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A trienone analog of curcumin, 1,7-bis(3-hydroxyphenyl)-1,4,6-heptatrien-3-one, possesses ROS- and caspase-mediated apoptosis in human oral squamous cell carcinoma cells in vitro


The leading causes of oral cancer treatment failure are cancer metastasis and chemotherapeutic resistance. Thus, developing novel anticancer agents that are effective against those aggressive cancer cells would be important for complementary or alternative treatments. The objective of this study was to investigate cytotoxicity and anticancer mechanisms of a synthetic trienone analog of curcumin, 1,7-bis(3-hydroxyphenyl)-1,4,6-heptatrien-3-one (trienone 11), against human oral squamous cell carcinoma (OSCC) cells exhibiting multidrug resistance (CLS-354/DX). The study of cytotoxicity showed that trienone 11 exerted threefold stronger cytotoxicity to CLS-354/DX cells than curcumin. Trienone 11 (15–30 μM) markedly induced intracellular reactive oxygen species (ROS) resulting in apoptotic cell death within 24 h, through activation of caspase-3/7 and caspase-9. A ROS inhibitor, N-acetylcysteine (NAC) prevented apoptotic cell death via decreasing caspase activation. Thus, the cytotoxicity of trienone 11 against CLS-354/DX cells was ROS-mediated intrinsic apoptosis. Overall, trienone 11 could be an interesting lead for developing anti-cancer agents against multidrug resistant OSCC cells.


Oral squamous cell carcinoma (OSCC) is particularly serious because this cancer type is often diagnosed when cancer metastasizes in its late stages [1, 2]. Late-stage cancer cells are rendered more resistant to chemotherapy by altering biological factors such as drug transportation, metabolic reprogramming, redox status, and DNA repair that may contribute to treatment failure [3, 4]. During the last few decades, extensive studies have examined new strategies to overcome multidrug-resistant cancer cells by restraining their escape via resistance mechanisms through the use of novel anticancer drugs [5]. Currently, new anticancer agents have been developed by structurally modifying a natural compound to create analogs or derivatives with increased anticancer efficacy and relatively weak side effects [6].

Curcumin, a major chemical constituent isolated from the rhizome of turmeric (Curcuma longa L.), has demonstrated broad-spectrum anti-inflammatory [7], antioxidant [8], and anticancer activities [9]. Curcumin exerts anticancer effects against various types of cancer by interacting with multiple intracellular proteins including transcription factors that potentiate the expression of multiple genes [10]. Thus, curcumin is a popular lead compound for new drug development [9]. However, the use of curcumin is restricted by poor bioavailability, stability, solubility, and absorption [11, 12]. Synthetic curcuminoid derivatives have, therefore, been developed by modifying the parent structure to improve anticancer potency. These analogs include DK1 [13], B63 [14], EF24 [15], WZ35 [16], and those with 1,4,6-trien-one function or trienones [17].

Trienone is a rare naturally occurring curcuminoid analog; its biological activities have not been comprehensively investigated. The 1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one (trienone 4) is a minor naturally occurring analog of curcumin that was initially isolated from C. domestica Val. [18]. Trienone 4 has been reported to exert anti-peroxidation [19] and anti-inflammatory activity [20], together with cytotoxicity against melanoma cells [21]. Recently, we demonstrated the successful synthesis of trienone 4 and its analogs in the laboratory to replace natural trienones for cytotoxicity evaluation in the KB cell line. Our results determined that a new trienone analog, 1,7-bis(3-hydroxyphenyl)-1,4,6-heptatrien-3-one (trienone 11), exerted interesting cytotoxicity against the KB cell line with a very high selectivity index, suggesting potential for anticancer activity [17]. However, the exact cytotoxic mechanism of trienone 11 remains unclear and further investigation of other cancer cell types is required.

Here, the underlying anticancer activity mechanism against the multidrug-resistant human OSCC cell line (CLS-354/DX) of trienone 11 was investigated. Potency against CLS-354/DX cells of trienone 11 was compared with curcumin and trienone 4, while mechanisms of cytotoxicity including apoptosis induction, reactive oxygen species (ROS) production, and caspase activation were also assessed. We proposed that trienone 11 is a promising candidate for developing agents against multidrug-resistant OSCCs.

Materials and methods

Curcumin, trienone, and trienone analog of curcuminoids

Curcumin was isolated from the rhizomes of C. longa as described previously [22]. The trienones 4 and 11 were synthesized and characterized by the literature method [17] (Fig. 1). The NMR spectra of these compounds are presented in Additional file 1. All these compounds were dissolved in dimethyl sulfoxide (DMSO) before use.

