Patent Application
20110224205
The present invention relates to use of curcumin or its analogues in cancer therapy utilizing an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), which reduces side effects resulting from the therapy and reduces doses of the EGFR-TKI needed for the therapy, particular in a patient resistant to treatment with the EGFR-TKI alone.
Cancer is the leading cause of death in the world. Recently, so called “target therapy” has been developed and agents that selectively target epidermal growth factor receptor (EGFR) have been shown to be of benefit clinically (Cancer Research 2004; 64(15):5355-62.). The EGFR pathway is a key driver in the regulation of cell growth and differentiation and acts via regulating the phosphorylation of intrinsic tyrosine kinases (Cancer Research 2003; 63(1):1-5). Over-expression of EGFR has been reported to occur in various malignant cells and is correlated with a poor prognosis (Oncologist 2004; 9(1):58-67). However, some critical issues still remain to limit the use of these agents.
Gefitinib (Iressa®), an orally active EGFR-TKI, is the first selective small molecular agent approved for non-small cell lung cancer (NSCLC) treatment (Lung Cancer 2003; 41 Suppl 1:S9-14; and Expert Review of Anticancer Therapy 2004; 4(1):5-17). Previous studies involving a multi-institutional clinical trial have been showed that the response to gefitinib is better in Asian patients compared to Caucasian patients and that women who are non-smokers and have adenocarcinoma are the most likely to benefit the most (Proceedings of the National Academy of Sciences of the United States of America 2004; 101(36):13306-11; and Lancet 2005; 366(9496):1527-37). Recent studies have indicated that in-frame deletions (ΔE746-A750) of exon 19 and L858R substitution in exon 21 of EGFR in NSCLC are highly correlated with gefitinib sensitivity (New England Journal of Medicine 2004; 350(21):2129-39; Science 2004; 304(5676):1497-500; and Oncologist 2008; 13(12):1276-84). However, the EGFR gene mutation rate of NSCLC patients has been found to range from 10% to 15% in Caucasians and from 30% to 40% in Asians (Clinical Cancer Research 2008; 14(10):2895-9; and Journal of the National Cancer Institute 2005; 97(5):339-46). Patients with a wild-type EGFR are still prominent in all NSCLC cases worldwide and this population shows a relatively poor response to gefitinib treatment. In addition, acquired resistance caused by a second site substitution, T790M in EGFR within exon 20, results in poor gefitinib activity (New England Journal of Medicine 2005; 352(8):786-92; and PLoS Medicine/Public Library of Science 2005; 2(3):e73).
Side effects are another limiting factor for the use of EGFR-TKIs. Gefitinib is known to cause side effects such as diarrhea (Journal of Clinical Oncology 2003; 21(12):2237-46; and Clinical Cancer Research 2004; 10(4):1212-8) and skin rash. The frequency of diarrhea caused by gefitinib was 67% in the 500 mg/day dose group and 48% in the 250 mg/day dose group during the clinical trials. A recent case report has indicated the concomitant use of EGFR-TKI and radiotherapy can cause unexpected toxicity and fatal diarrhea in a metastatic NSCLC patient (Lung Cancer 2008; 61(2):270-3). These adverse effects may lead to physical and psychosocial discomfort that can result in dose reduction or treatment interruption.
The present invention is based on the finding that curcumin is a potential agent to reduce side effects resulting from EGFR-TKI treatment and reduce doses of an EGFR-TKI needed for cancer therapy with the EGFR-TKI, particular in a patient resistant to treatment with the EGFR-TKI alone.
Accordingly, in one aspect, the present invention provides a method for reducing side effects resulting from treatment using an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), comprising administering curcumin or its analogue to a patient undergoing such treatment in an amount effective to reduce the side effects. In one embodiment, the side effects are EGFR-TKI induced adverse gastrointestinal effects, such as intestinal cell damage or growth inhibition.
