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Copper supplementation amplifies the anti-tumor effect of curcumin in oral cancer cells.

ABSTRACT

Background: Oral cancer is the sixth most common cancer worldwide and 90% of oral malignancies are caused by oral squamous cell carcinoma (OSCC). Curcumin, a phytocompound derived from turmeric (Curcuma longa) was observed to have anti-cancer activity which can be developed as an alternative treatment option for OSCC. However, OSCC cells with various clinical-pathological features respond differentially to curcumin treatment.

Hypothesis: Intracellular copper levels have been reported to correlate with tumor pathogenesis and affect the sensitivity of cancer cells to cytotoxic chemotherapy. We hypothesized that intracellular copper levels may affect the sensitivity of oral cancer cells to curcumin.

Methods: We analysed the correlation between intracellular copper levels and response to curcumin treatment in a panel of OSCC cell lines derived from oral cancer patients. Exogenous copper was supplemented in curcumin insensitive cell lines to observe the effect of copper on curcumin-mediated inhibition of cell viability and migration, as well as induction of oxidative stress and apoptosis. Protein markers of cell migration and oxidative stress were also analysed using Western blotting.

Results: Concentrations of curcumin which inhibited 50% OSCC cell viability ([IC.sub.50]) was reduced up to 5 times in the presence of 250 [micro]M copper. Increased copper level in curcumin-treated OSCC cells was accompanied by the induction of intracellular ROS and increased level of Nrf2 which regulates oxidative stress responses in cells. Supplemental copper also inhibited migration of curcumin-treated cells with enhanced level of E-cadherin and decreased vimentin, indications of suppressed epithelial-mesenchymal transition. Early apoptosis was observed in combined treatment but not in treatment with curcumin or copper alone.

Conclusion: Supplement of copper significantly enhanced the inhibitory effect of curcumin treatment on migration and viability of oral cancer cells. Together, these findings provide molecular insight into the role of copper in overcoming insensitivity of oral cancer cells to curcumin treatment, suggesting a new strategy for cancer therapy.

Keywords:

Copper

Anti-cancer

Oral cancer

Curcumin

Oxidative stress

Migratory phenotype

Introduction

Several plant-derived phytochemicals are now known to reduce the risk of cancer development. Among them, curcumin, the yellow pigment extracted from Curcuma longa, has been shown to have significant anti-cancer activities both in vitro and in vivo via targeting multiple oncogenic pathways (Kunnumakkara et al. 2008). Curcumin, like several other phytoagents, is known as an antioxidant that prevents cancer via antagonizing carcinogen-triggered oxidative stress by scavenging free radicals and/or activating endogenous defence systems such as Nrf2-regulated antioxidant genes or pathways. However, recent findings have suggested that the anti-cancer activity of curcumin may be attributed to the induction of ROS in cancer cells (Liang et al. 2014). Intriguingly, pro-oxidation of curcumin was recently shown to be enhanced in the presence of copper (Lou et al. 2010). Copper is a redox-active metal ion that fluctuates between the oxidized ([Cu.sup.2+]) and reduced ([Cu.sup.+]) states and is commonly utilized by organisms living in oxygen-rich environments (Ridge et al. 2008). More than 90% of serum copper is bound by ceruloplasmin, an oxidative enzyme (Hellman and Gitlin 2002). However, high levels of free form copper is part of the radical-reactive cellular environment and its role in cancer has long been the subject of speculation (Schwartz 1975). In this regard, there is a growing body of evidence shows that levels of copper are altered on the onset and progression of malignant diseases (Gupte and Mumper 2009, Khanna et al. 2013). Recently, high serum levels of copper were observed in patients with prostate cancer and the cytotoxic action of disulfiram in cancer cells was found to occur in a copper-dependent manner (Safi et al. 2014). When excessive concentrations of free form of metal ions exist, classic antioxidants such as curcumin, catalyze the redox cycling of metal ions by reducing their oxidized form. As a result, a burst of hydroxy! free radical production ensues and the phytoagents become prooxidants (Lee et al. 2013). This mechanism potentiates a novel therapeutic approach targeting elevated copper and related oxidative stress in aggressive tumors (Gupte and Mumper 2009, Trachootham et al. 2009).

