Novel broccoli sulforaphane-based analogues inhibit the progression of pancreatic cancer without side effects

Article abstract of DOI:10.3390/biom10050769

The naturally occurring isothiocyanate sulforaphane, found in Brassicaceae vegetables, is promising in cancer treatment, e.g., by the normalization of enhanced levels of NF-κB-signaling in tumor stem cells. We chemically synthesized seven sulforaphane ana

Full text of DOI:10.3390/biom10050769

biomolecules
Article
Novel Broccoli Sulforaphane-Based Analogues
Inhibit the Progression of Pancreatic Cancer without
Side Effects
Christina Georgikou 1,†, Laura Buglioni 2,†, Maximilian Bremerich 2, Nico Roubicek 2, Libo Yin 1,
Wolfgang Gross 1, Carsten Sticht 3, Carsten Bolm 2, ,‡
*
and Ingrid Herr 1, ,‡
*
1
Section Surgical Research, Molecular OncoSurgery, Department of General, Visceral and Transplantation
Surgery, University of Heidelberg, 69120 Heidelberg, Germany; christine.georgikou@outlook.com (C.G.);
yinlibo001@163.com (L.Y.); WGross@uni-hd.de (W.G.)
Institute of Organic Chemistry, RWTH Aachen University, 52056 Aachen, Germany; l.buglioni@tue.nl (L.B.);
maximilian.bremerich@rwth-aachen.de (M.B.); nico.roubicek@rwth-aachen.de (N.R.)
Medical Research Center, Medical Faculty Mannheim, University of Heidelberg, 69117 Mannheim, Germany;
carsten.sticht@medma.uni-heidelberg.de
2
3
*
Correspondence: carsten.bolm@oc.rwth-aachen.de (C.B.); i.herr@uni-heidelberg.de (I.H.);
Tel.: +49-241-809-4675 (C.B.); +49-6221-56-6401 (I.H.); Fax: +49-241-809-2391 (C.B.); +49-6221-56-6402 (I.H.)
Shared first authorship.
†
‡
Shard last authorship and corresponding authors.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 April 2020; Accepted: 12 May 2020; Published: 15 May 2020
Abstract: The naturally occurring isothiocyanate sulforaphane, found in Brassicaceae vegetables,
is promising in cancer treatment, e.g., by the normalization of enhanced levels of NF-κB-signaling
in tumor stem cells. We chemically synthesized seven sulforaphane analogues by substitution
of the sulfinyl group (S(O)) to either sulfimidoyl (S(NR)) or sulfonimidoyl (S (O) (NR)) groups,
and characterized them in the cell lines of pancreatic cancer and several other tumor entities, including
the NCI-60 cell panel. MTT and colony forming assays, flow cytometry, immunohistochemistry,
microRNA arrays, bioinformatics, tumor xenotransplantation, and Kaplan Meier survival curves were
performed. Compared to sulforaphane, the analogue SF102 was most efficient in inhibition of viability,
colony formation, tumor growth, and the induction of apoptosis, followed by SF134. Side effects were
not observed, as concluded from the body weight and liver histology of chick embryos and survival of
C. elegans nematodes. Among 6659 differentially regulated microRNAs, miR29b-1-5p, and miR-27b-5p
were downregulated by sulforaphane compared to controls, but upregulated by SF102 and SF134
compared to sulforaphane, suggesting differential signaling. Each substance was involved in the
regulation of several NF-κB-related target genes. In conclusion, sulforaphane analogues are promising
for the development of highly active new drugs in cancer treatment.
Keywords: bioactive agents; drug development; microRNA signaling; pancreatic cancer; NCI-60;
sulforaphane; sulfoximine
1. Introduction
Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer-related death in the US,
with a 5-year relative survival of 9% calculated for all stages, and of 3% when the tumor has spread [
The development of new therapeutic options is urgently needed.
1,2].
Epidemiological studies indicate that higher consumption of Brassicaceae vegetables is correlated
with a lower cancer risk [ ]. Recent pilot studies with sulforaphane-enriched broccoli sprout
supplementation in patients with advanced pancreatic or prostate cancer showed promising
3
Biomolecules 2020, 10, 769; doi:10.3390/biom10050769
www.mdpi.com/journal/biomolecules