In another aspect, the present invention provides a method for administering an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) to a patient in need of a cancer therapy using the EGFR-TKI, comprising administering to the patient a reduced dose of the EGFR-TKI in combination with curcumin or its analogue while efficacy of the EGFR-TKI with respect to the cancer therapy is substantially maintained as compared to that achieved with a standard dose of the EGFR-TKI without administration of the curcumin or its analogue. In one embodiment, the patient is diagnosed as EGFR-TKI resistant. In one embodiment, the reduced dose is about 50% or less of the standard dose of the EGFR-TKI.
In one embodiment, the patient to be treated is afflicted with non-small-cell lung cancer (NSCLC). In another embodiment, the patient to be treated is resistant to EGFR-TKI.
In one embodiment, the curcumin analogues are selected from the group consisting of:
In one embodiment, the EGFR-TKI is gefitinib (N-(3-Chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine).
In one embodiment, the curcumin or its analogue is administered concurrently with the EGFR-TKI.
Also provided is a method for treating a cancer patient with resistance to an EGFR-TKI, which comprises jointly administering to said patient an effective amount of the EGFR-TKI with curcumin or its analogue.
The various embodiments of the present invention are described in details below. Other characteristics of the present invention will be clearly presented by the following detailed descriptions and drawings about the various embodiments and claims.
It is believed that a person of ordinary knowledge in the art where the present invention belongs can utilize the present invention to its broadest scope based on the descriptions herein with no need of further illustration. Therefore, the following descriptions should be understood as of demonstrative purpose instead of limitative in any way to the scope of the present invention.
For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the preferred embodiments shown.
In the drawings:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sample” includes a plurality of such samples and equivalents thereof known to those skilled in the art.
As described above, it is found in the invention that administration of curcumin or its analogue can reduce side effects caused by EGFR-TKI treatment in a patient undergoing the treatment.
Therefore, in one aspect, the present invention provides a method for reducing side effects resulting from treatment using an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), comprising administering curcumin or its analogue to a patient undergoing such treatment in an amount effective to reduce the side effects.
Epidermal growth factor receptor (EGFR) is a 170 kilodalton (kDa) membrane-bound protein expressed on the surface of epithelial cells, which is known to involve regulation of cell growth and differentiation and act via regulating the phosphorylation of intrinsic tyrosin kinases. Over expression of EGFR has been reported to occur in various malignant cells and is correlated with a poor prognosis. As used herein, the EGFR protein is disclosed as GenBank accession no NM—005228 (SEQ ID NO: 1).
The term “EGFR-TKI” as used herein refers to an epidermal growth factor receptor tyrosine kinase inhibitor. Certain examples of EGFR-TKIs include gefitinib i.e. N-(3-Chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine (Iressa®) and erlotinib i.e. N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (Tarceva®), which are medicines for treating non-small cell lung cancer in clinical.
The term “side effects” as used herein refers to adverse effects induced by EGFR-TKIs such as adverse gastrointestinal effects (e.g. diarrhea, damage on intestine villi/cells, or growth inhibition of intestinal cells) or unfavorable skin conditions (e.g. rash or dry skin). In one embodiment, the side effects are EGFR-TKI induced intestinal cell damage or growth inhibition.
Curcumin (diferuloylmethane) is a highly active component extracted from the plant Curcuma longa, the formula of which (enol form) is as follows:
The term “curcumin” as used herein also includes its analogues, derivatives, or salts. A product made from curcumin, such as a food additive or supplement, is also included. In one embodiment, curcumin analogues are selected from the group consisting of LL-17, LL-18, LL-68 and LC-15, the formulae of which are shown below:
Curcumin or its analogue, derivative or salt thereof as used herein may be synthesized or isolated from natural sources according to common methods known in the art such as those described in Bioorganic & Medicinal Chemistry 2006; 14(8):2527-34; and Journal of Medicinal Chemistry 2006; 49(13):3963-72.
The terms “patient,” “subject” and “individual” are used interchangeably herein and particularly refer to a human subject for which cancer therapy is desired. In one embodiment, the subject is afflicted with non-small-cell lung cancer (NSCLC).
