SEARCH

SEARCH BY CITATION

Keywords:

  • proliferation;
  • apoptosis;
  • angiogenesis;
  • curcumin

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

Because a role for nuclear factor-κB (NF-κB) has been implicated in the pathogenesis of pancreatic carcinoma, this transcription factor is a potential target for the treatment of this devastating disease. Curcumin (diferuloylmethane) is a phytochemical with potent NF-κB-inhibitory activity. It is pharmacologically safe, but its bioavailability is poor after oral administration.

METHODS

The authors encapsulated curcumin in a liposomal delivery system that would allow intravenous administration. They studied the in vitro and in vivo effects of this compound on proliferation, apoptosis, signaling, and angiogenesis using human pancreatic carcinoma cells. NF-κB was constitutively active in all human pancreatic carcinoma cell lines evaluated and liposomal curcumin consistently suppressed NF-κB binding (electrophoretic mobility gel shift assay) and decreased the expression of NF-κB-regulated gene products, including cyclooxygenase-2 (immunoblots) and interleukin-8 (enzyme-linked immunoassay), both of which have been implicated in tumor growth/invasiveness. These in vitro changes were associated with concentration and time-dependent antiproliferative activity (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide assay [MTT assay]) and proapoptotic effects (annexin V/propidium iodide staining [fluorescence-activated cell sorting] and polyadenosine-5′-diphosphate-ribose-polymerase cleavage).

RESULTS

The activity of liposomal curcumin was equal to or better than that of free curcumin at equimolar concentrations. In vivo, curcumin suppressed pancreatic carcinoma growth in murine xenograft models and inhibited tumor angiogenesis.

CONCLUSIONS

Liposomal curcumin down-regulated the NF-κB machinery, suppressed growth, and induced apoptosis of human pancreatic cells in vitro. Antitumor and antiangiogenesis effects were observed in vivo. The experiments in the current study provide a biologic rationale for treatment of patients suffering from pancreatic carcinoma with this nontoxic phytochemical encapsulated in liposomes for systemic delivery. Cancer 2005. © 2005 American Cancer Society.

Pancreatic carcinoma is an aggressive, chemotherapy-resistant malignancy. As the 5th leading cause of cancer-related mortality, it remains a major public health problem, accounting for an estimated 30,000 deaths per year in the U.S.1 Surgical resection is the only curative therapy, but to the best of our knowledge virtually no patients survive 5 years. Gemcitabine, approved by the Food and Drug Administration in 1996, is the only chemotherapeutic agent licensed for use in pancreatic carcinoma. However, gemcitabine results in an objective tumor response in approximately 5% of patients and the impact on survival was minor.2 That it is considered the most active agent in this disease, despite such limited efficacy, highlights the desperate need for new therapeutic strategies.

Epidemiologic and animal studies have shown that the microchemicals present in the diet can be effective agents for the prevention of cancer.3–26 Some of these compounds have significant antitumor activity in vitro and in preclinical models. Because of their lack of toxicity, there is an increasing interest in plant-derived chemicals that exhibit such activity. The possibility of using phytochemicals with established chemopreventive activities and preclinical antitumor effects as a novel approach for management of established cancer merits exploration.

Curcumin (diferuloylmethane) is a food chemical present in tumeric (Curcuma longa). It has been found to be pharmacologically safe as indicated by its consumption as a dietary spice for centuries.6 This compound has potent antiproliferative and proapoptotic effects in vitro.18, 22, 23 In murine models, curcumin suppresses carcinogenesis of the skin,9–12 the forestomach,26 the breast,13 the colon,14–16 and the liver17 in mice.

Because curcumin has diverse effects on signaling molecules relevant to cancer, multiple mechanisms of action could account for its antitumor effect. Importantly, curcumin is a potent inhibitor of NF-κB, a transcriptor factor implicated in the pathogenesis of several malignancies including pancreatic carcinoma.27 NF-κB-regulated genes include IκBα, cyclooxygenase-2 (COX-2), and interleukin-8 (IL-8).28, 29 Pancreatic carcinoma cells often express high levels of IL-8 and COX-2, and these molecules appear to play a role in the proliferative and metastatic potential of this neoplasm.30, 31 Therefore, suppression of NF-κB and its downstream effectors may be an innovative strategy for treatment of pancreatic carcinoma.

In the current study, we investigated the in vitro and in vivo antitumor activity of liposomal curcumin against human pancreatic carcinoma cells. Our results demonstrate that all six pancreatic carcinoma cell lines studied expressed constitutively active NF-κB, which was suppressed by liposomal curcumin. NF-κB inhibition was associated with growth suppression, apoptosis, and down-regulation of expression of gene products (IL-8 and COX-2) regulated by NF-κB. In vivo, liposomal curcumin inhibited pancreatic carcinoma growth and demonstrated antiangiogenic effects.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Cell Lines

The human pancreatic carcinoma cell lines BxPC-3, Capan-1, Capan-2, ASPC-1, HS766-T, and MiaPaCa2 were purchased from American Type Culture Collection (Manassas, VA) and cultured using the manufacturer' instructions.

