Insights into the control of taxane metabolism: Molecular, cellular, and metabolic changes induced by elicitation in Taxus baccata cell suspensions

Abstract

More knowledge is needed about the molecular/cellular control of paclitaxel (PTX) production in Taxus spp. cell cultures. In this study, the yield of this anticancer agent in Taxus baccata cell suspensions was improved 11-fold after elicitation with coronatine (COR) compared to the untreated cells, and 18-fold when co-supplemented with methyl-β-cyclodextrins (β-CDs). In the dual treatment, the release of taxanes from the producer cells was greatly enhanced, with 81.6% of the total taxane content being found in the medium at the end of the experiment. The experimental conditions that caused the highest PTX production also induced its maximum excretion, and increased the expression of taxane biosynthetic genes, especially the flux-limiting BAPT and DBTNBT. The application of COR, which activates PTX biosynthesis, together with β - CDs, which form inclusion complexes with PTX and related taxanes, is evidently an efficient strategy for enhancing PTX production and release to the culture medium. Due to the recently described role of lipid droplets (LDs) in the trafficking and accumulation of hydrophobic taxanes in Taxus spp. cell cultures, the structure, number and taxane storage capacity of these organelles was also studied. In elicited cultures, the number of LDs increased and they mainly accumulated taxanes with a side chain, especially PTX. Thus, PTX constituted up to 50-70% of the total taxanes found in LDs throughout the experiment in the COR + β - CD-treated cultures. These results confirm that LDs can store taxanes and distribute them inside and outside cells.

Keywords: Taxus baccata; cell cultures; coronatine; gene expression; lipid droplets; paclitaxel; taxane accumulation.

FIGURE 1

(A) Average number of isolated LDs/cm² found in T. baccata cells, throughout 24 days, and maintained in Control, COR and CC conditions. Values presented are means ± SD (n = 10). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test. (B) Average size of LDs, measured in mm, found in T. baccata cells, throughout 24 days, and maintained in Control, COR and CC conditions. Values presented are means ± SD (n = 10). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 2

Example of detected isolated LDs under green and red fluorescence filters at 20 days of culture in COR (A), CC (B) and Control (C) conditions with a fluorescence microscope at 10X ocular and 20X objective. Image (D) shows a clump of T. baccata cells with stained LDs bordering the cell wall using a green fluorescence filter at the same magnifications and 20 days of culture in COR-treated samples.

FIGURE 3

Total taxanes produced in T. baccata cell cultures throughout 24 days of culture in control conditions, with the addition of coronatine (COR) or supplemented with COR + β-methyl-cyclodextrins (CC). Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 4

Total taxane contents found throughout the experiment inside the cells (A), liquid medium (B) and lipidic droplets (LDs) (C) in T. baccata cell cultures in control conditions and with the addition of 1 mM coronatine (COR) or 1 mM COR plus 50 mM β-cyclodextrins (CC). Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 5

Percentage of the total taxanes found inside the cell, liquid medium and lipid droplets (LDs). T. baccata cell cultures were maintained for 24 days in control conditions, or with the addition of coronatine (Cor) or Cor + β-cyclodextrins (CC). Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 6

Contents of the different taxanes studied (accumulated in the cells, in the liquid medium and in LDs) throughout the experiment in T. baccata cell cultures in elicited and control cultures. COR: 1 mM coronatine; CC: 1 mM coronatine + 50 mM β-methyl-cyclodextrins. Values presented are means ± SD (n = 3). Values followed by different symbols are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 7

Taxane content (expressed as mg L^–1) found inside the cells (A), in the culture medium (B) and within the LDs (C) of T. baccata cell cultures when maintained for 24 days in the optimum medium for production complemented or not with elicitors. COR: 1 mM coronatine; CC: 1 mM coronatine + 50 mM β-CDs; DABIII: 10-deacetylbaccatin III; BACIII: baccatin III; DAT: 10deacetylpaclitaxel; CEPH: cephalomannine; PTX: paclitaxel. Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 8

Percentage of each taxane in relation to the total accumulated in the LDs, in T. baccata cell cultures when maintained for 24 days in the optimum medium for production complemented or not with elicitors. COR: 1 mM coronatine; CC: 1 mM coronatine + 50 mM β-CDs; DABIII: 10-deacetylbaccatin III; BACIII: baccatin III; DAT: 10deacetylpaclitaxel; CEPH: cephalomannine; PTX: paclitaxel. Values presented are means ± SD (n = 3). Values followed by different symbols are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 9

Relative expression of TXS gene in T. baccata cell cultures grown in Control conditions and elicited with 1 mM coronatine (COR) or 1 mM coronatine + 50 mM methyl-β-cyclodextrins (CC). The relative gene expression levels were normalized with respect to the same cell line growing for 12 days in the growth medium (GM) without elicitors (reference value = 1). Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 10

Relative expression of the T7βOH gene in T. baccata cell cultures grown in Control conditions and elicited with 1 mM coronatine (COR) or 1 mM coronatine + 50 mM methyl-β-cyclodextrins (CC). Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 11

Relative expression of the BAPT gene in T. baccata cell cultures grown in Control conditions and elicited with 1 mM coronatine (COR) or 1 mM coronatine + 50 mM methyl-β-cyclodextrins (CC). Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.

FIGURE 12

Relative expression of the DBTNBT gene in T. baccata cell cultures grown in Control conditions and elicited with 1 mM coronatine (COR) or 1 mM coronatine + 50 mM methyl-β-cyclodextrins (CC). Values presented are means ± SD (n = 3). Values followed by different letters are significantly different (P ≤ 0.05) according to Tukey’s honestly significant difference test.