According to the invention, an EGFR-TKI and curcumin or its analogue may be administered in one therapeutic dosage form or in separate therapeutic dosages such as in separate capsules, tablets, containers, or injections. The EGFR-TKI and curcumin or its analogue can be administered simultaneously (concurrently) or sequentially. In one embodiment, the EGFR-TKI and curcumin or its analogue are administered concurrently.
To facilitate delivery, the EGFR-TKI and curcumin according to the invention may be, individually or in combination, formulated into a pharmaceutical composition with a pharmaceutically acceptable carrier. “Pharmaceutically acceptable” as used herein means that the carrier is compatible with the active ingredient contained in the composition, preferably capable of stabilizing the active ingredient, and not deleterious to the subject to be treated. The carrier may serve as a diluent, vehicle, excipient, or medium for the active ingredient. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The pharmaceutical composition can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
The pharmaceutical composition according to the invention can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and packaged powders.
The pharmaceutical composition of the invention may be delivered through any physiologically acceptable route such as orally, parentally (e.g. intramuscularly, intravenously, subcutaneously, interperitoneally), transdermally, rectally, by inhalation and the like. In one embodiment, the composition of the invention is orally administrated.
In the invention, it is also found that when an EGFR-TKI is administered to a patient in need thereof in combination with curcumin, the effective amount of EGFR-TKI can be reduced while the therapeutic efficacy is substantially maintained as compared to administering the EGFR-TKI alone.
Therefore, in another aspect, the present invention provides a method for administering an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) to a patient in need of a cancer therapy using the EGFR-TKI, comprising administering to the patient a reduced dose of the EGFR-TKI in combination with curcumin or its analogue while efficacy of the EGFR-TKI with respect to the cancer therapy is substantially maintained as compared to that achieved with a standard dose of the EGFR-TKI without administration of the curcumin or its analogue.
An “effective amount” or an “effective dose,” in connection with administration of a pharmacological agent, indicates an amount or dose that results in an intended pharmacological result, such as improvement of symptoms, reduction of side effects, extension of life or improvement of quality of life; in the case of a subject having a malignant tumor, for example, the rate of tumor growth is decreased, the volume of such tumor is reduced, or the tumor is eliminated entirely. The effective amount or dose of a pharmacological agent may vary depending on particular active ingredient employed, the mode of administration, and the age, size, and condition of the subject to be treated. Precise amounts of a pharmacological agent required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
The term “a standard dose” as used herein refers to an effective dose of a therapeutic agent that is recommended by authoritative sources in the pharmaceutical community including the Food and Drug Administration and often used in routine practice. The term “a reduced dose” as used herein refers to a dose that is lower than a standard dose but still retains substantially the same therapeutic effects of the same therapeutic agent. Specifically, according to the invention, a reduced dose of an EGFR-TKI is about 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, of standard therapeutic dose of the EGFR-TKI. In one embodiment of the invention, the reduced dose is about 50% or less of the standard dose of the EGFR-TKI.
It is known that some patients have a poor response to EGFR-TKI treatment and may need a high dose to achieve the required therapeutic effect, which however would cause unacceptable toxicity to the patient and lead to treatment interruption eventually. Surprisingly, the method of the invention is particularly effective in treatment of the patients with resistance to EGFR-TKI. When applied to EGFR-TKI resistant patients, the method of the invention allows a reduced dose of the EGFR-TKI to be administered to the patient while the therapeutic efficacy is substantially maintained as compared to administrating the EGFR-TKI alone. In one embodiment, the patients harbor mutation indicative of resistance to an EGFR-TKI such as T790M substitution in EGFR (SEQ ID NO: 1).
Also provided is a method for treating a cancer patient with resistance to an EGFR-TKI, which comprises jointly administering to said patient an effective amount of the EGFR-TKI with curcumin or its analogue.
The term “treating” as used herein refers to the application or administration of a composition including one or more active agents to a subject, who has a disease particularly to be treated by EGFR-TKI, including but are not limited to, tumor or cancer, a symptom of the disease, or a predisposition toward the disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of the disease, or the predisposition toward the disease.
The present invention will now be described more specifically with reference to the following embodiments, which are provided for the purpose of demonstration rather than limitation.