Materials

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and (DMPC/DMPG) (DMPG = 1,2-dimyristoyl-sn-glycero-3-[phospho-rac- (1-glycerol)] [sodium salt]) were obtained as dry powder from Avanti Polar Lipids (Alabaster, AL). Curcumin, dimethylsulfoxide (DMSO), acetone, and tert-butanol were obtained from Sigma Chemical Company (St. Louis, MO).

Liposome Preparation

Different total lipid curcumin ratios (weight/weight) ranging from 10:1 to 4:1 were tested before settling on a fixed ratio of 10:1. The lyophilization procedure involved several steps. First, curcumin was dissolved in 50 mg/mL DMSO. The lipid (e.g., DMPC) was dissolved in 20 mg/mL tert-butanol. The 2 solutions were mixed and filtered through a 0.22-μM filter for sterilization. Aliquots of this solution were placed in lyophilization vials. The vials were frozen in a dry ice acetone bath and lyophilized for 24 hours to remove all DMSO and tert-butanol. The vials were stored at −20 °C. The lipid formulation included DMPC or DMPC/DMPG in some experiments.

MTT Cell Proliferation Assay

Proliferation/survival of cells after liposomal curcumin exposure was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay using the manufacturer's instructions (Sigma-Aldrich Company, St. Louis, MO). Cells were incubated for 72 hours. The mean value and standard error for each treatment were determined, then converted to percent relative to control (empty liposomes). The concentration at which cell growth was inhibited by 50% (IC50) was determined by linear interpolation using the formula [(50% − low percentage)/(high percentage − low percentage)] × (high concentration − low concentration) + low concentration. IC90 was similarly determined, with 90% substituting for 50% in the above equation.

Cell Recovery Assay

To determine whether cells treated with liposomal curcumin recovered their proliferative capacity after removal of curcumin, the cell recovery assay was used. Cells were seeded at 5.0 × 105 cells per 100 μm plate and incubated overnight. Cells were treated with various concentrations of liposomal curcumin and incubated for 72 hours. Cells were trypsinized and counted, and the same number of cells for each treatment (3.0–5.0 × 103 cells per well, depending on the cell line) were then seeded in triplicate into 96-well plates in curcumin-free medium and incubated for 72 hours. The degree of cell recovery was determined via MTT assay.

Annexin V/Propidium Iodide Staining for Apoptotic Cells

Cells were seeded at 2.5–5.0 × 105 cells per 100 μm plate, depending on the cell line, and incubated overnight. Cells were treated with various concentrations of liposomal curcumin or free curcumin in DMSO (the final DMSO concentration was 0.1%) and incubated for 72 hours. The cells were harvested by quick (< 5 minutes) trypsinization to minimize potentially high annexin V background levels in adherent cells. Cells were then washed and stained with fluorescein 5(6)-isothiocyanate (FITC)/annexin V/propidium iodide (PI) as directed by the annexin V-FLUOS staining kit (Roche Diagnostics, Indianapolis, IN). Stained cells were placed on ice and protected from light until read via flow cytometry. Cells were analyzed on an Epics XL-MCL flow cytometer using the System II version 3.0 software (both hardware and software are from Beckman Coulter, Miami, FL), with the laser excitation wavelength set at 488 nanometers (nm). The green signal from FITC/annexin V was measured at 525 nm and the red signal from PI was measured at 620 nm. Cells staining negative for both annexin V and PI are viable. Cells that are annexin V+/PI are in early apoptosis, whereas cells that are necrotic or in late apoptosis are annexin V+/PI+.

Electrophoretic Mobility Shift Assay for Nuclear Factor-κB

Electrophoretic mobility shift assay (EMSA) was performed using standard procedures and was used to detect NF-κB binding before and after liposomal curcumin treatment. Cells seeded at 5.0 × 105 cells per 100 μm plate and incubated overnight were treated with IC50 concentrations of liposomal curcumin and incubated for 72 hours.

Nuclear extracts were thawed on ice, and protein concentration was determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). For supershifts, 2 μg of rabbit polyclonal anti-p50 or p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the nuclear extract and incubated at room temperature for 5 or 15 minutes before its addition in the binding reaction.

Quantitation of autoradiographs was performed on the Fluorchem 8900 imaging system using AlphaEase FC software (Alpha Innotech, San Leandro, CA).

Oligonucleotides

The consensus sequence wild-type and mutant blunt-end, double-stranded oligonucleotides used for the NF-κB EMSAs were purchased from Santa Cruz Biotechnology. The NF-κB wild-type sense strand sequence is 5′-AGTTGAGGGGACTTTCCCAGGC-3′ and the mutant sequence is 5′-GTTGAGGCGACTTTCCCAGGC-3′. Oligonucleotides were 32P-end-labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA), purified with Quick Spin G-50 Sephadex columns (Roche Diagnostics), and stored at −20 °C.