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results [
4,
5]. Brassicaceae are unique in their high content of glucosinolates [
6
]. A focus has
been placed on the glucosinolate glucoraphanin, which is found in high concentration in broccoli
and its sprouts. Glucoraphanin is converted to its active form, the isothiocyanate sulforaphane
[1-isothiocyanato-(4R)-(methylsulfinyl)-butane], by myrosinase-mediated hydrolysis [
is one of the best studied bioactive anti-inflammatory agents and suggested to induce detoxifying
enzymes, cell cycle arrest, apoptosis and epigenetic regulation [ ]. In pancreatic cancer, sulforaphane
normalized increased NF- B signaling in tumor stem cells by the induction of miRNA-365 expression,
7]. Sulforaphane
6
κ
which inhibited the expression of the NF-κB subunit c-Rel by binding to its 3´-UTR [8,9].
The development of highly active sulforaphane analogues suited for medication is urgently
required. However, the establishment of sulforaphane analogues by molecular single atom changes
in form of oxygen-to-nitrogen substitutions at the central sulfur, are unprecedented in the context of
sulforaphane chemistry. This is surprising, considering that a significant number of other structural
variations of sulforaphane are known [10–13], and that such one-atom modifications have recently led
to significant improvements in other medically relevant areas [14–20].
In the present study, we prepared seven analogues of sulforaphane by formal substitutions of
the sulfinyl group by sulfimidoyl or sulfoximidoyl moieties. The R substituent at the nitrogen was
acetyl, pentafluorobenzoyl, methyl, trifluoroacetyl, or carbamoyl. All products represented monoaza
analogues of sulforaphane or its sulfone, and were prepared as racemates. The analogue SF102 was
most effective in inhibition of viability, clonogenicity, and tumor growth, along with induction of
apoptosis in cell lines of various tumor entities. No obvious side effects were observed. miRNA
array profiling revealed differential signaling of sulforaphane, SF102, and SF134, but the regulation of
NF-κB-related target genes was common.
2. Materials and Methods
2.1. Chemical Syntheses of Sulforaphane Analogues
We prepared 7 monoaza racemate analogues of sulforaphane (SF85 and SF101) or its sulfone (SF86
,
SF102 SF113 SF134, and SF135), which differ from the parent compound by formal substitutions
,
,
of the sulfinyl (S = O) group by either sulfimidoyl (S(NR)) or sulfoximidoyl (S(O)(NR)) moieties.
The R substituent at the nitrogen is acetyl (as in SF85 with S(NAc) and SF86 with S(O)(NAc)),
pentafluorobenzoyl (as in SF101 with S(NC(O)C6F5) and SF102 with S(O)(NC(O)C6F5)), methyl (as in
SF113 with S(O)(NMe)), trifluoroacetyl (as in SF134 with S(O)(NCOCF3)), and carbamoyl (as in SF135
with S(O)(NCONH2)). The product purities were generally >95% (as determined by NMR spectroscopy
or HPLC). For the two most active compounds, the following purities were found: SF134 ≥ 99.5%
(as single batch); SF102 95.6%, 96.0%, and 92.3% (as three batches). The detailed description is provided
(Supplemental Information 1, Figure S1).
2.2. Human Tumor Cell Lines
Established cell lines from pancreas (BxPc-3, AsPC-1), ovary (OVM), prostate (PC3), breast (BT-20),
colorectum (SW707), glioblastoma (A172), liver (HepG2), neuroblastoma (IMR5), and T cell leukemia
(Jurkat) were obtained from ATCC (Manassas, VA, USA). Cervical (P5) and lung (P693) cell lines and
gemcitabine-resistant pancreatic cancer BxGEM cells are described [21,22]. Jurkat cells were cultured
in RPMI-1640 medium, supplemented with 10% heat-inactivated FCS (both from Sigma-Aldrich,
Deisenhoffen, Germany) and 25 mmol/L HEPES (PAA, Pasching, Austria). All other cancer cell
lines were cultured in a DMEM/high glucose medium, supplemented with 10% heat-inactivated FCS
(Sigma-Aldrich) and 25 mmol/L HEPES (PAA). Mycoplasma detection tests were performed monthly
by the use of PlasmoTest™ (InvivoGen, San Diego, CA, USA).