Materials and Methods
1. Reagents
Curcumin (purity by HPLC: 98.0%) for the in vitro studies was purchased from Calbiochem (Darmstadt, Germany). In vivo, the curcumin (purity˜70%) was from Sigma (St Louis, Mo.). Gefitinib (Iressa®), ZD1839, was kindly provided by Astra-Zeneca Pharmaceuticals (Macclesfield, UK). Stock solutions for curcumin and gefitinib were prepared in dimethyl sulfoxide (DMSO) and stored at −20° C. The compounds were diluted in fresh media before each experiment, and the final DMSO concentration was lower than 0.1%. Curcumin and gefitinib for the animals was prepared by fully suspending the drug in propylene glycol (J. T. Baker, Phillipsburg USA).
2. Cell Lines and Culture Conditions
The human lung adenocarcinoma cell line with highly invasive capacities (CL1-5) was established previously (American Journal of Respiratory Cell & Molecular Biology 1997; 17(3):353-60). The human lung carcinoma cell lines A549, H1299, H1650, and H1975 were obtained from American Type Culture Collection (Manassas, Va.). PC-9 was a kindly gift from Dr. Chih-Hsin Yang (National Taiwan University Hospital, Taiwan). These cells were grown in RPMI 1640 medium (Life Technologies Rockville, Md.). The IEC-18 rat intestinal epithelial cell line (BCRC 60230) was grown in DMEM medium (Life Technologies Rockville, Md.) supplemented with 10% fetal bovine serum (FBS) (Life Technologies). The media for each of the above contained penicillin and streptomycin (100 mg/ml each) and the cell lines were incubated at 37° C. in a humidified atmosphere with 5% CO2.
3. Proliferation Assay
A MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] (Sigma, St Louis, Mo.) assay was performed to determine cell proliferation. Briefly, CL1-5, A549, PC-9, H1650, H1975 and IEC-18 cells were plated in 96-well plates at a density of 5×103 cells/well. After incubating for 24 h, cells were then treated with different concentrations of curcumin and/or gefitinib for 72 h. In addition, the IEC-18 cells were treated with curcumin, BIRB 796 and/or gefitinib for 24 h incubation. MTT solution to a final concentration in the culture medium 0.5 mg/ml was then added to the wells. After a further 1.5 h of incubation, the medium was removed and DMSO was added to the plates. The color intensity of the solubilized formazan was measured at 570 nm using a multi-label plate reader (Vector3; Perkin-Elmer, USA).
4. Colony Formation Assay
CL1-5, A549 and H1975 cells were plated in 6-well plates (100 cells per well) with culture medium. After incubating for 24 hours, the cells were treated with gefitinib or curcumin alone or with a combined treatment as indicated. The cells were cultured with the agents for 5 days and then the medium completely changed; the cells were then incubated for a further 9 days. Colonies were then stained using 0.001% crystal violate and the number of colonies per well counted.
5. Western Blot Analysis
Western blotting was used to determine the protein expression levels of EGFR, pEGFR, Akt, pAkt, cyclin D1, PCNA, iNOS and various apoptosis related proteins (pro-caspase-3, 8, 9 and PARP). Cells were plated in 10-cm dish at a density of 1×106. After incubating overnight, the cells were serum starved for 24 h in medium with no FBS. Next the cells were treated with different concentrations of curcumin and/or gefitinib for 1 h under the serum-free conditions and then were stimulated with 20 ng/ml, EGF for 30 min. The IEC-18 cell was plated in 10-cm dish at a density of 1×106. After incubating overnight, the cells were treated with different concentrations of gefitinib and/or curcumin or BIRB 796 for further 24 h. These cells were washed three times with ice-cold PBS and their protein extracted. In addition, CL1-5 tumor tissue (100 mg) was harvest from each of the group and then minced in lysate buffer. The protein extracts were obtained using mammalian protein extraction reagent (Pierce, Rockford), which contains a protease inhibitor and a phosphatase inhibitor (Sigma, USA). SDS/PAGE using a 10% resolving gel was carried out to separate the proteins (25 mg/lane). Antibodies against phospho-EGFR (Tyr1068), phospho-Akt (Ser473), Akt, c-MET, caspase-3, caspase-8, caspase-9, PARP, active-p38 and p38 were purchased from Cell Signaling Technology (Beverly, Mass.). Antibodies against both forms of EGFR, cyclin D1, PCNA, and iNOS were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). The antibodies were used according to the conditions recommended by the manufacturer. Bound antibody was detected using the Enhanced Chemiluminescence System (Santa Cruz, Calif.). Chemiluminescent signals were captured using the Fujifilm LAS 3000 system (Fujifilm, Tokyo, Japan). All experiments were performed at least three times in duplicate.
