In vitro antiplasmodial activity of cepharanthine

Abstract

Background: New classes of anti-malarial drugs are needed to control the alarming Plasmodium falciparum resistance toward current anti-malarial therapy. The ethnopharmacological approach allows the discovery of original chemical structures from the vegetable biodiversity. Previous studies led to the selection of a bisbenzylisoquinoline, called cepharanthine and isolated from a Cambodian plant: Stephania rotunda. Cepharanthine could exert a mechanism of action different from commonly used drugs. Potential plasmodial targets are reported here.

Methods: To study the mechanism of action of cepharanthine, a combined approach using phenotypic and transcriptomic techniques was undertaken.

Results: Cepharanthine blocked P. falciparum development in ring stage. On a culture of synchronized ring stage, the comparisons of expression profiles showed that the samples treated with 5 μM of cepharanthine (IC90) were significantly closer to the initial controls than to the final ones. After a two-way ANOVA (p-value < 0.05) on the microarray results, 1,141 probes among 9,722 presented a significant differential expression.A gene ontology analysis showed that the Maurer's clefts seem particularly down-regulated by cepharanthine. The analysis of metabolic pathways showed an impact on cell-cell interactions (cytoadherence and rosetting), glycolysis and isoprenoid pathways. Organellar functions, more particularly constituted by apicoplast and mitochondrion, are targeted too.

Conclusion: The blockage at the ring stage by cepharanthine is described for the first time. Transcriptomic approach confirmed that cepharanthine might have a potential innovative antiplasmodial mechanism of action. Thus, cepharanthine might play an ongoing role in the progress on anti-malarial drug discovery efforts.

Figure 1

Design of the transcriptional assay. UT8 = untreated samples 8 hr post-invasion by merozoite (h pmi); UT16 = untreated samples 16 h pmi; T16 = samples treated by cepharanthine at 8 h pmi during eight hours; iRBC = infected red blood cells.

Figure 2

Hierarchical clustering showing gene expression before (UT8), after (T16) and without treatment with cepharanthine (UT16). The genes in blue are under-expressed, the genes in red are over-expressed, and the genes in yellow represent genes not affected by cepharanthine.

Figure 3

Functional classification of genes with expression modified by cepharanthine. The number of genes in each category is indicated in the outer circle (e.g. 21. Malaria Parasite Metabolic Pathways [http://priweb.cc.huji.ac.il/malaria/]).

Figure 4

Correlation of microarray and qRT-PCR results. The relative expression of 12 genes is represented in three histograms by FC (Figures 4, 5 and 6). The qRT-PCR results are in light grey and the microarray results are in dark grey. The positives FC are over the horizontal line and the negative ones are under the horizontal line. The names of genes tested are indicated in abscissa. The histogram compares UT16 and T16; the correlation assessed by a linear model allows obtaining p-value of 8.67 × 10^-6.

Figure 5

Correlation of microarray and qRT-PCR results. The relative expression of 12 genes is represented in these three histograms by FC (Figures 4, 5 and 6). The qRT-PCR results are in light grey and the microarray results are in dark grey. The positives FC are over the horizontal line and the negative ones are under the horizontal line. The names of genes tested are indicated in abscissa. The histogram compares UT8 and T16; the correlation assessed by a linear model allows obtaining p-value of 2.04 × 10^-6

Figure 6

Correlation of microarray and qRT-PCR results. The relative expression of 12 genes is represented in these three histograms by FC (Figures 4, 5 and 6). The qRT-PCR results are in light grey and the microarray results are in dark grey. The positives FC are over the horizontal line and the negative ones are under the horizontal line. The names of genes tested are indicated in abscissa. The histogram compares UT8 and UT16; the correlation assessed by a linear model allows obtaining p-value of 3.13 × 10^-7.