Interleukin-8 Enzyme-Linked Immunoadsorbent Assay

Levels of IL-8 protein in conditioned media before and after liposomal curcumin exposure were determined by enzyme-linked immunoadsorbent assay (ELISA) using the Quantikine human IL-8 immunoassay kit by R & D Systems (Minneapolis, MN). The lower limit of sensitivity of the assay is 10 pg/mL.

Immunoblotting

Immunoblotting using standard procedures was performed to assess steady-state levels of COX-2 protein and to determine polyadenosine-5′-diphosphate-ribose-polymerase (PARP) cleavage (the latter reflecting apoptosis). A protein assay to measure protein content was performed by using the bicihonimic acid (BCA) protein assay kit (Pierce Endogen, Rockford, IL). The samples were run on a 6–10% sodium dodecyl sulfate gel. The signal was detected by secondary antibody (horseradish peroxidase-conjugated antimouse immunoglobulin [Ig] or antirabbit Ig, as appropriate [1:5000]) (Amersham Pharmacia Biotech, Piscataway, NJ) and the enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech), and then autoradiographed.

Antibodies

Antibodies used for immunoblotting included monoclonal antitubulin antibody (Sigma Chemical Company), anti-PARP rabbit polyclonal (Cell Signaling, Beverly, MA), and anti-COX-2 monoclonal antibody (MoAb) (Cayman Chemical, Ann Arbor, MI). For EMSAs, rabbit polyclonal anti-p50 and anti-p65 (Santa Cruz Biotechnology) were used. Anti-vascular endothelial growth factor (anti-VEGF) MoAb was obtained from Oncogene (Boston, MA) and rat antimouse CD31 from BD Pharmingen (San Diego, CA).

Animals

Female athymic nu/nu mice (3–5 weeks old) obtained from Harlan Sprague Dawley (Indianapolis, IN) were maintained 5 per cage in microisolator units. Animals were given a commercial diet and water. Mice were quarantined for ≥ 1 week before experimental manipulation. All animal experiments were performed at The University of Texas M. D. Anderson Cancer Center (MDACC) (Houston, TX) under protocol 10-02-13231 approved by the Institutional Animal Care and Use Committee of The University of Texas MDACC.

Animal Models

A total of 5 × 106 BxPC-3 or MiaPaCa2 cells collected in 100 μL RPMI media in log phase growth were injected subcutaneously on 1 side of the abdomen of 3–5 week-old female nude mice. Once tumor masses became established, animals were randomized to receive intravenous liposomal curcumin (intravenous tail vein; 40 mg/kg body weight, 3 time per week; this is the maximum volume that could be injected), empty liposomes, or saline. Tumor size and body weight were measured with calipers three times a week. The tumor volume was calculated using the following formula: volume = (length × width 2)/2, in which width was the shortest measurement in millimeters.

Immunohistochemistry for Angiogenesis

Immunohistochemistry studies were performed by using formalin-fixed, paraffin embedded sections (5 μm), heat-induced antigen retrieval (Dako Corporation, Carpinteria, CA), and 1:200 monoclonal anti-VEGF antibody (Oncogene) or a 1:50 dilution of rabbit polyclonal anti-IL-8 antibody (Biosource International, Camarillo, CA). The frozen sections were cut from snap-frozen tissue and embedded in OCT compound (Miles, Elkhart, IN). Separate sections were incubated with 1:200 rat antimouse CD31 antibody (BD Pharmingen). The detection system was the LSAB2 detection kit (Dako Corporation). The secondary antibody is a biotinylated antibody (Dako Corporation) and forms a complex with peroxidase-labeled streptavidin. Counterstaining was performed by using Gill's hematoxylin (Sigma Chemical Company). Negative controls were performed by replacing the primary antibody with anti-IgG1 (the same isotype with the primary antibody) (Oncogene).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

We used the human pancreatic cell lines ASPC-1, BxPC-3, Capan-1, Capan-2, HS766-T, and MiaPaCa2 to investigate the effect of liposomal curcumin on cell proliferation and apoptosis and on constitutive NF-κB activity and NF-κB-regulated gene expression.

Liposomal Curcumin Inhibits Proliferation/Survival of Pancreatic Carcinoma Cell Lines In Vitro

Exposure to liposomal curcumin (for 72 hours) inhibited pancreatic cell growth in all 6 lines tested in a concentration and time-dependent manner (Fig. 1). Proliferation/survival was assessed by MTT assay. MTT is a pale yellow substrate that is cleaved by living cells to yield a dark blue formazan product. This colorimetric change reflects active cell proliferation/survival. IC50 concentrations varied from approximately 2.0 μM for Capan-1 1 to 37.8 μM for Capan-2. The IC50 for free curcumin ranged from 5.4 μM (BxPC-3 and Capan-1 cells) to 46 μM (Capan-2 cells) (Table 1). The antiproliferative effects of liposomal curcumin were equivalent or better than those free of free curcumin at equimolar concentrations in all cell lines.

thumbnail image

Figure 1. Effect of liposomal curcumin on proliferation/survival as assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay. Liposomal curcumin inhibited the growth of all five pancreatic cell lines tested. Values represent the mean ± the standard error of triplicate experiments after 72 hours of exposure to liposomal curcumin. (A) Dose-dependent inhibition. (B) Time-dependent inhibition of cells exposed to IC90 levels of curcumin (as determined in panel A). IC90: concentration at which cell growth was inhibited by 90%.

