Combination of 5-fluorouracil and thymoquinone targets stem cell gene signature in colorectal cancer cells

Article abstract of DOI:10.1038/s41419-019-1611-4

Cancer stem cells (CSCs) residing in colorectal cancer tissues have tumorigenic capacity and contribute to chemotherapeutic resistance and disease relapse. It is well known that the survival of colorectal CSCs after 5-fluorouracil (5-FU)-based therapy leads to cancer recurrence. Thus CSCs represent a promising drug target. Here, we designed and synthesized novel hybrid molecules linking 5-FU with the plant-derived compound thymoquinone (TQ) and tested the potential of individual compounds and their combination to eliminate colorectal CSCs. Both, Combi and SARB hybrid showed augmented cytotoxicity against colorectal cancer cells, but were non-toxic to organoids prepared from healthy murine small intestine. NanoString analysis revealed a unique signature of deregulated gene expression in response to the combination of TQ and 5-FU (Combi) and SARB treatment. Importantly, two principle stem cell regulatory pathways WNT/?-Catenin and PI3K/AKT were found to be downregulated after Combi and hybrid treatment. Furthermore, both treatments strikingly eliminated CD133+ CSC population, accompanying the depleted self-renewal capacity by eradicating long-term propagated 3D tumor cell spheres at sub-toxic doses. In vivo xenografts on chicken eggs of SARB-treated HCT116 cells showed a prominent nuclear ?-Catenin and E-cadherin staining. This was in line with the reduced transcriptional activity of ?-Catenin and diminished cell adhesion under SARB exposure. In contrast to 5-FU, both, Combi and SARB treatment effectively reduced the angiogenic capacity of the remaining resistant tumor cells. Taken together, combination or hybridization of single compounds target simultaneously a broader spectrum of oncogenic pathways leading to an effective eradication of colorectal cancer cells.

Full text of DOI:10.1038/s41419-019-1611-4

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https://doi.org/10.1038/s41419-019-1611-4
Cell Death & Disease
A R T I C L E
O p e n A c c e s s
Combination of 5-fluorouracil and
thymoquinone targets stem cell gene
signature in colorectal cancer cells
Benardina Ndreshkjana1,2, Aysun Çapci3, Volker Klein3, Pithi Chanvorachote4, Julienne K. Muenzner1,2,
Kerstin Huebner1,2, Sara Steinmann1,2, Katharina Erlenbach-Wuensch2, Carol I. Geppert2, Abbas Agaimy2, Farah Ballout5,
3
Chirine El-Baba1, Hala Gali-Muhtasib5, Adriana Vial Roehe6, Arndt Hartmann1,2, Svetlana B. Tsogoeva and
Regine Schneider-Stock1,2
Abstract
Cancer stem cells (CSCs) residing in colorectal cancer tissues have tumorigenic capacity and contribute to
chemotherapeutic resistance and disease relapse. It is well known that the survival of colorectal CSCs after 5-
fluorouracil (5-FU)-based therapy leads to cancer recurrence. Thus CSCs represent a promising drug target. Here, we
designed and synthesized novel hybrid molecules linking 5-FU with the plant-derived compound thymoquinone (TQ)
and tested the potential of individual compounds and their combination to eliminate colorectal CSCs. Both, Combi
and SARB hybrid showed augmented cytotoxicity against colorectal cancer cells, but were non-toxic to organoids
prepared from healthy murine small intestine. NanoString analysis revealed a unique signature of deregulated gene
expression in response to the combination of TQ and 5-FU (Combi) and SARB treatment. Importantly, two principle
stem cell regulatory pathways WNT/ß-Catenin and PI3K/AKT were found to be downregulated after Combi and hybrid
treatment. Furthermore, both treatments strikingly eliminated CD133+ CSC population, accompanying the depleted
self-renewal capacity by eradicating long-term propagated 3D tumor cell spheres at sub-toxic doses. In vivo xenografts
on chicken eggs of SARB-treated HCT116 cells showed a prominent nuclear ß-Catenin and E-cadherin staining. This
was in line with the reduced transcriptional activity of ß-Catenin and diminished cell adhesion under SARB exposure.
In contrast to 5-FU, both, Combi and SARB treatment effectively reduced the angiogenic capacity of the remaining
resistant tumor cells. Taken together, combination or hybridization of single compounds target simultaneously a
broader spectrum of oncogenic pathways leading to an effective eradication of colorectal cancer cells.
Introduction
CRC therapy are the resistance of tumor cells to che-
Colorectal cancer (CRC) remains a leading cause of motherapeutic drugs and their relapse after treatment,
cancer-related deaths worldwide1. Two major obstacles in limiting patient survival2. Accumulating evidence indi-
cates that cancer stem cells (CSCs) are the driving force of
cancer initiation, metastasis, recurrence, and they largely
contribute to chemoresistance3. Recent studies showed
Correspondence: Regine Schneider-Stock (regine.schneider-stock@uk-
erlangen.de)
that CSCs can be identified by the expression of CD133
cell surface marker. Indeed, CD133+ tumor cells are
capable of seeding new tumors4–7. Also WNT/β-Catenin
and PI3K/AKT signaling pathways play an essential role
in stem cell maintenance and are associated with an
1University Hospital, Friedrich-Alexander-University Erlangen-Nuremberg,
Experimental Tumorpathology, Institute of Pathology, Erlangen, Germany
2University Hospital, Friedrich-Alexander University of Erlangen-Nuremberg,
Institute of Pathology, Erlangen, Germany
Full list of author information is available at the end of the article.
These authors contributed equally: Benardina Ndreshkjana, Aysun Çapci
Edited by R. Aqeilan
© The Author(s) 2019
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If
material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
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enhanced tumorigenicity of CD133+ primary CRC cells8. exposing HCT116 and HT29 human colon cancer cells to
The WNT cascade is the dominant force in controlling different drug concentrations. We observed a clear
cell fate and the regulation of its key player ß-Catenin9–11
.
reduction of viability in both cell lines, when treating with
Blockage of WNT signaling was shown to inhibit angio- TQ or 5-FU alone, with HT29 cells being generally less
genesis and tumor growth in CRC12.
sensitive (Suppl. Fig. 1A, B). The combination of TQ with
5-FU-based chemotherapy is the common choice for 5-FU (Combi) increased the cytotoxic effect in HT29 cells
patients with CRC. Several combination strategies with (Suppl. Fig. 1C). Next, we synthesized three hybrids
plant-derived drugs have been proposed to overcome 5-FU bearing variable linkers (Fig. 1). Interestingly, only SARB
resistance and to reduce its side effects13–15. We and others hybrid was effective in HCT116 cells with an IC50
=
have demonstrated strong anticancer effects of thymoqui- 16 µM, whereas the two other hybrids, KV98 and AC29,
none (TQ), the main component of black seed16–20. Only a as well as SARB parent compounds, produced an IC50
few combination studies with 5-FU have been reported 100 µM (Suppl. Fig. 1D, E). Consequently, for all sub-
supporting the chemosensitization effects of TQ21,22
sequent experiments we focused on SARB hybrid. When
>
.