Head and neck neoplasias represent a major public health burden which accounts for 13.2% of all cancer incident among the population in Malaysia (Omar and Ibrahim Tamin 2011). One of the subtypes of head and neck cancer are the most common form of all oral malignancies (90%) arising in the oral cavity and collectively known as oral squamous cell carcinomas (OSCC) (Reis et al. 2011). It is commonly known that local recurrence, lymph node metastases and resistance to clinical drugs often cause the failure of oral cancer treatment (Rikiishi et al. 2007). Hence there is a pressing need to characterize the genetic and biochemical processes that underlie carcinogenesis and malignancy of OSCC in order to seek appropriate treatments. Pertinently, a recent study reported a progressive and elevated copper level in patients with oral OSCC when compared to the normal group, suggesting copper could be a potential biomarker for OSCC carcinogenesis (Khanna et al. 2013). Concordantly, a representative panel of OSCC cell lines which were derived from oral cancer patients with various clinicopathological characteristics was examined with different levels of endogenous copper in our laboratory. All the OSCC lines were found to possess higher copper content compared to normal oral keratinocytes (NOK). Whilst curcumin has been reported to inhibit oral cancer cell proliferation and invasion (Zhen et al. 2014), our data broadly demonstrated that the tested OSCC lines showed differential responses to curcumin treatment. In this study, we hypothesized intracellular copper levels may affect the sensitivity of cancer cells to cytotoxic chemotherapy and aimed to investigate the role of copper in regulating oral cancer cell response to a curcumin therapeutic regimen. Using a sub-set of cell lines show low copper content and insensitivity to curcumin to supplement with exogenous copper, our results revealed that copper demonstrated a remarkable effect in modulating prooxidation, anti-metastasis and cytotoxicity of curcumin in OSCC cells, potentially providing evidence for developing this natural product as a therapy option for OSCC.

Materials and methods

Herbal substance

Curcumin (mixture of curcumin, demethoxycurcumin, and bisdemethoxycurcumin), assay percentage range (98+%), Acros Organics (218580100).

Chemicals

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), curcumin, copper (II) chloride dehydrate (Cu[Cl.sub.2.] 2[H.sub.2]O), and N-acetyl cysteine (NAC) were purchased from Sigma-Aldrich (MO, USA). DCFDA, DMEM, DMEM, Nutrient Mixture F-12 (DMEM-F12) and Phen Green FL were from Invitrogen (CA, USA). Antibodies of Nrf2, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse were obtained from Santa Cruz (CA, USA). Antibody of PARP, E-cadherin and vimentin was from Cell Signaling Technology (MA, USA). Apoalert DNA Fragmentation Kit and Annexin-V/PI Apoptosis Kit were purchased from Clontech (CA, USA) and BD Biosciences (CA, USA) respectively.

Maintenance of cells

The H-series OSCC cell lines were obtained from European Collection of Cell Cultures (ECACC) and the ORL OSCC cell lines were kindly provided by Prof Sok-Ching Cheong (Cancer Research Malaysia, Malaysia). All the OSCC cell lines were grown in DMEM-F12 supplemented with 10% fetal bovine serum, 0.5pg/ml sodium hydrocortisone succinate, 100 units/ml of penicillin and 100 units/ml of streptomycin. Normal primary oral keratinocytes (NOK) 232 N and 337 N (by Prof Sok Ching from Cancer Research Malaysia, Malaysia) and immortalised nasopharyngeal epithelial cell lines (NP69, NP460: provided by Professor George Tsao from the Department of Anatomy, The University of Hong Kong) were cultured in keratinocyte serum free media (KSFM; GIBCO, Carlsbad, CA, USA) supplemented with 25pg/ml bovine pituitary extract, 0.2 ng/ml epidermal growth factor, 0.031 mM calcium chloride, 100 units/ml of streptomycin and 100 units/ml of penicillin. All the cell cultures were maintained at 37 [degrees]C with 5% carbon dioxide.

Determination of cell viability

OSCC and NOK cells were cultured in 96-well plates at a density of 3000-5000 cells per well and allowed to adhere overnight, and then treated with vehicle (0.05% DMSO) and curcumin at the indicated concentrations for 24 h and 48 h Cell viability was then measured using the MTT-based colorimetric assay. Briefly the cells were incubated using 5 mg/ml of MTT reagent in PBS for 3 h and after the medium removed and replaced with 100 pi of DMSO to solubilize the crystal formazan formed in the cells, followed by measuring absorbance at 570 nm using the Tecan Infinite 200 plate reader. All cell viability assays were performed in quadruplicate. The percentage of viable cells was calculated using the formula:

Cell viability (%) = (Absorbance of treated groups/Absorbance of untreated groups) x 100%

