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. 2017 Jun 15:805:25-35.
doi: 10.1016/j.ejphar.2017.03.034. Epub 2017 Mar 18.

A harmine-derived beta-carboline displays anti-cancer effects in vitro by targeting protein synthesis

Annelise Carvalho  1 , Jennifer Chu  2 , Céline Meinguet  3 , Robert Kiss  1 , Guy Vandenbussche  4 , Bernard Masereel  3 , Johan Wouters  3 , Alexander Kornienko  5 , Jerry Pelletier  2 , Véronique Mathieu  6
Affiliations

A harmine-derived beta-carboline displays anti-cancer effects in vitro by targeting protein synthesis

Annelise Carvalho et al. Eur J Pharmacol. .

Abstract

Growing evidence indicates that protein synthesis is deregulated in cancer onset and progression and targeting this process might be a selective way to combat cancers. While harmine is known to inhibit DYRK1A and intercalate into the DNA, tri-substitution was shown previously to modify its activity profile in favor of protein synthesis inhibition. In this study, we thus evaluated the optimized derivative CM16 in vitro anti-cancer effects unfolding its protein synthesis inhibition activity. Indeed, the growth inhibitory profile of CM16 in the NCI 60-cancer-cell-line-panel correlated with those of other compounds described as protein synthesis inhibitors. Accordingly, CM16 decreased in a time- and concentration-dependent manner the translation of neosynthesized proteins in vitro while it did not affect mRNA transcription. CM16 rapidly penetrated into the cell in the perinuclear region of the endoplasmic reticulum where it appears to target translation initiation as highlighted by ribosomal disorganization. More precisely, we found that the mRNA expression levels of the initiation factors EIF1AX, EIF3E and EIF3H differ when comparing resistant or sensitive cell models to CM16. Additionally, CM16 induced eIF2α phosphorylation. Those effects could explain, at least partly, the CM16 cytostatic anti-cancer effects observed in vitro while neither cell cycle arrest nor DNA intercalation could be demonstrated. Therefore, targeting protein synthesis initiation with CM16 could represent a new promising alternative to current cancer therapies due to the specific alterations of the translation machinery in cancer cells as recently evidenced with respect to EIF1AX and eIF3 complex, the potential targets identified in this present study.

Keywords: Beta-carboline; Cancer; Harmine; Protein synthesis; Translation initiation.

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Conflict of interest statement

Declaration of interests

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Chemical structure of the β-carboline derivative CM16.
Fig. 2
Fig. 2
In vitro anti-cancer effects of CM16 in the NCI 60-cell line panel. (Adapted presentation - shown with the permission of the NCI) A: Global growth inhibition [GI50] of each cell line after 48 h of culture with CM16. “-7” represents the mean GI50 of the 60 cell lines, i.e. 0.2 μM. Log10 differences are represented by the bars. B: Lethal concentration [LC50] of CM16 for each cell line compared to the mean LC50 [“-5”]. The scale of the bars is in log10 as for A and B.
Fig. 3
Fig. 3. CM16-induced cytostatic growth inhibition effects in cancer cells
A: Cell growth inhibition in cancer cells (solid line) versus non-cancerous cells (dashed line) treated with CM16 for 72 h. Cancerous cell lines: Hs683, SKMEL-28 and MDA-MB-231. Non-cancerous cell lines: NHLF and NHDF non-transformed fibroblasts. Data are expressed as the mean of viable cells relative to control (100%)±S.E.M. of the six replicates of one representative experiment. B: Global growth ratio in HS683, SKMEL-28 and MDA-MB-231 cells after 24 h, 48 h and 72 h treatments with CM16 at their GI50. Results are expressed as the mean growth ratio between treated cells relative to control (1)±S.E.M. of three replicates of one representative experiment. C: Videomicroscopy of CM16-induced in vitro effects in Hs683, SKMEL-28 and MDA-MB-231. Figures are representative of one experiment performed in three replicates.
Fig. 4
Fig. 4. CM16 cellular penetration and distribution in the ER and protein synthesis inhibitio
A: CM16 fluorescence properties allow its visualization in blue color (filter ex/em: 359–371/397 nm) in Hs683 cells over time after 5 μM treatment. B: CM16 parallel distribution to the endoplasmic reticulum fluorescent probe (ER-tracker) after a 3 h treatment in Hs683, C: SKMEL-28 and D: MDA-MB-231 cell lines. Exposure times for blue filter (ex/em) 359–371/397 nm: 40 ms (SKMEL-28) and 80 ms (Hs683 and MDA-MB-231); and for red filter (ex/em) 540–580/593–668 nm: 283 ms (SKMEL-28) and 850 ms (Hs683 and MDA-MB-231). All pictures were taken with a 40× objective. Illustrations are representative of one experiment performed in two replicates.
Fig. 5
Fig. 5
Analysis of the newly synthesized proteins in A: Hs683 and, B: SKMEL-28 and C: NHDF cells by fluorescent assay. Positive control: cycloheximide 0.1 mM for 1 h. Data are expressed as the mean neosynthezised protein amounts normalized to the control (100%)±S.E.M. of the six replicates of one representative experiment. D: Dose-dependent evaluation of [35S]-methionine labeling in MDA-MB-231and NHDF cells after 80 min of treatment. CPMs were normalized to total protein.
Fig. 6
Fig. 6. CM16 effects on ribosomal organization by sucrose gradient analysis
Effect of CM16 on ribosomal units and polysome organization after treatment. A: MDA-MB-231 cells incubated for 80 min with CM16 at 10 μM in comparison to the control. B: Cancerous cell lines Hs683 and C: SKMEL-28 and non-cancerous cell lines D: NHDF and E: NHLF incubated with CM16 at 0.5 μM or 5.0 μM for 3 h (grey lines) in comparison to the control (solid black line). Puromycin was used as positive control (1 h, 184 μM) to visualize the 80S peak containing fractions. Three or one independent experiments were performed, depending on the cell line. Each profile is representative of one experiment.
Fig. 7
Fig. 7. Initiation factors study
A: Effects of CM16 on the expression and phosphorylation status of eIF2α in MDA-MB231 cancerous and NHDF non-cancerous cells by western blot. Representative western blot of two experiments. B and C: Comparison of the transcriptomic expression levels of main translation initiation genes in the most (MS) versus least sensitive (LS) cell lines to CM16 effects among the NCI-cell-line-panel screening. The four least sensitive cell lines (i.e. those with a GI50 >1 μM) are HCT-15 [−5.72], NCI/ADR-RES [−5.36], Caki-1 [−5.85] and UO-31 [−5.80]. The five most sensitive cell lines (i.e. with a GI50 <0.1 μM) are A498 [−8.00], HL60 [−7.30], CCRF-CEM [−7.12], RPMI-8226 [−7.19] and T47D [−7.23]. B: Transcriptomic comparison of eIF2α, β and γ subunits between the most and least sensitive cell lines to the CM16 growth inhibitory effects identified in the NCI cell line panel. C: Transcriptomic comparison of the targets with significantly different expression levels between the cell lines most and least sensitive to CM16 growth inhibitory effects identified in the NCI cell line panel by means of t-test comparison. Increased expression levels as compared to the 60 cell line mean appear in black while decreased expression levels appear in grey. Results are expressed as Z scores as provided by the NCI database. Z scores are determined for each probe/cell line pair by the subtraction from its intensity of the probe mean (across the 60 cell lines), and division by the standard deviation of the probe (across the 60 cell lines). The z score average was then calculated as the mean across all probes and probe sets that passed quality control criteria.
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