Curcumin has demonstrated potential cytotoxicity across various cell lines despite its poor bioavailability and rapid metabolism. Therefore, our group have synthesized curcuminoid analogues with piperidone derivatives, FLDP-5 and FLDP-8 to overcome these limitations. In this study, the analogues were assessed on human glioblastoma LN-18 cells in comparison to curcumin. Results from cytotoxicity assessment showed that FLDP-5 and FLDP-8 curcuminoid analogues caused death in LN-18 cells in a concentration-dependent manner after 24-h treatment with much lower IC50 values of 2.5 µM and 4 µM respectively, which were more potent compared to curcumin with IC50 of 31 µM. Moreover, a significant increase (p<0.05) in the level of superoxide anion and hydrogen peroxide upon 2-h and 6-h treatment confirmed the oxidative stress involvement in the cell death process induced by these analogues. These analogues also showed potent anti-migratory effects through inhibition of LN-18 cells’ migration and invasion. In addition, cell cycle analysis showed that these analogues are capable of inducing S-phase cell cycle arrest during the 24-hour treatment, which explained the reduced proliferation indicated by MTT assay. In conclusion, these curcuminoid analogues exhibit potent anti-cancer effects with anti-proliferative and anti-migratory properties towards LN-18 cells as compared to curcumin.
Research Article
Curcumin piperidone derivatives induce anti-proliferative and anti-migratory effects in LN-18 human glioblastoma cells
https://doi.org/10.21203/rs.3.rs-1509729/v1
This work is licensed under a CC BY 4.0 License
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curcumin
curcuminoid analogues
cytotoxicity
anti-proliferation
anti-migration
Glioblastoma multiforme (GBM) is a grade IV astrocytoma characterized by rapid infiltrating growth and is the most common and aggressive malignant primary brain tumor in humans (Banu 2019; Nørøxe, Poulsen & Lassen 2016). GBM patients have a poor prognosis whereby most of the patients hardly survive for more than one year; with less than 3% of patients surviving for more than 5 years after diagnosis (Shahar et al. 2012). This is mainly due to the extensive heterogeneity at the cellular and molecular levels of the tumours and also the issue of the resistance towards the chemotherapeutic drug, temozolomide (Atkins et al. 2015; Van Tellingen et al. 2015; Zhang et al. 2012). Several studies had reported that glioblastoma cells, including LN-18 cells, were resistant to temozolomide via upregulation of methylguanine methyltransferase (MGMT) enzyme and deficiency in mismatch repair (MMR) mechanism (Ferreira et al. 2018; Zhang et al. 2012). The resistance and recurrence and also the side effects of existing chemotherapy have highlighted the need for new therapeutic alternatives.
Turmeric, a rich source of curcumin, has been used for centuries in traditional remedies to treat a variety of diseases. (Tan & Norhaizan 2019; Wong et al. 2021). With the advancement in technology over the years, the biological activities of curcumin and its molecular targets have been identified. Curcumin has demonstrated a wide range of biological activities, including anti-inflammatory, cytotoxicity and apoptosis induction on several cancer cell lines (Agrawal 2011; Wojcik et al. 2018; Wong et al. 2021). Numerous molecular targets for curcumin, for instance, tumour suppressor proteins (p53 & p21), oncoproteins (C-Myc, cyclin D1) and antiapoptotic proteins (survivin & Bcl-2), have been identified and reported crucial in curcumin-induced apoptosis (Al-Hujaily et al. 2011; Walker, Adhikari & Mittal 2021; Willenbacher et al. 2019). Despite this, curcumin has its own drawbacks, whereby clinical trials and animal studies on curcumin showed that this compound has poor bioavailability and weak pharmacokinetic profile, rendering it a poor drug candidate. The poor bioavailability is mainly contributed by poor absorption as well as rapid metabolism and elimination via sequential reduction and glucuronidation by the body (Anand et al. 2007; Sansalone et al. 2019; Thilakarathna & Vasantha Rupasinghe 2013).
Chemical synthesis and modification are commonly used to produce new derivatives of chemotherapeutic drugs with improved efficacy, bioavailability and selectivity (Yin 2014). Hence, our group has synthesized two curcuminoid analogues with two piperidone derivatives, namely FLDP-5 and FLDP-8 were created (Fig. 1). These newly synthesized analogues were used in this study to investigate the potential of anti-proliferative and anti-migratory effects against human GBM LN-18 cells, as well as to compare the efficacy with curcumin.
Reagents
Dulbecco’s Modified Eagle’s Medium (DMEM) Medium from Gibco Invitrogen, USA; penicillin/streptomycin from Nacalai Tesque Inc., Kyoto, Japan; fetal bovine serum (FBS) from Tico Europe, Amstelveen, Netherlands; phosphate buffer saline (PBS), (3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazolium bromide) (MTT) from Sigma-Aldrich, UK; dimethylsulphuxide (DMSO) and hydrochloric acid (HCl) form Fisher Scientific, UK; sodium hydrogen carbonate (NaHCO3) and potassium hydrogen phosphate (KH2PO4) from Systerm, Malaysia; sodium chloride (NaCl) and sodium dihydroxide (NaOH) from EMSURE, Jerman; disodium hydrogen phosphate (Na2HPO4) from HmbG Chemicals, German; potassium chloride (KCl) from J.T Baker, USA; dihidroethidium (DHE) stain and dichlorofluorescin-diacetate (DCFH-DA) stain from Eugene; Disodium ethylenediaminetetraacetate dihydrate (Na2EDTA), low-melting point agarose (LMA) and normal melting agarose (NMA) from Sigma-Aldrich, St. Louis, MO, USA; Tris from Bio-Rad Laboratories, Hercules, CA, USA; 70% alcohol and distilled water from the FSK laboratory.
Test compounds
Compounds 4-Peperidinone,3,5-bis[(4-hydroxy-3-methoxyphenyl) methylene]-,(3E,5E) (FLDP-5) and 4-Peperidinone, 3,5-bis[(4-hydroxy-3-methoxyphenyl) methylene]-1-Methyl(3E,5E) (FLDP-8) were synthesized and contributed by Dr. Lam Kok Wai from Centre for Drug and Herbal Development, Faculty of Pharmacy, Universiti Kebangsaan Malaysia (Kuala Lumpur, Malaysia). Curcumin and hydroquinone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of FLDP-5 and hydroquinone were prepared at 50 mM, while stock solutions for FLDP-8 and curcumin were prepared at 25 mM. All compounds were dissolved in solvent dimethyl sulfoxide (DMSO). Treatment for all compounds on LN-18 cells was performed in dark condition due to the compounds’ photosensitive characteristics.