Fig. 1
figure 1

Structures of curcumin (a), trienone 4 (b), and trienone 11 (c) determined by NMR (1H and 13C NMR, and 2D COSY, HMQC and HMBC) and mass spectroscopy

Cell lines and cell culture reagents

CLS-354/WT and CLS-354/DX cell lines were established from human OSCC cell line CLS-354 (CLS Cell line service, GmbH, Eppellheim, Germany) as has been described earlier [23, 24]. A monkey kidney epithelial cell line (Vero) was purchased from CLS Cell line service (GmbH, Eppellheim, Germany). All cell culture media and supplementary reagents were purchased from Gibco, Life Technology, USA. All cell culture materials were purchased from Nest Biotechnology, China.

Cell culture

CLS-354/DX, CLS-354/WT, and Vero cells were cultured in sterile conditions in RPMI-1640 supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM l-glutamine. The cells were maintained in a CO2 incubator with 5% CO2 and 95% humidity at 37 °C. Cell culture media were changed every 2 days in a sterile manner. The cells were sub-cultured when confluency was reached to 80% using 1:4 split ratio.

Cytotoxicity test

Cells were seeded in 96-well plates at a density of 1.0 × 104 cells/0.32 cm2. The cells were grown for 48 h before treatment to reach confluency at 70%. Curcumin, trienone 4, and trienone 11 solutions were diluted in complete RPMI-1640 to obtain a final concentration at 80 μM, and then serially diluted to 5 μM. The cells were treated with the compounds for 24 h. In some experiments, the cells were pre-incubated with 5 mM N-acetyl cysteine (NAC, Sigma-Aldrich Corp., St. Louis, MO, USA) for 1 h, followed by treatment with trienone 11 at various concentrations for 24 h in order to evaluate ROS-mediated cytotoxicity. After the 24 h-treatment, the spent media were removed, 200 μl of 0.5 mg/ml 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Invitrogen, Life Technologies, USA) solution was added to each well and incubated for 4 h. The formazan crystals were dissolved in DMSO. The absorbance (Abs) was detected at 560 nm and background at 670 nm was subtracted, using a microplate reader [23]. Cell viability was calculated as follows:

$$\% Cell\;\; viability = \left( {\frac{Abs. treated}{Abs. untreated}} \right) \times 100$$

Determination of half maximal inhibitory concentration (IC50) and selectivity index (SI)

IC50 was determined from a dose response curve (inhibitory effect) using Graphpad Prism 6.0 (Graphpad Software Inc., USA). Selectivity index (SI) was calculated by comparing IC50 values for the CLS-354/DX or CLS-354/WT cell lines against the IC50 of the same compound in a cell line of non-cancerous cells [25].

Measurement of intracellular ROS

Cells were seeded at 4.2 × 104/cm2 in a 6-well plate cultured for 48 h. The cells were treated with trienone 11 at various concentrations for 12 h. In some experiments, the cells were pre-treated with NAC for 1 h, followed by trienone 11 for 12 h. Intracellular ROS generation was monitored by a flow cytometer using the peroxide-sensitive fluorescent probe 2′,7′-dichlorofluorescin diacetate (H2DCFDA) as previously described [26]. In brief, after the incubation, cells were harvested, centrifuged at 1500 rpm for 5 min, and washed with phosphate buffer saline (PBS) twice. The suspension of cells was transferred to FACS tube, mixed with 10 μM of H2DCFDA (Invitrogen, Life Technologies, USA), further incubated at RT for 30 min and protected from light. The cell suspension was then subjected to a flow cytometer for ROS analysis. Intracellular ROS was quantitated using CellQuest™ Pro software (BD Biosciences, San Jose, CA, USA).

Caspase activity assay

Cells were treated with the compounds for 6 h. After treatment, the cells were harvested and washed twice in cold PBS. To extract protein, the cell pellet was lysed in lysis buffer and centrifuged at 13,000 rpm for 5 min. The supernatant was collected and measured for protein content using DC-Bradford assay kit (Bio-rad Inc., USA).

Activities of caspase-3/7, caspase-8, and caspase-9 were determined using Caspase Colorimetric Activity Assay Kit (Millipore, USA) [27]. Briefly, 100 μg protein was pipetted to a 96-well plate (70 μl), followed by 50 μl reaction buffer, and 10 μl substrate (IETD-pNA for caspase-8, LEHD-pNA for caspase-9, and DEVD-pNA for caspase-3). The mixture was incubated at 37 °C for 3 and 24 h. Formation of p-nitroaniline was measured with an ELISA-microplate reader (Biochrom EZ Read 400, UK).