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2.3. Reagents
Lyophilized powder of D,L-Sulforaphane (Sigma Aldrich) and its analogues were dissolved in
DMSO to stock concentrations of 100 mM. Aliquots were stored at –80 ◦C. Each stock was used only
once after thawing. DMSO alone was used as vehicle control diluted 1:1000 or higher.
2.4. Cell Viability Assay
Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), as previously described [23].
2.5. Colony Forming Assay
The cells were seeded at a density of 2–4
were treated, trypsinized 24 h later and analyzed as described [23].
×
104 cells/well in 6-well cell culture plates. The cells
2.6. NCI-60 Human Tumor Cell Lines Screen
This screen was performed by the NCI, Division of Cancer Treatment & Diagnosis (https:
//dtp.cancer.gov/discovery_development/nci-60/default.htm). It utilized 60 human tumor cell lines to
characterize the growth inhibitory or killing activities of a therapeutic subtstance.
2.7. Tumor Xenotransplantation on Fertilized Chicken Eggs
The tumor xenotransplantation to genetically identical hybrid Lohman Brown chicken eggs was
performed as previously described [24]. On day 9 of embryonic development, 7
cells were transplanted at a ratio of 1:1 with Matrigel onto the chorioallantoic membrane (CAM) of each
egg. Five days later, sulforaphane, its analogues were injected into CAM vessels (50 L/egg, diluted
in PBS). Control eggs were injected with 50 L PBS alone. The xenografts were resected on day 18,
×
105 BxPc3 or BxGEM
µ
µ
and the tumor volumes were evaluated 3-dimensionally by a USB microscope camera (eScope, Oitez,
Hongkong) and digital image editing, using a customized mount.
2.8. Immunohistochemical Staining
Staining was performed on 6-µm frozen tissue sections from the tumor xenografts, as previously
described [24]. A mouse monoclonal antibody against Ki67 (ab8191, 1:400, Abcam, Cambridge, UK)
was used, and positive signals were quantified with the ImageJ software (NIH, Bethesda, MD, USA).
2.9. Caenorhabditis Elegans Survival
◦
Caenorhabditis elegans (C. elegans) N2 nematodes (CGC, Minnesota, USA) were kept at 20 C.
L4-stage worms were picked (n = 100/group) and transferred into 60 mm medium-sized plates (TPP,
Trasadingen, Switzerland) containing S Basal medium, supplemented with E. coli OP50 bacteria [25].
Sulforaphane and analogues (400 µM), or DMSO alone, were added to S Basal medium for 48 h. Live
worms were transferred daily to new plates until they stopped laying eggs. Worms that died of causes
other than aging, such as internal hatching or vulva protrusion, were excluded. The survival rates
were documented by Kaplan-Meier curves.
2.10. miRNA Microarray Profiling
The miRNeasy Mini Kit was used for miRNA isolation, according to the manufacturer’s instructions
(Qiagen, Hilden, Germany). The microarray analysis was performed at the Microarray-Analytic Center
(Medical Faculty, Mannheim, Germany), using the Affymetrix GeneChip miRNA 4.0 Array with a total
of 30,424 microRNAs (Thermo Fisher Scientific, Dreieich, Germany).

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2.11. miRNA In Silico Analysis
The 500 most significantly differentially regulated miRNAs (p < 0.05) were selected from the
microarray raw data. Volcano plots and Venn diagrams were created by comparison of miRNA
expression between sulforaphane and control, SF102, or SF134 and between SF102 and SF134
miRNAs with –log10 p-Values >2 were included. To predict the target genes of the 3 most promising
miRNA candidates we used the online platform mirWalk (http://zmf.umm.uni-heidelberg.de/apps/
zmf/mirwalk2/) and its Target Mining option.
;
2.12. Statistical Analysis
The quantitative data are presented as the mean values with standard deviations from at least
three independent experiments performed in triplicate. The significance was analyzed using Student’s
t-test for parametric data corrected for multiple comparisons with the Bonferroni-Holm method.
For evaluation of the Kaplan Meyer analysis, the standard chi-square-based log-rank test and for
miRNA microarray, the JMP software (SAS, Heidelberg, Germany) were used. p < 0.05 was considered
statistically significant.
3. Results
3.1. Chemical Synthesis of Sulforaphane Derivatives
The sulforaphane analogues SF85
ruthenium-catalyzed imidations of erucin (1) and sulforaphane (2) (Figure 1A). Dioxalones 3a and 3b
served as nitrene precursors [26 28]. In general, the imidation reactions involving 1 proceeded better
, SF86, SF101, and SF102 were prepared by light-induced
–
than those with 2, providing sulfilimines SF85 and SF101 in higher yields (96% and 92%) than the
sulfoximines SF86 and SF102 (62% and 76%).
The syntheses of the sulforaphane analogues SF113, SF135, and SF134 started from N-Boc
protected sulfide 4 and sulfoxide 8 (Figure 1B) [29]. A one-pot imidation/oxidation of sulfide 4
provided N-methyl sulfoximine 5 in 30% yield [30]. As byproduct, 50% of the corresponding sulfone
was observed (not shown). Acidic Boc-cleavage of 5 was followed by Boa’s protocol for converting
amines into isothiocyanates [31]. Employing carbon disulfide and di-tert-butyl dicarbonate, SF113 was
synthesized in 75% yield. SF135 was prepared by a metal-free imidation of sulfide 4 to give N-cyano
sulfilimine 6 in 71% yield [32]. Oxidation of 6 with mCPBA provided 59% of the corresponding
sulfoximine 7. For 7, Boc-cleavage under acidic conditions was accompanied by hydrolysis of the
N-cyano group resulting in an intermediate with an N-carbamoyl moiety (not shown) [33,34]. Following
Boa’s protocol [31] as before led to sulforaphane analog SF135 in 83% yield. Key step in the synthesis of
SF134 was a rhodium-catalyzed imidation of sulfoxide 8, which provided N-trifluoroacetyl-protected
sulfoximine 9 in 65% yield [35]. Applying protocols in analogy to the ones detailed above for the
conversion of a Boc-protected amine into an isothiocyanate [31] afforded SF134 from 9% to 50% yield.
A streamlined and protecting group-free synthesis of the two most active derivatives (SF102 and
SF134) started from tetrahydrothiophene (10), which was alkylated with methyliodide (Figure 1C).
The corresponding S-methylthiophenium salt was isolated as its tetrafluoroborate (11), and the
subsequent ring-opening substitution with sodium azide afforded the (4-azidobutyl)(methyl)sulfide
(12) [36]. One-pot oxoimination with iodosobenzene diacetate (PIDA)/ammonium carbamate
furnished the NH-sulfoximine 13 [37], which was acylated to both the trifluoroacetyl- and
perfluorobenzoyl-derivatives 14 and 15. The azido terminus of the respective precursor was then
subjected to an in situ reduction (nBu3P in case of 14, Zn/NH4Cl in case of 15), and the resulting amine
was finally converted to targeted sulforaphane analogues SF134 and SF102.