Cell culture
LN-18 human GBM cells were established in 1976 from cells taken from a patient with a right temporal lobe glioma whereas HBEC-5i cells were established in 1994 which were derived from small fragments of human cerebral cortex obtained from patients who had died of various causes and were devoid of any pathologic abnormalities. Both cell lines were obtained from American Type Culture Collection (ATCC). The culture medium used throughout these experiments was Dulbecco’s Modified Eagle’s Medium (DMEM) medium (GIBCO) for both cell lines supplemented with fetal bovine serum (FBS) (5% for LN-18 cells and 10% for HBEC-5i cells) and 1% penicillin/streptomycin. Both LN-18 and HBEC-5i cells were used between passages 3-12 for the experiments and maintained at 37°C
and 5% CO2.
MTT cytotoxicity assay
The cytotoxic effects of curcumin and the curcuminoid analogues (FLDP-5 and FLDP-8) were assessed as previously described (Mosmann 1983). Briefly, LN-18 cells were seeded in a 96-well plate at a density of 5 × 104 cells per well in a volume of 200 µL and were treated with curcuminoid analogues (FLDP-5 and FLDP-8), curcumin and hydroquinone (positive control) with respective concentrations. The treated cells were incubated for 24-h under 5% CO2 at 37°C After 24-h incubation, 20 µL of 5 mg/mL MTT was added to each treated cells and further incubated for 4-h at 37°C . MTT salt was reduced forming a purple coloured crystal formazan by the active viable cells with dehydrogenase enzyme. Then, 180 µL of the medium was discarded from the treated cells before adding 180 µL of dimethyl sulfoxide (DMSO) to solubilize the crystal formazan, respectively. The plate was further incubated for 15 min to completely dissolve the crystal formazan, followed by gentle resuspension for each well. The cytotoxic effects of each compound were detected by measuring the absorbance of each well at 570 nm using iMarkTM microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The inhibitory concentration that kills 50% of the cell population (IC50) was obtained from the plotted of each compounds concentrations versus the percentage of the cell viability.
Selectivity index
The selective index (SI) of the compounds were calculated according to the equation as previously established (Popiołkiewicz et al. 2005). The calculation is done in order to determine the degree of selectivity of the compound tested against cancerous cells, in which values larger than “100” indicates the compound is selective toward cancerous cells and confers minimal toxicity in normal non-malignant cells. In this study, the SI values were determined by IC50 of normal human cerebral microvascular endothelial cell (HBEC-5i) divided by IC50 of LN-18 cells for each compound. It was simplified as follows:
Blood-brain barrier (BBB) and ADMET prediction
PubChem database was applied to get the smiles structures of FLDP-5 and FLDP-8 curcumioid analogues, and was further used for the BBB and ADMET prediction using two different online platforms, AlzPlatform (www.cbligand.org/AD/) and ADMETlab 2.0 (https://admetmesh.scbdd.com/) (Hu et al. 2020; Liu et al. 2014; Xiong et al. 2021; Zhao et al. 2007). AlzPlatform was built by using the support vector machine (SVM) and LiCABEDS algorithms on PubChem fingerprint from 1593 reported compounds. By entering the smiles structures of the compounds, the online predictor calculated the BBB score, which showed whether a compound could pass the blood-brain barrier (BBB+) or not (BBB-). ADMETlab 2.0 which is an integrated online platform, was used to computationally predict the pharmacokinetic properties of compounds such as absorption, distribution, metabolism, excretion, and toxicity (ADMET).
Reactive oxygen species assessment
The level of reactive oxygen species (ROS), specifically for superoxide anion and hydrogen peroxide were assessed as previously described (Chan et al. 2010; Ooi et al. 2021). Briefly, the treated LN-18 cells were administered at different time-point intervals before being harvested. The treated LN-18 cells were then collected by centrifugation at 220 × g for 5 mins. After the supernatant was discarded, the pellet was resuspended with 1 mL of fresh pre-warmed FBS-free DMEM media and with the addition of 1 µL of 10 mM DHE and 10 mM DCFH-DA stains. The cells suspension with DHE and DCFH-DA staining were incubated at 37°C
for 30 min. After the incubation period, the cells were centrifuged at 220 × g for 5 min. Then, the cells were washed with 1 mL chilled PBS, and the supernatant was discarded, followed by resuspension of the pellet by 500 µL of ice-cold PBS. The stained cells were transferred to flow cytometric analysis tubes and analyzed using FACSCanto II flow cytometer (BD Bioscience, USA) on 10,000 cells.
Alkaline comet assay
As previously described, the alkaline comet assay was performed to access DNA damage induced by curcumin and the curcuminoid analogues (FLDP-5 and FLDP-8) (Chan et al. 2006; Tan et al. 2020). Treated LN-18 cells were harvested and washed twice with Ca2+, Mg2+-free PBS. Cell pellets were then mixed thoroughly with 80 µL of 0.6% low melting point agarose (LMA) and laid on hardened 0.6% normal melting agarose (NMA). The agarose was allowed to solidify and subsequently placed in a chilled lysis buffer (2.5 M NaCl, 100 Mm EDTA, 10 mM Tris, and 1% Triton-X) for lysis to occur. Slides were then incubated in an electrophoresis buffer (0.3N NaOH, 1 Mm EDTA) for 20 minutes to facilitate DNA unwinding. Electrophoresis was performed under 25 V, 300 Ma for 20 minutes. Subsequently, slides were rinsed with neutralizing buffer (400 Mm Tris) thrice prior to staining with 50 µg/mL ethidium bromide solution. The slides were observed under Olympus BX51 fluorescence microscope (Olympus, Japan) equipped with 590 nm filter. The tail moment, the product of tail length and fraction of total DNA in the tail, was assessed using Comet ScoreTM software (TriTek Corp, Sumerduck, VA, USA) on 50 cells per slide.
Scratch/ wound-healing assay
A monolayer wound healing assay was performed following the protocol from previous studies with slight modifications (Nordin et al. 2021; Senft et al. 2010). LN-18 cells were seeded at a density of 2 × 105 cells per well in a 12-well plate (Nest Biotechnology, Jiangsu, China). After reaching 90% confluency, the cells monolayer were then scraped in a straight line creating a “scratch” using a sterile 200 uL pipette tip. The cells were then washed with PBS before taking photographs of the scratched area using a camera attached to an inverted phase contrast microscope (Olympus, Japan). The scratch area were photographed, and the location on the plate was noted. The cells were then treated with FLDP-5 curcuminoid analogue (1.25 and 2.5 µM), FLDP-8 curcuminoid analogue (2.5 and 5 µM) and curcumin (12.5 and 25 µM). Untreated cells were used as a control for the experiment. Cells were further incubated for 24-h and 48 h before the same area were photographed, and the cells migration area was measured using Image J software before the percentage of wound closure was calculated.