Detection of intracellular copper

Phen Green FL method was used to detect intracellular copper levels as described previously (Lou et al. 2010). OSCC and NOK cells were cultured in 96-well plates at a density of 3000-5000 cells per well and allowed to adhere overnight. For detection of basal copper level, all cells were incubated with 5 [micro]M of Phen Green FL in culture medium for 30 min at 37 [degrees]C. For detection of cellular copper level after treatment, OSCC cells were treated at indicated concentrations for 1 h before incubation with Phen Green FL. Then, the cells were washed three times with PBS and fluorescence measured using the Tecan Infinite 200 plate reader with excitation at 490 nm and emission at 529 nm. Binding of Phen Green FL to copper quenches its fluorescence. Therefore the fluorescence indicates free Phen Green FL which is in inversely proportional to cellular copper level. The measured fluorescence intensity in each well was normalized by viable cells as determined by MTT assay.

Detection of intracellular ROS

OSCC cells were cultured in 96-well plates at a density of 30005000 cells per well and allowed to adhere overnight. The cells were treated with curcumin and/or copper at the indicated concentrations for 1 h, then incubated with 10 [micro]M of DCFDA in DMEM-F12 for 30 min at 37 [degrees]C. The fluorescence was measured using the Tecan Infinite 200 plate reader with excitation at 495 nm and emission at 529 nm. The measured fluorescence intensity in each well was normalized by viable cells determined by MTT assay.

Measurement of cell migration

Cell migration assays were performed using Boyden chambers (6.5 mm diameter, 8 mm pore size; Costar, USA). Cells were seeded in the Boyden Chambers at a density of 1.5 x [10.sup.4] cells per insert and allowed to adhere overnight. The media in the upper wells (insert) with attached cells was replaced with media with 1% FBS and media containing 10% FBS as attractant was placed in the lower wells. Treatments were added into the upper wells and incubated for 6 h and 12 h at 37 [degrees]C. After, the non-migrating cells on the upper surface of the chamber were removed using a cotton swab. The cells remaining on the lower surface of the chamber were fixed with 4% formaldehyde, permeabilized with 70% methanol and stained with 0.1% crystal violet. The migrated cells were viewed and captured using a microscope equipped with a Canon 600D camera. The crystal violet was then dissolved using 0.1% Triton X-100 in PBS and absorbance was measured at 595 nm using the Biotek KC Junior imaging system (Vermont, USA).

Western blot analysis

OSCC cells were treated with curcumin and/or copper at the indicated concentrations and durations. After, the cells were lysed with RIPA buffer, sonicated and after centrifugation at 4[degrees]C for 15 min, the protein content determined using the Bradford assay. Next, 20 pg of total cellular protein content of each sample after denaturation was resolved using a 10% SDS-PAGE and after transferred to PVDF membranes. The membranes were then blocked with 5% (w/v) skim milk in Tris-buffered saline with 0.1% (v/v) Tween 20 (TBST) for 1 h, incubated with E-cadherin mouse monoclonal antibody, vimentin rabbit monoclonal or Nrf2 rabbit polyclonal antibody at a dilution of 1:1000 in 1% (w/v) BSA overnight at 4[degrees]C. After, membranes were washed with TBST each for 5 min and then probed with HRP-conjugated goat anti-mouse antibody or goat anti-rabbit antibody at a 1:10,000 in 5% (w/v) skim milk for 1 h at room temperature. Reacted protein bands were after visualized using enhanced chemiluminescence detection reagents (Thermo Scientific, MA, USA) with exposure to FluorChem Q System (Cell Biosciences, USA). The resulting images quantified using Image J.

Detection of apoptotic cells

OSCC cells were seeded in EZ 8-well glass slides (Merck Millipore, Germany) at a density of 1.5 x [10.sup.4] cells per well and allowed to adhere overnight. The cells were treated with curcumin and/or copper at the indicated concentrations for 6 h Terminal dUTP Nick End Labeling (TUNEL) assay was then carried out according to manufacturer's manual (Apoalert DNA Fragmentation Kit). In brief, treated cells on the slides were washed twice with PBS, fixed with 4% formaldehyde and permeated with pre-chilled 0.2% Triton X at 4[degrees]C. TdT incubation buffer was then added to stain the TUNEL-positive cells for 2 h The cells were also stained with DAPI for 7 min prior to washing, mounting and viewing using the fluorescence microscope AxioVision, version 4.8.2.0.

