Our previous studies on antiproliferative properties of genistein derivatives substituted at C7 hydroxyl group of the ring A revealed some compounds with antimitotic properties. The aim of this work was to synthesize their analogues substituted at the 4′-position of the ring B in genistein and to define their antiproliferative mechanism of action in selected cancer cell lines
Flavonoids are naturally occurring plant polyphenols found in abundance in fruits, vegetables, and plant-derived beverages [
Beneficial influence of genistein on human health, resulting from its interaction with multiple molecular targets [
There are many examples of genistein derivatives which show better pharmacological characteristics comparing to the parent compound genistein, including altered binding affinities to estrogen receptors (ERs) [
Hitherto, most of the described derivatives were obtained by a substitution of a C7 hydroxyl group hydrogen atom. The synthesis of C4′-substituted compounds was not studied frequently and biological activity of these compounds is poorly understood. It has been demonstrated [
In this paper, we presented the synthesis of genistein glycoconjugates substituted at the 4′-position of the ring B and described the biological effects of these compound on cancer cell lines. We showed that antiproliferative effects of C4′ substituted derivatives of genistein are related to the mechanism different than that described for C7 substituted regioisomers [
1H and 13C NMR spectra were recorded (DMSOd6 solution) with TMS internal standard on 600 MHz Varian Inova 600 MHz apparatus with Me4Si as internal reference. Optical rotations were measured with a Perkin-Elmer 141 polarimeter using a sodium lamp (589 nm) at room temperature. Mass spectra (HRMS) were recorded in the positive mode on a Mariner (PerSeptive Biosystems) detector using the electrospray-ionization (ESI) technique. Reactions were monitored by TLC on precoated plates of silica gel G (Merck); components were detected under UV light or by charring with 10% sulphuric acid in ethanol. Column chromatography was performed on silica gel 60 (70–230 mesh, E. Merck) developed with one of the toluene/ethyl acetate or hexane/ethyl acetate solvent systems. All evaporations were performed under diminished pressure at 50°C.
The reagents (chemicals), all being of A.R. grade, were purchased from Merck Company, 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one, Genistein (
Hydroxyalkyl derivatives of genistein were prepared according to procedures described elsewhere (Figure 5,7-dihydroxy-3-(4-(2-hydroxyethyloxy)phenyl) chromen-4 5,7-dihydroxy-3-(4-(3-hydroxypropyloxy)phenyl) chromen-4 5,7-dihydroxy-3-(4-(5-hydroxypentyloxy)phenyl) chromen-4
Synthesis of 4′-(hydroxyalkyl) genistein.
Synthesis of glycoconjugates derivatives of genistein.
HRMS (ESI): calculated C25H24O9 [M + Na]+ 491.1318, determined 491.1324.
HRMS (ESI): calculated C26H26O9 [M + Na]+ 505.1475, determined 505.2004.
HRMS (ESI) calculated C28H30O9 [M + Na]+ 533.1788, determined 533.2104.
Human prostate cancer cells DU 145 and human colon cancer cells HCT 116 wt were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). HCT 116 −/− p53 (both p53 alleles deleted by targeted homologous recombination) [
The assay was performed as described previously [
Cycle phases distribution after 24 h treatment with genistein derivatives was performed as described previously in [
p53, phospho-p53 (Ser15) and p21, phospho-ATM (Ser 1981) were detected as described previously in [
Immunocytochemistry was performed as described previously in [
Cells were plated in culture dishes
Cells were grown on coverslips in 3 cm plates for 24 h. Then the medium was replaced with the one containing cytochalasin B (2
PC3 cells were seeded in 8-well chamber slides (Nunc, NY, USA) and left for 24 h. Then the medium was aspirated from the chamber slide wells and replaced with warm medium with the tested compounds; cells were then incubated for 24 h. Z-stacks were acquired with the Zeiss LSM 710 inverted confocal microscope with the 488 nm excitation laser and Zeiss Plan-Apo oil immersion 63x objective. Consecutive optical sections were superimposed with Zen software in order to show vesicular pattern across the whole cell depth.
Genistein, like other flavonoid polyphenolic compounds, is a notoriously difficult substrate for chemical glycosylation, including Ferrier rearrangement of glycals, and regio- as well as stereoselectivity is practically unattainable [
In the first step we evaluated the influence of derivatives of genistein substituted at C4′ on the proliferation of HCT 116 and DU 145 cells after 72 h treatment with MTT assay (Figure
Inhibition of proliferation of cancer cells by genistein derivatives substituted at C4′.
On the basis of experimental data, we calculated the value of IC50 (proliferation inhibited to 50% of control) using CalcuSyn Software based on minimal square fitting to 4-parameter logistic curves. Mean IC50 value and standard deviation were then calculated from 3–5 independent experiments (Table
IC50 value of proliferation inhibition calculated on the base of MTT assay.
IC50 (µM) | ||
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HCT 116 wt | DU 145 | |
Gen-2′ | >100 | >100 |
Gen-3′ |
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Gen-5′ |
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Ram-2′ |
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Ram-3′ |
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Ram-5′ |
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In the next step we investigated the influence of the most potent derivatives on the cell-cycle after 24 h treatment. The DNA content was analyzed with the flow cytometer. The results shown in Figure
Cell-cycle phases distribution in HCT 116 and DU 145 cells treated with genistein derivatives substituted at C4′.
DU 145 cells harbor mutations in two alleles of
Cell-cycle phases distribution in HCT 116 −/− p53 cells treated with genistein derivatives substituted at C4′.
Stress factors, among them genotoxic stress, block the cell-cycle in both p53-wild-type and p53-deficient cells [
We did not observe either single- or double-strand breaks measured as a moment of a tail moment in a comet assay performed in HCT 116 treated with tested derivatives (Table
Single-strand breaks (SSB) and double-strand breaks (DSB) detected with comet assays in HCT 116 cells.
