Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-
Curcumin, also known as diferuloyl methane, a natural component of the rhizome of
A variety of molecular mechanisms have been proposed to mediate anticancer effects of curcumin, but its ability to inhibit the growth of various types of cancer cells at various stages of cancer progression is probably due to its potential to act on multiple targets [
Several preclinical and clinical studies suggest that curcumin may represent a novel strategy to treat cancer patients alone or in combination with already existing therapeutic regimens [
Different synthetic concepts have been therefore developed to expand the molecular diversity, from the side-chain and diketone transformations to alkyl and alkenyl functionalizations on C-4 in the central position or with different substituents at the 4 positions of the phenyl group of curcumin [
Previous reports demonstrated that bortezomib (Velcade, formerly known as PS-341), a potent and selective proteasome inhibitor approved by the FDA for the treatment of patients with multiple myeloma, is able to block chemotherapy-induced NF-
NF-
Beside the canonical pathway, several studies show a critical role for the noncanonical NF-
It has been shown that bortezomib possesses in vitro and in vivo activity against a variety of malignancies, including acute and chronic lymphocytic leukemia, prostate cancer, pancreatic cancer, and colon cancer [
Several studies reported enhanced cytotoxicity of bortezomib in multiple myeloma cells when combined with curcumin or its analogues both in vitro and in vivo [
In this report, we studied curcumin and a novel Mannich-type curcumin analogue C-150 alone and in combination with bortezomib. Effects of cotreatment were studied on cytotoxicity, NF-
Human leukemia cells (HL60) were obtained from ATCC, USA. Cells were cultured at 37°C under 5% CO2 and 100% humidity in LeviTubes designed for use in the bench top bioreactor-incubator hybrid BioLevitator (Hamilton, Hungary). During cultivation, cells were proliferated in suspension with rotation without the use of microcarriers. Rotation period was 2 seconds with 1 second pause at 65 rpm. LeviTubes were filled with 40 mL RPMI cell culture medium containing 10% fetal bovine serum, 1x GlutaMAX, and penicillin-streptomycin antibiotics (Life Technologies, Hungary). Culture medium was changed after 48 hours by removing half of the volume of the culture medium and replacing it with fresh medium.
Viability of HL60 human leukemia cells was determined by the colorimetric MTS assay (CellTiter 96 AQueous Assay, Promega, Madison, WI) and by the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI). The cells were seeded into 96-well plates at 7000 cells/well density and cultured overnight before treatment. Effects of curcumin (5, 10, 25, and 50
Viability was calculated with relation to untreated control cells from three parallel measurements for all conditions. For statistical significance, two-tailed Student’s
Mouse B16 melanoma cell line was obtained from ATCC, USA. Cells were cultured in RPMI medium (Life Technologies, Hungary) supplemented with 10% FCS (Life Technologies, Hungary). NF-
B16/NF-
HL60 cells (105) were plated in 24-well tissue culture plates (Corning Life Sciences). Cells were treated with curcumin (25
HL60 leukemia cells (106) were plated on 10 cm2 tissue culture plates (Corning Life Sciences). Cells were treated with curcumin, C-150, bortezomib, and their combinations. 48 h after treatment, cells were collected and centrifuged (3000 rpm, 5 min) and total RNA was purified from treated and control (0.2% DMSO) cells using AccuPrep Viral RNA Extraction kit (Bioneer Corp., Korea) with a modified protocol as described earlier [
Identical reaction volumes were prepared by the Agilent Bravo Liquid handling Platform (Agilent Technologies) in a 16 × 96-well design according to Agilent and Roche’s recommendations. Each 2
Data was collected and processed using the LightCycler 1536 SW 1.0 software. Curves were analyzed by using dynamic tube and slope correction methods. Relative expression of the analyzed genes was normalized to the mean value of the reference genes.