Measurement of cell apoptosis

OSCC cells were stained with Annexin V and propidium iodide (PI) according to the manufacturer's manual (Annexin-V/PI Apoptosis Kit, BD Biosciences). OSCC cells treated for 24 h were trypsinized, washed twice with ice-cold PBS and resuspended in 500 [micro]l binding buffer. Five microliters of Annexin V-FITC and 5 [micro]l of 100 [micro]g/ml PI working solution were added to stain the cells in the dark at room temperature for 15 min. The emission of Annexin V and PI from stained cells were then detected via flow cytometer BD LSR II (CA, USA) using filters of 525 and 575 nm, respectively. The data obtained were analysed using BD Accuri Software FACS Diva Version 6.1.3. The results were shown as quadrant dot plots with stainless live cells (Annextin V-/PI-), Annexin V stained early apoptotic cells (Annextin V+/PI-), late apoptotic cells with double staining (Annextin V+/PI+) and PI stained necrotic cells (Annextin V-/PI+).

Statistical analysis

All data are expressed as mean [+ or -] standard deviation (SD) and were analyzed using Tukey Honest Significant Difference (HSD) from SPSS 16.0 software (SPSS, USA). A p-value of less than 0.05 was considered statistically significant.

Results

Intracellular copper and sensitivity of oral cancer cells in curcumin treatment

Endogenous copper in twelve human OSCC cell lines, two normal oral keratinocytes (NOK) and two nasopharyngeal epithelial cell lines without any treatment, was detected using a copper-reactive fluorescent dye Phen Green FL and the relative copper level was derived for each cell line (Fig. 1A & Table A.1). In parallel, MTT assay was used to evaluate the viability of these cell lines treated with various concentrations of curcumin for 24 h The concentration which caused 50% inhibition of the viability of each cell line ([IC.sub.50]) was then determined (Table 1). From our data, H357 cells demonstrated the lowest levels of copper (relative value = 2.02 [+ or -] 0.57), which was significantly higher than that observed in NOK cells (relative values = 0.001 [+ or -] 0.82 to 1.00 [+ or -] 0.36) (Fig. 1A). A moderate levels of copper were observed in H314 (relative value = 2.55 [+ or -] 0.62) and H376 cells (relative value = 2.29 [+ or -]0.45), while a significantly high level of copper was observed in H103 (relative value = 4.61 [+ or -] 0.13) and H400 cells (relative value = 4.15 [+ or -] 0.30). In ORL OSCC cell lines, the relative levels of intracellular copper (ranged from 2.08 [+ or -] 0.39 to 3.99 [+ or -] 0.20) were all significantly higher than those found in NOK cells (232N, 337N) and nasopharyngeal epithelial cell lines (NP69, NP460) (Fig. 1A). Pearson correlation coefficient was used to relate the intracellular copper and sensitivity of tested cell lines to curcumin ([IC.sub.50]). A weak negative correlation (coefficient value 0.527, P < 0.05) was observed between these two values, as shown in Fig. 1B. Together, these data indicate that intracellular copper might play a role in regulating sensitivity to curcumin treatment for most, but not all the OSCC cell lines.

Exogenous copper sensitizes oral cancer cells to curcumin treatment

The H314 and ORL-115 oral cancer cell lines were chosen for subsequent experiments based on their low level of copper and high insensitivity to curcumin treatment. To determine the proper working concentration of copper, H314 cells treated with curcumin at [IC.sub.50] (50 [micro]M for 24 h and 25 [micro]M for 48 h) were supplemented with 0, 50, 100, 250 and 500 [micro]M of Cu[Cl.sub.2.] 2[H.sub.2]O. The viability of H314 cells was measured by MTT assay. From these experiments, we observed that 250 [micro]M Cu[Cl.sub.2.] 2[H.sub.2]O further inhibited the viability of curcumin-treated H314 ceils from 50% to 36.8% and 19.4% at 24 and 48 h, respectively. Doubling the dose (500 [micro]M of Cu[Cl.sub.2]. 2[H.sub.2]O) resulted in a similar inhibitory effect (Fig. A.1). Therefore we used 250 [micro]M copper in subsequent experiments to study its effect in H314 and ORL-115 cells. To evaluate the effect of copper in curcumin-mediated toxicity, H314 cells with or without addition of copper were treated with different concentrations of curcumin. We observed that copper significantly suppressed the viability of H314 cells in treatment with curcumin from 35 to 50 [micro]M for 24 h while viability of cells dropped nearly 50% upon treatment with both copper and curcumin at doses as low as 5 [micro]M for 48 h (Fig. 2A & B). With addition of copper, the concentration of curcumin that was required to inhibit 50% of H314 cell viability ([IC.sub.50]) was reduced from 50 [micro]M to 40.3 [micro]M at 24 h and 25 [micro]M to 5.3 [micro]M (a five-fold change) at 48 h (Table 2). The suppressive effect of copper on curcumin-treated oral cancer cells was notably diminished in the presence of 250 [micro]M metal chelator ethylenediaminetetraacetic acid (EDTA) (Fig. 2A & 2B, Table 2). A similar response was observed in the ORL-115 cell line which was noted to be less sensitive to curcumin. In the presence of 250 [micro]M copper, [IC.sub.50] of ORL-115 cells was remarkably reduced from 105.5 [micro]M to 36.1 [micro]M (a three-fold change) at 24 h and 69.8 [micro]M to 25.8 [micro]M (a 2.5-fold change) at 48 h Treatment of 250 [micro]M EDTA significantly diminished the sensitizing effect of copper but a full recovery of curcumin-mediated toxicity by EDTA was not observed in ORL-115 as in H314 cells (Fig. 2C & D, Table 3). Together these results suggest that copper has a prominent role in sensitizing oral cancer cells in response to curcumin treatment.