6. Flow Cytometry
Cells were seeded in 6-mm culture plate at the density of 105 cells per dish. After incubating for 24 h, the cells were serum starved overnight. The cells were then treated with curcumin and/or gefitinib for further 72 h. Adherent and floating cells were collected separately and resuspended in cold 1×PBS for further analysis. The cells were stained with an Annexin V-FITC Apoptosis Kit (BD Pharmingen, USA) to monitor apoptosis cells and propidium iodide (PI) to detect dead cells. The samples were analyzed on a FC 500 Flow Cytometry Systems (Beckman Coulter). Unstained cells were classified as “live”, cells stained for annexin V only were classified as “early apoptotic”, cells stained for both annexin V and PI were classified as “late apoptotic” and cells stained for PI only were classified as “dead”.
7. Real-Time Quantitative RT-PCR
The expression level of EGFR was detected by real-time PCR on an ABI prism 7900 sequence detection system (Applied Biosystems). The EGFR primers used were as follows: forward primer EGFR-F, 5′-GTGACCGTTTGGGAGTTGATGA-3′; and reverse primer EGFR-R, 5′-GGCTGAGGGAGGCGTTCTC-3′. The TATA-box binding protein (TBP) was used as the internal control (GenBank X54993). The primers and probe used for the quantitative RT-PCR of TBP mRNA were as described previously (24, 25). The relative expression level of EGFR compared with that of TBP was defined as −ΔCT=−[CTEGFR−CTTBP]. The EGFR mRNA/TBP mRNA ratio was calculated as 2−ΔCT×K, in which K is a constant. All experiments were performed three times in triplicate.
8. In Vivo Study Protocol
We perform the in vivo study in mice according to the protocols approved by the National Yang-Ming University Animal Care and Use Committee. The CL1-5 cells were calculate in terms of cell survival and cell number using trypan blue and then 1×106 live CL1-5 cells in 100 μl HBSS were injected subcutaneously into 6-weeks-old SCID mice (supplied by the animal center in the College of Medicine, National Taiwan University, Taipei, Taiwan). To examine whether curcumin can enhance the antitumor effects of gefitinib, the mice were randomized into five groups (n=9) at 1 week after cell injection; these were: (1) vehicle control; (2) curcumin alone (1 g/kg); (3) gefitinib alone (120 mg/kg); (4) 60 mg/kg gefitinib plus 1 g/kg curcumin; and (5) 120 mg/kg gefitinib plus 1 g/kg curcumin. Curcumin and gefitinib were fed to the animals by oral administration once daily at indicated treatment dose. Tumor sizes were monitored every 4 days by electronic vernier caliper and the tumor volume was calculated using the formula V=0.4×ab2, where a and b are the longest and shortest diameters of the tumors, respectively. After 3 weeks, the mice were sacrificed, the subcutaneous tumors were excised; they were then frozen in liquid nitrogen and finally stored at −80° C. Intestine samples from the mice in each group were fixed using Bouin's fluid and paraffin embedded for routine hematoxylin and eosin staining.
9. Immunohistochemical Staining for PCNA, and the Cell Apoptosis Detection Assay
Cell proliferation analysis was performed on the paraffin-embedded tumor tissue samples using PCNA staining. Briefly, a rabbit anti-human PCNA polyclonal antibody (Santa Cruz, Calif.) was used in the primary reaction. The DAKO EnVision System, containing a secondary horseradish peroxidase-conjugated anti-mouse antibody complex, was used with 3,3′-diaminobenzidine to detect the PCNA.