The generation of hybrids between natural products and comparing Combi and SARB, both showed increased
conventional chemotherapeutics is a novel approach to cytotoxicity in HCT116 with almost similar IC50, whereas
obtain new anticancer compounds facilitating adminis- in HT29 cells Combi produced a lower IC50 (Suppl. Fig.
tration and allowing easier prediction of pharmacokinetic 1C, E). Next, we tested the cytotoxic effects of SARB
features23–25. In previous studies, we showed the impor- against the HCEC nonmalignant colon epithelial cells.
tance of the hybridization concept by linking two natural Intriguingly, SARB exerted higher IC50 values on normal
compounds, TQ and artemisinin, along with other cells (IC50 = 52 µM), suggesting specificity to cancer cells
bioactive natural products being highly potent anti- (Suppl. Fig. 1F). In order to evaluate the toxicity of the
malarial, anticancer, and antiviral compounds26–30
.
compounds under physiological condition, organoid cul-
In this present study, we analyzed the effects of novel 5- tures were treated with both individual compounds,
FU/TQ hybrids in CRC cells in vitro and in vivo. A Combi and SARB. In the control group, the organoids
NanoString-based gene expression analysis was performed to were found to gradually enlarge with a sharp morphology
compare the anticancer effect of the SARB hybrid with the and pronounced villi. The organoid architecture was
effect of the individual compounds as well as Combi treat- nearly unaffected after TQ treatment and the clear villi-
ment. In comparison to the single drug treatments we lumen structure was only slightly disrupted under 5-FU,
identified additional novel targets exceeding the known Combi, or SARB exposure at a dose up to 50 µM. Viability
mechanisms of action of 5-FU and TQ. We show that both measurements showed only a minor reduction in viable
combination strategies are highly effective against CD133+ cells to ~70–80% when individual treatments were used,
CSC populations in CRC and simultaneously inhibit the while in response to the Combi and SARB, the vitality
WNT/ß-Catenin and PI3K/AKT signaling pathways. Thus, varied from 60% to 80% (Suppl. Fig. 2A, B). To determine
our findings strongly support the idea of combination ther- whether the growth-inhibitory effect was related to the
apy between the clinical drug 5-FU and the plant-derived induction of apoptosis, we performed an Annexin/PI
compound TQ and suggest the hybridization concept as a analysis. The cells were exposed to the IC50 of single
promising strategy to develop new drug candidates for CRC. compounds and IC65 of Combi and SARB. The apoptotic
cell fraction was significantly increased when cells were
Results
treated with SARB in HCT116 cells; however, in HT29
cells the Combi and SARB exhibited comparable effects to
the single drugs (Suppl. Fig. 2C, D). Altogether, these
Generation of hybrid compounds of the natural product
TQ (1) and 5-FU (2)
We designed hybrid molecules based on 5-FU and TQ. observations suggest the superior action of our designed
The parent compounds (3–8) of hybrids were synthesized drug combination strategy which we aimed to further
from TQ and 5-FU. Hybrids were obtained via three dif- investigate at the molecular level.
ferent chemical reactions; esterification (SARB), amide
coupling (AC29), and click reaction (KV98) (Fig. 1). All Gene expression profiling identifies WNT/ß-Catenin and
hybrid structures were identified with spectroscopic ana- PI3K/AKT signaling pathways as the major targets of SARB
lysis and their purity was confirmed via elemental analysis
hybrid treatment
(see supplementary information).
To identify a specific pattern of gene alterations caused
by single drugs, Combi or SARB, we performed a gene
expression profiling using the NanoString PanCancer
Panel. TQ-treated and 5-FU-treated cells displayed a
Combination and hybrid treatments potentiate the
cytotoxic effects of the individual compounds
First, we examined the anti-proliferative properties of different gene expression profile compared to the
the single drugs and their combination in a 1:1 mixture by untreated control as shown in the scatter plot diagram,
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Fig. 1 Generation of hybrid compounds from the natural product Thymoquinone (TQ, 1) and 5-Fluorouracil (5-FU, 2). All parent compounds (3–8) of
hybrids (SARB, AC29, and KV98) were synthesized from Thymoquinone and 5-Fluorouracil
where each point represents a particular gene (Suppl. Fig. frequency of potentially synergistic effects detectable for
3A). Using STRING database we found that the affected SPP1, EGF, SPRY1, and IGFBP3. In case of the FGF sig-
downregulated genes under TQ belong to the RAS, RAP1, naling antagonist SPRY1 and the metastasis suppressor
PI3K, and WNT signaling pathways (Suppl. Fig. 3B), as gene IGFBP334, it seems to be an inhibition of an onco-
previously described31–33. The most significantly altered genic pathway whereas the upregulation of the two
pathways in TQ upregulated genes are given in Suppl. Fig. secreted factors SPP1 and EGF seems to be an undesired
3C and include mostly the MAPK, PI3K, focal adhesion, side effect of treatment reinforcing the PI3K signaling.
and p53 signaling pathways. As expected, 5-FU down- Nevertheless, we identified a specific gene target panel
regulated components of the cell cycle pathway (Suppl. whose expression was altered by Combi treatment
Fig. 3D) and led to the upregulation of p53-target genes including genes, such as FST, PLA2G10, NGF, PAK7
associated with DNA damage response (GADD45A), cell DNMT1, and BIRC3. Although many genes were
cycle regulation (CDKN1A), and intrinsic apoptotic sig- deregulated in same direction as observed for the single
naling (FAS, BAX, PML, SFN, EPHA2). The most sig- treatments, we provide evidence that the combination
nificantly altered upregulated pathways in 5-FU are given treatment also targets a characteristic gene profile (Suppl.
in Suppl. Fig. 3E.
Table 1). Next, in a scatter plot, we confirmed dissim-
Compared to the individual treatments, the Combi ilarities among SARB and Combi-treated samples, espe-
treatment reinforced the downregulation of NGFR, cially in the downregulated genes (Fig. 2A).
NPM1, HIST13H, JUN, and PLA2G4C in a possible
Although KEGG pathway analysis of the downregulated
additive manner (Suppl. Table 1). Considering the upre- genes revealed a significant cell cycle inhibition for both
gulated genes in Combi, we found a remarkable higher treatments, there was a specific set of downregulated
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Fig. 2 Gene expression profiling identifies WNT/ß-Catenin and PI3K/AKT signaling pathways as the major targets of SARB hybrid
treatment. (A) Scatter plot of the whole NanoString gene set of HCT116 cells after 24 h of treatment with Combi and SARB hybrid. Normalized
grouped data are shown. Log10 data are shown as treatment vs. untreated samples (n = 3). Pearson's correlation was calculated using GraphPad
Prism 7.0. KEGG pathway was performed using STRING database (version 10.5) on the significantly downregulated genes under (B) Combi and under
(C) SARB treatment. (D) Venn diagram of significantly up and (E) downregulated genes upon treatments. A p-value of ≤ 0.05 and a fold change of
≥ / ≤ 1.5-fold are selected for the analysis. (F) RT-qPCR validation of WNT target genes and FOS gene in HCT116 cells. Gene expression fold-change
differences in Combi and SARB hybrid after 24 h of treatment. Values represent means SEM (n = 3), (*p < 0.05; **p < 0.01; ***p < 0.001; One way-
ANOVA). B2M was used as a housekeeping gene. (G) Western Blot analysis of key proteins involved in PI3K/AKT signaling in HCT116 cells after 24 h of
treatment with TQ 40, 5-FU 15, Combi, and SARB at same concentration of 35 µM. Western Blot images are representative of at least two independent
experiments. The quantification values are presented as relative to GAPDH housekeeping protein
WNT-associated genes only in the SARB-treatment group samples (Fig. 2D, E). When examining these 25 genes by
(Fig. 2B, C). We believe, this signature derives from TQ, KEGG pathway analysis, we distinguished another key
since we could confirm the respective panel of genes with oncogenic pathway, the PI3K pathway, as the most
WN16, TBL1XR1, RAC1, PPP3CA, PPP3CB, PPKACB, downregulated pathway under SARB exposure. These
and CTNNB1 both significantly downregulated under TQ unique 30 genes in SARB are given in Suppl. Table 2.
