Natural remedies have been used since ancient times, and are still in use today. In addition to being a vital part of traditional herbal medicine—due to their effectiveness and minimal side-effects—they are also being used to develop new drugs. Turmeric has been receiving increased attention from researchers due to its anticancer, antibacterial, antiseptic, antioxidatory, and anti-inflammatory activity, as well as its role in treating autoimmune diseases. Turmeric is an orange-yellow pigment extracted from the rhizomes of Curcuma longa
, a green leafy plant from the Zingiberaceae
family. It exhibits pharmacological activity, mainly attributed to the curcuminoids found within it. Curcuminoids are composed of three compounds (Figure 1
): curcumin, desmethoxycurcumin (DMC), and bisdesmethoxycurcumin (BDMC) [1
]. The chemotherapeutic and chemopreventive potential of curcuminoids makes them a promising candidate for the treatment of cancer. In addition, curcuminoids are able to regulate a wide number of biological targets without inducing side-effects [5
], and target multiple signaling pathways involved in survival, cell cycle regulation, metastasis, and angiogenesis in many cancers. The acceptable daily intake of curcumin, as defined by the World Health Organization (WHO), is around 0–3 mg/kg. Curcuminoids are also widely used as a coloring agent and food additive that can prolong the shelf life of food due to its antioxidant and antibacterial activities [1
]. Curcumin is non-toxic, even at relatively high doses. It has no lethal effect on healthy organs at doses as high as 8 g/day; however, cancer cells are sensitive to the cytotoxic activity of curcumin [8
]. Curcuminoids have been listed as safe by the Food and Drug Administration (FDA) and are included in pharmacopeia in Europe, U.K., U.S., Japan, China, and India.
Ovarian cancer is the eighth most commonly occurring cancer in women worldwide and the 18th
most commonly occurring cancer overall, according to World Cancer Report 2018. In most cases, women with ovarian cancer are diagnosed at advanced stages due to the absence of early symptoms. Thus, ovarian cancer has a five-year survival rate of 46% [9
]. Standard treatment regimens depend on the stage of the disease. However, the usual treatment after surgical cytoreduction involves a combination of platinum (Cisplatin and Carboplatin) and Taxane-based therapy. Although this régime has been found to be effective in 60–80% of cases, most women with advanced ovarian cancer face recurrence of the disease [11
]. Hence, there is an increased demand for effective therapeutic approaches and alternative healing agents for advanced-stage drug-resistant ovarian cancer.
Curcuminoid compounds have been shown to exhibit cancer growth suppression, both in vitro and in vivo, and inhibit tumorigenesis and cell proliferation in several cancer cell lines [13
]. Commercially available pure curcumin, DMC, and BDMC are not accessible as food supplements. Therefore, this study aimed to assess the effect of the three curcuminoids together as one sample against the epithelial ovarian cancer cell line SKOV-3, which is highly resistant to various cytotoxic drugs. To the best of our knowledge, this is the first report studying the three curcuminoid components as one sample—with the aim to investigate their combined effects on proliferation, apoptosis, cell cycle, and migration in cultured human ovarian cancer cells.
2. Materials and Methods
2.1. Preparation of Curcuminoids Solutions
The curcuminoids mixture from India Glycols Ltd. was kindly provided as a gift by Jamjoom Pharmaceuticals, SA. A stock solution of curcuminoids (10 mM) was prepared in dimethyl-sulfoxide (DMSO) and stored at −20°C in the dark. Subsequent dilutions were prepared in Dulbecco’s modified Eagle’s medium (DMEM) (UFC-Biotech) to obtain different concentrations.
2.2. Analysis of Curcuminoid Compositions by HPLC
The analysis of total curcuminoid composition was carried out by reversed-phase high performance liquid chromatography (RP-HPLC) on a chromatographic column Phenomenex (4.6 mm × 25 cm, 5 µm). In brief, 20 mg of curcuminoids was dissolved in acetone to a total volume of 50 mL. The generated solution was subjected to sonication for 30 min, followed by centrifugation at 400× g for 5 min. Before the HPLC experiment, the stock solution was diluted with a mobile phase consisting of Tetrahydrofuran (THF) and 1 mg/mL of citric acid in water (4:6). Then, 20 μL of the sample was injected automatically and the mobile phase used for the elution of curcuminoids. The amount of curcuminoid compound was quantified by UV detection at 420 nm. The compositional distribution of curcuminoids in the sample was compared to the peak area correlated with the United States Pharmacopeia (USP) Reference Standard.