AnnexinV-FITC/Propidium iodide double staining assay

After that the cells were incubated with the compounds at various concentrations for 24 h, they were trypsinized, collected, washed with PBS, and stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer’s instructions (Roche Diagnostics Deutschland GmbH, Mannheim, Germany). After staining, the stained cells were subjected to a flow cytometer (BD Biosciences 2 Laser FacsCalibur, USA) using FL-1 and FL-2. Percentages of Annexin V-FITC- and Annexin V-FITC/PI-positive cells were analyzed as apoptotic cells by CellQuest™ Pro (BD Biosciences) [23].

Statistical analysis

Data are expressed as mean ± standard deviation (SD) from at least three independent experiments. Statistical comparisons were performed using Student’s t-test for differences between two groups and by one-way ANOVA with Bonferroni Post Hoc test for differences among multiple groups. Significance was assigned for p values < 0.05 using GraphPad Prism 6.0 (GraphPad Software, USA).


Effect of trienone 11 on cell viability and cytotoxicity in CLS-354 cells

Human oral squamous carcinoma cell lines CLS-354/DX and CLS-354/WT were established as in vitro models for drug responsiveness studies. CLS-354/DX cells were found to be more aggressive and more multidrug-resistant than CLS-354/WT [23, 24]. To examine responsiveness to curcumin, trienone 4, and trienone 11 (Fig. 1), we performed MTT assay after 24 h treatment of the three compounds at concentrations ranging from 0.01 to 80 μM. Cell proliferation of CLS-354/DX cells (Fig. 2a) and CLS-354/WT (Fig. 2b) was inhibited by curcumin, trienone 4, and trienone 11 in a dose-dependent manner. In both cell lines, cell viability drastically decreased by 70–75% upon treatment with 20 μM of trienone 11 (Fig. 2a, b, lower panel) but not so with curcumin (Fig. 2a, b, upper panel) and trienone 4 (Fig. 2a, b, middle panel), indicating comparatively low responsiveness to curcumin and trienone 4. Half-maximal inhibitory concentration (IC50) and selectivity index (SI) were examined to compare the potency and safety margin of the compounds. As shown in Table 1, the IC50 concentrations of curcumin, trienone 4, and trienone 11 observed in CLS-354/DX cells were 47.54 ± 1.42, 36.58 ± 1.02, and 16.50 ± 1.03 μM, respectively, while IC50 values observed in CLS-354/WT were 13.31 ± 1.24, 23.81 ± 1.03, and 10.09 ± 1.06 μM, respectively (Table 1). DMSO (< 0.5%) was used as the treatment vehicle and did not affect cell viability. Results suggested that trienone 11 treatment provided a significantly greater IC50 value than curcumin (threefold) or trienone 4 (twofold) in CLS-354/DX cells (p < 0.05). This finding indicated that curcumin had low potential to inhibit multidrug-resistant cancer but was effective against CLS-354/WT which is non-aggressive. To evaluate the cytotoxicity of the three compounds against normal cells, African green monkey kidney cells (Vero cells) were tested as representative normal epithelial cells. We found that trienone 11 exerted cytotoxicity to Vero cells at the IC50 concentration of 33.89 ± 6.83 μM, which was significantly weaker than the IC50 observed for both cancer cell lines. Moreover, trienone 11 provided the best selectivity index (SI) in both cell lines at 2.0 in CLS-354/DX and 3.4 in CLS-354/WT (Table 1). These data suggested trienone 11 as the most potent compound against CLS-354/DX cells.

Fig. 2
figure 2

The effects of curcumin, trienone 4, and trienone 11 on cell viability of human squamous cell carcinoma CLS-354/DX and CLS-354/WT cells. CLS-354/DX cells (a) and CLS-354/WT cells (b) were treated with 0.01- 80 μM of curcumin, trienone 4, or trienone 11 for 24 h. Cell viability was measured by MTT assay after treatment. Values are expressed as mean ± standard deviation (SD) of duplicate samples in three independent experiments. Different letters above the bar indicate the significant difference (p < 0.05)

Table 1 The IC50 and selectivity index (SI) of curcumin, trienone 4, and trienone 11 in CLS-354/DX and CLS-354/WT

Effect of trienone 11 on apoptosis induction in CLS-354/DX cells

CLS-354/DX cells were exposed to trienone 11 for 24 h and changes in cell morphology were observed including cell floating, cell shrinkage, and blebbing (Additional file 1: Fig. S2) as associated characteristics of apoptotic cells. Apoptosis induction was then investigated using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining method. Treatment with 15-30 μM of trienone 11 in CLS-354/DX cells for 24 h increased the proportions of Annexin V-FITC-positive cells (early apoptotic cells) from 2.57 to 11.60%, and Annexin V-FITC-/PI positive cells (late apoptotic cells) from 10.01 to 27.64% (Fig. 3a), while total apoptosis in CLS-354/DX cells significantly increased to 40% of the total cell population (Fig. 3b). Early apoptotic and late apoptotic CLS-354/WT cells also increased from 7.24 to 17.52% and from 5.05 to 14.56%, respectively (Fig. 3c), while total induced apoptosis rate was 30% (Fig. 3d). This data indicated that trienone 11 induced apoptosis, leading to CLS-354/DX cell death.