and SARB treatment. The upregulated genes mostly Surprisingly, we identified the stress oncogene FOS to be
affected the PI3K/AKT, focal adhesion, and p53 pathways massively upregulated under Combi and SARB did not
(Suppl. Fig. 3C, E, G).
alter its expression, which was confirmed by RT-qPCR
To identify specific gene signatures, we set another (Fig. 2F).
strong selection criterion by overlapping all the treat-
Next, we validated the unique WNT and PI3K/AKT
ments and focusing on the uniquely deregulated genes. gene signature obtained by NanoString analysis using RT-
From 89 downregulated genes in TQ and 5-FU, 31 genes qPCR and Western Blot analysis. We verified a reduced
were overlapping, whereas in 25 and 75 upregulated genes expression of AXIN2, the most prominent transcriptional
in TQ and 5-FU, respectively, 12 genes were similarly target of the canonical WNT signaling, as well as other
altered (Fig. 2D, E). We could reduce the target panel to WNT target genes such as DKK2, FGF9, ID2, and c-Myc
25 downregulated and 5 upregulated genes that were (Fig. 2F). Interestingly, Combi treatment also affected the
uniquely and significantly altered in SARB-treated WNT pathway; however, with SARB being more effective
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in suppressing the three oncogenes AXIN2, DKK2, FGF9.
Since nuclear localization of ß-Catenin reflects an
Regarding the PI3K/AKT pathway we selected 16 genes activated WNT pathway, we investigated the ß-Catenin
that were significantly downregulated after SARB treat- distribution in HCT116 and HT29 cells before and after
ment whereas only four of them were deregulated in the treatment with SARB. HCT116 and HT29 untreated cells
same manner after Combi treatment (Suppl. Table 3).
showed nuclear ß-Catenin localization in a sub-
To validate that the changes in the gene expression population of cells in addition to pronounced membra-
pattern affect also the protein level, we investigated nous and cytosolic localization. Interestingly, when
treatment effects on the key players of the PI3K/AKT treating HCT116 cells with low concentrations of SARB
pathway. All treatments did not show strong effects on (10 µM), ß-Catenin was expressed in apoptotic nuclei
the total AKT and MEK levels. Individual compounds and with condensed chromatin and cells gain a more spindle
Combi led to increased levels of pAKT and pMEK, cell-like phenotype, an effect that was lost upon treatment
whereas SARB did not target or even slightly down- with 50 µM SARB (Fig. 3A). SARB treatment had no
regulated these proteins suggesting that the hybridization significant effect on cell migration, which supports our
strategy is superior to the single or Combi treatments at findings of no prominent alteration of cell differentiation
this point (Fig. 2G).
(Suppl. Fig. 4A). ß-Catenin was mostly lost from the
Fig. 3 WNT/ß-Catenin signaling pathway is a major target of SARB hybrid treatment. (A) Representative images of immunofluorescence
staining of ß-Catenin in HCT116 cells after treatment for 48 h with SARB 10 and 50 µM. Scale bar—25 µm. The lower panel shows images for the
control, SARB 10 µM, and 50 µM group which were digitally enlarged. (B) Representative images of immunofluorescence staining of ß-Catenin in
HT29 cells after treatment for 48 h with SARB 10 and 50 µM. Scale bar—25 µm. The lower panel shows images for the control, SARB 10 and 50 µM
group which were digitally enlarged. An additional image (derived from another replicate) of Hoechst-stained cells was added to this panel to show
the presence of micronuclei when treated with 50 µM of SARB. All images are representative for at least two independent experiments. (C) Western
Blot analysis of ß-Catenin in HCT116 and HT29 cells treated with different concentrations of SARB for 48 h. The star (*) indicates that the GAPDH blot is
the same as in Fig. 5f. (D) Western Blot analysis of ß-Catenin in SW620 and SW837 cells treated with different concentrations of SARB for 48 h. (E)
Western Blot analysis of ß-Catenin in HCT116 and HT29 cells treated with 35 µM of Combi or SARB for 48 h. The star (*) indicates that two other
treatments were excluded from this blot. The entire original blot is given in supplementary information. Western Blot images are representative for at
least two independent experiments. GAPDH immunoblots served as loading control. (F) HCT116 cells transfected with TOP-FOP-FLASH and ß-
galactosidase either treated with SARB for 24 h or left untreated and the data is shown as a percentage relative to DMSO. The reporter assay results
represent the average of two independent transfection experiments. RLU relative light units
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plasma membrane of the surviving population of HCT116 SW620, and SW837 (Fig. 3C, D) and seemed to be more
cells treated with SARB, suggesting that SARB might effective than Combi (Fig. 3E). This decrease in ß-Catenin
selectively disrupt the cell–cell contact in these cells. This protein levels was accompanied by a significant reduction
effect was less pronounced in HT29 cells where cell in transcriptional activity of ß-Catenin using a TCF/LEF
aggregates were still visible possibly leading to the higher reporter plasmid (Fig. 3F), suggesting that SARB hybrid
resistance against SARB (Fig. 3B). When evaluating the has the capability to interfere with WNT/ß-Catenin sig-
NanoString data for the available 30 ECM and adhesion- naling and to block its transcriptional activity.
associated genes we extracted six significantlly upregu-
lated genes (>1.5 fold; p-value ≤ 0.05), i.e. ITGA6, CD133 is targeted by Combi and SARB hybrid treatments
NOTCH1, LAMA3, LAMB3, THBS1, TGFB1 for SARB
treatment.
Since SARB targeted the CSC-related WNT/ß-Catenin
and PI3K/AKT-signaling pathways36, we next focused on
Interestingly, both cell lines showed nuclear anomalities the most common stem cell marker in CRC, namely
visible as micronuclei or nuclear buds, a phenomenon CD133 (PROM1), which was found to be significantly
35
described by Nersesyan et al. as a genotoxic effect (Fig. down-regulated by 5-FU, Combi, and SARB treatment in
3A, B). SARB effectively decreased the ß-Catenin levels in both cell lines (Fig. 4A, B). FACS analysis confirmed the
different colorectal tumor cell lines HCT116, HT29, significant reduction in CD133-positive tumor cell
Fig. 4 CD133 is targeted by the Combi and SARB hybrid treatments. (A) Western Blot analysis of CD133 marker in HCT116 and (B) in HT29 cells
after 48 h of treatment with TQ 40, 5-FU 15, Combi, and SARB 35 µM. (C) FACS analysis of CD133 population after treatment of HCT116 cells and (D)
HT29 cells with Combi and SARB for 48 h. Error bars indicate SEM calculated from three and two independent experiments for HCT116 and HT29,
respectively. (E) Protein–protein interaction of the genes which were downregulated under SARB and which belong to PI3K/AKT and WNT signaling
pathways generated via STRING. (F) Venn diagram of GEO dataset overlapping with the NanoString PanCancer Pathway Panel dataset. All 770 genes
were used from NanoString. (G) Venn diagram of the 77 overlapping genes from (f) with the downregulated genes (green line) and upregulated
genes (red line) in CD133− population deriving from the GEO dataset. (J) Table with the four downregulated overlapping genes and five
upregulated overlapping genes in Combi and SARB treatment.(H) Western Blot analysis of CD133 in HCT116 parental cells and the seven HCT116 CSC
enriched-sub-clones. (I) Western Blot analysis of CD133 expression in ST3 clone after 48 h of treatment with Combi and SARB at different drug
concentrations. GAPDH immunoblots served as a loading control. All Western Blot images are representative for at least two independent
experiments
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population after Combi and SARB treatment of HCT116 numbers by more than two-fold, however not significantly
cells (Fig. 4C). Using our FACS-specific CD133 antibody, (Suppl. Fig. 5D–G).