2.3. DPPH Radical Scavenging Assay
The antioxidant activity of curcuminoids was evaluated using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) (Sigma, St. Louis, MO, USA). A 0.1 mM DPPH solution was freshly prepared in pure ethanol and mixed with curcuminoids at different concentrations (1, 2, 4, 6, 8, 10 µM). The reaction mixture was shaken vigorously and left in the dark for 30 minutes at room temperature. As a control, a DPPH solution with the absence of curcuminoids was prepared; absorbance for this solution and the samples was measured at 517 nm. The percentage of DPPH scavenging radical activity was calculated using the following equation:
value was calculated using a four-parameter logistic nonlinear regression model by plotting the percentage of DPPH scavenging radical activity against the concentrations of the curcuminoids [14
]. Ascorbic acid was used as a standard reference compound.
2.4. Cell Culture
SKOV-3 cells were obtained from the American Type Culture Collection (ATCC). SKOV-3 cells were cultured in DMEM and complemented with 10% fetal bovine serum (FBS) (Gibco), 100 I.U/mL penicillin, and 100 µg/mL streptomycin (Sigma). The cells were grown in a humidified incubator at 37 °C and 5% CO2.
2.5. Cell Morphological Analysis
SKOV-3 cells were plated at a seeding density of 2.5 × 103 cells/well in a 6-well plate and tested with curcuminoids (0, 10, 30, 70 µM). Both untreated and treated cells were then cultured at 37 °C in a 5% CO2 incubator for 24 h. For morphological apoptotic evaluation, the SKOV-3 cells were stained with the Wright–Giemsa stain following the manufacturer’s instructions (Rapi-Diff II Stain Kit, Atom Scientific Ltd., Manchester, UK). Any visible changes in cell morphology following the experimental procedure were captured by microscopic images.
2.6. WST-1 Assay for Cell Viability
The cytotoxicity of curcuminoids was examined using the water-soluble tetrazolium salt (WST-1) assay (Abcam, Cambridge, UK) according to the manufacturer’s instructions. SKOV-3 cells were seeded into 96-well plates at a density of 5 × 103 cells/well in a 200 μL medium. After a culture time of 24 h, the culture supernatant was discarded and fresh medium containing various concentrations of curcuminoids was added and incubated for 1 h, 3 h, 6 h, 24 h, and 48 h. At each time point, 10 μL WST-1 was added to each well and the cultures were incubated for an additional 3 h at 37 °C. Absorbance was measured at 450 nm using an ELISA reader.
2.7. Quantitative Analysis of Cell Apoptosis by Flow Cytometry
The apoptotic activity of the SKOV-3 cells in response to curcuminoids was determined by Annexin APC, as per the manufacturer’s instructions (BD Biosciences). Briefly, the SKOV-3 cells were grown in a 25 cm2 flask at a density of 3 × 105 cells/well. The induction of apoptosis was investigated in untreated and treated SKOV-3 cells with curcuminoids at a concentration of 30 µM for 6 h, 12 h, 24 h, and 48 h. After harvesting by trypsinization and washing with PBS, the cells were stained with 5 µL Annexin APC and incubated for 15 min in the dark, then immediately analyzed using a FACS flow cytometer (BD Biosciences).
2.8. Analysis of Cell Cycle by Flow Cytometry
The SKOV-3 cells were seeded in cell culture flasks at a density of 2.5 × 106 cells per 25 cm2. After 6 h, 24 h, and 48 h, the culture medium was replaced by a fresh one containing 30 µM curcuminoids. The untreated cells were left as negative control. After harvesting by trypsinization and washing with PBS, the cells were fixed with 70% ethanol and stained with Hoechst 33342 (Thermo Fisher Scientific), which is a DNA dye used to assess DNA content. After 45 min incubation at 37 °C in the dark, the cells were resuspended in PBS and analyzed using a FACS flow cytometer.
2.9. Wound Healing Assay
The SKOV-3 cells (2.5 × 103
) were plated in a 24-well plate for 24 h and allowed to attach overnight. The confluence monolayers were scratched using a pipette tip to create a linear wound devoid of cells, which was subsequently washed with PBS. Images were captured at 0 h and 24 h using a camera attached to a microscope. The migration was evaluated microscopically and the wound area quantified by image J [15
]. The captured images were converted to grayscale and the edges of the wound areas were automatically identified using an MRI wound healing tool after image sharpening. The size of the initial wound area was compared to that after 24 h in both untreated cells and cells treated with curcuminoids. The migration index was calculated as follows:
2.10. Cytokine ELISA
The SKOV-3 cells were treated with 30 µM curcuminoids. Cell culture supernatants were collected after 24 h, 48 h, and 72 h of culture. Tumor necrosis factor-alpha (TNF-α) and interleukin 10 (IL-10) levels were quantified using human ELISA kits as per the manufacturer’s instructions (Invitrogen, Frederick, MD, USA). The analytical sensitivity of the assay was 1.7 pg/mL and <1 pg/mL for human TNF-α and human IL-10, respectively.
