Lung cancer is still in the first place in terms of both incidence and mortality. In the present study, we demonstrated the effect of curcumin, a phytochemical of the plant
Worldwide, lung cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths (
The large receptor tyrosine kinase (RTK) family in the human genome contains 58 RTKs and is divided into 20 subfamilies. One of the subfamilies is TAM family composed of three RTK members which are Tyro3 (also referred to Brt, Dtk, Rse, Sky or Tif ), Axl (also referred to Ark, Tyro7 or Ufo) and Mer (also referred to Eyk, Nyk or Tyro12) (
Since the first identification of Axl in 1988,
Curcumin, a polyphenolic compound extracted from
In the present study, we examined the effect of curcumin on expression and activation of Axl RTK in NSCLC cells, which subsequently inhibits cell proliferation and overcomes chemo-resistance via both induction of p21 and reduction of X-linked inhibitor of apoptosis (XIAP), suggesting that Axl RTK is a novel target of curcumin to exert its anticancer activity.
Curcumin was obtained from Sigma-Aldrich (St. Louis, MO, USA). A549 and H460 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Primers for Axl were synthesized by the domestic company, Bioneer Corp. (Daejeon, Korea). TRI reagent was obtained from Solgent Co., Ltd. (Daejeon, Korea). AmpliTaq DNA polymerase and Lipofectamine 2000 were obtained from Roche Diagnostics Corp. (Indianapolis, IN, USA) and Invitrogen (Carlsbad, CA, USA), respectively. G418 was from Gibco-BRL (Gaithersburg, MD, USA). The plasmid, pGL3-basic vector and the Dual-Glo luciferase assay kit were purchased from Promega Corp. (Madison, WI, USA). For western blot analysis, specific antibodies against Axl, cyclin D1, p21, XIAP and GAPDH, as well as secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
The A549 and H460 cells were grown in RPMI-1640 medium (Gibco-BRL) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 U/ml penicillin and 10 g/ml streptomycin at 37ºC in 5% CO2 in a water-saturated atmosphere. The variants of A549 and H460 cells which are resistant to cisplatin (A549/CisR and H460/CisR) or paclitaxel (A549/TR and H460/TR cells) were established by stepwise exposure of the parental cells to escalating concentrations of cisplatin (ranging from 0.5 to 2 mM) and paclitaxel (ranging from 3 to 24 nM), respectively.
Cells (2×105) were seeded in a 60-mm culture dish and grown overnight. They were then treated with the indicated concentrations (0, 5, 10 and 20 μM) of curcumin for 24 h. Total RNA was extracted using TRI reagent and subjected to cDNA synthesis and PCR. The specific primers were as follows: Axl sense, 5′-AACCTT CAACTCCTGCCTTCTCG-3′ and antisense, 5′-CAGCTTCT CCTTCAGCTCTTCAC-3′; GAPDH sense, 5′-GGAGCCAA AAGGGTCATCAT-3′ and antisense, 5′-GTGATGGCATG GACTGTGGT-3′. The mRNA level of Axl was normalized to that of GAPDH.
The promoter reporter plasmid, pGL3-Axl, which contains the
Total cell lysates were prepared from the parental or chemoresistant cells treated with the indicated concentrations (0, 5, 10 and 20 μM) of curcumin using lysis buffer [1% Triton X-100, 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM PMSF, 1 mM Na3VO4 and protease inhibitor cocktail]. Untreated cells were used as controls. Protein concentrations were determined using Bio-Rad protein assays. Proteins from the cell lysates (20–40 μg) were separated by 12% SDS-PAGE, and electrotransferred onto nitrocellulose membranes. The membranes were blocked for 30 min at room temperature in Tris-buffered saline with 0.05% Tween-20 (TTBS) containing 5% non-fat dry milk, and then incubated with TTBS containing a primary antibody for 4 h at room temperature. After 3×10 min washes in TTBS, the membranes were incubated with peroxidase-conjugated secondary antibody for 1 h. Following 3 additional 10-min washes with TTBS, the protein bands of interest were visualized using an enhanced chemiluminescence detection system (Amersham™ ECL™ Prime Western Blotting Detection reagent; GE Healthcare, Piscataway, NJ, USA).
To assess cell viability, the number of viable cells was counted using Trypan blue. Briefly, 3×103 cells were seeded into 60-mm culture dish, grown overnight and then treated with the indicated concentrations (0, 5, 10 and 20 μM) of curcumin for 24 h. After curcumin treatment, cells were harvested and stained with 0.4% Trypan blue solution. Dye-excluding viable cells were counted under the microscope. Cell viability was also expressed as a percentage of the viable cells with respect to untreated control cells.
Cells were seeded into 24-well plates (1×102 cells/well) and treated with the indicated concentrations (0, 5, 10 and 20 μM) of curcumin for 24 h. Curcumin-treated cells were then cultured for the next 7–10 days to form colonies. Colonies of >50 cells were stained with Crystal violet (in 60% methanol; Junsei Chemical Co., Ltd., Tokyo, Japan) and images were acquired using the RAS-3000 Image Analysis System (FujiFilm, Tokyo, Japan).