Fig. 3
figure 3

The effects of trienone 11 on the induction of apoptosis in CLS-354/DX and CLS-354/WT cells. CLS-354/DX cells (a, b) and CLS-354/WT (c, d) were treated with trienone 11 at 15–30 μM and 15 μM, respectively, for 24 h. Apoptosis was determined by flow cytometric analysis using double staining with Annexin V-FITC and propidium iodide, and the results are represented as scatter plots (a, c) and as percentages of apoptotic cells (b, d). Values are expressed as mean ± standard deviation (SD) of duplicate samples in three independent experiments. Different letters above the bar indicate the significant difference (p < 0.05)

Effect of trienone 11 on activation of caspases

Caspases are a family of protease enzymes that play a significant role in programmed cell death by initiating two distinct apoptotic pathways as the death receptor-mediated (extrinsic) pathway or the mitochondrial-mediated (intrinsic) pathway [28]. Caspase-9 and caspase-8 are key players in the intrinsic and extrinsic pathways, respectively. These two caspases subsequently activate caspase-3 or caspase-7 (executioner), contributing to degradation of cellular components for apoptosis [29]. To investigate whether caspase was involved in apoptosis, we measured the catalytic activities of the caspases. The cells were exposed to trienone 11 for 6 h. After that, catalytic activities of caspase-3/7, caspase-8, and caspase-9 were monitored in the cell lysate at 3 and 24 h. Results showed that catalytic activity of caspase-3/7 was rapidly elevated within 3 h at 3.3-fold compared to basal level. After 24 h of incubation, activities of caspase-3/7, caspase-8, and caspase-9 significantly increased by 5.2-, 2.0- and 1.9-fold, respectively (Fig. 4). Thus, trienone 11 induced apoptosis in CLS-354/DX cells through both intrinsic and extrinsic pathways.

Fig. 4
figure 4

The effects of trienone 11 on the activation of caspase-3/7, caspase-8, and caspase-9 in CLS-354/DX cells. The cells were treated with trienone 11 (30 μM) for 6 h. After treatment, protein lysates (100 μg protein) were incubated with the substrates of caspase-3/7, caspase-8, and caspase-9 for indicated times at 37 °C for 3 and 24 h. Activities of caspase-3/7 (a), caspase-8 (b), and caspase-9 (c) were measured by a microplate reader. Values are expressed as mean ± standard deviation (SD) from four independent treatments. Different letters above the bar indicate the significant difference (p < 0.05)

Effect of trienone 11 on intracellular ROS production

ROS are well-known signaling molecules that play a pivotal role in controlling cell death as well as cell survival. Here, intracellular ROS levels were examined after trienone treatment. Results indicated that trienone 11 at 15 and 30 μM increased ROS levels in CLS-354/DX cells dose-dependently, as shown by right-shifted signals (Fig. 5a). Elevated intracellular ROS levels significantly increased 1.5- and 3.1-fold after treatment with 15 and 30 μM of trienone 11, respectively (Fig. 5b). Furthermore, N-acetyl cysteine (NAC), an antioxidant, was used as a ROS inhibitor to confirm trienone 11-induced ROS formation in CLS-354/DX cells. We found that NAC treatment reversed ROS levels to the basal level (Fig. 5a, b), suggesting that trienone 11 had ROS-inducing potency. We further compared the ROS-inducing potency between trienone 11 and trienone 4. Results determined that trienone 4 was not able to induce ROS (Additional file 1: Fig. S3), while trienone 11 was successful.

Fig. 5
figure 5

The effects of trienone 11 on production of reactive oxygen species (ROS) in CLS-354/DX cells. CLS-354/DX cells were treated with 15–30 μM trienone 11 for 12 h with/without 5 mM N-acetyl cysteine (NAC) pre-treatment. Intracellular ROS was detected using H2DCFDA probe followed by flow cytometric analysis. ROS levels are represented in a histogram (a) and mean ± standard deviation (SD) of fluorescent intensities (MFI) (b). Values are expressed as mean ± standard deviation (SD) of duplicate samples in three independent experiments. Different letters above the bar indicate the significant difference (p < 0.05)