2.11. Statistical Analysis
The data are presented as the mean ± standard deviation (SD) of the mean from two or three different replicates for individual assays. Statistical significance was determined using SPSS 22.0. *p < 0.05, ** p < 0.01, and *** p < 0.001 indicated statistical significance, compared to the untreated control cells.
Curcuminoids have been extensively used in ancient Chinese and Indian medicine. Moreover, they still attract great attention in modern medicine due to their ability to treat and prevent several diseases and conditions such as cancer, inflammation, and wounds. Pure curcumin, DMC, and BDMC are not available in the market; however, commercial curcumin consists of a mixture of these three curcuminoids [17
]. Thus, the present study aimed to investigate the combined effect of these three curcuminoids compounds on ovarian cancer cells. Since the contents of the three curcuminoids may vary among different curcuminoid sources, it was important to quantify the amount of each analogue by HPLC. The three curcuminoids were resolved as individual peaks with no interface with other compounds. The sample was found to consist of 81.76% curcumin, 15.16% DMC, and 3.08% BDMC. These results show curcumin to be the main component among the three curcuminoids, which meets the requirements of pharmacopeia in Europe and China.
Turmeric contains powerful natural antioxidants, and thus acts as a singlet oxygen quencher and a radical scavenger that can arrest the generation of free radicals after oxidative stress and can lower the molecular damage to the cell [18
]. Due to the ability of DPPH to assess the free radical scavenging activity of antioxidant substances, it has been used to investigate the efficiency of curcuminoids to scavenge the free radicals formed by reactive oxygen species (ROS). The antiradical power of curcuminoids can be detected by recording the decrease of DPPH absorbance values. At 517 nm, DPPH has an absorbance that disappears or decreases when it accepts an electron or hydrogen radical from an antioxidant [21
]. Thus, the decrease of absorbance values is attributed to the scavenging effect of curcuminoids and the formation of stable non-radical DPPH molecules. Previous works showed different IC50
values for DPPH radical-scavenging activity for curcuminoids. For example, curcumin, DMC, and BDMC displayed free radical scavenging activity with IC50
values 31.8, 92.5, and 104.4 μM, respectively [23
]. In another study by Akter et al., (2019), the IC50
values were 18, 47, and 198 μM for curcumin, DMC, and BDMC, respectively [24
]. In the present study, curcuminoids effectively exhibited DPPH scavenging activity with IC50
= 9.14 µM in a cell-free system. Here, a low IC50
value indicates high free radical scavenging activity of DPPH.
For cytologic details, the effect of curcuminoids was tested on ovarian cancer cells, as ovarian cancer is the most aggressive gynecological cancer [25
]. The ovarian cancer cell line SKOV-3 was chosen in the current study because it is one of the most invasive cell lines [26
]. Morphological alterations and nuclear fragmentation are important to be investigated because these changes are attributed to cell response and sensitivity toward drugs. Besides reflecting the growth and fission of cells, these biological characteristics of cell morphology reflect the apoptotic phenotype. The main morphological hallmarks of apoptotic cells are membrane blebs and cell shrinkage [27
]. Therefore, cellular morphological changes can be investigated to evaluate cell apoptosis qualitatively. In this study, treating SKOV-3 cells with curcuminoids leads to morphological changes and reduction in size and cell number. Further, treated cells exhibited the distinctive morphological features of apoptotic cell death, including membrane damage, cell shrinkage, and subsequent cell death. The cellular changes were concentration-dependent.
Cellular migration is critical in cancer as it contributes to the development of cancer metastasis. The scratch wound healing assay is a simple and cost-effective strategy used to investigate cell migratory activity. Several studies have confirmed the ability of curcuminoid members to suppress cancer cell migration and invasion. For example, curcumin inhibits migrated cells from entering into the wound area of lung [28
], breast [29
], and bladder [30
] cancer cells, while DMC inhibits the motility of breast [31
] and prostate [32
] cancer cells. Further, BDMC inhibits the migration of ovarian [33
] and lung [34
] cancer cells. In the current work, the percentage closure was calculated to assess the effect of curcuminoids on wound healing. The wound closure reached 100% after 24 h in untreated cells, while treatment with curcuminoids diminished the wound area to 36.77%. These results confirm the ability of curcuminoids to suppress SKOV-3 cell motility and scratch closure.