To ectopically express Axl, the recombinant plasmid, pcDNA3-Axl, was constructed by cloning the Axl cDNA into the
To reduce Axl expression, RNA interference-mediated gene silencing was performed. Cells (3×105) were seeded in 60-mm culture dishes, grown overnight and then transfected with 50 nM siRNA targeting Axl (sense, 5′-AAGAUUUGGAGAdACACACUGA-3′ and antisense, 5′-UCAGUGUGUUCUCCAAAUCUU-3′), as previously described (
Data were expressed as the means ± SD of triplicate samples or at least three independent experiments. To determine statistical significance, the Student's t-test was used with a P-value threshold of <0.05.
We first examined if curcumin alters expression of Axl receptor tyrosine kinase (RTK) in the lung cancer A549 and H460 cells. After treatment of cells with 5, 10 and 20 μM curcumin for 24 h, Axl protein level was determined by western blot analysis. The results of western blot analysis showed that Axl protein level in curcumin-treated cells was reduced in the dose-dependent manner (
Downregulation of Axl expression by curcumin was further demonstrated by RT-PCR. Consistent with western blot results, Axl mRNA levels of A549 and H460 cells were also markedly and dose-dependently diminished by the indicated concentrations of curcumin (
Binding of growth arrest-specific gene 6 (Gas6), a validated ligand, to Axl results in its activation, that is the phosphorylation of tyrosine residues at intracellular kinase domain (
Serum-starved A549 and H460 cells were treated with Gas6 for 15, 30, 60 and 120 min and phosphorylated Axl levels were determined by western blot analysis. As illustrated in
Since overexpression and activation of Axl had been reported to be involved in oncogenesis, cell survival, proliferation and anti-apoptosis (
The anti-proliferative effects of curcumin on the A549 and H460 cells were also observed by clonogenic assay. The cells were exposed to the indicated concentrations of curcumin for 24 h and then allowed to grow for the next 10 days. Curcumin treatment was found to result in the dose-dependent decline of colony formation (
To further confirm the involvement of Axl in the antiproliferative effects of curcumin, we examined the cytotoxic effect of curcumin on cells manipulated to enhance or reduce Axl expression. As shown in
Next, we asked if curcumin could be cytotoxic in the cisplatin- and paclitaxel-resistant NSCLC cells which are the variants of A549 and H460 cells. Each of the variants was established by stepwise exposure of parental cells to increasing concentrations of cisplatin (A549/CisR and H460/CisR cells) or paclitaxel (A549/TR and H460/TR cells), respectively. As shown in
In accordance with the western blot results, the viability of both paclitaxel- and cisplatin-resistant cells were dose-dependently declined by curcumin treatment. Especially, exposure of cells with 20 μM curcumin for 24 h was found to result in only 41.3% (A549/TR), 62.8% (A549/CisR), 15.6% (H460/TR), 27% (H460/CisR) survival of these cells, respectively (
To demonstrate the intracellular effectors which are involved in the curcumin-mediated downregulation of Axl expression and result in the inhibition of cell proliferation, we assessed the levels of cell cycle regulator, p21 and apoptosis related protein, the X-linked inhibitor of apoptosis protein (XIAP). The H460, H460/TR and H460/CisR cells were treated with 10 μM curcumin for 24 h and western blot analysis showed that curcumin induced the expression of the cyclin dependent kinase inhibitor p21, which causes cell cycle arrest, but reduced that of XIAP, which inhibits apoptosis (
Standard chemotherapy for NSCLC has been the combination of platinum-based agents (cisplatin or carboplatin) and a second drug (pemetrexed, gemstabine, paclitaxel or vinorelbine), but low response rate (20–35%) and eventual development of chemoresistance among initial responders have been the main causes of poor prognosis (
In the present study, we observed that curcumin had inhibitory effects on Axl expression, Gas6-dependent Axl phosphorylation, and
Several reports have shown that curcumin could affect each stage of cancer such as initiation, promotion and progression, ingestion of curcumin was even found to significantly inhibit the activity of lymphocytic glutathione S-transferase, a phase II detoxification enzyme, involved in the development of chemoresistance (
In summary, our data indicate that curcumin has inhibitory effects on Axl expression and the activation in response to Gas6 binding, which are associated with its anti-proliferative activity in parental as well as each type of cisplatin/paclitaxel-resistant NSCLC cells. Thus, Axl seems to be a potent therapeutic target of curcumin to inhibit cell proliferation and to overcome chemoresistance of NSCLC cells.