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Figure 1. Scheme of chemical synthesis of sulforaphane derivatives. (A) Imidations of erucin (1)
and sulforaphane (2) by light-induced ruthenium catalysis leading to the respective sulforaphane
analogues SF85 SF86 SF101, and SF102. ( ) Syntheses of sulforaphane analogues SF113 SF135,
,
,
B
,
and SF134. Procedures: (a) 1. MeNH2 (3.0 equiv.); Br2 (2.0 equiv.), MeOH, r.t. 20 min.; 2. KMnO4
(3.0 equiv.), K2CO3 (2.0 equiv.), acetone, r.t., 12 h; (b) 4N HCl in dioxane (3.0 equiv.), dry DCM, r.t.,
4 h; (c) 1. TEA (2.0 equiv.), CS2 (10.0 equiv.), dry EtOH, r.t., 60 min; 2. Boc2O (1.0 equiv.), DMAP
(2 mol%), r.t., 60 min; NH2CN (1.5 equiv.), (d) PhI(OAc)2 (1.1 equiv.), CH3CN, r.t., 16 h; (e) mCPBA
(1.5 equiv.), K2CO3 (3.0 equiv.), DCM; r.t., 16 h; (f) TFA (13.0 equiv.), Et3SiH (2.5 equiv.), DCM, r.t.,
16 h; (g) Rh2(OAc)4 (2.5 mol%), CF3C(O)NH2 (2.0 equiv.), MgO (4.0 equiv.); PhI(OAc)2 (1.5 equiv.),
DCM, r.t., 16 h. (C) Protecting group-free syntheses of the sulforaphane analogues SF102 and SF134.
Procedures: (a) 1. MeI (1.0 equiv.), 50 ◦C, 1 h, ultrasound, NaBF4 (1.0 equiv.), EtOH, reflux, 30 min;
(b) NaN3 (3.0 eq.), DMF, 80 ◦C, 16 h; (c) PhI(OAc)2 (2.5 equiv.), ammonium carbamate (4.0 equiv.),
MeOH, r.t., 90 min; (d) trifluoroacetic anhydride (1.2 equiv.), Et3N (1.3 equiv.), 4-DMAP (0.1 equiv.),
DCM, r.t., 16 h; (e) perfluorobenzoyl chloride (1.5 equiv.), Et3N (1.5 equiv.), DCM, r.t., 16 h; (f) 1. nBu3P
(1.1 equiv.), THF, r.t., 30 min; 2. CS2 (3.0 equiv.), Et3N (3.5 equiv.), r.t., 90 min; 3. MsCl (1.2 equiv.), r.t.,
40 min; (g) 1. Zn powder (10 equiv.), NH4Cl (2.5 equiv.), MeOH, r.t., 30 min; 2. CS2 (1.5 equiv.), Et3N
(4.5 equiv.), THF, r.t., 90 min; 3. MsCl (1.1 equiv.), r.t., 40 min.
3.2. SF102 and SF134 Are Cytotoxic in All the Several Evaluated Tumor Cells
The pancreatic cancer cell lines BxPc3, BxGEM, and AsPC-1 were treated with sulforaphane or the
analogues SF85, SF86, SF101, SF102, SF113, SF134, and SF135, in concentrations of 10 µM and 30 µM.
Twenty-four hours after treatment, the cell viability was measured by MTT assay and documented by
microscopy. Sulforaphane strongly reduced the viability in all cell lines, compared to solvent DMSO