Boyden chamber invasion assay
The principle of this assay is based on two medium containing chambers separated by a porous membrane through which cells transmigrate (BOYDEN 1962). This assay was conducted following the protocol provided by QCMTM Collagen Cell Invasion Assay, 24-well (8 μm), Colorimetric kit purchased from Merck, Germany. Generally, LN-18 cells were starved for 24-h prior to assay in serum-free DMEM medium. Then, 250 μL of harvested cell suspension with concentration of 7 × 105 cells/mL in chemo-attractant free media was added to the insert/upper chamber of the well containing collagen-coated membrane. After that, 500 μL of DMEM containing respective compounds treatment will be added to the bottom chamber of the well. After 24-h incubation, the insert coated with the membrane will be fixed and stained with 400 μL Cell Stain. A cotton-tipped swab was used to remove the non-invading cells/collagen layer from the interior of the insert. The stained insert was transferred to a new well containing 200 μL of Extraction Buffer, and 100 μL of the dye mixture was transferred into a 96-well plate. The Optical Density (OD) of invaded cells were measured using iMarkTM microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at 560nm.
Cell cycle analysis
Cell cycle distribution was determined following protocol as previously described (Leong et al. 2016). Cells were seeded at 2 × 105 cells per well in a 6-well plate before being treated with curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin for 24-h. The treated cells will be harvested and washed with chilled PBS before being fixed with 70 % alcohol for at least overnight before staining. After fixing, cells will be washed with PBS and later stained with PI/RNase staining buffer (500 μL) (BD Bioscience) for 15 minutes at room temperature. Stained cells will then be analysed by using FACSCanto II flow cytometer (BD Bioscience, USA) on 20,000 cells, and the content of DNA will be determined by using ModFit LT™ software (Verity Software House).
Statistical analysis
The data are expressed as the mean ± standard error of mean (S.E.M.) from at least three independent experiments. The statistical significance was evaluated using one-way ANOVA with the Dunnet post hoc test to assess significance difference with negative control (NEG) or Tukey post hoc test to determine the significance of differences between multiple treatment groups. Differences were considered statistically significant with a probability level of p<0.05.
Curcuminoid analogues (FLDP-5 and FLDP-8) induced cytotoxicity on LN-18 human GBM cells and HBEC-5i cells
The cytotoxic effects of curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin were determined using MTT cytotoxicity assay. The results showed that the curcuminoid analogues, FLDP-5 and FLDP-8, including curcumin, induced cytotoxicity in LN-18 cells in a concentration-dependent manner after 24-h treatment. Interestingly, the IC50 values observed for the FLDP-5 and FLDP-8 curcuminoid analogues were 2.4 µM (Fig. 2) and 4 µM (Fig. 2), respectively, which were more potent in comparison to curcumin with an IC50 value of 31 µM (Fig. 3). Evaluation of FLDP-5 and FLDP-8 curcuminoid analogues toxicity on the non-cancerous HBEC-5i cell line, showed that much higher doses were required to cause HBEC-5i cell viability to decrease by 50% compared to the LN-18 cancer cell lines. The IC50 values of curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin in HBEC-5i were determined as 5.6 ± 0.5 (Fig.4), 9 ± 0.66 (Fig.4), and 192 ± 4.67 (Fig.5), respectively. As shown previously (Paulraj et al. 2015; Popiołkiewicz et al. 2005), the IC50 values were used to calculate the selective index, SI (Table 1), a baseline used to assess the selective toxicity of the analogues and curcumin towards cancerous cells over normal cells. It was noteworthy that the SI for both analogues in HBEC-5i were 2.33-fold and 2.25-fold higher (Table 1) respectively compared to the baseline (100) showing the selectivity of these analogues towards cancerous cells than normal cells. Hydroquinone (HQ) is used as the positive control in this study, where the IC50 value is 10 µM following 24-h treatment in LN-18 cells
Table 1
IC50 of the compounds in LN-18 cells and HBEC-5i cells including the selectivity index (SI)
|
Compounds |
IC50 (µM) ± S.E.M |
SI |
|
|
LN-18 cells |
HBEC-5i cells |
||
|
FLDP-5 |
2.4 ± 0.12 |
5.6 ± 0.5 |
233 |
|
FLDP-8 |
4 ± 0.12 |
9 ± 0.66 |
225 |
|
Curcumin |
31 ± 5.46 |
192 ± 4.67 |
619 |
FLDP-5 and FLDP-8 curcuminoid analogues were predicted to be BBB permeable
The probability of the analogues penetrating the BBB were first estimated by an online platform: AlzPlatform with cloud computing and sourcing functions. The analysis showed that FLDP-5 and FLDP-8 curcuminoid analogues including curcumin had positive BBB scores (Fig. S1), implying that these analogues and curcumin are permeable to the BBB. We further confirmed this prediction using another online predictor which is ADMETlab 2.0 that were usually used in predicting the pharmacokinetic properties of compounds such as absorption, distribution, metabolism, excretion, and toxicity (ADMET). The results from distribution section of the reports stated the similar predictions with the previous predictor which showed that curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin were capable to cross the BBB with the output value of the probability of being BBB+ were 0.029, 0.38 and 0.155 (Table S1) respectively. It is depicted that FLDP-5 crucuminoid analogue and curcumin showed excellent output values of the probability of being BBB+ compared to FLDP-8 curcuminoid analogue with medium output value of the probability of being BBB+.
FLDP-5 and FLDP-8 curcuminoid analogues induce both superoxide and hydrogen peroxide in LN-18 cell death
Intracellular ROS, specifically superoxide anion and hydrogen peroxide, were assessed using HE and DCFH-DA staining and detected through flow cytometer. The detection was performed to determine the ROS involvement in inducing oxidative stress and consecutively cell death in LN-18-treated cells. Our study demonstrated curcuminoid analogues (FLDP-5 and FLDP-8), and curcumin caused a significant elevated level of superoxide anion as compared to negative control cells with respective 1.42-fold, 1.26-fold and 1.83-fold increase at 2-h time-point treatment and persisted up to 6-h (Fig.6A). Interestingly, Fig.6B showed that FLDP-5 and FLDP-8 curcuminoid analogues were also able to induce a significant level of hydrogen peroxide with 2.93-fold and 3.45-fold increase respectively at 6-h time-point treatment in comparison to negative control cells. Consequently, these analogues showed a significant difference in which different reactions compared to parent compound curcumin as hydrogen peroxide level was not induced in curcumin-treated LN-18 cells.