Effect of exogenous copper in redox regulation of curcumin-treated oral cancer cells

Phen Green fluorescence emission in the cells was measured with addition of increasing amounts of exogenous copper and a significant correlation ([R.sub.2] = 0.996) was observed (Fig. A.2). Therefore, in subsequent experiments, we estimated the change in cellular copper level using the corresponding fluorescence intensity. Treatment with curcumin alone increased the intracellular copper of H314 (129.5 [+ or -] 36.6 [micro]M) and ORL-115 cells (172.5 [+ or -] 30.2 [micro]M) in comparison to the basal copper level in the untreated group. Meanwhile, ROS in treated H314 cells was reduced by low concentration of curcumin (5-10 [micro]M) but was stimulated in treatment with concentrations higher than 15 [micro]M (20-50 [micro]M) within 1 h (Fig. 3B). Notably in H314 cells, curcumin at [IC.sub.50] levels (50 [micro]M) demonstrated a role as a prooxidant. In both cell lines, addition of exogenous copper in curcumin-treated cells doubled the increase of intracellular copper (385.1 [+ or -] 49.3 [micro]M in H314 and 336.9 [+ or -] 31.7 [micro]M in ORL-115 compared to basal copper level in the untreated groups, respectively) accompanied by significant induction of ROS which could be diminished by chelator EDTA as well as ROS scavenger NAC. Intriguingly, treatment with copper alone could increase intracellular copper level but not induce additional ROS (Fig. 3A & C). As shown in Fig. 3D, treatment with both curcumin and copper significantly increased the level of Nrf2, a master regulator of oxidative stress in mammalian cells (2.1-fold increase in H314 and 3.4 fold increase in ORL-115 compared to the untreated groups respectively) while the increase was not observed in control and treatments with inhibitors. Together, these observations suggest that additional copper augments the pro-oxidative activity of curcumin in causing stress in oral cancer cells.

Co-treatment with curcumin and copper inhibits migration of oral cancer cells

The Boyden Chamber assay was used to measure H314 cell migration via quantification of cells that migrated to the other side of the chamber. The migrated cells quantified in the control group were considered as 100% in this study. A remarkable inhibition, up to 54% reduction in migrated cells, was observed after 12 h treatment with curcumin and copper compared to the control group. On the other hand, a 57% increase in migrated H314 cells was observed upon 12 h treatment with copper only (Fig. 4A & B, Fig. A.3). Of note, copper alone stimulated oral cancer cell migration but could effectively suppress such activity when combined with curcumin. In good agreement with the results of the migration assay, the inhibitory effect on H314 oral cancer cell migration by curcumin and copper was followed by a 2.0-fold increase in E-Cadherin protein expression together with suppressed expression of vimentin, both common markers of EMT (Fig. 4C). On the other hand, lower levels of E-cadherin levels (ratio 0.4:1) and slightly higher levels of vimentin (ratio 1.1:1) compared to the control group were observed in H314 cells treated with copper only, a treatment which caused a vigorous migratory phenotype (Fig. 4). A similar regulation of migration was observed in ORL-115 cells with various treatments of copper and curcumin (Fig. A.4-Fig. A.7). These results suggest a correlation between the migratory phenotype and the EMT process in OSCC cells with the indicate treatments, a finding that supports the supplementary role of copper in augmenting the inhibitory effect of curcumin on oral cancer cell migration.