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controls or untreated cells, as expected (Figure 2A, Figure S2A). The effects of SF102 were comparable
to sulforaphane, whereas SF134 was less potent. All other derivatives had no significant effects, and
consequently, they were excluded from further testing.
Figure 2. SF102 and SF134 repress viability and colony formation.
(A) BxPc-3, BxGEM, and AsPC-1
cells were treated with sulforaphane (SF, 10 M or 30 M) and its analogues SF85, SF86, SF101,
µ
µ
SF102, SF113, SF134, and SF135, or were left untreated (CO). After 24 h, the viability was measured by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. DMSO was used as a vehicle
control. Data are means SD of at least three independent experiments. (B) BxPc-3 and AsPC-1 cells were
±
treated with sulforaphane, SF102 or SF134 as described above, followed by plating of viable cells 24 h
later at a low density (BxPc-3: 500 cells/well, AsPC-1: 1000 cells/well) in 6-well plates. After two weeks,
colonies were fixed with 4% paraformaldehyde (PFA) and Coomassie-stained. Colonies containing
more than 50 cells were quantified under a dissecting microscope and photographed. Representative
images are shown on the right. The percentage of plating efficiency was calculated by setting the
number of surviving colonies in the control group to 1. The diagrams on the left show the survival
fraction (1st Gen). The surviving cells were harvested from non-fixed and non-Coomassie-stained
duplicate plates of the first generation (1st Gen) of colony formation, and re-plated at the same density
in 6-well plates. Two weeks later, the clonogenic survival of the second generation (2nd Gen) was
evaluated as described above. The means ±SD are shown. ** p < 0.01, * p < 0.05.
To study the ability to interfere with clonogenicity, which is a typical tumor stem cell feature,
BxPc-3 and AsPC-1 cells were treated with sulforaphane, SF102 or SF134. After 24 h, a colony-forming

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assay was performed, and the number of surviving cells was evaluated by microscopy (Figure 2B).
SF102 and SF134 significantly reduced the number of colonies even in chemoresistant AsPC-1 cells,
although the effect of sulforaphane was more pronounced. In BxPc-3 cells, SF102 was most potent in
decreasing colony formation, followed by sulforaphane and SF134. To test if the effect on clonogenicity
is long lasting, we isolated single live cells from the colonies, and re-seeded them without additional
treatment. In the resulting second generation, the number of colonies was induced even stronger,
suggesting that the treatment indeed eliminated the more aggressive, colony-forming, tumor stem
cell-like cancer cells.
The observed therapeutic effects also occurred in cancer cells from cervix ovary, prostate, breast,
colorectum, and lung, as well as in hepatocellular, neuroblastoma, T-cell leukemia, and glioblastoma
cell lines, as detected by MTT assay 24 h after treatment (Figure 3). By the use of the NCI-60 cancer cell
line panel, we confirmed the high potency of SF102 in induction of growth inhibition and lethality
in cancer cell lines of leukemia, melanoma, non-small-cell lung carcinoma, and cancers of the brain,
ovary, breast, colon, kidney, and prostate (Figure 4). Likewise, SF101 was effective in the NCI-60
panel test (Figure S3), which is consistent with the observed reduction of viability in pancreatic cancer
cells (compare Figure 2A), although these data were not statistically significant. These results were
confirmed in pancreatic cancer cells, where SF102 most potentially induced apoptosis, even in resistant
and highly aggressive BxGEM and AsPC-1 cells (Figure S2B).
Figure 3. SF102 and SF134 repress viability in various cancer entities. Ovary (OVM), prostate (PC3),
breast (BT-20), colorectal (SW707), glioblastoma (A172), hepatocellular (HepG2), neuroblastoma (IMR5),
and T cell leukemia (Jurkat) cell lines were treated with SF101 or SF102 in concentrations of 10
30 M. After 24 h, the viability was measured by MTT assay. DMSO served as a vehicle control. Data
are means ±SD of at least three independent experiments. * p < 0.05, ** p < 0.01.
µM and
µ

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Figure 4. SF102 is active in 60 cancer cell lines of the NCI-60 cell panel. These studies were
performed by the Services of the Discovery & Developmental Therapeutics Program at the NIH
(http://dtp.cancer.gov). SF102 was applied at a single dose (10 M) to the 60 cancer cell lines
µ
representing leukemia, melanoma, non-small-cell lung carcinoma, and cancers of the brain, ovary,
breast, colon, kidney, and prostate, as indicated. The growth inhibition (values between 0 and 100) and
lethality (values less than 0) were measured. For example, a value of 100 means no growth inhibition.
A value of 40 means 60% growth inhibition. A value of 0 means no net growth over the course of the
experiment. A value of −40 means 40% lethality. A value of −100 means all cells are dead.
3.3. SF102 and SF134 Inhibit Tumor Growth without Obvious Side Effects
To evaluate the in vivo relevance, we performed xenograft studies by transplanting BxPc-3
cells onto the CAM of fertilized chicken eggs on day 9 of chick development. Five days later,
sulforaphane, SF102, SF134, or a PBS control were injected into CAM vessels, followed by the
resection of the tumor xenografts on day 18. Although visual inspection showed an apparent tumor
suppressing effect for sulforaphane, SF102 and SF134, these differences were not significant (Figure 5A).
To examine the proliferation, xenograft cryosections were stained with the marker Ki67, followed
by immunohistochemistry and evaluation of the fluorescence intensity. Representative pictures and
quantitative analysis demonstrated that SF102 significantly reduced the proliferation to about 25%
(Figure 5B), followed by sulforaphane and SF134, although the latter were not statistically significant.