Curcuminoid analogues (FLDP-5 and FLDP-8) induced higher severity of DNA damage compared to curcumin in LN-18 treated cells
In this study, the occurrence of DNA damage induced by the curcuminoid analogues and curcumin on LN-18 cells were assessed using alkaline comet assay. The scoring for DNA damage was based on the length of the formed tail, which represents the migration of DNA with strand breakage during electrophoresis following treatment. The images were captured using a fluorescence microscope and depicted in Fig. 7. In the negative control group, only a small number of cells showed DNA damage with tail moment of 0.28 ± 0.03. However, the DNA damage in all treated compounds progressively increased in a time-dependent manner following respective treatments using IC50 values (Fig. 7A). In contrast to the negative control, severe damages at 6-h time-point treatment with a significantly higher tail moment up to 192-fold, 211-fold and 122-fold increase respectively were observed in curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin. Curcuminoid analogues (FLDP-5 and FLDP-8) were also observed to have a significantly higher tail moment (p<0.05) with respective values of 53.97 ± 4.54 and 59.23 ± 4.71 compared to parent compound curcumin with tail moment of 34.35 ± 4.9 following 6-h treatment.
Curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin potentiate anti-migration in LN-18 Cells
The anti-migratory effects of curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin on LN-18 cells were investigated through wound scratch assay. The migration rate of treated LN-18 cells with respective compounds was calculated based on the closure of the wound/ scratch on the monolayer LN-18 cells. The images of the closure for each treatment were captured using a camera attached an inverted microscope and represented in Fig. 8C. It could be observed that treatment with IC50 and IC25 of curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin showed a significant concentration-dependent decrease in the percentage of wound closure compared to the untreated group after 24-h and 48 h incubation time. The percentage of wound closure in the untreated group reached 100%, as indicated in Fig. 8B and Fig. 8C after 48 h incubation time, indicating the high rate of motility and rapid growth characteristics of LN-18 glioblastoma cells (Johansen et al. 2016). Our results demonstrated that curcumin and FLDP-5 curcuminoid analogue showed a higher potential in inhibiting the migration of LN-18 cells when treated with values of IC25 and IC50 compared to curcuminoid analogue FLDP-8. After 48 h treatment, percentage of wound closure of curcumin and FLDP-5 curcuminoid analogue when treated with IC25 concentration is 42.74% ± 8.32 and 56.43% ± 6.28 respectively, which were lower than the percentage of wound closure of FLDP-8 curcuminoid analogue treated with IC25 concentration, which was 82.69% ± 7.02 (Fig. 8B). Identically, the percentage of wound closure when treated with IC50 values of curcumin and FLDP-5 curcuminoid analogue after 48 h were also much lower than FLDP-8 curcuminoid analogue with the percentage of wound closure of 5.69% ± 1.56, 3.17% ± 0.71 and 42.6% ± 9.1 respectively (Fig. 8B).
FLDP-5 and FLDP-8 curcuminoid analogues and curcumin inhibit the invasion of LN-18 cells
To study the effects of curcuminoid analogues (FLDP-5 and FLDP-8) and curcumin on the invasive behaviours of LN-18 cells, we performed a transwell invasion assay with modified Boyden chambers. Our findings were consistent in corresponding to the wound healing experiments, with significant decline in the invasiveness of cells following the treatment of curcuminoid analogues and curcumin. All treatment compounds were able to reduce the percentage of relative invasion in LN-18 cells in a significant dose-dependent fashion compared to the untreated group after 24-h treatment (Fig. 9A). Treatment of FLDP-5 curcuminoid analogue at IC50 concentration showed the lowest relative invasion at 2.48-fold decrease with a percentage of 40.32% ± 3.14. The untreated group without chemo-attractant (serum: FBS) was used as an indicator that invasive properties of LN-18 were affected through the absence or presence of chemo-attractant (Fig. 9A and Fig. 9B).
FLDP-5 and FLDP-8 curcuminoid analogues induced cell cycle arrest in LN-18 treated cells
Cell cycle analysis was conducted to determine the involvement of cell cycle arrest in FLDP-5 and FLDP-8 curcuminoid analogues mechanism of action on LN-18 cells. Flow cytometric assessment was performed following 24-h treatment using IC25 and IC12.5 of all compounds to detect the cell population of each phase. Our study demonstrated that FLDP-5 and FLDP-8 curcuminoid analogues were able to induce arrest in S phase in a concentration-dependent manner, but a significant arrest in S phase with 1.5-fold increase for both analogues were observed when LN-18 cells when treated with IC25 values by accumulating 63.38% ± 4.42 and 61.59% ± 5.66 respectively (Fig. 10A and 10B). Contradictory results were observed in curcumin-treated LN-18 cells where the cell cycle inhibition occurred significantly at the G2/M phase (Fig. 10C). Curcumin induced arrest in the G2/M phase in a concentration-dependent manner and reached significant comparison with the untreated group after treatment of IC25 value with a cell population of 62.99% ± 2.38.
The available standard treatments for GBM have been found to be ineffective due to the inherent resistance of GBM cells to radiotherapy and chemotherapy, with the addition to the invasive behaviour of GBM cells, causing the effectiveness of surgery to be limited (Maiti et al. 2017; Ramirez et al. 2013). Moreover, because of the high frequency of drug resistance, GBM remains challenging to deal with through the drug-mediated therapy. Due to the poor efficacy of crossing the BBB, most chemotherapy medicines, such as doxorubicin and cisplatin, have failed to treat this tumour (Chen et al. 2020; Hassannia, Vandenabeele & Vanden Berghe 2019). Therefore, finding novel approaches is an urgent priority for the improvement of patients’ prognosis. With the aim for the drug-mediated therapy to pursue, identifying a potent compound that could defeat the resistance of GBM in addition to BBB-crossing ability is desperately needed. Recently, natural polyphenol curcumin has been found to be able to attenuate GBM growth, proliferation, and metastasis in vitro and in vivo models of glioma (Maiti et al. 2017; Weissenberger et al. 2010). However, the major concern regarding utilizing curcumin in treating GBM is its problems which having poor solubility, rapid degradation, and limited bioavailability, as reported by few researchers. These drawbacks may limit the efficacy of curcumin therapy in GBM (He et al. 2018; Vyas et al. 2013). Therefore, in the present study, we have compared the effiectiveness of newly synthesized curcumin derivatives, namely FLDP-5 and FLDP-8 curcuminoid analogues and natural curcumin on GBM cell lines derived from human (LN-18). This study demonstrated that FLDP-5 and FLDP-8 curcuminoid analogues exhibit a highly potent tumour-suppressive effect on LN-18 human GBM cell line compared to curcumin. These curcuminoid analogues gave higher cytotoxicity towards LN-18 human GBM cell line with more production of ROS and significantly severe DNA damage.