Download figure to PowerPoint

Table 1. Inhibitory Concentration of Liposomal Curcumina
Pancreatic carcinoma cell lineASPC-1BxPC-3Capan-1Capan-2HS766-TMiaPaCa2
  • IC50: concentration at which 50%; growth inhibition occurred; IC90: concentration at which 90% growth inhibition occurred.

  • a

    Mean of triplicate experiments (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide [MTT] assay after 72 hours of exposure to curcumin).

IC505 μM2.5 μM2.0 μM37.8 μM6.75 μM4.8 μM
IC9027 μM10.5 μM6.75 μM94.5 μM21.6 μM20.0 μM

Growth-Inhibitory Effects of Liposomal Curcumin Are at Least Partially Irreversible

After exposure to liposomal curcumin for 72 hours, pancreatic carcinoma cells were replated in fresh media, and recovery of proliferation/survival was assessed (by MTT assay) after an additional 72 hours. (The concentrations of liposomal curcumin were approximately the IC50 and IC90 for each cell line.) There was a concentration-dependent loss of ability to recover in all six cell lines, although this was not complete in all cell lines. These results suggest that the cells had at least partially undergone irreversible changes such as apoptosis (Fig. 2).

thumbnail image

Figure 2. (A–C) Failure of proliferation/survival (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide [MTT] assay) to recover after liposomal curcumin. The antiproliferative effects of liposomal curcumin on pancreatic cells were irreversible. Five × 103 cells were plated at Time 0. After 72 hours of growth with and without curcumin, 5 × 103 cells were replated in fresh media. At 72 hours, MTT assay was performed. Liposomal curcumin-treated cells failed to recover. Data represent the mean ± the standard error of triplicate experiments. Black bars: 72-hour treatment; gray bars: 72-hour treatment and 72-hour recovery.

Download figure to PowerPoint

Liposomal Curcumin Induces Apoptosis in Pancreatic Carcinoma Cell Lines

Apoptosis was first assessed by annexin V/PI staining (FACS analysis) after 72 hours of exposure to liposomal curcumin. Annexin V binds to cells that express phosphatidylserine on the outer layer of the cell membrane, a characteristic feature of cells entering apoptosis. This allows discrimination of live cells (unstained by either fluorochrome) from apoptotic cells (stained with annexin V). There was a dose-related increase in apoptosis after liposomal curcumin exposure and the effects of liposomal curcumin were equal to or better than those of free curcumin at equimolar concentrations (Fig. 3A).

thumbnail image

Figure 3. Effect of liposomal curcumin on apoptosis. Liposomal curcumin induced apoptosis of pancreatic cells and its effect was equivalent or better compared with free curcumin at equimolar concentrations. Programmed cell death was assessed by annexin V/propidium iodide staining (fluorescence-activated cell sorting analysis) after 72 hours of exposure to liposomal curcumin. (A) The mean ± the standard error of the percent apoptotic cells in triplicate experiments. Black bars: liposomal curcumin; gray bars: free curcumin. (B) Apoptosis was also assessed by Western blot analysis for polyadenosine-5′-diphosphate-ribose-polymerase cleavage after 72 hours of exposure to liposomal curcumin.

Download figure to PowerPoint

A hallmark of apoptosis is cleavage of PARP from the native 116 kilodalton (kD) to 85 kD. PARP is an enzyme involved in DNA damage and repair mechanisms. It synthesizes the poly(ADP-ribose) polymer to chromatin and other target proteins. During apoptosis, PARP is cleaved by the protease, caspase-3, an important downstream apoptotic caspase. Western blot analysis using anti-PARP antibody demonstrated PARP cleavage after 72 hours of incubation with IC50 to IC90 levels of liposomal curcumin (Fig. 3B and data not shown).

Liposomal Curcumin Inhibits Activation of the NF-κB Transcription Factor in Pancreatic Carcinoma Cell Lines

NF-κB is a transcription factor that has been implicated in the growth of diverse neoplasms including pancreatic carcinoma.27 The NF-κB pathway regulates numerous downstream oncogenic and growth signals. We have previously demonstrated, with the use of EMSAs, constitutive binding of NF-κB transcription factor in all pancreatic carcinoma cell lines tested.32 Liposomal curcumin inhibited NF-κB activation in a dose-dependent manner in all of these lines (representative data on Fig. 4). At equimolar concentrations, liposomal curcumin was as effective or better than free curcumin at suppressing NF-κB activity (Fig. 4).

thumbnail image

Figure 4. Effect of liposomal curcumin on nuclear factor-κB (NF-κB) binding as assessed by electrophoretic mobility gel shift assay. Liposomal curcumin decreased NF-κB binding in pancreatic cell lines. Cells were exposed to IC75 levels of liposomal or free curcumin for 72 hours. Equal amounts of protein (8 μg) were used in each lane. Supershifts using anti-p50 and anti-p65 antibodies confirmed that the band shown contained p50 and p65 subunits of NF-κB. IC75: concentration at which cell growth was inhibited by 75%.

