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. 2015 Jan 20;16(1):2174-86.
doi: 10.3390/ijms16012174.

Transgenic plants as low-cost platform for chemotherapeutic drugs screening

Daniele Vergara  1 , Stefania de Domenico  2 , Michele Maffia  3 , Gabriella Piro  4 , Gian-Pietro Di Sansebastiano  5
Affiliations

Transgenic plants as low-cost platform for chemotherapeutic drugs screening

Daniele Vergara et al. Int J Mol Sci. .

Abstract

In this work we explored the possibility of using genetically modified Arabidopsis thaliana plants as a rapid and low-cost screening tool for evaluating human anticancer drugs action and efficacy. Here, four different inhibitors with a validated anticancer effect in humans and distinct mechanism of action were screened in the plant model for their ability to interfere with the cytoskeletal and endomembrane networks. We used plants expressing a green fluorescent protein (GFP) tagged microtubule-protein (TUA6-GFP), and three soluble GFPs differently sorted to reside in the endoplasmic reticulum (GFPKDEL) or to accumulate in the vacuole through a COPII dependent (AleuGFP) or independent (GFPChi) mechanism. Our results demonstrated that drugs tested alone or in combination differentially influenced the monitored cellular processes including cytoskeletal organization and endomembrane trafficking. In conclusion, we demonstrated that A. thaliana plants are sensitive to the action of human chemotherapeutics and can be used for preliminary screening of drugs efficacy. The cost-effective subcellular imaging in plant cell may contribute to better clarify drugs subcellular targets and their anticancer effects.

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Figures

Figure 1
Figure 1
Fluorescent patterns of GFP-tagged marker proteins in transgenic Arabidopsis tissues in control conditions or treated with drugs targeting the cytoskeleton. (A) Normal fluorescent pattern of transgenes of (A.1) and (A.2) microtubules marker GFP-TUA6 in elongated petiole cells; (A.3) ER marker GFPKDEL in leaf epidermal cells; (A.4) Marker of the lytic vacuole, AleuGFP, in the lower face of the leaf; (A.5) AleuGFP, in the upper face of the leaf; and (A.6) Vacuolar marker characteristic of the direct transport ER-to-vacuole GFPChi; (B) Effect of treatment with 30 μM Paclitaxel on fluorescent pattern of (B.1) GFP-TUA6 in petiole cells; (B.2) GFPKDEL in leaf epidermis; (B.3) GFPChi in leaf epidermis; and (C) Effect of treatment with 200 μM Y-27632 on fluorescent pattern of (C.1) GFP-TUA6 in petiole cells showing no significant alteration; (C.2) GFP-TUA6 showing stronger effects; (C.3) GFPKDEL in leaf epidermis; (C.4) GFPChi faint fluorescence in leaf epidermis; and (C.5) GFPChi fluorescence secreted in the intercellular spaces of leaf mesophyll. GFP fluorescence and chlorophyll autofluorescence are shown in green and blue, respectively. Scale bar = 20 μm.
Figure 2
Figure 2
(A) Fluorescent patterns of GFP-tagged marker proteins in transgenic Arabidopsis tissues in control conditions (A.1) GFP-TUA6 in petiole cells, (A.2) GFPKDEL, (A.3) AleuGFP, and (A.4) GFPChi in leaf epidermis or treated with drugs targeting the endomembranes; (B) Effect of treatment with Crizotinib at the concentration of 0.2 μM (B.1) or 20 μM (B.2) on fluorescent pattern of GFP-TUA6 in petiole cells; (B3) effect of 20 μM in leaf epidermis on GFPKDEL; (B.4) AleuGFP and (B.5) GFPChi distribution; and (C) Effect of treatment with Sorafenib at the concentration of (C.1) 2 μM on GFP-TUA6 in petiole cells or 1 μM on GFPKDEL (C.2) and AleuGFP (C.3) in leaf epidermis. GFP fluorescence and chlorophyll autofluorescence are shown in green and blue, respectively. Scale bar = 20 μm.
Figure 3
Figure 3
Fluorescent patterns of GFPChi in transgenic Arabidopsis leaf epidermis treated with low doses of two combined drugs. Treatment with 0.2 μM Crizotinib had a moderate effect on GFPChi (A); but it was enhanced when combined with 10 μM Paclitaxel disturbing intermediate steps of its sorting (B) or causing mis-sorting to the apoplast when combined with 25 μM Y-27632 (C); combined treatment with 0.01 μM Sorafenib caused the increase of fluorescence in the central and small vacuoles (D). GFP fluorescence and chlorophyll autofluorescence are shown in green and blue, respectively. Scale bar = 20 μm.
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