Dysregulation in the expression of cellular proliferation, apoptosis, and cell cycle are distinctive marks of cancer cells. Cancer cells are resistant to antiproliferation signals and begin proliferation in a dysregulation manner in the absence of exogenous mitogenic growth factor signals. Indeed, irregular apoptosis is mainly attributed to uncontrolled cell numbers. However, cancer cells have several mechanisms to skip regulatory proliferative and apoptosis signals. Furthermore, the initiation and growth of cancer is associated with dysregulation in the regulatory pathways of the cell cycle, which involves abnormalities in oncogenes and tumor suppressor genes [35
]. While curcuminoids are composed of three main members—curcumin, DMC, and BDMC—all have shown efficient anticancer effects against various malignancies and induce apoptosis, but with different potencies. Although many studies present curcumin as the active ingredient in turmeric, with a strong ability to kill different cancer cells such as hepatic [37
], lung [28
], breast [38
], pancreas [27
], bladder [30
], and ovarian [39
] cancer cells, DMC, which is more stable than curcumin, was reported by Simon et al. [40
] to exhibit the strongest antitumor activity among the three curcuminoids against breast cancer cells. DMC has also shown ability to inhibit the growth of prostate cancer cells [32
]. Furthermore, BDMC, the minor constitute, has shown the highest stability compared to the other two curcuminoids and was found to inhibit tumor growth in various cell lines such as HeLa and ovarian cancer cells [33
]. In the present study, the three members of curcuminoids jointly exhibited a toxic effect against SKOV-3 cells with an IC50
of 30 µM. This low value indicates that the tested curcuminoid mixture has high anticancer potency against ovarian cancer cells and can effectively inhibit cell proliferation. Besides the classic apoptotic phenotype, treated SKOV-3 cells quantitatively showed the ability of curcuminoids to induce apoptosis in ovarian cancer cells. The number of apoptotic cells measured by flow cytometry increased markedly with increasing treatment time. The cellular damage induced by curcuminoids may contribute to the inhibition of growth activity and subsequent cell death. Moreover, the cell cycle analysis showed an increasingly high population of cells in the sub-G1 phase in a time-dependent manner, which may be attributed to cell death via apoptosis. Interestingly, curcumin and DMC induce G2/M cell cycle arrest in hepatic [37
] and prostate [32
] cancer cells, respectively, while BDMC induces S phase arrest in the lung [43
]. Due to curcumin ability to exhibit dynamically biphasic dose-response on a broad range of cells, a recent study by Calabrese et al. (2019) showed that curcumin prompts hormetic responses in neural stem cells and further improves the biological resilience. These findings show that the hormetic responses of curcumin may be independent of biological model, cell type, endpoint, and inducing agent and mechanism [44
It has become widely clear that epithelial cells as a first line of defense mechanism can play a crucial role in modulating the function of immune cells through production of cytokines and other mediators, which ultimately maintain immune homeostasis [45
]. Although epithelial cells modulate immune responses via their own ability to produce a plethora of cytokines in vivo, not all cell lines produce constitutive amounts of cytokines in vitro [16
]. In vivo assays have shown that highly elevated levels of the inflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 are detected in ovarian cancer [48
]. In contrast, in in vitro assays, the expression of TNF-α and IL-10 in ovarian cancer cell lines is not a common event. For example, IGROV-1 and TOV21G cell lines release a detectable amount of TNF-α, while OVCAR-3 cell line releases IL-10. In contrast, other human ovarian cancer cell lines such as TOV112D, CAOV-3, and OAW-42 do not produce TNF-α or IL-10 [16
]. Further, it has been shown that the induction of TNF-α protein by TNF-α is common in malignant but not normal ovarian surface epithelial cells [51
]. Several polyphenols such as curcumin, quercetin, and catechins exhibit their effects on the balance between secretion of pro-inflammatory and anti-inflammatory cytokines. They inhibit the release of pro-inflammatory cytokines and activate anti-inflammatory cytokines’ production [52
]. However, curcumin has shown the ability to inhibit the production of TNF-α and some pro-inflammatory interleukins such as IL-1 and IL-6, while increasing the production of anti-inflammatory IL-10 [49
]. In the current study, to investigate whether curcuminoids may influence pro-inflammatory or anti-inflammatory activation, SKOV-3 cells were cultured in a basal medium without an additional stimulation agent in the absence and presence of curcuminoids. The obtained results exhibit that SKOV-3 cells are similar to the normal ovarian surface epithelial cells that do not produce TNF-α or IL-10, and the curcuminoids mixture has no activation effect on cytokine secretion.