HT29 cells expressed CD133 only in <2% of cells (Fig. 4D).
In a second approach, we studied the ability of HCT116,
In SW620 cells we did not see any dose-dependent effect HT29, and 5-FU-resistant HCT116 cells to form 3D non-
of SARB on CD133 expression and the rectal SW837 cells adherent spheroids under different drug pre-treatments
did not express CD133 at all (data not shown). Next, (Fig. 5A). As expected for this model, HCT116 and HT29-
when PROM1 was additionally included in the STRING derived 3D spheres showed enrichment in the CD133
PI3K/AKT/WNT network, it showed a direct interaction CSC marker when compared to HCT116 cells grown in
with ß-Catenin (CTNNB1) (Fig. 4E).
2D (Fig. 5B). When CRC cells were pretreated with dif-
We next used the public GEO database (GSE3405337) to ferent concentrations of SARB, we observed a clear dose-
extract specific gene expression profiles of CD133+ and dependent attenuation of CSC-derived spheroids for all
CD133− colorectal tumor cells. When matching the gene three cell lines (Fig. 5C–E). As expected, 5-FU-resistant
panel of GSE34053 with our NanoString panel, we could cells showed the highest ability to form spheres (Fig. 5E).
acquire 77 genes that were included in both setups 5-FU treatment of HCT116 cells led to the formation of a
(Fig. 4F). We hypothesized that the gene signature after low number of larger spheres, whereas Combi treatment
SARB treatment might resemble the gene signature of the at 35 µM was as effective as SARB treatment at the same
CD133− population. From the 77 common genes, 27 concentration (Suppl. Fig. 6A). SARB treatment led to a
genes were overlapping with the downregulated genes in dose-dependent decrease in CD133 protein levels in all
the CD133− population (Fig. 4G). Four of these 27 genes three CRC lines (Fig. 5F–J).
were significantly downregulated also in SARB and
included the oncogene KRAS, the membranous protein SARB hybrid diminishes the in vivo growth of CAM
kinase PRKAR2B, which is known to regulate the MAP2 xenografts
kinase, the receptor for fibronectin ITGB8 and PROM1
In a next step, we studied the effects of all compounds
(Fig. 4J). From this panel, only two genes were sig- in vivo by employing the CAM assay, a model that has
nificantly downregulated in Combi. A similar result was been widely established for drug screening39. Tumor cells
achieved when comparing upregulated genes in CD133− were treated prior to CAM transfer since we hypothesize
and HCT116 cells under SARB exposure with four genes, that targeting CSCs should reduce the potential to
including inhibitor of angiogenesis THBS1 and PDGFD, develop vital tumors. Ex ovo and histological images of
which plays an important role in wound healing by HCT116 xenografts pretreated with the drugs for 48 h are
macrophage recruitment, calcium channel protein presented in Fig. 6A1–E2. We observed that HCT116
CACNB2, and SOCS3, an inhibitor of the JAK/STAT control cells developed substantially well-vascularized
pathway (Fig. 4j). Thus, we hypothesize that the two tumor masses, while tumor xenografts from 5-FU,
combination modalities, Combi and SARB treatments, Combi, and SARB pre-treated cells showed extended
might provide a selective advantage against CD133- areas of dead cells and calcification. TQ-treated cells
enriched tumor populations. To study this, we analyzed formed rather well growing vital tumors. Comparing
CD133-enriched HCT116 clones derived from spheroids vessel infiltration into the tumor cell matrix, we observed
by single cell dilution strategy as previously described38 a high vessel density with intense bleeding in 5-FU group
(Fig. 4H). Indeed, Combi and SARB treatments were (Fig. 6C2), whereas vascularization in CAM tumors of
highly effective in eradicating the CD133+ population in Combi or SARB pre-treated HCT116 cells was relatively
the CD133-enriched clone 3 (Fig. 4I).
low or moderate (Figs. 6F1, F2, G1, G2). The size of CAM
tumors derived from SARB pre-treated cells was strikingly
smaller when compared to the control group, whereas all
SARB hybrid targets self-renewal capacity of CRC cells
Since WNT and PI3K/AKT-abrogation is known to other treatments did not show any significant effects on
decrease the stem-cell like features of tumor cells we the tumor volume (Fig. 6H). There were significantly
evaluated the effect of SARB in sub-toxic doses using the reduced numbers of vital tumor cells in 5-FU, Combi, and
sphere propagation assay (Supp. Fig. 5A). Here, HCT116 SARB-treatment groups, where SARB seemed to be more
cells were continuously exposed to different sub-doses of effective than Combi (Fig. 6I). In contrast to SARB, Combi
SARB hybrid immediately after plating the cells. pre-treated tumors showed an increased peritumoral
Impressively, we found that even a low dose of 3 µM lymphoplasmocytic infiltration, some immune cells and
SARB was enough to significantly reduce the propagated mild fibrosis (Figs. 6F1, F2).
spheres already at the first generation, and spheroids were
When quantifying the number of mitoses, we observed
even completely eradicated at 6 and 10 µM (Suppl. Fig. cells that were still proliferating in the Combi-derived
5B, C). When following sphere formation over time, a xenografts, whereas SARB-derived tumors basically did
dose as low as 1 µM SARB was capable to decrease sphere not show any sign of mitotic activity (Fig. 6D2, E2, J), but
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Fig. 5 SARB hybrid targets self-renewal capacity of CRC cells. (A) Schematic overview of the protocol used for spheroid formation assay (SFA).
(B) Representative immunofluorescence images of CD133 staining (red) in 3D HCT116 and HT29 cells (n ≥ 2). Hoechst staining (blue) is used to stain the
nuclei. Scale bar—100 µm. Representative bright-field images of spheroids in the control group and in the treated ones with increasing concentration of
SARB in HCT116 (C), HT29 (D) and 5-FU resistant cells (E). Scale bar—250 µm. Sphere number quantification in the control and SARB-treated groups.
Data represent the mean SEM from two independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001; One way-ANOVA). Western blot analysis of
CD133 in (F) HCT116, (G) HT29, and (J) 5-FU-resistant cells after SARB treatment. GAPDH served as a loading control. Western Blot images are
representative for at least two independent experiments
Ki67 staining verified vital proliferating cells in both, Discussion
Combi and SARB-treated xenografts (Fig. 7A). When
Acquired therapeutic resistance of tumor cells con-
qualitatively evaluating ß-Catenin staining pattern we tributes to severe clinical restrictions in CRC. An addi-
observed a more membranous distribution after Combi tional limitation is the fact that many available drugs
and a more prominent nuclear ß-Catenin staining after target the fast dividing cells and not the CSCs, the drivers
SARB treatment (Fig. 7B). To assess a possible drug- of post chemotherapy recurrence40. Recent reports
dependent EMT induction we stained CAM xenografts showed that 5-FU treatment might even selectively enrich
with E-cadherin. In general the E-cadherin levels did not a subset of CSCs41,42. Here natural plant-derived com-
remakably change after Combi and SARB treatment pounds, such as TQ, could be potentially promising43.