Download figure to PowerPoint

Because NF-κB is a family of proteins, various combinations of the Rel/NF-κB protein can constitute an active NF-κB heterodimer that binds to a specific sequence in DNA. To show that the retarded band visualized by EMSA in pancreatic carcinoma cells was indeed NF-κB, we incubated nuclear extracts from pancreatic carcinoma cells with antibody to either the p50 (NF-κB1) or the p65 (RelA) subunit of NF-κB. Both shifted the band to a higher molecular mass (Fig. 4), thus suggesting that the major NF-κB band in pancreatic carcinoma cells consisted of p50 and p65 subunits. A nonspecific minor band was also observed. This was not supershifted by the antibody. Preimmune serum had no effect and excess unlabeled NF-κB (100-fold) caused complete disappearance of the band (data not shown). Use of mutated NF-κB oligonucleotide (instead of the NF-κB oligonucleotide) failed to yield the NF-κB bands (data not shown).

Liposomal Curcumin Decreases the Steady-State Level of NF-κB-Regulated Gene Products (Interleukin-8 and Cyclooxygenase-2)

IL-8 has been implicated in the growth and metastatic potential of pancreatic carcinoma.31 IL-8 expression is regulated by NF-κB. In our current experiments, 72 hours of exposure to IC50 levels of curcumin substantially decreased IL-8 levels (35–90%) as assessed by ELISA in all 6 lines tested (Fig. 5).

thumbnail image

Figure 5. Effect of liposomal curcumin on interleukin-8 (IL-8) levels as assessed by enzyme-linked immunoadsorbent assay (ELISA). Liposomal curcumin exposure substantially decreased IL-8 levels in conditional media of all 5 pancreatic carcinoma cell lines, as assessed by ELISA (R & D Systems, Minneapolis, MN). Cells were exposed to IC50 levels of curcumin for 72 hours. The decrease in levels ranged from 29–79%. The mean ± the standard error of triplicate values are shown. IL-8 levels of controls were 16,525 pg/mL (ASPC-1 cells); 2666 pg/mL (BxPC-3 cells); 21,649 pg/mL (Capan-1 cells); 7329 pg/mL (Capan-2 cells); 473 pg/mL (HS766-T cells); and 5415 pg/mL (MiaPaCa2 cells).

Download figure to PowerPoint

COX-2 has been implicated in the growth of diverse tumors including pancreatic carcinoma8, 33–35 and its expression is regulated by NF-κB.29 Steady-state levels of COX-2 protein were assessed by Western blotting of protein extracts (100 μg per lane) derived from pancreatic carcinoma cell lines, both before and after exposure to IC90 levels of curcumin for 72 hours. BxPC-3, Capan-1, and Capan-2 expressed COX-2, but ASPC-1 and HS766T did not.32 In COX-2–expressing lines, a dose-dependent decrease in COX-2 expression was seen after exposure to liposomal curcumin (Fig. 6). These changes were dose dependent (data not shown).

thumbnail image

Figure 6. Effect of liposomal curcumin on cyclooxygenase-2 levels as assessed by Western blot analysis. Steady-state levels were decreased after 72 hours of exposure to IC90 levels of liposomal curcumin. Tubulin levels remained unchanged. IC90: concentration at which cell growth was inhibited by 90%.

Download figure to PowerPoint

For both IL-8 and COX-2, the suppressive effect of liposomal curcumin was equal to or greater than that of free curcumin at equimolar concentrations.

Liposomal Curcumin Inhibits Pancreatic Tumor Xenograft Growth in Murine Models

Liposomal curcumin suppressed the growth of both BxPC-3 and MiaPaCa2 tumors in murine models (Fig. 7). Animals received 40 mg/kg of liposomal curcumin intravenously 3 times weekly. This dose was the maximum that could be delivered based on volume injected. The mice demonstrated no overt drug-related toxicity.

thumbnail image

Figure 7. Effect of liposomal curcumin on in vivo growth of pancreatic tumor xenografts. Liposomal curcumin had significant growth suppressive effects compared with control vehicles (saline or empty liposomes) (P < 0.05) Five mice were treated in each group. Measurements are expressed as the mean ± the standard error of tumor volume. (A) BxPC-3 model. (B) MiaPaCa2 model. Heavy dashed line: intravenous saline; small dashed line: intravenous empty liposomes; straight line: intravenous liposomal curcumin.