We first investigated these analogues’ cytotoxicity potential through MTT assay as these curcuminoid analogues are the newly synthesized novel compounds. Interestingly, our results found that these analogues were able to reduce the viability of LN-18 cells in dose-dependent fashion with higher toxicity than curcumin. FLDP-5 and FLDP-8 curcuminoid analogues were synthesized through the addition of 4-piperidinone group to the curcumin skeleton. In relation to this, we hypothesized that these findings whereby the FLDP-5 and FLDP-8 curcuminoid analogues appeared to have higher potential than curcumin could be resulted from the added 4-piperidinone group. Our results were in agreement with previous studies by Eryanti et al. whereby their group synthesized analogues of curcumin with the addition of 4-piperidone group, namely (N-methyl-(3E,5E)-3,5-bis-(2-chlorobenzylidene)-4-piperidone and N-methyl-(3E,5E)-3,5-bis-(3-bromobenzylidene)-4-piperidone. They found that both analogues were able to inhibit proliferation in breast cancer cells (T47D) with IC50 values of 8 μM and 4 μM, respectively, following 24-h treatment. There were no reported curcumin-treated T47D cells in their study (Eryanti et al. 2018). But then, an earlier study by Nejati-Koshki et al. reported that curcumin-induced cytotoxicity in T476D cell line after 24-h treatment at 28 μM (Nejati-Koshki et al. 2014). Thus, we could see that analogues added with 4-piperidone group were able to induce higher cytotoxicity in the cancer cell line corresponding to our findings.
The selectivity of FLDP-5 and FLDP-8 curcuminoid analoges were verified by comparing the IC50 of both analogues against normal human cerebral microvascular endothelial cell (HBEC-5i). Both analogues depicted SI values in which higher than the ‘100’ baseline by several folds suggesting that the analogues were more selective towards cancerous cells than normal cells. However, further investigation should be carried out in future using normal glial and astrocytes cells to strongly confirm the selectivity of both analogues between brain cancerous and normal cells. We reported that curcumin have a high SI value with several folds higher compared to the baseline and also the analogues showing its selectivity. This results are in line with the previous study by Zanotto-Filho et al. that reported curcumin depicted a much higher value of IC50 in the normal cells compare to cancer cells indicating the cytototoxic effects of curcumin was selectively targeted at GBM (Zanotto-Filho et al. 2012).
Limited and heterogeneous drug distribution across the BBB is a primary cause of treatment failure for otherwise promising novel drug treatments in GBM. Drug distribution through an intact BBB into the brain is a necessary first step in developing effective GBM therapeutics and must be a highlight concern in any clinical trial design of GBM (Sarkaria et al. 2018). In concern of that, we decided to investigate the permeability of these analogues across the BBB using two different online prediction tools. The predictions suggested that both FLDP-5 and FLDP-8 curcuminoid analogues were capable to cross the BBB in which an excellent output value of probability was observed in FLDP-5 curcuminoid analogue indicating its great potential. The prediction for curcumin with excellent probability of crossing the BBB was in agreement in previous studies that stated that curcumin was BBB permeable and was found in cerebrospinal fluid (CSF) due to its highly lipophilic property (Mishra & Palanivelu 2008; Sordillo et al. 2015).
Then, we further performed the DHE and DCFH-DA staining assays in order to investigate the role of ROS in inducing the cell death of LN-18 cells. Our findings suggested that curcumin, FLDP-5 and FLDP-8 curcuminoid analogues induced oxidative stress through the generation of ROS superoxide anion in a time-dependent manner. Significant ROS hydrogen peroxide production was also observed induced by these analogues at 6-h time treatment which differed with curcumin. Our curcumin data were consistent with a study reported by Yin research group where they found a non-significant effect of hydrogen peroxide on U87MG GBM cells (Yin et al. 2014). In other previous studies, curcumin was confirmed to cause cell death in cancer cells through ROS production, where studies on gastric cancer cells and osteosarcoma cells reported that ROS induced apoptosis signal-regulating kinase 1 (ASK1)/ MAPK kinase (MKK) 4/ c-Jun N-terminal kinase (JNK) signalling pathway and mitochondrial cytochrome c/ caspase 3 apoptotic pathway respectively (Chang, Xing & Yu 2014; Liang et al. 2014) leading to apoptosis. In respect to that, further research should be carried out on FLDP-5 and FLDP-8 analogues to fully confirm the mechanism regarding involvement of ROS in inducing cell death in LN-18 cancer cell line.
ROS accumulation could directly damage DNA and cause oxidative lesions. In regards to that, our study demonstrated that FLDP-5 and FLDP-8 curcuminoid analogues induced DNA damage in LN-18 cells in a time-dependent manner. DNA damage was induced in accords with the elevated ROS production resulting in oxidative stress, as confirmed from the ROS assessment. The 6-h time-point for both treated FLDP-5 and FLDP-8 curcuminoid analogues appeared to have the most severe damages suggested that the severity could have resulted from the presence of both superoxide anion and hydrogen peroxide as demonstrated in the DHE and DCFH-DA staining experiment results. Particularly, hydrogen peroxide effects should be highlighted as the brain contains high amounts of unsaturated fatty acids (UFA), which are mainly found in the membrane phospholipids of the brain resulting in the brain to be especially vulnerable to damage from peroxides (Tanriverdi et al. 2007). Detoxification of hydrogen peroxides usually occurs with presence of enzymes catalase and glutathione peroxidases (GPx), where the peroxides will be reduced into water and oxygen. But, several studies have demonstrated that catalase and GPx activity were greatly decreased in brain tumour causing the scavenging activity of peroxides to decline (Guo et al. 2021; Ramírez-Expósito & Martínez-Martos 2018). This explain as to the reason behind the severe damage that occurred in the 6-h time-point induced by both curcuminoid analogues.
In GBM, the spreading of this tumour is mainly due to its highly invasive nature and high rate of motility, leading to migration. Glioma cells can spread widely beyond the primary tumour and even pass into the contralateral hemisphere, making total surgical removal of GBM impossible (Altieri et al. 2015; Johansen et al. 2016). Curcumin has been reported in various studies to have anti-migratory effects on GBM (Abbas et al. 2019; Park et al. 2019; Shabaninejad et al. 2020b). Therefore, in this study, we decided to investigate the ability of these analogues to inhibit the migration and invasion of LN-18 cells, and our findings revealed that the potential of these analogues in inhibiting migration and invasion in LN-18 cells. Curcumin reported to inhibit migration and invasion in GBM in in-vitro studies as well as in in-vivo models of GBM cells through various pathways such as regulation of proteins MMP-2/9, fascin expression, SHH/GLI1 pathway and miRNA (Park et al. 2019; Shabaninejad et al. 2020b; Yin et al. 2018). So, a thorough research investigating the pathways that caused the inhibition of migration and invasion which may be through regulation of MMP-2/9 as illustrated in Fig.11 of these novel analogues should be conducted as it could greatly strengthen the understanding of anti-migratory effects of these analogues.