Liquid phase C-150 containing liposomes was produced by dissolving lipid powder of CHOL/PC/DSPE-mPEG (62/28/0.6 mol%) and C-150 (9.2 mol%) in ethanol. The solution was transferred to a rotary evaporator and dried overnight in vacuum. The lipid film was hydrated and redispersed in PBS solution to a final C-150 concentration of 0.5 mM for 25 min over the phase transition temperature of the organic components. The dispersion was subjected to size extrusion (0.45
8-week-old male NOD CB17-
For the induction of rodent model of human leukemia 106 HL60, human leukemia cells were injected in 100
Four different groups, each containing 8 mice, were studied: PBS control, bortezomib (0.15 mg/kg), C-150 (3 mg/kg), and C-150 (3 mg/kg) + bortezomib (0.15 mg/kg). All animals were treated moribund and were euthanized at the observation of the first sign of torment. The study was performed according to the Institutional and National Animal Experimentation and Ethics Guidelines in possession of an ethical clearance (XXIX./3610/2012).
All data presented are means ± standard deviation (SD) as indicated in the text. The statistical comparisons were performed by two-tailed Student’s
We have utilised two common viability assays to assess the cytotoxic effects of curcumin and the novel analogue C-150 on HL60 cells. Combinations of curcumin or C-150 with bortezomib were also investigated. The colorimetric MTS assay and the luminescence based CellTiter-Glo (CTG) assay yielded comparable results; however, additive effects could be registered at different concentrations (Tables
Cell viability measurements (MTS) of curcumin (C), C-150, and bortezomib (BTZ) alone and combined. Curcumin and its analogue C-150 and bortezomib significantly decreased the cell viability of HL-60 human leukemia cells in a dose-dependent manner. C-150 exhibited dose-dependent effect on HL60 cells in submicromolar concentrations and was effective at 27 times lower concentration than curcumin. Both curcumin and its analogue exhibited additive effects with bortezomib (bold data with significant values) (
— | BTZ |
BTZ |
BTZ |
BTZ | |
---|---|---|---|---|---|
— | 0.35 | 0.45 | 1.26 | 1.34 | |
C (50 |
0.34 | 0.33 | 0.35 | 0.39 | 0.37 |
C (25 |
0.26 | 0.23 | 0.31 | 0.28 | 0.27 |
C (10 |
0.48 |
|
|
0.42 | 0.60 |
C (5 |
1.10 | 0.33 | 0.43 | 1.21 | 1.26 |
C-150 (1 |
0.25 | 0.21 |
|
0.23 | 0.25 |
C-150 (0.5 |
0.40 |
|
0.27 |
|
0.33 |
C-150 (0.1 |
0.94 | 0.25 | 0.41 | 0.83 | 1.02 |
C-150 (0.05 |
1.16 | 0.32 | 0.39 | 1.13 | 1.22 |
Cell viability measurements of curcumin (C), C-150, and bortezomib (BTZ) alone and in combination with CellTiter-Glo assay. Curcumin and its analogue C-150 significantly potentiated the effect of bortezomib (
— | BTZ |
BTZ |
BTZ |
BTZ | |
---|---|---|---|---|---|
— | 0.11 | 0.16 | 0.91 | 0.94 | |
C (50 |
0.12 | 0.11 | 0.12 | 0.12 | 0.11 |
C (25 |
0.17 | 0.15 | 0.13 |
|
|
C (10 |
0.99 | 0.11 | 0.15 | 0.52 |
0.87 |
C (5 |
1.08 | 0.11 | 0.17 | 0.79 | 1.05 |
C-150 (1 |
0.09 | 0.10 |
|
0.08 | 0.08 |
C-150 (0.5 |
0.16 | 0.10 | 0.10 | 0.12 | 0.16 |
C-150 (0.1 |
0.99 |
|
0.12 |
|
0.90 |
C-150 (0.05 |
0.93 | 0.10 | 0.15 |
|
1.02 |
We found that curcumin had a dose-dependent cytotoxic effect on HL60 cells (MTS IC50 = 8.21
At the highest applied concentrations, both curcumin and bortezomib alone produced robust cell death, which could not be further enhanced by the combined treatment. At the lowest applied concentration, both curcumin and bortezomib showed no cytotoxic effect neither alone nor in combination. Interestingly, only the CTG assay detected appreciable and statistically significant additive effects. Viability dramatically decreased after combination of 10
Combination of C-150 and bortezomib also showed additive effects. Both methods detected a statistically significant decrease in viability following the combination of 0.1
In conclusion, cytotoxic effects of curcumin and its analogue were detected by two cell viability measurements. Furthermore, we showed that both curcumin and C-150 additively potentiated the effect of bortezomib.