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Figure 5. SF102 and SF134 inhibit tumor xenograft growth without obvious side effects. (A) BxPc-3
cells were seeded onto the CAM of fertilized chicken eggs on day 9 of embryonal development.
Sulforaphane (SF, n = 6), SF102 (n = 9), or SF134 (n = 6), 10 M each, were injected in 50 l PBS into
µ
µ
the CAM vessels on day 14 of development. The tumor xenografts were resected on day 18, and the
tumor volumes were calculated as described in the Methods part. The control (CO, n = 8) group
refers to xenografts that have received a PBS injection only. A representative image of a xenograft
on the CAM (black arrow) on day 18 is shown. The volumes of individual tumors (black dots) and
the mean tumor volumes of each experimental group (black lines) are shown. Statistical significance
according to Bonferroni-Holm correction for multiple tests was not obtained and the single P values
were: P = 0.646 for CO versus SF; P = 0.098 for CO versus SF102; P = 0.649 for CO versus SF134
) Frozen xenograft tissue sections were stained with the proliferation marker Ki67 (brown), and
representative immunofluorescence pictures are shown on the left under 400 magnification. The scale
bar indicates 50 m. The intensity of the Ki67 immunofluorescence signal was quantified in 10 randomly
chosen vision fields using ImageJ and the means SD are shown in the diagram on the right. ** p < 0.01
) Hematoxylin staining of embryonal liver sections of embryos derived from fertilized chicken eggs,
which were treated as described above. The scale bar indicates 25 m. ( ) The mean embryonal
weight of the chick embryos was determined by weighting—the means of 15 embryos per group SD
are shown. ( ) Wild type C. elegans nematodes were treated with sulforaphane (SF), SF102, or SF134
.
(
B
×
µ
±
.
(
C
µ
D
±
E
for 48 h, or were left untreated (CO) and Kaplan Meier analysis was performed. A representative
microscopy image from a C. elegans worm is shown on the right. The scale bar indicates 100 µm.

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To further investigate toxic side effects, the liver morphology of the chicken embryos and their body
weight were analyzed at day 18 of development. As indicated by hematoxylin staining, no necrotic
areas were detected in liver tissue (Figure 5C), and the body weight between 12 to 14 g was consistent
between groups (Figure 5D). To further rule out any side effects, we used the C. elegans nematode
model and treated the worms with sulforaphane, SF102, SF134, or the vehicle control DMSO for
48 h. The survival of the worms over a period of 35 days was evaluated by Kaplan Meier analysis.
We did not observe a shorter survival of worms after treatment with sulforaphane, SF102, and SF134
compared to the control (Figure 5E), suggesting that no apparent side effects regarding reproduction
and development occurred.
3.4. Sulforaphane, SF102 and SF134 Induce Differential microRNA Expression and NF-κB Signaling
To further highlight the underlying signaling pathways responsible for the observed effects,
we treated AsPC-1 cells with sulforaphane, SF102, or SF134 or let them untreated. The RNA was
isolated 24 h later and examined by miRNA microarray expression profiling, followed by bioinformatic
evaluation. We identified 500 significantly (p < 0.05) differentially regulated miRNAs compared to the
control. As presented in a heat map, there were many similar clusters but also obvious differences in
miRNA expression following sulforaphane, SF102, and SF134 treatment (Figure 6A). Next, the top 10
most significantly differentially regulated miRNAs were compared between control and sulforaphane,
control and SF102, and control and SF134 (Figure 6B). By this method, we identified miR2278 as
common and most significantly downregulated miRNA following sulforaphane, SF102, and SF134
treatment (Table S1). To further highlight differences in miRNA expression, we created volcano plots
from a comparison of the most significantly differentially regulated miRNAs with –log10 P values >2.
The topmost interesting candidates were miR27b-5p and miR29b-1-5p, which were downregulated
after sulforaphane treatment compared to the control, but upregulated by SF102 and SF134 compared
to the sulforaphane (Figure 6C, Table S2). The number of target miRNAs regulated by sulforaphane,
SF102, and SF134 is schematically presented in a Venn diagram, and the names of the identified miRNA
candidates are provided in a table (Figure 6D, Table S3).
To obtain information about target genes regulated by the three candidate miRNAs, a bioinformatics
analysis using the mirWalk online platform and its Target Mining tool was performed. Based on
the highest predicted binding probability of miRNAs to target gene sites, we identified 309 target
genes that are individually or commonly regulated by the three miRNA candidates, as visualized in a
network plot (Figure 7A). To follow up on our previous studies showing that sulforaphane normalizes
enhanced NF-
Table S4). The top candidate was miR2278, because it was predicted to regulate the highest number of
NF- B-related target genes, followed by miR27b-5p and miR29b-1-5p, which partially overlapped in
regulation of NF-κB-related target genes.
κB activity by miRNA signaling [8,23], several candidate genes were identified (Figure 7B,
κ