Download figure to PowerPoint

Liposomal Curcumin Inhibits Angiogenesis in Vivo

Treatment with liposomal curcumin resulted in reduced tumor size and visible blanching of tumors (Fig. 8A). In addition, expression of CD31 (endothelial cell marker), as well as VEGF and IL-8, was decreased in both BxPC-3 and MiaPaCa2 tumor xenografts (by immunohistochemistry analysis), consistent with an antiangiogenic effect (Fig. 8B and data not shown).

thumbnail image

Figure 8. Antiangiogenic effect of liposomal curcumin in vivo. Mice bearing MiaPaCa2 tumor xenografts were treated with saline, empty liposomes, or liposomal curcumin. Approximately 2 weeks after the start of treatment, mice were killed. (A) Dissected tumors are shown. Smaller size and blanching of tumors from mice treated with liposomal curcumin are evident. Tumors on the left side versus the right side represent tumors from two different mice. (B) Immunohistochemistry of tumor sections using anti-CD31, anti-vascular endothelial growth factor (VEGF), and anti–interleukin-8 (IL-8) antibodies. Tumors from mice treated with liposomal curcumin showed decreases in CD31, VEGF, and IL-8 expression, suggesting an antiangiogenic effect.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Plant-derived chemicals have been used successfully for a wide spectrum of medicinal purposes. Their salutary effects often derive from a favorable therapeutic to toxicity index. Curcumin (diferuloylmethane) is a component of the root of the plant Curcuma longa. A wealth of data indicates that this compound has potent antitumor effects against a variety of cancer cell lines in vitro, and chemopreventive effects in murine carcinoma models.26, 33–35 Furthermore, curcumin is virtually devoid of side effects in animals as well as in early Phase I trials in humans.36 Free curcumin is, however, highly hydrophobic and cannot be administered systemically. Liposome encapsulation of curcumin makes this agent amenable to intravenous dosing and circumvents the problem of poor oral availability that limits the utility of free curcumin.

Because of the central role of NF-κB in cell survival and proliferation, we explored this transcription factor as a target for liposomal curcumin. During normal homeostasis, NF-κB is present in the cytoplasm as an inactive heterotrimer composed of the p50, p65, and IκBα subunits.37 After activation, IκBα undergoes phosphorylation and ubiquitination-dependent degradation by the proteosome. As a result, nuclear localization signals on the p50-p65 heterodimer are exposed, leading to nuclear translocation and binding to a specific consensus sequence found in the promoters of diverse, growth-regulatory genes. This binding activates gene transcription. Considerable data now indicate that NF-κB regulates the expression of various genes that play critical roles in apoptosis, proliferation, and transformation.38 We have previously reported that NF-κB is constitutively active in all the human pancreatic cell lines evaluated.32 Liposomal curcumin decreased NF-κB binding and its effects were as potent as these of free curcumin (Fig. 4). NF-κB inhibition was accompanied by marked in vitro growth suppression and apoptosis (Figs. 1–3).

Several mechanisms could explain why NF-κB down-regulation by liposomal curcumin attenuates proliferation of cancer cells.39–42 First, the constitutive transcription of various angiogenic and tumorigenic chemokines modulated by NF-κB facilitates proliferation and survival of malignant cells.42 In our studies, down-regulation of expression of the gene products COX-2 and IL-8, whose synthesis is known to be regulated by NF-κB, was observed (Figs. 5 and 6). Importantly, in this regard, high levels of COX-2 have been implicated in the growth of several malignancies including pancreatic carcinoma30, 43, 44 and ectopic COX-2 expression suppresses apoptosis.39, 40 The down-regulation of COX-2 by liposomal curcumin is most likely mediated by the attenuation of NF-κB binding, which is needed for COX-2 expression.29 Liposomal curcumin also decreased IL-8 expression (Fig. 6). This is noteworthy because IL-8 is a pleiotropic cytokine that promotes tumor growth and vascularization.9 IL-8 transcription is controlled, at least in part, by NF-κB.31

The inhibitory effects of liposomal curcumin on the NF-κB apparatus were associated with substantial antiproliferative activity against all six of our human pancreatic carcinoma cell lines (Table 1). Consistent failure of the cells to recover after removal of liposomal curcumin from the media suggested a cytotoxic, rather than cytostatic, effect (Fig. 2). Marked programmed cell death was observed after 72 hours of treatment with liposomal curcumin (Fig. 3).

Liposomal curcumin was also active in in vivo models. We treated animals bearing human pancreatic carcinoma xenografts (BxPC-3 and MiaPaCa2) with systemically administered liposomal curcumin. Significant tumor growth inhibition without overt host toxicity was observed (Fig. 7). Tumors from animals treated with liposomal curcumin demonstrated an antiangiogenic effect, including obvious blanching of tumors on visual inspection and attenuation of CD31 (an endothelial marker) and VEGF and IL-8 expression (Fig. 8). These results are consistent with those of Arbiser et al.,45 who demonstrated that curcumin suppresses corneal neovascularization in mice.