Involvement of ROS in trienone 11-induced apoptosis

To further demonstrate the involvement of ROS in caspase activation and its anti-proliferative effects, we determined whether NAC prevented caspase catalytic activity and cell death in CLS-354/DX cells. All caspase catalytic activities significantly increased after trienone 11 treatment at 2–5-fold from the basal level (Fig. 6). When the cells were exposed to trienone 11 (30 μM) in the presence of NAC (5 mM) for 6 h, catalytic activities of caspase-3/7 and caspase-9 significantly reduced, similar to observation levels in untreated cells (Fig. 6a). Furthermore, the presence of NAC (2.5–10 mM) completely prevented trienone 11-induced cell death with significantly increased cell viability from 50 to 90% (Fig. 6b). These findings indicated that ROS induced by trienone 11 was decisively involved in the mitochondrial-mediated (intrinsic) pathway of apoptosis.

Fig. 6
figure 6

Trienone 11-induced cell death is ROS mediated and caspase-3/7, -9 dependent in CLS-354/DX. The activities of caspase-3/7, -8, and -9 of CLS-354/DX cells upon pre-treatment with 5 mM N-acetyl cysteine (NAC), followed by 30 μM trienone 11 for 12 h (a). Values are expressed as mean ± standard deviation (SD) from four independent treatments. Cell viability (b) of CLS-354/DX cells upon pre-treatment with NAC at the indicated concentrations and trienone 11 (30 μM) treatment for 24 h was measured using MTT assay. Values are expressed as mean ± standard deviation (SD) of triplicate samples in three independent experiments. Different letters above the bar indicate the significant difference (p < 0.05). The proposed cytotoxic mechanism (c) of trienone 11 in CLS-354/DX cells; ❶ trienone 11 markedly induced intracellular ROS production, which may directly damage mitochondria or DNA resulting in caspase-9 and -3/7 activation through intrinsic pathway of apoptosis; ❷ trienone 11 can trigger extrinsic pathway of apoptosis via ROS-independent caspase-8 activation


Extensive studies have demonstrated that curcumin exhibits various biological activities including anticancer activity [7,8,9,10]; however, curcumin had moderate-to-low cytotoxic efficacy against human cancer cells due to its poor bioavailability [11, 12]. Recently, structural modification of curcumin has offered a potential approach for optimizing new leads for anticancer drug development [30]. Structural modification studies of curcumin have suggested β-diketone moiety as the position leading to chemical instability, while modification to mono-ketone moiety improved stability as well as cytotoxicity [31]. Accordingly, curcuminoid analogs exerting mono-ketone moiety such as trienones could be used as curcumin-based leads for anticancer drug development. Trienones are found in very small amounts in nature [18] but some have been synthesized in the laboratory by various methods including key fragment substituted 4-phenylbut-3-en-2-ones and 3-phenylacrylaldehydes [17], dehydrogenation of the enone with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and THF [22], and Wittig reaction and aldol condensation [32], thereby overcoming the limitation of trienone preparation.

Several trienones and modified analogs have demonstrated more potent cytotoxicity than curcumin toward cancer cells including melanoma cells (B16F10) [21], prostate cancer cells (PC-3, DU-45, and LNCaP) [32], cervical cancer cells (HeLa) [32] and the KB cell line [17]. Recently, we found that trienone 11 as modified trienone 4 exerted the highest cytotoxic potency in KB cells [17]. Here, trienone 11 was investigated for its anticancer effect against the multidrug-resistant OSCC cell line CLS-354/DX and the parental cell line CLS-354/WT in comparison to curcumin and trienone 4. We found that curcumin poorly inhibited multidrug-resistant cancer cells with the high IC50 at 47.54 μM, whereas curcumin was still active in parental cells with IC50 at 13.31 μM. This occurred because CLS-354/DX cells are more aggressive than CLS-354/WT cells by expressing multidrug-resistant protein (MRP), increased drug pump activity, and reduced redox status [22, 23]. As expected, trienone 11 was more potent than both trienone 4 and curcumin in inhibiting cell growth in CLS-354/DX cells, similar to results for KB cells in our previous report [17]. Interestingly, the IC50 of trienone 11 inhibiting CLS-354/DX cells (16.5 μM) indicated higher potency than cisplatin (a platinum-based anticancer drug) (33.2 μM) [22]. This finding suggested the notion that trienone 11 may be useful for eliminating multidrug-resistant cancer cells.