(Fig. 7C), an effect that we also confirmed in Western Indeed, TQ treatment potentiates the effects of conven-
Blotting of 2D cell culture (data not shown). Interestingly, tional drugs in various cancers17,44
E-cadherin staining after Combi treatment was mainly We extended our study from single and Combi treat-
.
visible in the membrane and cytoplasm, whereas SARB ments to the treatment with new hybrid compounds. We
treatment resulted in a mostly nuclear pattern, especially identified the SARB hybrid as the most promising com-
in single cells and not when cells were clustered (Fig. 7C). pound against CSC-like cells. Although SARB showed
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Fig. 6 SARB hybrid diminishes the in vivo growth of CAM xenografts. (A1–E1) Representative light microscopy images of CAM tumors derived
from untreated (control) and pre-treated (single drugs, Combi and SARB) HCT116 cells and the respective H&E histological staining (A2–E2, ×10). The
ruler segments represent a length of 1 mm. (D2, E2) Computer-enlarged images of H&E staining’s show a rest of mitotic cells after combination
treatment (white arrows). H&E staining of micrografts derived from Combi pre-treated cells (F1, ×10) and (F2, ×40). H&E staining of micrografts
derived from SARB pre-treated cells (G1, ×10) and (G2, ×40). White arrows indicate areas of vital tumor cells, rosa color represents Matrigel. (H) Tumor
volume calculation in HCT116 CAM samples (*p < 0.05; One way-ANOVA). (I) Fraction of vital tumor cells and (J) evaluation of mitotic activity in CAM
sections. Mitosis in the vital tumor areas (average of five HPFs) (*p < 0.05; **p < 0.01; ***p < 0.001; One way-ANOVA) is given. Mann–Whitney t-test was
used for comparison of Combi and SARB (n ≥ 7). Scale bar—100 µm
similar antitumor effects as the Combi treatment in vitro, associated with loss of cell–cell contact thus diminishing
it was more effective in vivo in the CAM assay. Combi and cell adhesion. We selected six deregulated genes that are
SARB showed commonly deregulated genes of the cell associated with ECM and cell adhesion. The significance
cycle and PI3K pathway, an oncogenic trigger that is of this gene panel is discussed very controversy in CRC.
closely associated with tumor aggressiveness45. One As an example, THBSP1 decreased tumor cell adhesion
drawback for Combi treatment was the massive up- via promotion of the urokinase plasminogen activator48.
regulation of the proto-oncogene FOS, which in contrast In contrast, THBSP1 can also override these intrinsic pro-
was not affected under SARB exposure. Moreover, we adhesive effects, promoting the detachment of tumor cells
uncovered the WNT signaling pathway as the distinctive from primary tumor. Since these six genes were deregu-
pathway targeted only by SARB. Although this WNT lated in both Combi and SARB treatment group, their
effect appears to arise from TQ mediated signaling, it is function is context/stimulus dependent or might be not
not targeted by Combi treatment in the same manner. responsible for the observed effect on cell–cell adhesion
Target genes AXIN2, DKK2 and FGF9, well known for at all. Since migration capability and E-cadherin levels
their potential to promote tumor formation and pro- were unaffected by SARB, we exclude dramatic alterations
gression in vitro as well as in vivo in CRC, were sig- in cell plasticity and strongly believe that the tumor
nificantly downregulated in response to SARB46,47
.
simply dissociates. This is also in agreement with the
ß-Catenin has a bidirectional role also as cell adhesion inability of tumor cells to form spheroids or xenografts
molecule at the plasma membrane via interaction with E- after SARB treatment. The tumor grafts appear as single
Cadherin. Thus, SARB-induced loss of ß-Catenin together distributed cells with prominent nuclear E-cadherin and
with loss of E-cadherin at the plasma membrane might be ß-Catenin. In a first view ß-Catenin shuttling to the
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Fig. 7 Combination treatments affect proliferation and adhesion in vivo. Immunohistochemical stainings of (A) Ki67, (B) ß-Catenin, and (C) E-
cadherin of CAM-xenografts derived from control, Combi and SARB-treated HCT116 tumor cells. Upper panel shows ×20 magnifications; Scale bar—
50 µm. Lower panel shows ×40 magnifications; Scale bar—20 µm
nucleus would insist on an activation of WNT signaling. inhibitor p21 after Combi and SARB treatment might
However, nuclear E-cadherin is a potent inhibitor of ß- suggest an induction of senescence-like phenotype, we
Catenin transcriptional activity49, which we confirmed by found that in CAM tumors the survived cells were Ki67
a reduction of ß-Catenin on its TCF-responsive genes positive.
after SARB treatment. The higher resistance to SARB in
Another stem cell-related pathway, the PI3K/AKT
HT29 cells or in the surviving cell clusters in HCT116 pathway, was uncovered to be downregulated under
xenografts of Combi treatment could be caused at least SARB50. The key players of this pathway, such as pAKT,
partially by the consistent level of ß-Catenin in the cell AKT, and MEK proteins were found to be slightly
membranes. Although the upregulation of the cell cycle downregulated
under
SARB
treatment.
More
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convincingly 16 genes associated with the PI3K/AKT 600 MHz. ESI mass spectra were recorded on a Bruker
pathway extracted from the NanoString panel were micrOTOF II focus TOF MS-spectrometer. Elemental
deregulated under SARB exposure but only four of them analysis (C, H, N), carried out with an Euro EA 3000 (Euro
under Combi. Conversely, the single compounds and Vector) machine and an Elementar vario MICRO cube
Combi treatment led even to increased phospho-AKT machine, calculated values confirm a purity of >95%. We
levels. This SARB-specific effect is promising since recent obtained the crystals of SARB hybrid in solvent acetoni-
clinical data of Phase 1 and 2 support the potent impact of trile and molecular structure was unambiguously deter-
PI3K/AKT or WNT/B-Catenin inhibitors51,52
.
mined by X-ray crystallography (Fig. 1). The data for the
Given that WNT signaling pathway and CD133 are crystal structure of SARB hybrid can be found free of
interconnected in promoting resistance to conventional charge in: The Cambridge Crystallographic Data Centre
therapies10,41,53–55, we showed dramatically decreased (via www.cam.ac.uk/data_request/cif with the deposition
CD133 level after SARB and Combi treatment. This number CCDC-1856382).
suggests that SARB might target a broader spectrum of
CSC-related players. Indeed the self-renewal capacity was
targeted by SARB in two different functional assays, the
Cell lines and reagents
Human colon carcinoma HCT116 and HT29 cells were
spheroid formations assay and the propagation assay. In purchased from ATCC and maintained in RPMI 1640
the CAM model a pre-treatment protocol was used and (PAN Biotech) supplemented with 10% FBS (PAN Biotech)
only the resistant tumor cell population was transplanted. and 1% penicillin and streptomycin (PAN Biotech). Rectal
If CSCs were eradicated, the tumor cells should have lost SW837 cells were kindly provided by Dr. Kaulfuß (Institute
the potential of self-renewal and should not grow well on of Human Genetics in Göttingen) and mantained in the
the CAM. Both, Combi and SARB treatment significantly same conditions as HCT116 and HT29. SW620 cells
reduced the tumor growth, only small cell aggregates or derived from metastatic site, were purchased from ATCC
single cells were visible. The reduction in ß-Catenin levels and maintained in DMEM medium (Gibco/Life Tecnolo-
after SARB treatment was a general effect whereas the gies) supplemented with 10% FBS and 1% penicillin and
reduction of the CD133+ population was cell line specific. streptomycin. Normal human intestinal epithelial HCEC
Thus, we suggest that SARB has the potential to reduce at cells were grown in basal HCEC medium (PAN Biotech)
least one of these two stemness players. We also cannot supplemented with L-glutamine (Gibco/Life Tecnologies),
exclude treatment effects on other CSC-related markers.