Pancreatic carcinoma is a devastating illness, and virtually all patients die, usually within 1 year. This malignancy is highly resistant to chemotherapy. The best agent available, gemcitabine, improved survival by only 6 weeks in a pivotal, randomized trial.2 Centuries of use of curcumin in Far Eastern countries demonstrate that this phytochemical is pharmacologically safe. In addition, in Phase I clinical trials, humans can tolerate ≤ 8 g per day when free curcumin is ingested.36 The bioavailability of oral curcumin is, however, poor.36, 46 A liposome-encapsulated formation of curcumin circumvents this problem by permitting intravenous administration. The results presented in the current study demonstrate that liposomal and free curcumin are equipotent in their ability to suppress NF-κB activity, COX-2, and IL-8 expression, as well as cell proliferation/survival of pancreatic carcinoma cells. In vivo, liposomal curcumin inhibits pancreatic cell growth in murine xenograft models and these effects are accompanied by a potent antiangiogenic response. No overt host toxicity is noted when maximal volumes are administered to mice. Taken together with the dismal outlook for patients with pancreatic carcinoma, our observations suggest that liposomal curcumin should be investigated in the clinical setting.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • 1
    Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin. 2002; 52: 2347.
  • 2
    Burris HA III, Moore MJ, Andersen J, et al. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol. 1997; 15: 24032413.
  • 3
    Willett WC. Diet and breast cancer. J Intern Med. 2001; 249: 395411.
  • 4
    Kelloff GJ. Perspectives on cancer chemoprevention research and drug development. Adv Cancer Res. 2000; 78: 199334.
  • 5
    el-Bayoumy K. Evaluation of chemopreventive agents against breast cancer and proposed strategies for future clinical intervention trials. Carcinogenesis. 1994; 15: 23952420.
  • 6
    Ammon HP, Wahl MA. Pharmacology of Curcuma longa. Planta Med. 1991; 57: 17.
  • 7
    Huang M-T, Wang ZY, Georgiadis CA, Laskin JD, Conney AH. Inhibitory effects of curcumin on tumor initiation by benzo[a]pyrene and 7,12-dimethylbenz[a] anthracene. Carcinogenesis. 1992; 13: 21832186.
  • 8
    Conney AH, Lysz T, Ferraro T, et al. Inhibitory effect of curcumin and some related dietary compounds on tumor promotion and arachidonic acid metabolism in mouse skin. Adv Enzyme Regul 1991; 31: 385396.
  • 9
    Lu YP, Chang RL, Lou YR, et al. Effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate- and ultraviolet B light-induced expression of c-Jun and c-Fos in JB6 cells and in mouse epidermis. Carcinogenesis. 1994; 15: 23632370.
  • 10
    Limtrakul P, Lipigorngoson S, Namwong O, Apisariyakul A, Dunn FW. Inhibitory effect of dietary curcumin on skin carcinogenesis in mice. Cancer Lett. 1997; 116: 197203.
  • 11
    Huang MT, Ma W, Yen P, et al. Inhibitory effects of topical application of low doses of curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion and oxidized DNA bases in mouse epidermis. Carcinogenesis. 1997; 18: 8388.
  • 12
    Huang MT, Lysz T, Ferraro T, Abidi TF, Laskin JD, Conney AH. Inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis. Cancer Res. 1991; 51: 813819.
  • 13
    Huang MT, Lou YR, Xie JG, et al. Effect of dietary curcumin and dibenzoylmethane on formation of 7,12-dimethylbenz[a]anthracene-induced mammary tumors and lymphomas/leukemias in Sencar mice. Carcinogenesis 1998; 19: 16971700.
  • 14
    Kim JM, Araki S, Kim DJ, et al. Chemo-preventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethyl-hydrazine initiation. Carcinogenesis. 1998; 19: 8185.
  • 15
    Rao CV, Rivenson A, Simi B, Reddy BS. Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res. 1995; 55: 259266.
  • 16
    Kawamori T, Lubet R, Steele VE, et al. Chemopreventive effect of curcumin, a naturally occurring anti-inflammatory agent, during the promotion/progression stages of colon cancer. Cancer Res. 1999; 59: 597601.
  • 17
    Chuang SE, Kuo ML, Hsu CH, et al. Curcumin-containing diet inhibits diethylnitrosamine-induced murine hepatocarcinogenesis. Carcinogenesis. 2000; 21: 331335.
  • 18
    Kuo ML, Huang TS, Lin JK. Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells. Biochim Biophys Acta. 1996; 1317: 95100.
  • 19
    Ranjan D, Johnston TD, Reddy KS, Wu G, Bondada S, Chen C. Enhanced apoptosis mediates inhibition of EBV-transformed lymphoblastoid cell line proliferation by curcumin. J Surg Res. 1999; 87: 15.
  • 20
    Piwocka K, Zablocki K, Wieckowski MR, et al. A novel apoptosis-like pathway, independent of mitochondria and caspases, induced by curcumin in human lymphoblastoid T (Jurkat) cells. Exp Cell Res. 1999; 249: 299307.