Several studies reported that curcuminoids induced apoptotic cell death by increasing the intracellular ROS level over the threshold in OSCC cell lines [33,34,35]. Similar to our observation, trienone 11 treatment increased intracellular ROS levels and cell death which could be reversed by supplementation with NAC, indicating ROS-mediated cell death. ROS-metabolic enzymes have been proposed as the main biological targets of curcuminoids. Curcuminoids and analogs can bind to several ROS-metabolic enzymes including carbonyl reductase 1, glutathione-S-transferase, glyoxalase I, NAD(P)H dehydrogenase, quinone 1, and thioredoxin reductase 1 (TrxR1), thereby inactivating the enzymes and leading to ROS accumulation [16, 36]. Additionally, a differential ROS-inducing ability was related, in part, to the compound’s structure. Trienone 11 possesses the free phenolic hydroxyl group at the meta-position which can activate stronger oxidation than analogs having a methoxy group [17]. This may induce mitochondrial membrane damage and subsequently mitochondrial ROS leakage.

When the ROS level climbs above cellular tolerability thresholds, excessive ROS causes oxidative damage to deoxyribonucleic acid (DNA), lipids, and proteins, leading to activation of cell death processes mainly as the mitochondrial pathway of apoptosis [37]. Initially, damaged DNA leads to activation of p53 protein and the p53 signaling pathway to upregulate pro-apoptotic proteins and downregulate anti-apoptotic proteins in the mitochondria. Overexpression of pro-apoptotic Bax induces cytochrome c release from the mitochondria to form complexes with Apaf-1 and caspase-9 that consequently activate caspase-3 and apoptosis [37]. ROS-derived curcuminoid treatment causes DNA damage, change in mitochondrial membrane potential (MMP), increased expression of pro-apoptotic proteins and decreased expression of anti-apoptotic proteins, leading to caspase-9 and -3 activation in oral cancer SAS cells [33]. Thus, caspase-9 and -3 activation could be an indicator of the mitochondrial pathway of apoptosis. Our results showed that ROS-derived trienone 11 revealed activation of caspase-9 and caspase-3, suggesting that trienone 11 might induce apoptosis through a mitochondrial pathway [36]. Furthermore, curcumin has also been reported to induce apoptosis via a Fas receptor/caspase-8 or extrinsic pathway [38]. Consistent with our findings, trienone 11 also increased caspase-8 catalytic activity; however, it is ROS-independent. Trienone 11 may interact with the Fas receptor and form a complex on the cell surface that directly triggers a signaling cascade to activate caspase-8. The proposed cytotoxic mechanism of trienone 11 in CLS-354/DX cells is illustrated in Fig. 6c.

Our studies are the first to indicate that the trienone 11 analog of curcumin shows promise as a potent cytotoxic agent, more powerful than curcumin and the natural trienone 4 against the multidrug-resistant OSCC cell line CLS-354/DX. The compound induced apoptotic cell death via ROS and caspase-3/7, -8, and -9 activations. We also demonstrated the mechanism by which trienone 11 activates ROS to mediate caspase activation and eventually apoptosis via the intrinsic pathway. To further develop trienone 11 as an effective anticancer agent for OSCCs, the therapeutic potential of an in vivo model, as well as the main molecular targets, require a more detailed and comprehensive analysis.

Availability of data and materials

Not applicable.


  1. Massano J, Regateiro FS, Januário G, Ferreira A (2006) Oral squamous cell carcinoma: review of prognostic and predictive factors. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 102(1):67–76

    Article  Google Scholar 

  2. Warnakulasuriya S (2009) Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 45(4–5):309–316

    Article  Google Scholar 

  3. Chang CW, Chen YS, Chou SH, Han CL, Chen YJ, Yang CC, Huang CY, Lo JF (2014) Distinct subpopulations of head and neck cancer cells with different levels of intracellular reactive oxygen species exhibit diverse stemness, proliferation, and chemosensitivity. Cancer Res 74(21):6291–6305

    Article  CAS  Google Scholar 

  4. Wang C, Liu XQ, Hou JS, Wang JN, Huang HZ (2016) Molecular mechanisms of chemoresistance in oral cancer. Chin J Dent Res. 19(1):25–33

    PubMed  Google Scholar 

  5. Saraswathy M, Gong S (2013) Different strategies to overcome multidrug resistance in cancer. Biotechnol Adv 31(8):1397–1407

    Article  CAS  Google Scholar 

  6. Xiao Z, Morris-Natschke SL, Lee KH (2016) Strategies for the optimization of natural leads to anticancer drugs or drug candidates. Med Res Rev 36(1):32–91

    Article  CAS  Google Scholar 

  7. Ghosh S, Banerjee S, Sil PC (2015) The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: a recent update. Food Chem Toxicol 83:111–124

    Article  CAS  Google Scholar 

  8. Trujillo J, Chirino YI, Molina-Jijón E, Andérica-Romero AC, Tapia E, Pedraza-Chaverrí J (2013) Renoprotective effect of the antioxidant curcumin: recent findings. Redox Biol. 1:448–456

