried out by scientists at the University of Stanford has allowed for the discovery of the “formula” used to create this powerful antitumour agent with Podophyllum hexandrum.

 

James Steakley (Wikimedia)

 

According to a recent publication in Science, it does so through six enzymes that complete the biosynthetic pathway to etoposide. Studying this endangered plant using transcriptome tools has allowed for the identification of genes responsible for the biosynthesis of podophyllotoxin. Subsequently, researchers selected 29 candidate genes to express them in the tobacco plant (Nicotiana benthamiana), a transformation mediated through Agrobacterium. They thus discovered six key enzymes in the production of this compound, including oxoglutarate-dependent dioxygenase which, according to scientists, “closes” the cyclohexane ring of aryltetralin lignans, such as podophyllotoxin. By co-expressing ten genes in the tobacco plant - these six in addition to four others discovered beforehand - the researchers were able to reconstruct the synthesis pathway to 4-demethyl-epipodophyllotoxin, the lignan which is the immediate precursor to etoposide itself, known for being a powerful inhibitor of topoisomerase.

How were they able to discover the candidate genes? To achieve this, the scientists at Stanford sapped the RNA sequencing data from this plant. These data can be consulted by the public, thanks to the Medicinal Plants Consortium. They checked that the genes related to podophyllotoxin had a high expression in the rhizome, stalk and leaf tissue, and then selected those candidate genes with a similar gene expression profile. Subsequently, they also employed liquid chromatography-mass spectrometry (LC-MS) to determine the consumption of one of the precursors of etoposide in the leaves, with the objective of reconstructing the biosynthesis pathway. This technique was also employed in order to discover the accumulation and maximum concentration of the metabolites related to this pathway. 

 

Wikimedia

 

With the aim of checking the six candidate genes, the researchers carried out a transcriptome analysis of Podophyllum hexandrum leaves. RNA sequencing and measuring the levels of expression were key to classifying the genes according to how they were already known in this biosynthetic pathway: candidate genes or genes identified as de novo. This was how they finally discovered the six missing enzymes needed to complete the "formula" for the immediate precursor of etoposide in this endangered plant. With the expression of this pathway in the tobacco plant, the researchers indicate that we could “bypass the cultivation of Podophyllum hexandrum and the subsequent semi-synthetic epimerisation and demethylation”. These are reactions that are currently necessary to produce this powerful antitumour agent. Thanks to transcriptomics, the production and development of cancer-fighting drugs, such as etoposide, has been made easier, with biodiversity being protected at the same time.