Colorimetric immunohistochemical staining for apoptotic cell death (TUNEL) was performed on the paraffin-embedded tumor and intestine tissue sections using the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Germany); the sections were also counterstained with Gill's hematoxylin. TUNEL-positive cells were examined in 10 random fields from three intestines of each of the treatment groups and then expressed as the mean number of TUNEL-positive cells ±SE per high-power field (×400 magnification).
10. Measurement of Caspase Activity
Caspase activity was detected by using Caspase-Glo® 3/7 assay kit (Promega Corporation, Australia). Briefly, The IEC-18 cell was seeded in 96-well white luminometer assay plates at a density of 1×104 cells per well and incubated at 37° C. After incubating for 24 h, cells were then treated with different concentrations of gefitinib and/or curcumin for further 24 h. 100 μl caspase 3/7 reagents were added to each well and incubated for 1 h on rotary shaker at room temperature. The luminescence intensity for each well was measured using a multi-label plate reader (Vector3; Perkin-Elmer, USA).
11. Statistical Analysis
All experiments were performed in triplicate and analyzed by ANOVA (Excel, Microsoft; Taipei, Taiwan). Comparisons were made using a two-tailed Student's t test and significant differences were defined as p<0.05. Where appropriate, the data are presented as the mean±SD.
Results
1. Curcumin can Inhibit Lung Adenocarcinoma Cell Proliferation, EGFR and AKT Protein Expression and Phosphorylation
To develop new agents or compounds for enhancing the anti-tumor effects, reducing the dosage, or overcoming the resistance of gefitinib in NSCLC patients, a high-throughput drug screening system with different gefitinib resistant cell lines was applied for screening hundreds of compounds from herbs in our laboratory. Table 1 shows the EGFR status and ethnicities of the NSCLC cell lines used herein.
Curcumin was selected as a potential candidate. We confirmed that curcumin exhibited significantly inhibitory effects on cell proliferation in the gefitinib resistant NSCLC cell lines, including CL1-5 (wt-EGFR), A549 (wt-EGFR), H1299 (wt-EGFR), H1650 (in-frame deletions ΔE746-A750 of exon 19 in EGFR with PTEN loss), and H1975 (L858R and T790M mutations in EGFR). As shown in
The EGFR signaling pathway is known to be highly correlated with tumor progression and therefore the effect of curcumin on the expression level and activity (phosphorylation) of EGFR and AKT in CL1-5, A549 and H1975 cells was examined (
2. Binding Activity of Curcumin on EGFR
We investigated the binding activity of curcumin on EGFR and compared it with that of gefitinib. The results show that predicted 3-D conformation of curcumin have relatively high score bound to the open-form wild-type EGFR protein by LIBDOCK (curcumin's score=89.5; gefitinib's score=80.3). In addition,
3. Curcumin Accelerates EGFR Degradation and Downregulates EGFR Protein Level
We examined whether curcumin was able to accelerate EGFR degradation in the translational level. Lung adenocarcinoma cells were pre-treated with or without MG132 for 3 h and then treated with curcumin as indicated. Our data showed that curcumin was able to inhibit the EGFR protein expression in a dose-dependent manner; however, while the cells were pre-treated with MG132, a proteasome inhibitor, the EGFR protein level can be recovered (
We also found that EGFR protein level in the gefitinib combined with curcumin group were lower than curcumin alone. Thus, we processed the immunoprecipitation assay to examine the observations. The results showed that gefitinib (10 μM) didn't alter the EGFR ubiquitin level, however, in the combine treatment group, gefitinib (1 μM) and curcumin (10 μM) was able to increase the EGFR protein ubiquitination compare to the other single treatment group (
4. Combining Curcumin with Gefitinib is Able to Improve the Anti-Tumor Effects of Gefitinib in NSCLC Cells with Either a Wild-Type or Mutant EGFR