ascorbic acid (Sigma), dexamethasone (Sigma Aldrich),
From NanoString analysis, we learned that retinoic acid (Sigma Aldrich), and bovine pituitary extract
angiogenesis-associated genes, such as FZD3, MAPK3, (PromoCell). 5-FU resistant HCT116 cells were obtained
AKT3, VEGFA, CTNNB1, and FOS, were significantly from the group of Prof. Lee M. Ellis (MD Anderson, TX,
downregulated after SARB treatment. These anti-vascular USA) and cultured in minimal essential medium (MEM,
properties of SARB− and partly of Combi were visible in Sigma-Aldrich) enriched with 10% FBS, 1% sodium
the CAM model. Interestingly 5-FU, led to remarkable pyruvate 100 mM (Gibco/Life Tecnologies), 1%
bleeding in the grafts, a well-known side effect that could MEM NEAA non-essential amino acids 100× solution
be associated with the spreading of tumor cells56. In (Gibco/Life Tecnologies), MEM Vitamin solution
Combi-treated grafts the tumor environment showed an 100× (Gibco/Life Tecnologies), 1% L-glutamine 200 mM
accumulation of desmoplasia with increased peritumoral 100x (Gibco/Life Tecnologies), and 1% P/S (Gibco/Life
lymphoplasmocytic infiltration and fibrosis. Thus, we Tecnologies). In order to maintain the resistance, 7.7 µM of
suggest that SARB might have a lower side-effect spec- 5-FU were added to the cells with each medium change.
trum than the Combi or single drug treatments.
The cell lines were mycoplasma free and were authenticated
This work supports the hypothesis that linking two using Multiplex Cell Authentication by Multiplexion.
molecules into a single compound did not simply reflect
the Combi-treatment effects, but rather showed targeting
Chemical compounds
of a completely new gene signature. Our findings already
The single compounds, 5-fluorouracil (5-FU) and thy-
give valuable insights into the potential mechanism of moquinone (TQ) were purchased from Sigma Aldrich. All
action of the novel hybrid drug candidate SARB for CRC. investigated compounds were dissolved in 100% dimethyl
sulfoxide (DMSO) (Pan Biotech) to 50–200 mM stock
Materials and methods
Chemical evaluation
solutions, and stored at −20 °C. The stock solutions were
further diluted in cell culture medium.
Detailed information about synthesis of hybrid com-
1
pounds are given in the supplementary information. H Crystal violet assay
NMR and 13C NMR spectra were recorded at RT on a
Crystal violet was performed as previously described26.
Bruker Avance spectrometer operating at 300, 500, and Briefly, HCT116, HT29, and HCEC cells were seeded at a
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density of 7.5 × 103 cells/well in 100 μl of growth medium in 50% Matrigel and 10 µl of the suspension were seeded
in 96-well plates (Corning). After 24 h the cells were into pre-warmed 96-well plates. After solidifying of the
treated with the compounds at various concentrations for Matrigel, the crypt droplets were overlaid with 100 µl
48 h. IC50 values were calculated from at least two bio- IntestiCult™ organoid growth medium. Three days after
logical replicates each performed in at least three tech- seeding (passage 2) the organoids were treated (passage 2,
nical triplicates.
day 3–5 after seeding) with the corresponding drugs; TQ
40, 5-FU 15, Combi 35, SARB 35, and SARB 50 µM for
48 h. The viability of the organoids was assessed by
Annexin V assay for apoptosis detection
Apoptosis was quantitated by flow cytometry using the CellTiter-Glo® 3D Cell Viability Assay (#G9682, Promega)
Annexin-V-FITC staining kit (Miltenyi), according to the according to the manufacturer’s instructions.
manufacturer´s protocol. Shortly, 5 × 105 HCT116 and
HT29 cells were seeded in 6 cm cell culture dishes and
Collection of the cell pellets for RT-qPCR and NanoString
treated after 24 h of adherence, with TQ 40, 5-FU 15, analysis
Combi 35 + 35, and SARB 35 μM. Floating and adherent
HCT116 cells were used for NanoString analysis and
cells were harvested after 48 h of treatment by trypsini- RT-qPCR. The cells were treated at 50–60% confluency
zation. After inactivation of trypsin with 2 ml of growth with TQ 40, 5-FU 15, Combi 35, and SARB 35 µM for
medium, cells were centrifuged, washed in binding buffer 24 h. The collection of cell pellet was performed on ice
(included in the kit), stained with Annexin-V-staining and the cells were scraped from the plates and the
solution (10 μl Annexin-solution in 100 μl of binding respective supernatants with the cells were transferred to
buffer) and incubated for 15 min at RT in the dark. The a 50-ml tube, washed with ice cold PBS and the remaining
washing step was repeated, the cells were resuspended in cells were scraped and centrifuged for 5 min, 5000 rpm at
500 μl binding buffer and stained with 5 μl propidium 4 °C. The supernatants were then discarded and the cells
iodide (PI) directly before analysis with a BD FACSCanto® were washed with 1 ml cold PBS, transferred to 1.5 ml
II flow cytometer (BD Biosciences) and FlowJo software. tube and centrifuged another time with the same settings.
Unstained cells served as a background reference and The supernatants were then discarded, the pellets were
untreated cells as negative controls.
frozen in liquid nitrogen and stored at −80 °C.
Drug treatment of ex vivo crypt-derived organoids
In order to evaluate the cytotoxicity of our compounds
RT-qPCR analysis
RNA was isolated using QIAzol® Lysis Reagent (Qiagen)
in normal cells, we treated murine intestinal crypt-derived in combination with RNeasy Mini Kit (Qiagen) according
organoids that were grown ex vivo. We used the protocol to the manufacturer’s protocols. cDNA synthesis was
of Toshiro Sato57 with minor variations. The small performed with the QuantiTect Reverse Transcription Kit
intestine from 8-week-old C57BL/6J was harvested and (Qiagen) according to the manufacturer. CFX96TM Real-
washed with a syringe to remove feces. After several Time System (Bio-Rad) and the C1000TM Thermal Cycler
washing steps, the SI was opened longitudinally and villi (Bio-Rad) were used to measure the Ct values for inves-
were scraped off using a cover slip. Then the SI was cut tigated genes which were normalized to human B2M
into 2–4 mm large fragments and put into PBS. Four to expression. The primers used in this study are given in
five washing steps with fresh PBS were done to remove Suppl. Table S4.
any villi or fecal contamination. Afterwards, the fragments
were incubated for 15 min at RT with the gentle dis- NanoString gene expression analysis
sociation solution (STEMCELL Technologies) and crypts
The NanoString PanCancer Pathway panel gene
were liberated by vigorous shaking. The fragments were expression assay was performed according to the manu-
removed and the crypts in the supernatant were pelleted. facturer´s instructions with 50 ng of total RNA. Data
The crypts were resuspended in Matrigel and 20 µl of the processing was performed using nSolver Analysis Soft-
crypt–Matrigel suspension were seeded in a 48-well plate. ware 3.0 (NanoString Technologies). All samples were
The Matrigel droplet was incubated for 10 min at 37 °C normalized using the geometric mean of the house-
and finally overlaid with 250 µl of IntestiCult™ organoid keeping genes. Differentially expressed genes between the
growth medium (Mouse) (STEMCELL Technlogies). treated and the untreated control cells were given as fold
Fresh medium was supplied every 2–3 days and organoids change expression. Deregulated KEGG pathways were
were passaged every 5–7 days. After the first passage, fully identified via STRING software (http://string-db.org). The
developed organoids were liberated from the Matrigel and Gene Expression Omnibus (GEO) database (GSE34053
washed with PBS several times. Organoids were then dataset) was used to identify the expression profile of key
physically disrupted by pipetting up and down to release genes in CD133 colon cancer populations. The GEO2R
single crypts. Single crypts were then equally resuspended web tool was used to identify differentially expressed
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genes. NanoString results are deposited at GEO and manufacturer´s suggestion with mouse anti-human
GSE122860 accession number provides access to the raw CD133/1 (AC133) conjugated with VioBright FITC
and processed data.