    Article  CAS  Google Scholar 

  9. Aggarwal BB, Kumar A, Bharti AC (2003) Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res 23(1A):363–398

    CAS  PubMed  Google Scholar 

  10. Kunnumakkara AB, Bordoloi D, Harsha C, Banik K, Gupta SC, Aggarwal BB (2017) Curcumin mediates anticancer effects by modulating multiple cell signaling pathways. Clin Sci 131(15):1781–1799

    Article  CAS  Google Scholar 

  11. Kharat M, Du Z, Zhang G, McClements DJ (2017) Physical and chemical stability of curcumin in aqueous solutions and emulsions: impact of pH, temperature, and molecular environment. J Agric Food Chem 65(8):1525–1532

    Article  CAS  Google Scholar 

  12. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB (2007) Bioavailability of curcumin: problems and promises. Mol Pharm 4(6):807–818

    Article  CAS  Google Scholar 

  13. Aziz MNM, Hussin Y, Che Rahim NF, Nordin N, Mohamad NE, Yeap SK et al (2018) Curcumin analog DK1 induces apoptosis in human osteosarcoma cells in vitro through mitochondria-dependent signaling pathway. Molecules.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Chen X, Chen X, Zhang X, Wang L, Cao P, Rajamanickam V et al (2019) Curcuminoid B63 induces ROS-mediated paraptosis-like cell death by targeting TrxR1 in gastric cells. Redox Biol. 21:101061

    Article  Google Scholar 

  15. He Y, Li W, Hu G, Sun H, Kong Q (2018) Bioactivities of EF24, a novel curcumin analog: a review. Front Oncol. 8:614

    Article  Google Scholar 

  16. He W, Xia Y, Cao P, Hong L, Zhang T, Shen X et al (2019) Curcuminoid WZ35 synergize with cisplatin by inducing ROS production and inhibiting TrxR1 activity in gastric cancer cells. J Exp Clin Cancer Res. 38(1):207

    Article  Google Scholar 

  17. Chuprajob T, Changtam C, Chokchaisiri R, Chunglok W, Sornkaew N, Suksamrarn A (2014) Synthesis, cytotoxicity against human oral cancer KB cells and structure-activity relationship studies of trienone analogues of curcuminoids. Bioorg Med Chem Lett 24(13):2839–2844

    Article  CAS  Google Scholar 

  18. Nakayama R, Tamura Y, Yamanaka H, Kikuzaki H, Nakatani N (1993) Two curcuminoid pigments from Curcuma domestica. Phytochemistry 33(2):501–502

    Article  CAS  Google Scholar 

  19. Mohamad H, Lajis NH, Abas F, Ali AM, Sukari MA, Kikuzaki H, Nakatani N (2005) Antioxidative constituents of Etlingera elatior. J Nat Prod 68(2):285–288

    Article  CAS  Google Scholar 

  20. Jang MK, Sohn DH, Ryu JH (2001) A curcuminoid and sesquiterpenes as inhibitors of macrophage TNF-alpha release from Curcuma zedoaria. Planta Med 67(6):550–552

    Article  CAS  Google Scholar 

  21. Lo CY, Liu PL, Lin LC, Chen YT, Hseu YC, Wen ZH, Wang HM (2013) Antimelanoma and antityrosinase from Alpinia galangal constituents. ScientificWorldJournal. 2013:186505

    Article  Google Scholar 

  22. Changtam C, de Koning HP, Ibrahim H, Sajid MS, Gould MK, Suksamrarn A (2010) Curcuminoid analogs with potent activity against Trypanosoma and Leishmania species. Eur J Med Chem 45(3):941–956

    Article  CAS  Google Scholar 

  23. Utaipan T, Athipornchai A, Suksamrarn A, Chunsrivirot S, Chunglok W (2017) Isomahanine induces endoplasmic reticulum stress and simultaneously triggers p38 MAPK-mediated apoptosis and autophagy in multidrug-resistant human oral squamous cell carcinoma cells. Oncol Rep 37(2):1243–1252

    Article  CAS  Google Scholar 

  24. Utaipan T, Sattayakhom A, Prachongsai I, Charong N, Chunglok W (2018) Reduction of intracellular-reactive oxygen species and diminished mitogen-activated protein kinases (MAPKs) activation are associated with oral squamous cell carcinoma cell aggressiveness. Walailak J Sci Technol 15(2):131–141