Co-treatment with curcumin and copper induces late apoptosis in oral cancer cells

The inhibitory effect of the combination of curcumin and copper on H314 cell viability was apparent as only 41% and 19% of treated cells were found viable after 6 h and 12 h treatments, respectively, while no significant difference was observed between treatment with copper-only and the control group (Fig 5A). In addition, the inhibited cell viability in the combined treatment was accompanied by the cleavage of PARP, an indicator of apoptosis induction in treated cells. Intriguingly, the cleaved form of PARP was not observed in any other treatment except for the combined curcumin and copper with addition of NAC. This observation suggests that ROS scavenging could not rescue H314 cells from induced apoptosis. The combined treatment had a similar inhibitory effect on cell viability and apoptosis induction in ORL-115 cells (Fig. A.8 and Fig. A.9). To further verify and to elucidate the effect of copper on curcumin-induced apoptosis in H314 cells, two assays (TUNEL staining and Annexin V & PI double staining) were conducted. At 6h, a significant amount of TUNEL-positive apoptotic cells (~41%) were observed in cells treated with curcumin and copper but not in the other experimental groups (Fig. 5C & Fig. A.10). Apoptosis can also be detected via positive staining of Annexin V at the extracellular side of the plasma membrane. Flow cytometric results showed that longer treatment duration (24 h) increased the apoptosis in H314 cells treated with curcumin alone (Annexin V+, 62.2%) and combined treatment with EDTA (Annexin V+, 56.2%) while only low amounts of apoptotic cells were observed in the untreated group (Annexin V+, 4.50%) and copper only treatment group (Annexin V+, 10%) (Fig. 5D). Propidium iodide (PI) does not stain live or early apoptotic cells but the disintegrated plasma membrane of late apoptotic/necrotic cells allows PI to pass through membranes and stain nucleic acids. Apparently, curcumin alone caused early apoptosis (Annexin V+/PI-, 53.9%; Annexin V+/PI+, 8.3%) in cells treated for 24 h while late apoptotic cells was increased in the presence of copper (Annexin V+/PI-, 54.0%; Annexin V+/PI+, 14.9%) (Fig. A.11). Results from the two assays clearly demonstrated that curcumin together with copper induced apoptotic DNA fragmentation as early as at 6 h treatment and subsequently compromised cell membrane integrity at 24 h as a late event in apoptosis (Fig. 5). In another words, curcumin could induce apoptosis more efficiently with the presence of copper in oral cancer cells.

Discussion

Cancer is patho-biologically complex and exhibits substantial heterogeneity leading to extremely varied clinical behaviour and treatment responses (Swanton 2012). Therefore, to start with, we wished to address the heterogeneity of oral cancer cells that are derived from various sites of the oral cavity of different patients. In this we opted to use a representative panel of OSCC cell lines derived from distinct cohorts of oral cancer patients: The H-series cell lines which were established and characterized in UK by 1990 and the ORL lines which were recently derived from an Asian population (Prime et al. 1990, Hidayatullah Fadlullah et al. 2016). This is the first report to characterize the relative copper level of these oral cancer cell lines in comparison to normal oral keratinocytes (NOK) and immortalised nasopharyngeal epithelial cells. Consistent with previous finding (Khanna et al. 2013), we demonstrated that normal immortalised cells contained the lowest cellular copper while the copper levels found in the OSCC cell lines were significantly higher than those found in normal cell lines. Further we observed a moderate correlation between the responses of these cell lines to the therapeutic agent curcumin and the corresponding cellular copper level. Cytotoxicity of curcumin in cancer cells may involve ROS induction and copper is a transient metal that regulates cellular redox balance (Ridge et al. 2008, Liang et al. 2014). The correlation observed in this study could possibly be due to the elevated copper level compromising the capability of cancer cells to withstand excessive oxidative stress induced by curcumin. Among the cell lines that showed remarkably high insensitivity in curcumin treatment, H314 cells are derived from the poorly differ entiated mouth floor tumor of a non-smoker (Yeudall et al. 1995) while ORL-115 was established from the gingiva-derived tumor of a betel quid user (Hidayatullah Fadlullah et al. 2016). These cell lines were derived from different cohorts but both contained low intracellular copper content. We propose that this insensitivity may be because there is insufficient copper to trigger apoptosis in treated cells and supplemental copper replenishes the ion pool which restores the therapeutic response to oral cancer cells. Notable sensitizing effects were observed in curcumin-treated H314 and ORL115 cells with copper supplement thus backing up this supposition.