(#130-105-226, 1:11; Miltenyi Biotec), or the IgG isotype
control after blocking the Fc receptors with the FcR-
blocking reagent (# 130‑059-901). The analysis was made
Western blot analysis
Western blot analysis was performed as previously with a BD FACSCanto® II flow cytometer (BD Bios-
described38. Briefly, 30 or 40 μg of total protein were ciences) and FlowJo software. All events were included in
separated by 10% denaturing SDS–PAGE and transferred the analysis.
onto 0.45 μm nitrocellulose membranes overnight. The
membranes were incubated with primary ß-Catenin (6B3)
(1:1000, Cell Signaling, #9582), CD133 (1:250, Miltenyi,
Wound healing assay
HCT116 cells were seeded in culture plates (six-well
#130-092-395), p-AKT (Ser 473) (D9E) XP (1:1000, Cell plates) and allowed to adhere for minimum one day in the
Signaling, #4060), AKT (1:1000, Cell Signaling, #9272), p- respective medium. The scratch was done in three dif-
MEK1/2 (Ser217/221) (1:1000, Cell Signaling, #9121), or ferent area of the well when the cells were completely
MEK1/2 (1:1000, Cell Signaling, #9122) antibodies over- confluent, using a sterile 200 µl tip and the plates were
night at 4 °C. Secondary horseradish-peroxidase-coupled washed with PBS. The medium alone or with the com-
antibodies goat-anti-mouse IgG (H + L) (1:10,000, pounds (Combi 35 µM or SARB 35 µM) was added to the
Thermo Fisher Scientific) or anti-rabbit IgG (H + L) cells and the plates were monitored immediately after the
(1:10,000, Thermo Fisher Scientific), and anti-biotin, treatment, corresponding to 0 h, as well as at 24 and 48 h.
HRP-linked antibody (1:5000, Cell Signaling) were then The data is presented as the percentage of the wound
applied for 1 h at RT. Hybridization with GAPDH-HRP closure which was calculated: (wound length at 0 h)
(6C5) (1:10,000–20,000, Abnova, #MAB5476) coupled −(wound length at 24 or 48 h)/(wound length at 0 h) ×
antibody was performed for 30 min at RT as a house- 100. The data are calculated from three independent
keeping gene. Antibodies were visualized using the experiments, each done in duplicate.
Immobilon Western Chemiluminescent horseradish per-
oxidase (HRP) substrate kit (Merck Millipore). Images Spheroid formation assay (SFA)
were generated using Gene Genome Syngene Bio Imaging
To investigate the capacity of CRC cells to grow under
and quantified using Image J (Rasband, W.S., U.S. non-adherent conditions, cells were seeded at a density of
National Institutes of Health).
1.5 × 106 cells in 10 cm plates. After 24 h of recovery, the
cells were treated with increasing concentrations of SARB
(10, 20, 50 μM) or Combi 35 µM. Incubation continued
TOP/FOP-FLASH Luciferase Reporter assay
HCT116 cells (20 × 103) were co-transfected for 6 h for another 48 h, then the cells were washed with PBS,
before 24 h treatment with 10, 20, 50 µM SARB hybrid trypsinized and 5 × 104 cells were then seeded in ultra-low
either with 100 ng Super8xTOP-FLASH or 100 ng attachment (ULA) six-well plates (Corning) in 2 ml of
Super8xFOP-FLASH, respectively, and 10 ng ß-galactosi- CSC medium (PromoCell) and 40 μl Matrigel with growth
dase reporter plasmid for normalization of protein factors (Corning). Spheroid formation was monitored
amounts. Plasmids were kindly provided by Prof. Trevor 7 days after seeding and then the spheroids were trans-
Dale, Cardiff University School of Biosciences. The ferred to 15 ml falcon tubes, washed with PBS, trypsinzed,
reporter activity was assessed by adding 50 µl of Beta-Glo and 2.5 × 104 cells were seeded in ULA six-well plates
(#E4720, Promega) or 50 µl of Bright-Glo (#E2610, Pro- with 2 ml CSC medium and 40 μl Matrigel. Cells were let
mega) to the 50 µl of lysed samples, following the man- grow for another 14 days and fresh medium was added
ufacturer instructions and measured by a luminometer every 2–3 days. At day 21, the spheroid growth was
(VictorTM X3 Multilabel reader). The results were nor- documented by light microscopy using a Nikon Eclipse
malized to total protein amounts and were expressed Ti–S microscope or Leica DMI1 light microscope. The
relative to DMSO controls.
images were then analyzed using a macro (given in SI)
which was imported to ImageJ (modified from ref. 58) to
determine spheroid number.
Flow cytometry analysis of CD133
HT29 and HCT116 cells were treated at 50–60% con-
fluence for 48 h with Combi 35 µM, SARB 35 µM or were
left untreated. After trypsinization the cells were collected
Propagation assay
For the propagation assay, single cell suspensions of
in 100 µL of buffer solution containing PBS, 0.5% BSA, HCT116 cells were suspended in Matrigel (Growth Factor
and 2 mM EDTA by diluting MACS BSA Stock Solution Reduced) and in serum-free RPMI medium (1:1) at a
(# 130‑091‑376, 1:20) with auto MACS rinsing solution concentration of 2000 cells/well in a total volume of 50 µl.
(#130‑091‑222) and were incubated according to the The solution was plated in 24-well plate and was allowed
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to solidify for 1 h at 37 °C. The RPMI medium supple- incubated with a 1:250 goat anti-mouse dilution of the
mented with 5% FBS and 1% P/S without or with the drug secondary antibody (Alexa Fluor 555, Invitrogen), where
at the indicated concentrations was added in triplicate. the cells labeled with ß-Catenin were incubated with
The medium containing the drug was refreshed every 1:100 goat anti-rabbit IgG (Alexa Fluor 555, Invitrogen)
2–3 days. The spheres were counted and harvested for 1 h RT, washed again, stained with 1:1000 Hoechst
between 9 and 13 days after plating (generation 1). For the 33342 (Sigma) in PBS (1 mg/ml stock solution) per well
propagation, the spheres were collected and the Matrigel and imaged with a Nikon Eclipse Ti–S.
was digested by incubation in 500 µl of serum-free media
containing dispase (1 mg/ml) (Corning) for 1 h at 37 °C. Chorioallantoic membrane (CAM) assay and
The resulting sphere containing solution was collected,
centrifuged and the resulting pellet was re-suspended in
immunohistochemical staining
The chick embryo CAM xenograft assay was performed
Trypsin (5–10 min) and later inactivated by adding RPMI as previously described37,38. Shortly, 1 × 106 HCT116 cells
media (5% FBS). The cells were than centrifuged, washed, were pretreated with the compounds and were applied in
suspended in serum-free RPMI and the number of viable a 1:1 medium-Matrigel mixture (Corning) onto the CAM.
cells was determined using trypan blue. Cells were again After 5 days of incubation the generated micro-tumors
suspended in Matrigel-medium mixture and were plated were harvested. The CAM tumors were photographed
as described above. Spheres were propagated up to five and the tumor volume was calculated using the formula:
generations and the number of spheres was calculated for V = (L × W × H)/2 (n ≥ 9 for all groups). The CAM
each generation using the same macro as for the SFA.
microtumours were then fixed in 4% phosphate buffered
formalin for 24 h, dehydrated and embedded in paraffin.