    Google Scholar 

  25. Peña-Morán OA, Villarreal ML, Álvarez-Berber L, Meneses-Acosta A, Rodríguez-López V (2016) Cytotoxicity, post-treatment recovery, and selectivity analysis of naturally occurring podophyllotoxins from Bursera fagaroides var. fagaroides on breast cancer cell lines. Molecules.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Zhang X, Chen M, Zou P, Kanchana K, Weng Q, Chen W et al (2015) Curcumin analog WZ35 induced cell death via ROS-dependent ER stress and G2/M cell cycle arrest in human prostate cancer cells. BMC Cancer. 15:866

    Article  Google Scholar 

  27. McStay GP, Green DR (2014) Assaying caspase activity in vitro. Cold Spring Harb Protoc. 2014(7):774–777

    PubMed  Google Scholar 

  28. Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25(34):4798–4811

    Article  CAS  Google Scholar 

  29. Zimmermann KC, Bonzon C, Green DR (2001) The machinery of programmed cell death. Pharmacol Ther 92(1):57–70

    Article  CAS  Google Scholar 

  30. Mbese Z, Khwaza V, Aderibigbe BA (2019) Curcumin and its derivatives as potential therapeutic agents in prostate, colon and breast cancers. Molecules.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Thakur A, Manohar S, Gerena CE, Zayas B, Kumar V, Malhotra SV, Rawat DS (2014) Novel 3,5-bis(arylidiene)-4-piperidone based monocarbonyl analogs of curcumin: anticancer activity evaluation and mode of action study. MedChemComm. 5(5):576–586

    Article  CAS  Google Scholar 

  32. Wang R, Zhang X, Chen C, Chen G, Zhong Q, Zhang Q et al (2016) Synthesis and evaluation of 1,7-diheteroarylhepta-1,4,6-trien-3-ones as curcumin-based anticancer agents. Eur J Med Chem 110:164–180

    Article  CAS  Google Scholar 

  33. Hsiao YT, Kuo CL, Chueh FS, Liu KC, Bau DT, Chung JG (2018) Curcuminoids induce reactive oxygen species and autophagy to enhance apoptosis in human oral cancer cells. Am J Chin Med 46(5):1145–1168

    Article  CAS  Google Scholar 

  34. Kim JY, Cho TJ, Woo BH, Choi KU, Lee CH, Ryu MH, Park HR (2012) Curcumin-induced autophagy contributes to the decreased survival of oral cancer cells. Arch Oral Biol 57(8):1018–1025

    Article  CAS  Google Scholar 

  35. Chang PY, Peng SF, Lee CY, Lu CC, Tsai SC, Shieh TM et al (2013) Curcumin-loaded nanoparticles induce apoptotic cell death through regulation of the function of MDR1 and reactive oxygen species in cisplatin-resistant CAR human oral cancer cells. Int J Oncol 43(4):1141–1150

    Article  CAS  Google Scholar 

  36. Larasati YA, Yoneda-Kato N, Nakamae I, Yokoyama T, Meiyanto E, Kato JY (2018) Curcumin targets multiple enzymes involved in the ROS metabolic pathway to suppress tumor cell growth. Sci Rep. 8(1):2039

    Article  Google Scholar 

  37. Yang Y, Karakhanova S, Hartwig W, D’Haese JG, Philippov PP, Werner J, Bazhin AV (2016) Mitochondria and mitochondrial ROS in cancer: novel targets for anticancer therapy. J Cell Physiol 231(12):2570–2581

    Article  CAS  Google Scholar 

  38. Bush JA, Cheung KJ Jr, Li G (2001) Curcumin induces apoptosis in human melanoma cells through a Fas receptor/caspase-8 pathway independent of p53. Exp Cell Res 271(2):305–314

    Article  CAS  Google Scholar 

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We would like to thank Assoc. Prof. Dr. Seppo Karrila for English language editing and reviewing of this article.


This work was supported by the Faculty of Science and Technology Research Fund, Prince of Songkla University, Pattani Campus, Contract no. SAT610461S and The Thailand Research Fund, Contract no. DBG6180030.

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TU, TC, WC, performed experiments and wrote the paper. PB and AS helped the preparation of experiments. TU, WC, AS, PB revised the manuscript. AS, WC supervised the work. All authors read and approved the final manuscript.

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Correspondence to Tanyarath Utaipan.

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Additional file 1.

1H and 13C NMR spectra of compounds (Fig. S1), cell morphology (Fig. S2), and intracellular ROS levels in CLS-354/DX cells upon treatment with trienone 4 (Fig. S3).

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Utaipan, T., Boonyanuphong, P., Chuprajob, T. et al. A trienone analog of curcumin, 1,7-bis(3-hydroxyphenyl)-1,4,6-heptatrien-3-one, possesses ROS- and caspase-mediated apoptosis in human oral squamous cell carcinoma cells in vitro. Appl Biol Chem 63, 7 (2020).

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