In the same concentration ranges as in the viability tests, we determined the effect of curcumin, C-150, and bortezomib on NF-
In vitro effect of curcumin and C-150 on NF-
— | BTZ |
BTZ |
BTZ |
BTZ | |
---|---|---|---|---|---|
— | 0.36 | 0.44 | 0.54 | 0.94 | |
C (50 |
0.19 | 0.20 | 0.22 | 0.28 | 0.28 |
C (25 |
0.20 | 0.24 | 0.31 | 0.44 | 0.34 |
C (10 |
0.43 | 0.32 | 0.25 | 0.47 | 0.54 |
C (5 |
1.05 | 0.54 | 0.61 | 0.64 | 0.94 |
C-150 (1 |
0.58 | 0.37 | 0.41 |
|
0.65 |
C-150 (0.5 |
0.82 | 0.36 | 0.50 | 0.56 | 0.72 |
C-150 (0.1 |
1.17 |
|
0.54 | 0.58 |
|
C-150 (0.05 |
0.99 | 0.38 | 0.54 |
|
0.95 |
Several studies show a critical role for the NF-
Using FACS analysis and annexin V and PI staining, the percentage of cells in early or late apoptosis was determined.
Two doses of curcumin (15 and 25
Summary of FACS analysis. Percentage of cells in early and late stages of apoptosis and total apoptotic cells as determined by annexin V and PI staining. The curcumin analogue C-150 induced a similar rate of apoptosis at 50-fold lower concentration than curcumin. Furthermore, it retained the ability to potentiate the effect of bortezomib.
Early apoptotic cells |
Late apoptotic cells |
Total apoptotic cells | ||||
---|---|---|---|---|---|---|
% | +/− | % | +/− | % | +/− | |
Control | 4.5 | 0.5 | 3.4 | 0.6 | 7.9 | 1.1 |
BTZ 20 nM | 21.4 | 0.4 | 16.5 | 4.2 | 37.9 | 4.6 |
C 15 |
18.6 | 0.8 | 21.9 | 4.2 | 40.5 | 5.0 |
C 15 |
21.3 | 1.3 | 29.1 | 1.6 | 50.4 | 2.9 |
C 25 |
45.6 | 5.0 | 54.1 | 4.9 | 99.6 | 9.9 |
C 25 |
53.3 | 8.0 | 46.5 | 7.9 | 99.7 | 15.9 |
C-150 0.3 |
23.8 | 4.1 | 7.0 | 2.7 | 30.8 | 6.9 |
C-150 0.3 |
25.4 | 0.1 | 21.4 | 4.4 | 46.9 | 4.5 |
C-150 0.6 |
24.5 | 1.0 | 45.9 | 3.9 | 70.4 | 4.9 |
C-150 0.6 |
24.2 | 2.0 | 45.7 | 3.2 | 69.8 | 5.3 |
At the lower curcumin concentration (15
Effect of curcumin (C) or C-150 treatments alone or in combination with bortezomib (BTZ) on cell viability. C-150 (0.3
From these results, we can conclude that the novel curcumin analogue induced a similar level of apoptosis at 50-fold lower concentration than curcumin, while retaining the ability to potentiate the effect of bortezomib. In addition, we found that both curcumin and C-150 induced apoptosis and not necrosis, since the only propidium iodide positive population did not change (data not shown).
We have analysed the effects of curcumin on the expression of apoptosis and cell cycle related genes using high-throughput QPCR. Figure
Gene expression analysis of curcumin (C) and bortezomib (BTZ) treatment. Curcumin influenced the expression of a wide range of cell cycle and apoptosis related genes.