Moreover, our study also found that FLDP-5 and FLDP-8 curcuminoid analogues were able to induce cell cycle arrest through inhibition at the S phase in LN-18 cells. The inhibition at the S phase suggested that the cells may be prevented from progressing to G2/M phase, thus preventing the cells to undergo mitosis (Shabaninejad et al. 2020a). Our results demonstrated a differed findings of cell cycle arrest between curcuminoid analogues and curcumin in which curcumin showed inhibition at G2/M phase. Our curcumin data were in agreement with previous studies that reported curcumin significantly inhibits GBM cell growth and proliferation via the suppression of cell cycle progression in different human glioma cell lines at G2/M phase (Liu et al. 2007; Wu et al. 2013; Zanotto-Filho et al. 2015). Curcumin caused G2/M cell cycle arrest in a p53-dependent manner, according to Liu et al. In fact, curcumin increases p53 protein levels in U251 glioma cells, followed by induction of CDK inhibitor /cell-cycle regulator p21 and tumor suppressor ING4, thus resulting in cell cycle arrest. Another study found that U251-treated cells are inhibited in the G2/M phase due to increased expression of the tumour suppressor death-associated protein kinase 1 (DAPK1), which is accompanied by suppression of the NF-B and STAT3 pathways, as well as caspase 3 activation (Wu et al. 2013). Anyhow, the pathway that induced by these novel analogues should be studied more in depth as it gives a new perspective contradict with previous curcumin data.
In recent years, a significant research effort has also been focused on synthesizing new panel analogues of curcumin in overcoming its drawbacks. The low cancer-killing potency of curcumin, its multiple biological effects, and its low bioavailability were the major factors curcumin analogues with similar safety profiles but increased anti-cancer activity and solubility were designed. EF24 (diphenyl difluoroketone) is one such analogue that recently gained a high interest as this analogue exhibits potent anti-cancer activity in colon and gastric cancer (Yang et al. 2013). Nonetheless, taken together, our results have proven that these analogues possessed potent anti-cancer activity as summarized in the schematic representation in Fig. 11, making it a worthwhile study to pursue.
Overall, our findings elucidate the potential of FLDP-5 and FLDP-8 curcuminoid analogues in human GBM LN-18 cells. This study has demonstrated that these curcuminoid analogues exhibited anti-cancer effects with anti-proliferative, anti-migratory and BBB permeable properties in GBM with higher potency compared to curcumin. However, further investigation into the underlying mechanism that causes cell death should be carried out as it could greatly enhance the understanding of the anti-cancer role of these compounds.
Authors’ contributions
NSCR carried out the experiments. The principle investigator of this project is CKM. NSCR wrote the manuscript with the support of CKM. CKM, LKW, NFR, ARAJ and NFK supervised the project. All authors contribute the feedbacks in finalizing the manuscript
Funding
This work was supported by a grant from Universiti Kebangsaan Malaysia (UKM) through the fundamental research grant scheme (FRGS) (FRGS/1/2017/SKK10/UKM/02/2) funded by the Ministry of Higher Education of Malaysia.
Data availability
The data that support the findings of this study are available from the corresponding author upon request.
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Ethics approval
Not applicable.
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Code availability
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- Abbas, M.N., Kausar, S. & Cui, H. 2019. Therapeutic potential of natural products in glioblastoma treatment : targeting key glioblastoma signaling pathways and epigenetic alterations. Clinical and Translational Oncology.
- Agrawal, A.D. 2011. Pharmacological Activities of Flavonoids: A Review. International Journal of Pharmaceutical Sciences and Nanotechnology 4(2).
- Al-Hujaily, E.M., Mohamed, A.G., Al-Sharif, I., Youssef, K.M., Manogaran, P.S., Al-Otaibi, B., Al-Haza’A, A., Al-Jammaz, I., Al-Hussein, K. & Aboussekhra, A. 2011. PAC, a novel curcumin analogue, has anti-breast cancer properties with higher efficiency on ER-negative cells. Breast Cancer Research and Treatment 128(1).
- Altieri, R., Zenga, F., Fontanella, M.M. ari., Cofano, F., Agnoletti, A., Spena, G., Crobeddu, E., Fornaro, R., Ducati, A. & Garbossa, D. 2015. Glioma Surgery: Technological Advances to Achieve a Maximal Safe Resection.
- Anand, P., Kunnumakkara, A.B., Newman, R.A. & Aggarwal, B.B. 2007. Bioavailability of curcumin: Problems and promises.
- Atkins, R.J., Ng, W., Stylli, S.S., Hovens, C.M. & Kaye, A.H. 2015. Repair mechanisms help glioblastoma resist treatment.
- Banu, Z. 2019. Glioblastoma Multiforme: a Review of Its Pathogenesis and Treatment. International Research Journal Of Pharmacy 9(12): 7–12.
- BOYDEN, S. 1962. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. The Journal of experimental medicine 115.
- Chan, K.M., Rajab, N.F., Ishak, M.H.A., Ali, A.M., Yusoff, K., Din, L.B. & Inayat-Hussain, S.H. 2006. Goniothalamin induces apoptosis in vascular smooth muscle cells. Chemico-Biological Interactions 159(2).
- Chan, K.M., Rajab, N.F., Siegel, D., Din, L. Bin, Ross, D. & Inayat-Hussain, S.H. 2010. Goniothalamin induces coronary artery smooth muscle cells apoptosis: The p53-dependent caspase-2 activation pathway. Toxicological Sciences 116(2).
- Chang, Z., Xing, J. & Yu, X. 2014. Curcumin induces osteosarcoma MG63 cells apoptosis via ROS/Cyto-C/Caspase-3 pathway. Tumor Biology 35(1).
- Chen, T.-C., Chuang, J.-Y., Ko, C.-Y., Kao, T.-J., Yang, P.-Y., Yu, C.-H., Liu, M.-S., Hu, S.-L., Tsai, Y.-T., Chan, H., Chang, W.-C. & Hsu, T.-I. 2020. AR ubiquitination induced by the curcumin analog suppresses growth of temozolomide-resistant glioblastoma through disrupting GPX4-Mediated redox homeostasis. Redox biology 30: 101413.
- Eryanti, Y., Hendra, R., Herlina, T., Zamri, A. & Supratman, U. 2018. Synthesis of N-methyl-4-piperidone curcumin analogues and their cytotoxicity activity against T47D cell lines. Indonesian Journal of Chemistry 18(2).
- Ferreira, J., Ramos, A.A., Almeida, T., Azqueta, A. & Rocha, E. 2018. Drug resistance in glioblastoma and cytotoxicity of seaweed compounds, alone and in combination with anticancer drugs: A mini review. Phytomedicine 48: 84–93.
- Guo, B., Liao, W. & Wang, S. 2021. The clinical significance of glutathione peroxidase 2 in glioblastoma multiforme. Translational Neuroscience 12(1).