Compound (µM) | Alkaline lysis—SSB | Neutral lysis—DSB |
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Tail moment (%) | ||
Control ( |
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Gen-5′ ( |
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Ram-2′ ( |
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Ram-3′ ( |
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Ram-5′ ( |
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Etoposide ( |
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Genistein [ |
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Camptothecin ( |
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Treatment (µM) | Fold change of |
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Etoposide ( |
2.45 |
Genistein ( |
1.4 |
Gen-5′ ( |
0.92 |
Ram-2′ ( |
0.70 |
Ram-3′ ( |
0.66 |
Ram-5′ ( |
0.91 |
Next, we assessed the genotoxic effects evoked by genistein derivatives with the use of micronucleus assay. The assay was performed routinely with cytochalasin B, the mycotoxin inhibiting the division of a cytoplasm, without influencing the nuclear division, by blocking the formation of actin microfilaments necessary for cytokinesis [
Index of binucleated cells representing completed nuclear divisions counted after 24 h treatment with genistein or its C4′-substituted derivatives used at concentrations corresponding to 2x IC50 and 3x IC50.
Next, we compared the level of ATM phosphorylation on serine 1981 in HCT 116 cells after treatment with genistein, camptothecin (CPT) and C4′-substituted derivatives (Figure
ATM activation in HCT 116 wt cells treated with genistein (100
The activation of ATM by genistein and camptothecin (Figure
The level of total p53, phosphorylated p53 (ser 15), p21 and MDM2 in HCT 116 wt cells treated for 24 h with the tested genistein derivatives used at the concentration equivalent to 3x IC50 (Ram-2′ (3
The pattern of p53 phosphorylation corresponded to p21 induction after treatment with the tested compounds (Figure
In parallel, we tested the level of MDM2, the cellular regulator of p53 level. Genistein did not influence the level of MDM-2 in relation to control. We observed depletion of MDM-2 by camtothecin, reported previously in [
Altogether, the pattern of p53, p21, and MDM-2 expression after treatment with genistein derivatives bears striking resemblance to the pattern characteristic for AICAR, the pharmacological activator of AMP kinase [
The restriction of nutrients is a stress signal that affects cell-cycle decisions, causing the cell to arrest in G1. Usually, cells subjected to a nutrient stress induce autophagy, a process of degradation of its own organelles within lysosomes to reconstitute cellular components and supply the cell with energy [
We studied the influence of C4′-substituted derivatives on the process of autophagy induction using PC3 prostate cancer cell line stably expressing EGFP-LC3 protein. During autophagy, the cytoplasmic form of LC3 is processed and recruited to the autophagosomes. The hallmark of autophagic activation is the formation of cellular autophagosome puncta decorated by LC3 [
In control cells we observed typical, evenly distributed fluorescence of EGFP-LC3. In cells treated with genistein and genistein derivatives the induction of autophagy, visible as vesicular LC3 pattern, was detected 24 h after addition of a tested substance. The intensity of a process, expressed as the number of cells with autophagic morphology was much higher in the presence of C4′-substituted genistein derivatives than genistein (Figure
The percentage number of autophagic cells in PC3 cell line.
Treatment (µM) | Autophagic cells (%) |
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Control ( |
18.5 |
Genistein ( |
33.2 |
Gen-5′ ( |
64.0 |
Ram-2′ ( |
84.6 |
Ram-3′ ( |
94.1 |
Ram-5′ ( |
55.5 |
Autophagy in PC3 prostate cancer cell line stably expressing EGFP-LC3 (green fluorescence). Series of optical sectiones (Z-stack) were combined together. (a) control; (b) genistein (100
The vast literature implicating antitumor activity of genistein, including induction of cancer cell differentiation, stimulation of apoptosis, inhibition of angiogenesis, and inhibition of tumor cell proliferation points tyrosine kinases and topoisomerase II as primary molecular targets of this compound [
In this paper, we focused on the influence of C4′-substituted genistein derivatives on a cell-cycle and compared the results with the effects obtained previously for C7-substituted derivatives [
The type of cell-cycle arrest by flavonoids dependent on the structural groups was critically reviewed by Casagrande and Darbon [
Our results indicate that regioisomeric substitutions at C4′ and C7 are key regulators of the mode of isoflavonoid action. Here we show that genistein derivatives substituted with a bulky group at C4′ block a cell-cycle at G1 phase, despite the presence of a hydroxyl at C5. Our previous work showed that the substitution of genistein at C7 (with unmodified hydroxyls present at C5 and C4′) causes cell-cycle arrest in G2 phase [
Cell-cycle arrest by differently substituted genistein derivatives.
The cell-cycle block in G2 phase caused by genistein, followed by the process of apoptosis, is related to activation of DNA damage checkpoint pathways [
Although the results are still too fragmentary to give comprehensive mechanistic explanation of activity of C4′-substituted genistein derivatives, we can with high probability exclude the induction of DNA double-strand breaks. The observation of autophagy induction would rather suggest that cell-cycle progression is stopped by insufficient supply with nutrients. In cells under metabolic stress the progression into S phase is stopped and, after prolonged starvation, the process of self-eating, called autophagy, is induced [
Regardless the unknown yet mechanism, the C4′-substituted compounds may be useful as non-genotoxic compounds lowering the rate of proliferation of cancer cells.
This work was financed by the European Union Structural Funds in Poland no. UDA-POKL. 04.01.01-00-114/09-01, the Grant from Polish National Science Centre no. DEC-2011/01/N/NZ4/01141, and the Grant from the European Science Foundation (ESF) for the activity entitled “The Euroglycoscience Forum.” The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or nonfinancial interest in the subject matter or materials discussed in this paper.