We can conclude that both apoptosis and cell cycle related genes were differentially expressed by curcumin and by its combination with bortezomib.
We have also investigated the effects of the novel analogue C-150 alone and in combination on genes related to apoptosis and cell cycle. Figure
Gene expression analysis of curcumin analogue C-150 and bortezomib treatment. C-150 influenced the expression of a wide range of cell cycle and apoptosis related genes at 40–80-fold lower concentration compared to curcumin.
In conclusion, C-150 possessed dose-dependent effects on certain apoptosis and cell cycle related genes which could be enhanced by coadministration with bortezomib. In comparison with cotreatment of bortezomib and curcumin or C-150, similar gene expression changes were detected, which suggests a similar mechanism of action.
When gene expression values from curcumin and C-150 treated cells were plotted against each other and their correlation was investigated, we concluded that genes most affected by treatment behaved in a similar fashion suggesting that the related compounds act with similar mechanism of action (Figure
Correlation of gene expression changes following curcumin and C-150 treatment (a). Correlation of gene expression values of the combined treatments of curcumin + bortezomib and C-150 (0.6
While fewer genes responded to C-150 treatment alone compared to curcumin alone (with at least 2-fold induction or repression), combined treatment of C-150 and bortezomib affected the same genes with a similar profile as curcumin and bortezomib together (Figure
It seems that, among the tested members of the tumor necrosis factor receptor superfamily TNFRSF10A, B and C were differentially affected by curcumin and C-150. All three genes were upregulated by curcumin, while C-150 did not alter their expression. GADD45A (growth arrest and DNA-damage-inducible, alpha) was also differentially affected by curcumin and its analog. This stress response gene also mediates the activation of the JNK pathway [
The effective therapy of acute myelocytic leukemia still remains problematic. Single treatment of leukemia and similar hematological cancer types has shown modest improvement. Despite chemotherapy, nearly half of the patients with myelocytic leukemias will fall prey to the disease because of the maturation of multiresistant subclones of leukemia cells [
We found that curcumin and its synthetic analogue C-150 can improve the effect of bortezomib in vitro. From the evidence published by Bai and Zhang and from our findings, curcumin and its analogue C-150 can modulate and inactivate the NF-
We found that 1/8 mice survived in the control group, while both bortezomib and C-150 treatment alone was effective; 3/8 survived after 90 days in both groups. When C-150 and bortezomib were administered in combination, 5/8 mice survived. In addition, we found that this survival ratio did not change until 120 days, when the experiment was terminated (Figure
Survival of HL-60 xenograft mice treated with C-150 (3 mg/kg), bortezomib (0.15 mg/kg), or their combination. The curcumin analogue C-150 and bortezomib alone exhibited antitumor activity in HL-60 xenograft SCID mice, whereas combined treatment (bortezomib + C-150) developed a greater antitumor activity compared to single treatments.
Consistent with the results obtained in vitro, bortezomib or liposome formulated C-150 alone exhibited antitumor activity in HL-60 xenograft SCID mice, whereas combined bortezomib and C-150 treatment resulted in greater antitumor activity than single treatments. These results suggest that the combined use of bortezomib and C-150 could be developed into an effective option in the treatment of AML.
The multitarget action of curcumin and its analog, C-150, showed additive cytotoxic effects with bortezomib. Curcumin and its analogue induced expression changes in apoptotic and cell cycle related genes as determined by high-throughput QPCR. Most of the affected genes showed similar changes suggesting that the related compounds act through similar signaling pathways. Differences in tumor necrosis factor receptor superfamily and GADD45a gene activity profiles induced by curcumin and C-150 could be explained by differences in their target affinity or more likely, effectiveness of the applied concentrations. The enhanced proliferation inhibition capability of curcumin and C-150 analogue with bortezomib was confirmed by in vitro as well as in vivo studies. Pronounced positive effects of the liposomal formulated curcumin analogue on survival of HL60 xenograft mice would render this molecule a potent clinical candidate against leukemia alone or in combination with other antineoplastic agents.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by the following Grant: GOP-1.1.1-11-2011-0003.