The present study was supported by the 2014 Yeungnam University Research Grant (no. 214A380116).
epidermal growth factor receptor
growth arrest-specific 6
non-small cell lung cancer
receptor tyrosine kinase
X-linked inhibitor of apoptosis
Curcumin decreases Axl expression at transcriptional level. Cells (3×103 cells/dish) were seeded onto 60-mm dishes, grown overnight and exposed to curcumin. (A) A549 or H460 cells were treated with 5, 10 and 20 μM curcumin for 24 h and then cells were harvested. (B) A549 or H460 cells were treated with 20 μM curcumin for 6, 12 and 24 h and then cells were harvested. Axl protein levels were determined by western blot analysis. GAPDH was used as a loading control. The result shown is a representative of three independent experiments. (C) For RT-PCR, total RNAs from the cells treated with the indicated concentrations of curcumin for 12 h were isolated and used to determine
Curcumin inhibits Axl activation upon Gas6 stimulation. Cells (3×103 cells/dish) were seeded onto 60-mm dishes, serum-starved overnight, and treated with 250 ng/ml Gas6. (A) A549 or H460 cells were treated with Gas6 for 15, 30, 60 and 120 min and then harvested. (B) H460 cells were pre-incubated with 20 μM curcumin for 60 min, followed with Gas6 treatment and then harvested. Phosphorylated Axl protein levels were determined by western blot analysis. Total Axl protein level was used as a loading control. The result shown is a representative of three independent experiments.
Curcumin inhibits cell proliferation and its anti-proliferative effect is reduced or augmented by Axl protein level. A549 or H460 cells (3×103 cells/dish) were seeded onto 60-mm dishes, grown overnight. (A) Cells were treated with 5, 10 and 20 μM curcumin for 24 h and then harvested. The number of viable cells were counted using trypan blue exclusion assay. Data are represented as mean ± SD of at least three independent experiments. The asterisks indicate a significant difference compared to the control value (*P<0.05 vs. untreated group). (B) Cells (2×103 cells/dish) were seeded onto 35-mm dishes, treated with the indicated dose of curcumin for 24 h and allowed to grow for next 7–10 days. Colonies were visualized by crystal violet staining. The data shown are representative of three independent experiments. (C) A549 cells were transfected with pcDNA3 or pcDNA3-Axl plasmid using Lipofectamine 2000. The transfected cells were treated with the indicated doses of curcumin for 24 h, then harvested, and stained with tryphan blue to count the viable cells. Data are expressed as the means ± SD from three independent experiments. The asterisks indicate the significant difference compared to the control value (*P<0.05 vs. untreated group). (D) A549/pc-DNA3 and A549/pc-DNA3-Axl cells were treated with the indicated concentrations of curcumin for 24 h. The total cell lysates were prepared and Axl protein level was determined by western blot analysis. (E) Colonogenic assay was also conducted with A549/pc-DNA3 and A549/pc-DNA3-Axl cells exposed to the indicated concentrations of curcumin for 24 h. Colonies formed during 7–10 days of culture were visualized by crystal violet staining. (F) H460 cells (3×104 cells) were transfected with Axl specific siRNA or control siRNA, respectively. Cells were harvested 24 h post-transfection, divided into two groups, and grown for 24 h in the presence or absence of 10 μM curcumin. The total cell lysates were prepared and Axl protein levels were determined by western blot analysis. GAPDH was used as a loading control and results shown are representative of at least three independent experiments. (G) After curcumin treatment, cells were harvested and stained with tryphan blue to count viable cells. Data are represented as mean ± SD of at least three independent experiments. The asterisks indicate a significant difference compared to the control value (*P<0.05 vs. untreated group).
Curcumin inhibits proliferation of cisplatin- and paclitaxel-resistant lung cancer cells and causes induction of p21 as well as reduction of XIAP. Each variants of A549 and H460 cells, which are cisplatin- (A549/CisR and H460/CisR) and paclitaxel-resistant (A549/TR and H460/TR), respectively, were seeded onto 60-mm dishes, and grown overnight. (A) Cells (3×103 cells/dish) were treated with 5, 10 and 20 μM curcumin for 24 h and then harvested. Axl protein levels were examined by western blot analysis. GAPDH was used as a loading control. The result shown is a representative of three independent experiments. (B and C) Cells (1×103 cells/dish) were seeded onto 96-well plates and treated with the indicated concentrations of curcumin for 24 h. Cell proliferation was determined by CCK-8 assay. The data shown are representative of at least three independent experiments. Data are represented as mean ± SD. The asterisks indicate a significant difference compared to the control value (*P<0.05 vs. untreated group). (D) Cells (2×103 cells/dish) were seeded onto 35-mm dishes and treated with 5, 10 and 20 μM curcumin for 24 h, and allowed to grow for 7–10 days. The colonies were visualized by crystal violet staining. The data shown are representative of at least three independent experiments. (E) H460, H460/CisR and H460/TR cells (3×103 cells/dish) were treated with 10 μM curcumin for 24 h and then harvested. The levels of p21, cyclin D1 and XIAP protein were determined by western blot analysis. GAPDH was used as a loading control. The result shown is a representative of three independent experiments.