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Figure 6. miRNA expression profiling reveals differential expression induced by sulforaphane,
SF102 or SF134. ) AsPC-1 cells were treated with 10 M sulforaphane (SF), SF102, or SF134, or were
(A
µ
left untreated (CO). After 24 h, RNA was isolated and analyzed by a GeneChip miRNA 4.0 Array in
triplicates. The heatmap presents the top significantly regulated miRNAs. The red color marks high
expression, and the green color marks low expression within a scale from 0.5 to –1, as indicated. (
3 heatmaps present the top 10 significantly regulated miRNAs. miR2278 is highlighted in pink color.
) Volcano plots show the miRNA distribution. On the y-axis, the –log10 p value is plotted, and on the
x-axis, the fold change is plotted. ( ) A Venn diagram shows the distribution of differentially expressed
B) The
(C
D
miRNAs among three groups: SF—CO, SF102—SF, and SF134—SF. The overlapping region identified
two miRNAs, miR27b-5p and miR29b-1-5p, which are differentially expressed between sulforaphane,
SF102 and SF134.

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Figure 7. Identification of NF-κB-related miRNA-target genes. (A) Using the Target Mining option of
the mirWalk online platform, 309 target genes of miR2278, miR27b-5p, and miR29b-1-5p were identified
and displayed as a network graph. Each orange dot is associated to a target gene. The black lines
symbolize, e.g., the joint target gene CCDC141 of miR2278 and miR29b-1-5p, the joint target gene
CREB5 of miR-29b-1-5p, and the joint target genes RPRD1A, CLMN, and LRRC8C of miR-2278 and
miR27b-5p. (B) An miRNA-target gene analysis with focus to NF-
κB signaling identified the most
significantly NF-
κ
B-related target genes of miR29b-1-5p, miR2278, and miR27b-5p. TIRAP and DDX58
are target genes of miR2278 and miR29b-1-5p, CFLAR, ERC1, and PRKCB are target genes of miR2278
and miR27b-5p, and PLCG1 and TRIM25 are target genes of miR29b-1-5p and miR27b-5p.
4. Discussion
Here, we prepared a series of unprecedented analogues of sulforaphane with sulfur core variations.
We examined the anti-cancer potential of seven analogues and compared them to sulforaphane. Viability

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assays in pancreatic cancer cell lines allowed to identify the two most promising derivatives, SF102 and
SF134. They inhibited colony-formation, xenograft growth, and induced apoptosis in a variety of cancer
cells of various tumor entities. SF102 was partially more active than sulforaphane. Most importantly,
obvious side effects in vivo were not observed. As detected by miRNA array analysis and bioinformatics
evaluation, there were significant differences in signal transduction between sulforaphane, SF102, and
SF134. However, all of them had in common the regulation of NF-κB-related target genes.
To the best of our knowledge, this is the first time that the cytotoxicity of synthetic sulforaphane
analogues has been tested and evaluated in pancreatic cancer. Previously, other sulforaphane derivatives
with modifications in the carbon substituents at the sulfur core were shown to inhibit the proliferation
of melanoma cells [13], or to induce cell cycle arrest and apoptosis in hepatocellular carcinoma cells [38].
In this regard, Shi et al. demonstrated that sulforaphane heterocyclic analogues enhanced apoptosis
and eliminated the tumor stem cell population in breast cancer cells [39]. These data are in line
with our results, as the most effective SF102 analogue did not only inhibit the viability of pancreatic
cancer cells but also of breast, hepatocellular, and several cancer types, suggesting a broad range of
anti-tumor activity.
Our results suggest that SF102 and SF134 decrease the viability, clonogenicity, and tumor growth,
and induce apoptosis in pancreatic cancer cells as potent or, in the case of SF134, even better than
sulforaphane. As already known, sulforaphane normalizes enhanced NF-
cells and thereby mediates chemosensitization [23], among several other activities. Our bioinformatic
analysis of miRNA array data now predict that sulforaphane, SF102, and SF134 affect NF- B signaling
by the induction of miR2278, which is involved in the regulation of many NF- B target genes.
κB signaling in tumor stem
κ
κ
Additionally, while sulforaphane inhibits the expression of miR27b-5p and miR29b-1-5p compared to
untreated control cells, SF102 and SF134 rather induce these miRNAs compared to their expression in
sulforaphane-treated cells. This finding is of high relevance, because, according to our in silico analysis,
miR27b-5p and miR29b-1-5p are also regulators NF-κB-related target genes. According to the Human
Protein Atlas, the predicted target genes PLCG1 (https://www.proteinatlas.org/ENSG00000124181-
PLCG1/pathology) and TRIM25 (https://www.proteinatlas.org/ENSG00000121060-TRIM25/pathology)
are of high relevance in pancreatic cancer, because patients with high PLCG1 (n = 110) or TRIM25
(n = 121) expression survive longer than those with low expression (n = 66, N = 55, respectively).
Interestingly, our miRNA candidates miR2278, miR27b-5p and miR29b-1-5p have never before been
documented in pancreatic cancer, as far as we know. However, Kim et al. demonstrated both miR27b-5p
and miR29b-1-5p as markers for gastric cancer progression [40], while miR29b-1-5p overexpression
induced epithelial-mesenchymal transition in oral squamous cell carcinoma [41]. The only available
information about miR2278 and cancer is that its upregulation is associated with the inhibition of
leukemic cell proliferation and enhanced apoptosis [42].
To confirm an in vivo activity of sulforaphane, we used tumor xenotransplantation to fertilized
chicken eggs, an animal model that offers an ethical alternative to mammalian assays and has frequently
been used in cancer research for xenotransplantation and drug delivery studies [43]. Our results
suggest that the derivatives SF102 and SF134 decrease the tumor volume as sulforaphane, whereas
SF102 significantly inhibited the proliferation. Sulforaphane, SF102, and SF134 caused no obvious side
effects, as liver necrosis or abnormal weight loss of chicken embryos was not detectable. Our results
obtained by the use of the animal model C. elegans demonstrated that sulforaphane and its analogues
did not reduce the lifespan of the nematodes. In contrast, SF102 even significantly induced longevity,
a process that has been proven to be anticarcinogenic [44]. These observations could lead to the
conclusion that SF102 has the highest activity.
Regarding the clinical relevance of sulforaphane and its derivatives, a pilot study with freeze-dried,
pulverized broccoli sprouts was performed in our Department of Surgery of the University Clinic
Heidelberg. Forty patients were involved, suffering from advanced, non-resectable pancreatic cancer.
The results indicated that the patients in the broccoli sprout group survived longer compared to the
placebo group. However, these data were not significant, which might be due to the small number of