- Hassannia, B., Vandenabeele, P. & Vanden Berghe, T. 2019. Targeting Ferroptosis to Iron Out Cancer.
- He, Y., Li, W., Hu, G., Sun, H. & Kong, Q. 2018. Bioactivities of EF24, a novel curcumin analog: A review.
- Hu, Y., Hu, X., Lu, Y., Shi, S., Yang, D. & Yao, T. 2020. New Strategy for Reducing Tau Aggregation Cytologically by A Hairpinlike Molecular Inhibitor, Tannic Acid Encapsulated in Liposome. ACS Chemical Neuroscience 11(21).
- Johansen, M.D., Rochat, P., Law, I., Scheie, D., Poulsen, H.S. & Muhic, A. 2016. Presentation of Two Cases with Early Extracranial Metastases from Glioblastoma and Review of the Literature. Case Reports in Oncological Medicine 2016.
- Leong, L.M., Chan, K.M., Hamid, A., Latip, J. & Rajab, N.F. 2016. Herbal formulation C168 attenuates proliferation and induces apoptosis in HCT 116-human colorectal carcinoma cells: Role of oxidative stress and DNA damage. Evidence-based Complementary and Alternative Medicine 2016.
- Liang, T., Zhang, X., Xue, W., Zhao, S., Zhang, X. & Pei, J. 2014. Curcumin induced human gastric cancer BGC-823 cells apoptosis by ROS-mediated ASK1-MKK4-JNK stress signaling pathway. International journal of molecular sciences 15(9): 15754–15765.
- Liu, E., Wu, J., Cao, W., Zhang, J., Liu, W., Jiang, X. & Zhang, X. 2007. Curcumin induces G2/M cell cycle arrest in a p53-dependent manner and upregulates ING4 expression in human glioma. Journal of Neuro-Oncology 85(3).
- Liu, H., Wang, L., Lv, M., Pei, R., Li, P., Pei, Z., Wang, Y., Su, W. & Xie, X.Q. 2014. AlzPlatform: An Alzheimer’s disease domain-specific chemogenomics knowledgebase for polypharmacology and target identification research. Journal of Chemical Information and Modeling 54(4).
- Maiti, P., Al-Gharaibeh, A., Kolli, N. & Dunbar, G.L. 2017. Solid Lipid Curcumin Particles Induce More DNA Fragmentation and Cell Death in Cultured Human Glioblastoma Cells than Does Natural Curcumin. Oxidative medicine and cellular longevity 2017: 9656719.
- Mishra, S. & Palanivelu, K. 2008. The effect of curcumin (turmeric) on Alzheimer’s disease: An overview.
- Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65(1–2).
- Nejati-Koshki, K., Akbarzadeh, A. & Pourhassan-Moghaddam, M. 2014. Curcumin inhibits leptin gene expression and secretion in breast cancer cells by estrogen receptors. Cancer Cell International 14(1).
- Nordin, F.J., Pearanpan, L., Chan, K.M., Kumolosasi, E., Yong, Y.K., Shaari, K. & Rajab, N.F. 2021. Immunomodulatory potential of Clinacanthus nutans extracts in the co-culture of triplenegative breast cancer cells, MDA-MB-231, and THP-1 macrophages. PLoS ONE 16(8 August).
- Nørøxe, D.S., Poulsen, H.S. & Lassen, U. 2016. Hallmarks of glioblastoma: A systematic review. ESMO Open 1(6): 1–9.
- Ooi, T.C., Ibrahim, F.W., Ahmad, S., Chan, K.M., Leong, L.M., Mohammad, N., Siew, E.L. & Rajab, N.F. 2021. Antimutagenic, cytoprotective and antioxidant properties of ficus deltoidea aqueous extract in vitro. Molecules 26(11).
- Park, K., Yoon, S., Park, S. & Hwang, J. 2019. Anti-Migration and Anti-Invasion Effects of Curcumin via Suppression of Fascin Expression in Glioblastoma Cells 7(1): 16–24.
- Paulraj, F., Abas, F., Lajis, N.H., Othman, I., Hassan, S.S. & Naidu, R. 2015. The curcumin analogue 1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one induces apoptosis and downregulates E6 and E7 oncogene expression in HPV16 and HPV18-infected cervical cancer cells. Molecules 20(7).
- Popiołkiewicz, J., Polkowski, K., Skierski, J.S. & Mazurek, A.P. 2005. In vitro toxicity evaluation in the development of new anticancer drugs - Genistein glycosides. Cancer Letters 229(1).
- Ramírez-Expósito, M.J. & Martínez-Martos, J.M. 2018. The Delicate Equilibrium between Oxidants and Antioxidants in Brain Glioma. Current Neuropharmacology 17(4).
- Ramirez, Y.P., Weatherbee, J.L., Wheelhouse, R.T. & Ross, A.H. 2013. Glioblastoma multiforme therapy and mechanisms of resistance.
- Sansalone, L., Veliz, E.A., Myrthil, N.G., Stathias, V., Walters, W., Torrens, I.I., Schürer, S.C., Vanni, S., Leblanc, R.M. & Graham, R.M. 2019. Novel Curcumin Inspired Bis-Chalcone Promotes Endoplasmic Reticulum Stress and Glioblastoma Neurosphere Cell Death. Cancers 11(3).
- Sarkaria, J.N., Hu, L.S., Parney, I.F., Pafundi, D.H., Brinkmann, D.H., Laack, N.N., Giannini, C., Burns, T.C., Kizilbash, S.H., Laramy, J.K., Swanson, K.R., Kaufmann, T.J., Brown, P.D., Agar, N.Y.R., Galanis, E., Buckner, J.C. & Elmquist, W.F. 2018. Is the blood-brain barrier really disrupted in all glioblastomas? A critical assessment of existing clinical data. Neuro-Oncology 20(2).
- Senft, C., Polacin, M., Priester, M., Seifert, V., Kögel, D. & Weissenberger, J. 2010. The nontoxic natural compound Curcumin exerts anti-proliferative, anti-migratory, and anti-invasive properties against malignant gliomas. BMC cancer 10: 491.
- Shabaninejad, Z., Pourhanifeh, M.H., Movahedpour, A., Mottaghi, R., Nickdasti, A., Mortezapour, E., Shafiee, A., Hajighadimi, S., Moradizarmehri, S., Sadeghian, M., Mousavi, S.M. & Mirzaei, H. 2020a. Therapeutic potentials of curcumin in the treatment of glioblstoma. European journal of medicinal chemistry 188: 112040.