Curcumin may play a role as an anti-oxidant to scavenge reactive radicals and maintain the levels of anti-oxidant enzymes in the presence of copper (Leung et al. 2013). However, recent studies have found that increased generation of reactive oxygen species (ROS) was correlated to escalated malignancy in cancer cells (Wondrak 2009). The process of carcinogenesis alters redox status in the cells which leads to altered biochemical properties of cancer cells in comparison to other normal cell types (Trachootham et al. 2009). Previously, we reviewed the "double-edged sword" role of phytoagents in cellular redox regulation. With evidence from various recent studies, we proposed that phytoagents function as antioxidants in normal cells to scavenge harmful ROS in order to maintain redox homeostasis. However excessive ROS and free form of metal ions, e.g., copper, in cancer cells alter the redox status in which classic antioxidants such as curcumin catalyze the redox cycling of metal ions by reducing their oxidized form. As a result, a burst of hydroxyl free radical production ensues and the phytoagents become prooxidants (Lee et al. 2013). Significant increased expression of Nrf2, an indicator of oxidative stress, was observed in both cell lines with co-treatment, which could be suppressed by chelator EDTA or antioxidant NAC or both the inhibitors. In ORL-115 cells, decrease of Nrf2 via chelation of copper by EDTA is more apparent (ratio to control = 0.7:1.0) in comparison to the decrease caused by ROS scavenged by NAC (ratio to control = 2.1:1.0) (Fig. 3D). Further, the cleavage of PARP indicating apoptosis induction in co-treated H314 cells was mostly abolished by EDTA but not by NAC (Fig. 5B). Together, the results suggest that the metal ions induced oxidative stress likely play a role in the anti-cancer effect of combined treatment of curcumin and copper. However, the scavenging of ROS by NAC might not entirely eradicate the induced oxidative stress, or other metal ion activities may be involved.

Without additional supplement of copper, increased cellular copper was observed in oral cancer cells in the presence of curcumin. This result is in good agreement with a previous study demonstrating that curcumin can act as an ionophore to carry copper into cancer cells (Lou et al. 2010). On the other hand, we did observe that copper alone significantly stimulated transwell migration and in the oral cancer cell lines H314 (Fig. 4A & B) and ORL-115 (Fig. A.5 & A.6) accompanied by a decrease in E-cadherin as well as an increase in vimentin (Fig. 4C and Fig. A.7). Both these molecular events indicate an enhanced invasiveness of cancer cells. This observation is consistent with a previous finding on copper-mediated progression of oral cancer via regulating the activity of lysyl oxidase (LOX) (Shih et al. 2013). However, an opposite effect was observed when copper was used to treat oral cancer cells together with curcumin. A significant inhibitory effect of co-treatment of copper and curcumin on viability and motility of H314 and ORL-115 cells was consistently observed throughout the study. Together these observations show that the presence of curcumin critically affects the role of copper in cancer cells. Leung et al. (2013) who reviewed the medicinal effects of curcumin has suggested that by forming a complex with copper, curcumin could induce DNA damage and inhibition of prominent signaling pathways in cancers which subsequently leads to apoptosis. We consistently observed early apoptotic DNA damage and late apoptosis exclusively in H314 cells treated with both copper and curcumin.

Epithelial-mesenchymal transition (EMT) signaling is associated with progression of several cancer types. Upon the activation of the EMT program, loss of cell-to-cell adhesion is one of the first critical steps of cancer metastasis, invasion, and progression. Therefore the loss of epithelial markers such as E-cadherin, a major constituent of the adherens junctions, is considered to be a key event to activate EMT. In most of the recent studies of invasiveness in oral cancers, decrease of epithelial E-cadherin is one of the most commonly-used indicators of EMT induction which was significantly proved to correlate well with metastatic and malignant phenotypes of oral cancer cells as well as clinical samples (da Silva et al. 2015, Ghosh et al. 2016, Huang et al. 2016, Kimura et al. 2016). Decrease of E-cadherin was accompanied by acquisition of mesenchymal markers in carcinoma cells, such as vimentin, N-cadherin, and fibronectin in the processes of EMT (De Craene and Berx 2013). In good agreement with this phenomenon, we observed both up-regulation of E-cadherin and down-regulation of vimentin in H314 cells with suppressed mobility caused by co-treatment with curcumin and copper (Fig 4). The remarkable changes in both the epithelial and mesenchymal markers imply curcumin in combination with copper should intervene in EMT progress in OSCC cells to a certain degree. Many studies have attempted to seek antimetastatic agents and recently a prooxidant was reported to inhibit the EMT process with an increase in E-cadherin (Das et al. 2014). Curcumin coupled with copper in this study possibly induced ROS that significantly suppressed migratory phenotypes of curcumin-treated cells via impeding EMT as indicated by up-regulation of E-cadherin in both H314 and ORL-115 cells. In short this study suggests copper is important in the anticancer activities of curcumin, as curcumin combined with copper induces fatal oxidative stress which could effectively inhibit migration and induce apoptosis in curcumin insensitive oral cancer cells. We believe our findings provide important information to enhance the therapeutic efficacy of curcumin.