The HE staining and the scanning of the slides were done
as previously described38. The percentage of vital tumor
Generation of spheroid-derived CD133 enriched
sub-clones
We used HCT116 cells which were seeded into 30–35% cell areas and mitosis was evaluated (n = 7 control-CAM,
Matrigel (growth factor reduced, Corning) in RPMI n = 7 TQ-CAM, n = 7 5 FU-CAM, n = 8 Combi-CAM,
medium (supplemented with 10% FBS + 1% P/S; 1× and n = 8 SARB-CAM microtumors).
volume Matrigel + 2× volume medium). A total volume
The rate of mitotic cells was determined using HE-
of 2 × 103 single cells/well was seeded into one well of a stained sections of the CAM micro-tumors and the high-
six-well plate, incubated at 37 °C for 15 min until the power-field (HPF) method. Immunohistochemical label-
Matrigel solidified and covered with 300–500 µl medium. ing for β-Catenin (1:50, BD Bioscences), E-cadherin
The medium was changed every 2–3 days and after 7 days (1:2000, BD Bioscences), and Ki67 (1:100, Dako) was
of incubation, single cell colonies were picked gently with performed according to standard routine procedures for
a tip of a sterile toothpick. The individual colony was then immunohistochemical analysis.
transferred into one well of a 96-well plate, which was
filled with 100 µl RPMI medium beforehand. The cells Statistical analysis
were cultivated until a colony started to grow and only
All statistical analyses (t-test and one-way ANOVA)
wells with single colonies were used. Seven cell lines were performed using GraphPad Prism 7 (version 7.0,
(ST1–ST7) were generated from these clones, which were GraphPad Software Inc., La Jolla, CA, USA).
then used for further experiments. The generated clones
Acknowledgements
We thank the research group of Dr. Sendurai A. Mani (MD Anderson Cancer
Center) for their helpful discussions. B.N. was supported by a traveling grant of
were monitored by microscopic images (Leica DMi1,
Leica Microsystems).
the Boehringer-Ingelheim-Foundation. The present work was performed in
partial fulfillment of the requirements for obtaining the degree Dr. rer. nat. for
B.N. and A.C. at the FAU Erlangen-Nuremberg. R.S.-S. and S.B.T. thank the
Wilhelm Sander Stiftung for financially supporting this project (No. 2014.019.1).
Immunofluorescence staining
2D HCT116 and HT29 cells were grown (8 × 104 cells/
well) in eight-well chambered coverslips (Ibidi) and trea-
ted after 2 days of adherence for 48 h with increasing
concentrations of SARB (10, 20, 50 µM). Spheroids (3D)
at day 21 derived from SFA and 2D cells were washed
with PBS, fixed with 4% paraformaldehyde for 20 min at
RT, permeabilized with 0.1% TritonX in PBS (15 min at
RT), blocked for 30 min with 3% BSA (w/v) in PBS, and
stained with a 1:250 dilution of primary antibody against
CD133 (Miltenyi Biotec) or 1:100 of ß-Catenin (D10A8)
XP (Cell Signaling) overnight at 4 °C. The next day, after a
washing step, the spheroids stained with CD133 were
Financial support by the German Academic Exchange Service DAAD (doctoral
research fellowship for Aysun Çapci) is also gratefully acknowledged. This
article is based upon work from COST Action CA17118, supported by COST
(European Cooperation in Science and Technology). www.cost.eu. Partial
support was provided by the Medical Practice Plan of the American University
of Beirut for propagation assay. We thank Rudolph Jung, Martina Morgenroth,
Adrian Koch and Ingrid Mons for their excellent technical support.
Author details
1University Hospital, Friedrich-Alexander-University Erlangen-Nuremberg,
Experimental Tumorpathology, Institute of Pathology, Erlangen, Germany.
2University Hospital, Friedrich-Alexander University of Erlangen-Nuremberg,
Institute of Pathology, Erlangen, Germany. 3Organic Chemistry Chair I and
Interdisciplinary Center for Molecular Materials (ICMM), Friedrich-Alexander
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University Erlangen-Nuremberg, Erlangen, Germany. 4Department of
Pharmacology and Physiology, Faculty of Pharmaceutical Sciences,
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15. Toden, S., Tran, H. M., Tovar-Camargo, O. A., Okugawa, Y. & Goel, A.
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Chulalongkorn University, Bangkok, Thailand. 5Department of Biology and
Center for Drug Discovery, American University of Beirut, Beirut, Lebanon.
6Department of Pathology, Federal University of Health Sciences of Porto
Alegre (UFCSPA), Porto Alegre, Brazil
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Author contributions
B.N. and A.C. performed the experimental work. B.N., A.C., R.S.-S. and S.B.T.
wrote the paper. S.B.T. and A.C. designed the compounds for biological
studies. A.C. and V.K. performed chemical synthesis of the hybrids under
supervision of S.B.T. P.C. assisted with the spheroid assay. J.K.M. contributed to
the experimental work regarding the CAM assay. K.H. prepared the organoid
cultures. S.S. generated the HCT116 clones. K.E.-W., A.A., A.H., and A.V.R.
performed the immunohistological analysis of CAM xenografts and C.I.G.
assisted with the digital analysis. F.B. assisted with the propagation assay. C.E.-B.
was involved in initial experiments for the combination study. H.G.-M. critically
reviewed and edited the manuscript. R.S.-S. and S.B.T. designed and supervised
the experimental outline of the study.
Conflict of interest
21. Kensara, O. A. et al. Thymoquinone subdues tumor growth and potentiates
the chemopreventive effect of 5-fluorouracil on the early stages of colorectal
carcinogenesis in rats. Drug Des. Dev. Ther. 10, 2239–2253 (2016).
22. Lei, X. et al. Thymoquinone inhibits growth and augments 5-fluorouracil-
induced apoptosis in gastric cancer cells both in vitro and in vivo. Biochem.
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approach towards new molecular entities. Chem. Soc. Rev. 31, 324–334 (2002).
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for drug discovery. Angew. Chem. Int. Ed. Engl. 42, 3996–4028 (2003).
25. Tsogoeva, S. B. Recent progress in the development of synthetic hybrids of
natural or unnatural bioactive compounds for medicinal chemistry. Mini Rev.
Med. Chem. 10, 773–793 (2010).
The authors declare that they have no conflict of interest.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary information accompanies this paper at (https://doi.org/
10.1038/s41419-019-1611-4).
Received: 6 December 2018 Revised: 6 April 2019 Accepted: 17 April 2019
26. Frohlich, T. et al. Synthesis of novel hybrids of thymoquinone and artemisinin
with high activity and selectivity against colon cancer. ChemMedChem. 12,
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