  • 21
    Han SS, Chung ST, Robertson DA, Ranjan D, Bodada S. Curcumin causes the growth arrest and apoptosis of B cell lymphoma by downregulation of egr-1, c-myc, bcl-XL, NF-kappa B, and p53. Clin Immunol. 1999; 93: 152161.
  • 22
    Chen H, Zhang ZS, Zhang YL, Zhou DY. Curcumin inhibits cell proliferation by interfering with the cell cycle and inducing apoptosis in colon carcinoma cells. Anticancer Res. 1999; 19: 36753680.
  • 23
    Mehta K, Pantazis P, McQueen T, Aggarwal BB. Curcumin (diferuloyl methane) is an antiproliferative agent against human breast tumor cell lines. Anticancer Drugs. 1997; 8: 470481.
  • 24
    Hidaka H, Ishiko T, Furuhashi T, et al. Curcumin inhibits interleukin-8 production and enhances interleukin-8 receptor expression on the cell surface: impact on human pancreatic carcinoma cell growth by autocrine regulation. Cancer. 2000; 95: 12061214.
  • 25
    Korutla L, Kumar R. Inhibitory effect of curcumin on epidermal growth factor receptor kinase activity in A431 cells. Biochim Biophys Acta. 1994; 1224: 597600.
  • 26
    Huang MT, Lou YR, Ma W, Newmark HL, Reuhl KR, Conney AH. Inhibitory effects of dietary curcumin on forestomach, duodenal and colon carcinogenesis in mice. Cancer Res. 1994; 54: 58415847.
  • 27
    Wang W, Abbruzzese JL, Evans DB, Larry L, Cleary KR, Chiao PJ. The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. Clin Cancer Res. 1999; 5: 119127.
  • 28
    Jobin C, Bradham CA, Russo MP, et al. Curcumin blocks cytokine-mediated NF-kappa B activation and proinflammatory gene expression by inhibiting inhibitory factor I-kappa B kinase activity. J Immunol. 1999; 163: 34743483.
  • 29
    Plummer SM, Holloway KA, Manson MM, et al. Inhibition of cyclo-oxygenase 2 expression in colon cells by the chemopreventive agent curcumin involves inhibition of NF-kappaB activation via the NIK/IKK signalling complex. Oncogene. 1999; 18: 60136020.
  • 30
    Yip-Schneider MT, Barnard DS, Billings SD, et al. Cyxlooxygenase-2 expression in human pancreatic adenocarcinomas. Carcinogenesis. 2000; 21: 139146.
  • 31
    Le X, Shi Q, Wang B, et al. Molecular regulation of constitutive expression of interleukin-8 in human pancreatic adenocarcinoma. J Interferon Cytokine Res. 2000; 20: 935946.
  • 32
    Li L, Aggarwal BB, Shishodia S, Abbruzzese J, Kurzrock R. Nuclear factor-κB and IκB kinase are constitutively active in human pancreatic cells and their downregulation by curcumin (diferuloyl methane) is associated with suppression of proliferation and induction of apoptosis. Cancer. In press.
  • 33
    Moradoga L, Jaszewski R, Majumdar AN. Curcumin induced modulation of cell cycle and apoptosis in gastric and colon cancer cells. Anticancer Res. 2001; 21: 873878.
  • 34
    Huang MT, Smart RC, Won CQ, Conney AH. Inhibitory effect of curcumin, chlorogenic acid, caffeic acid and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoyl phorbol-13-acetate. Cancer Res. 1988; 48: 59415946.
  • 35
    Tanaka T, Makita H, Ohnishi M, et al. Chemoprevention of 4-nitro-quinlone 1-oxide-induced oral carcinogenesis by dietary curcumin and hesperidin: comparison with the protective effect of betacarotine. Cancer Res. 1994; 54: 46534659.
  • 36
    Cheng AL, Shu CH, Lin JK, et al. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res. 2001; 21: 28952900.
  • 37
    Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol. 2000; 18: 621663.
  • 38
    Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene. 1999; 18: 68536866.
  • 39
    Williams CS, Mann M, DuBois RN. The role of cyclooxygenases in inflammation, cancer, and development. Oncogene. 1999; 18: 79087916.
  • 40
    Reddy BS, Hirose Y, Lubet R, et al. Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis. Cancer Res. 2000; 15; 60: 293297.
  • 41
    Xiong HQ, Abbruzzese JL, Lin E, Wang L, Zheng L, Xie K. NF-kappaB activity blockade impairs the angiogenic potential of human pancreatic cancer cells. Int J Cancer. 2004; 10; 108: 181188.
  • 42
    Richmond A. NF-κB, chemokine gene transcription and tumour growth. Nat Rev Immunol. 2002; 2: 664674.
  • 43
    Fournier DB, Gordon GB. COX-2 and colon cancer: potential targets for chemoprevention. J Cell Biochem. 2000; 77: 97102.
  • 44
    Hida T, Yatabe Y, Achiwa H, et al. Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer Res. 1998; 58: 37613764.
  • 45
    Arbiser JL, Klauber N, Rohan R, et al. Curcumin is an in vivo inhibitor of angiogenesis. Mol Med. 1998; 4: 376383.
  • 46
    Garcea G, Jones DJ, Singh R, et al. Detection of curcumin and its metabolites in hepatic tissue and portal blood of patients following oral administration. Br J Cancer. 2004; 8; 90: 10111015.