ARTICLE INFO

Article history:

Received 20 July 2015

Revised 4 August 2016

Accepted 4 September 2016

Conflict of interest

The authors declare that they have no conflict of interest. Acknowledgments

We gratefully acknowledge the Fundamental Research Grant Scheme grants (FRGS/1/2014/SKK01/MUSM/03/2) and (FRGS/1 /2015/SKK08/MUSM/02/3) from the Ministry of Higher Education Malaysia for financial support and the Taiwan International Graduate Program-International Internship Program for enabling the international collaboration between Academia Sinica Taiwan and the University Monash Malaysia. We would like to show gratitude to Prof Cheong Sok Ching from Cancer Research Malaysia and Prof Ian Paterson from University Malaya for providing us OSCC and NOK cell lines.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.09.005.

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Hui-Mei Lee (a), Vyomesh Patel (a), Lie-Fen Shyur (b,c,d), Wai-Leng Lee (e), *

(a) Nasopharyngeal Cancer Research Team, Cancer Research Malaysia, Selangor 47600, Malaysia

(b) Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan

(c) Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan

(d) Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan

(e) School of Science, Monash University Malaysia, Selangor 46150, Malaysia

Abbreviations: DAPI. 4.6-diamidine-2-phenylindole; DCFDA, dichlorofluorescin diacetate; DM EM. dulbecco's minimal essential medium: DMSO, dimethyl sulfoxide: EMT. epithelial-to-mesenchymal transition; FITC. fluorescein isothiocyanate; NAC. N.acetyl cysteine; NOK. normal oral keratinocyte; Nrf2, nuclear factor E2-related factor 2; PBS. phosphate-buffered saline; PI. propidium iodide: OSCC, oral squamous cell carcinoma; ROS, reactive oxygen species.

* Corresponding author. Fax: +603-55146184.

E-mail address: lee.wai.leng@monash.edu (W.-L Lee).

http://dx.doi.org/ 10.1016/j.phymed.2016.09.005

Table 1 Concentration of curcumin ([micro]M) that is required to
inhibit 50% cell viability ([IC.sub.50]) in different cell lines of
OSCC and NOK at 24 h.

Cell lines   [IC.sub.50]   [+ or -] SD

232N         100.0         13.4
337N          96.7          4.1
NP69          80.1          8.3
NP460         72.1          7.2
H103          13.9          3.8
H157          11.1          3.7
H314          50.0          8.2
H357          30.0          2.3
H376          10.8          1.1
H400           7.9          0.5
H413          25.8          3.4
ORL48         55.9          3.7
ORL115       103.3          2.9
ORL204        89.9          2.8
ORL214        70.8          2.8
ORL215        33.7          4.4

Table 2
Effect of copper on [IC.sub.50] of curcumin ([micro]M) in treatment
of H314 cells.

Treatments                              IC50 ([micro]M)
                                        of curcumin

                                        24 h   48 h

Curcumin only                           50.0   25.5
Curcumin + 250 [micro]M copper          40.3   5.3
Curcumin + 250 [micro]M copper + 250    49.4   28.4
  [micro]M EDTA

Table 3
Effect of copper on [IC.sub.50] of curcumin ([micro]M) in treatment
of ORL115 cells.

Treatments                              IC50 ([micro]M)
                                        of curcumin

                                        24 h    48 h

Curcumin only                           105.5   69.8
Curcumin + 250 [micro]M copper           36.1   25.8
Curcumin + 250 [micro]M copper + 250    62.4    41.6
  [micro]M EDTA


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Author:Lee, Hui-Mei; Patel, Vyomesh; Shyur, Lie-Fen; Lee, Wai-Leng
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9TAIW
Date:Nov 15, 2016
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