- Shabaninejad, Z., Pourhanifeh, M.H., Movahedpour, A., Mottaghi, R.R., Nickdasti, A., Mortezapour, E., Shafiee, A., Hajighadimi, S., Moradizarmehri, S., Sadeghian, M., Mousavi, S.M. & Mirzaei, H. 2020b. Therapeutic potentials of curcumin in the treatment of glioblstoma. European Journal of Medicinal Chemistry: 112040.
- Shahar, T., Nossek, E., Steinberg, D.M., Rozovski, U., Blumenthal, D.T., Bokstein, F., Sitt, R., Freedman, S., Corn, B.W., Kanner, A.A. & Ram, Z. 2012. The impact of enrollment in clinical trials on survival of patients with glioblastoma. Journal of Clinical Neuroscience 19(11).
- Sordillo, L.A., Sordillo, P.P. & Helson, L. 2015. Curcumin for the Treatment of Glioblastoma. Anticancer research 35(12): 6373–6378.
- Tan, B.L. & Norhaizan, M.E. 2019. Curcumin Combination Chemotherapy: The Implication and Efficacy in Cancer. Molecules (Basel, Switzerland) 24(14).
- Tan, H.H., Thomas, N.F., Inayat-Hussain, S.H. & Chan, K.M. 2020. Cytoprotective effects of (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2carboxamide (BK3C231) against 4-nitroquinoline 1-oxide-induced damage in CCD-18Co human colon fibroblast cells. PLoS ONE 15(5).
- Tanriverdi, T., Hanimoglu, H., Kacira, T., Sanus, G.Z., Kemerdere, R., Atukeren, P., Gumustas, K., Canbaz, B. & Kaynar, M.Y. 2007. Glutathione peroxidase, glutathione reductase and protein oxidation in patients with glioblastoma multiforme and transitional meningioma. Journal of Cancer Research and Clinical Oncology 133(9).
- Van Tellingen, O., Yetkin-Arik, B., De Gooijer, M.C., Wesseling, P., Wurdinger, T. & De Vries, H.E. 2015. Overcoming the blood-brain tumor barrier for effective glioblastoma treatment.
- Thilakarathna, S.H. & Vasantha Rupasinghe, H.P. 2013. Flavonoid bioavailability and attempts for bioavailability enhancement.
- Vyas, A., Dandawate, P., Padhye, S., Ahmad, A. & Sarkar, F. 2013. Perspectives on New Synthetic Curcumin Analogs and their Potential Anticancer Properties. Current Pharmaceutical Design 19(11): 2047–2069.
- Walker, B.C., Adhikari, S. & Mittal, S. 2021. Therapeutic Potential of Curcumin for the Treatment of Malignant Gliomas. Dlm. Debinski, W. (pnyt.). hlm. Brisbane (AU).
- Weissenberger, J., Priester, M., Bernreuther, C., Rakel, S., Glatzel, M., Seifert, V. & Kögel, D. 2010. Dietary curcumin attenuates glioma growth in a syngeneic mouse model by inhibition of the JAK1,2/STAT3 signaling pathway. Clinical cancer research : an official journal of the American Association for Cancer Research 16(23): 5781–5795.
- Willenbacher, E., Khan, S.Z., Mujica, S.C.A., Trapani, D., Hussain, S., Wolf, D., Willenbacher, W., Spizzo, G. & Seeber, A. 2019. Curcumin: New Insights into an Ancient Ingredient against Cancer. International journal of molecular sciences 20(8).
- Wojcik, M., Krawczyk, M., Wojcik, P., Cypryk, K. & Wozniak, L.A. 2018. Molecular Mechanisms Underlying Curcumin-Mediated Therapeutic Effects in Type 2 Diabetes and Cancer. Oxidative medicine and cellular longevity 2018: 9698258.
- Wong, S.C., Kamarudin, M.N.A. & Naidu, R. 2021. Anticancer Mechanism of Curcumin on Human Glioblastoma. Nutrients 13(3).
- Wu, B., Yao, H., Wang, S. & Xu, R. 2013. DAPK1 modulates a curcumin-induced G2/M arrest and apoptosis by regulating STAT3, NF-κB, and caspase-3 activation. Biochemical and Biophysical Research Communications 434(1).
- Xiong, G., Wu, Z., Yi, J., Fu, L., Yang, Z., Hsieh, C., Yin, M., Zeng, X., Wu, C., Lu, A., Chen, X., Hou, T. & Cao, D. 2021. ADMETlab 2.0: An integrated online platform for accurate and comprehensive predictions of ADMET properties. Nucleic Acids Research 49(W1).
- Yang, C.H., Yue, J., Sims, M. & Pfeffer, L.M. 2013. The Curcumin Analog EF24 Targets NF-κB and miRNA-21, and Has Potent Anticancer Activity In Vitro and In Vivo. PLoS ONE 8(8).
- Yin, H., Zhou, Y., Wen, C., Zhou, C., Zhang, W., Hu, X., Wang, L., You, C. & Shao, J. 2014. Curcumin sensitizes glioblastoma to temozolomide by simultaneously generating ROS and disrupting AKT/mTOR signaling. Oncology Reports 32(4): 1610–1616.
- Yin, N. 2014. Enhancing the Oral Bioavailability of Peptide Drugs by using Chemical Modification and Other Approaches. Medicinal Chemistry.
- Yin, S., Du, W., Wang, F., Han, B., Cui, Y., Yang, D., Chen, H., Liu, D. & Liu, X. 2018. MicroRNA-326 sensitizes human glioblastoma cells to curcumin via the SHH / GLI1 signaling pathway 19(4): 260–270.
- Zanotto-Filho, A., Braganhol, E., Edelweiss, M.I., Behr, G.A., Zanin, R., Schröder, R., Simões-Pires, A., Battastini, A.M.O. & Moreira, J.C.F. 2012. The curry spice curcumin selectively inhibits cancer cells growth in vitro and in preclinical model of glioblastoma. The Journal of nutritional biochemistry 23(6): 591–601.
- Zanotto-Filho, A., Braganhol, E., Klafke, K., Figueiró, F., Terra, S.R., Paludo, F.J., Morrone, M., Bristot, I.J., Battastini, A.M., Forcelini, C.M., Bishop, A.J.R., Gelain, D.P. & Moreira, J.C.F. 2015. Autophagy inhibition improves the efficacy of curcumin/temozolomide combination therapy in glioblastomas. Cancer letters 358(2): 220–231.
- Zhang, J., F.G. Stevens, M. & D. Bradshaw, T. 2012. Temozolomide: Mechanisms of Action, Repair and Resistance. Current Molecular Pharmacology.
- Zhao, Y.H., Abraham, M.H., Ibrahim, A., Fish, P. V., Cole, S., Lewis, M.L., De Groot, M.J. & Reynolds, D.P. 2007. Predicting penetration across the blood-brain barrier from simple descriptors and fragmentation schemes. Journal of Chemical Information and Modeling 47(1).
No competing interests reported.