The CL1-5, A549, H1299, H1650 and H1975 cell lines, with either a wild-type or mutant EGFR, were used to evaluate whether curcumin can increase the antitumor effects of gefitinib in various gefitinib-resistant NSCLC cells that differ in their EGFR status. The cell proliferation assay showed that gefitinib (≦10 μM) or curcumin (≦15 μM) treatment alone only produced a slight inhibition of cell proliferation (
In addition, we investigated whether curcumin is able to increase the amount of apoptosis caused by gefitinib in the CL1-5, A549 and H1975 cell lines. A flow cytometry assay using propidium iodide/annexin-V, staining indicated that gefitinib (1 μM) combined with curcumin (15 μM) induced a higher level of apoptosis than a high concentration of gefitinib alone (20 μM) in CL1-5 and A549 cells (
As the next step, we examined whether curcumin was able to enhance the anti-tumorigenicity of gefitinib in CL1-5, A549 and H1975 cells using a colony formation assay. The results were similar to the MTT assay and it was found that a combination of curcumin and gefitinib significantly inhibited colony formation by CL1-5, A549 and H1975 compared to the drug-free control and either drug treatment alone (
5. Curcumin Enhances the Antitumor Properties of Gefitinib in Human Lung Adenocarcinoma Cell Xenografts In Vivo.
The next step was to investigate whether curcumin is able to enhance the antitumor activity of gefitinib in vivo. To do this, CL1-5 cells were transplanted subcutaneously into SCID mice. After one week, when the tumors were palpable (3-5 mm), the mice were randomized into five groups (
6. Curcumin Enhances the In Vivo Antitumor Effect of Gefitinib by Reducing EGFR-Related Signaling and Affecting the Regulation of Apoptosis
In order to investigate the molecular mechanisms involved in the antitumor activity of combined curcumin and gefitinib treated mice, the protein lysates from the various tumor tissues were analyzed by Western blot analysis; this approach was used to measure the protein levels of EGFR, AKT, cyclin D1, c-MET, PCNA, and iNOS in the tumors.
We next examined the proliferation marker PCNA using immunohistochemical staining and cell death by the TUNEL assay; this was done in paraffin-embedded tumor tissue samples. The results in
7. Curcumin Attenuates the Adverse Gastrointestinal Effects of Gefitinib
During the in vivo xenograft study, the gefitinib-treated mice showed dramatic body weight lost (data not shown) and there was also an obviously diarrhea side effect; this is similar to previous reports in clinical literature and was even severe enough to result in death within the group. Interestingly, when gefitinib was combined with curcumin, it was able to prevent the body weight loss and it also significantly reduced number of deaths among the mice (the survival rate with the combined therapy was 78% compared to 33% for the gefitinib therapy) (
Finally, we investigated the protective effect of curcumin on gefitinib-induced intestinal epithelial cell apoptosis in vitro using the non-transformed intestinal epithelial cell line IEC-18. The Caspase-Glo® 3/7 assay showed that gefitinib (IC25 at 30 μM and IC50 at 40 μM of gefitinib for IEC-18 cell, respectively) was able to induce caspase 3/7 activities in IEC-18 cell, whereas, 5 μM curcumin (non-toxic dosage) could significantly inhibit the gefitinib-induced caspase 3/7 activities (
In conclusion, our results showed the prominent activity of curcumin as an enhancer of gefitinib's anti-tumor abilities. The agent also attenuates the diarrheal side effects of gefitinib and thus may be a good adjuvant for lung cancer patients. Clinically, the price of lung cancer targeted therapy is quite high and adverse effects are always the critical issues during treatment. Curcumin is a common and cheaper agent and it seems to be able to enhance the effectiveness of gefitinib and thus reduce the costs related to the medical and patient financial burden. To reduce the dosage of gefitinib, cut-down the costs, and prevent the side effects, we suggest that curcumin should be a good adjuvant for NSCLC cancer patients during gefitinib treatment.
This application claims priority to U.S. Provisional Application No. 61/303,593, filed on Feb. 11, 2010, the content of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61303593 | Feb 2010 | US |