Biomolecules 2020, 10, 769
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patients [
4]. Noteworthy, the daily intake of 15 capsules (90 mg/508
µmol) in order to reach a high
sulforaphane concentration was unpleasant for the patients, because it increased pre-existing digestion
problems in some patients and caused a higher drop-out rate. The development of a drug with highly
active sulforaphane or a derivative thereof is urgently required to circumvent the unpleasant side effects
of broccoli consumption, and to enable the intake of a high sulforaphane amount by a single capsule.
In conclusion, we identified two sulforaphane analogues, which showed promising inhibitory
properties in all of several examined tumor entities. Although our data are convincing, we suggest
taking them with caution as well. For example, product purity and stability must be regarded as critical
factor, in particular, as we found in a previous study that different sulforaphane lots from a commercial
supplier led to data deviations (Herr et al., unpublished data). Furthermore, trace impurities and
byproducts stemming from the syntheses and compound degradations during their application might
play a role. Notably, all compounds applied here—including sulforaphane, which in nature occurs as
(R)-enantiomer—were racemic, and it is reasonable to assume that the use of single enantiomers would
affect the inhibitory properties of the molecules. If so, the results would indeed vary. At the same
time, however, new opportunities could arise by exclusively using the more effective stereoisomer
allowing a dose reduction in subsequent treatments. Finally, a detailed analysis of the absorption,
distribution, metabolism, excretion and toxicity (ADMET) properties of the new sulforaphane analogs
is still lacking. Certainly, factors such as solubility and transport will significantly impact the biological
activity of the compounds. Overall, we are convinced that chemical modifications of the sulforaphane
scaffold are promising in opening unidentified opportunities for the future development of new
sulforaphane-related drugs.
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/2218-273X/10/5/769/s1.
Author Contributions: I.H., C.B., C.G: Concept and design; C.G., L.B., M.B., N.R.: Development of synthetic
methodology; sample preparation; C.G., L.Y., C.S.: Acquisition of data; C.G., L.Y., C.S., W.G.: Analysis and
interpretation of data; C.G., I.H., C.B.: Writing, review and/or revision of the manuscript. All authors have read
and agreed to the published version of the manuscript.
Funding: This study was supported by grants to I. Herr from the German Cancer Aid (Deutsche Krebshilfe
111299), German Research Council (DFG HE 3186/15-1), Heidelberger Stiftung Chirurgie, Dietmar Hopp-Stiftung,
Klaus Tschira Stiftung and the Hanns A. Pielenz-Stiftung.
Acknowledgments: We thank the Discovery & Development Services of the NIH for testing SF101 and SF102 in
the NCI-60 human cancer cell line screen. We are grateful to S. Karakhanova for helpful discussion, M. Le for
providing a microscope image of C. elegans and S. Bauer for excellent technical assistance.
Conflicts of Interest: The authors have no conflicts of interest to disclose regarding the publication of the
present manuscript.
Abbreviations
Sulforaphane analogue 102 (SF102), Sulforaphane analogue 134 (SF134).
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