2.1. Effect of Individual 6G and γT3 and in Combination on Cell Viability
MTS assays of individual 6G and γ-T3 were carried out on both HT-29 and SW837 cells at concentrations ranging from 0 to 300 µg/mL for 6G and 0 to 150 µg/mL for γ-T3. Both compounds caused a concentration-dependent decrease in cell viability in HT-29 and SW837 cells (
Figure 1). IC
50 values obtained for 6G on HT-29 was 254.0 ± 42.0 and 158.4 ± 20.5 for SW837, while they were 138.9 ± 9 and 57.7 ± 5.8 µg/mL for HT-29 and SW837 after treatment with γ-T3 (
Table 1).
Table 1.
MTS assay results for individual 6-gingerol and γ-tocotrienol treatments on each cell line. Data are expressed as mean ± SD, in three independent experiments (n = 3).
Table 1.
MTS assay results for individual 6-gingerol and γ-tocotrienol treatments on each cell line. Data are expressed as mean ± SD, in three independent experiments (n = 3).
Cell Lines | Bioactive Compound | IC50 Value (µg/mL) | * Cell Viability, % |
---|
HT-29 | 6-Gingerol (6G) | 254.0 ± 42.0 | 40.1 ± 18.0 |
γ-Tocotrienol (γ-T3) | 138.9 ± 8.7 | 41.1 ± 8.4 |
SW837 | 6-Gingerol (6G) | 158.4 ± 20.5 | 13.4 ± 2.8 |
γ-Tocotrienol (γ-T3) | 57.7 ± 5.8 | 8.0 ± 1.9 |
Figure 1.
Cell viability assay of individual 6G and γ-T3 of both untreated and treated HT 29 (A,C) and SW 837 (B,D) cells after 24 h treatment. HT 29 and SW 837 cells were treated with 6G (0, 50, 100, 150, 200, and 300 µg/mL) and γ-T3 (0, 20, 40, 60,100, 150 µg/mL). Cell viability was observed using ELISA microplate reader at 405nm. Values are expressed as mean ± SEM from three independent experiments in triplicates (n = 3). * significant when compared with untreated cells (p < 0.05).
Figure 1.
Cell viability assay of individual 6G and γ-T3 of both untreated and treated HT 29 (A,C) and SW 837 (B,D) cells after 24 h treatment. HT 29 and SW 837 cells were treated with 6G (0, 50, 100, 150, 200, and 300 µg/mL) and γ-T3 (0, 20, 40, 60,100, 150 µg/mL). Cell viability was observed using ELISA microplate reader at 405nm. Values are expressed as mean ± SEM from three independent experiments in triplicates (n = 3). * significant when compared with untreated cells (p < 0.05).
Subsequent cell viability tests were done by using sub-half maximal individual 6G concentrations, which was 105 for HT-29 and 70 µg/mL for SW837, in combination with γ-T3 at varying doses (0, 5, 20, 50 and 100 µg/mL). The new IC
50 values obtained for 6G+γ-T3 combined were 105 + 67 µg/mL and 70 + 20 µg/mL for HT 29 and SW 837 cells, respectively. The combination index was also calculated (
Table 2). The combination treatment showed inhibitory effects in a concentration-dependent manner (
Figure 2). Normal hepatic WRL-68 cells were unaffected when treated with the IC
50 concentration of 6G+γT3 obtained from both HT-29 and SW837 results (
Figure 3).
Table 2.
Cell viability, IC50 value, and combination index for combined 6G+γ-T3 on each cell lines. Data are expressed as mean ± SD, of three dependent experiments (n = 3).
Table 2.
Cell viability, IC50 value, and combination index for combined 6G+γ-T3 on each cell lines. Data are expressed as mean ± SD, of three dependent experiments (n = 3).
Cell Lines | | γT3 (µg/mL) | Combination Index, C.I |
---|
0 | 5 | 20 | 50 | 100 | IC50 Value |
---|
HT-29 (105 µg/mL 6G) | 100 | 88.6 ± 9.2 | 81.6 ± 6.9 | 62.8 ± 8 | 26.7 ±6.5 | 67.0 ± 3.0 | 0.89 |
SW837 (70 µg/mL 6G) | 100 | 70.9 ± 9 | 51.1 ± 9.7 | 21.9 ± 10.9 | 4.9 ± 3.9 | 20.1 ± 8.8 | 0.79 |
Figure 2.
Cell viability assay of combined 6G and γ-T3 on cells after 24 h treatment. HT 29 cells were treated with 105 µg/mL 6G while SW 837 treated with 70 µg/mL 6G in combinations of γ-T3 (0, 5, 20, 50, and 100 µg/mL). Cell viability was measured using ELISA microplate reader at 405 nm. Values are expressed as mean ± SEM from three independent experiments (n = 3). * Compared with untreated HT-29 cells (p < 0.05), # compared with untreated SW 837 cells (p < 0.05).
Figure 2.
Cell viability assay of combined 6G and γ-T3 on cells after 24 h treatment. HT 29 cells were treated with 105 µg/mL 6G while SW 837 treated with 70 µg/mL 6G in combinations of γ-T3 (0, 5, 20, 50, and 100 µg/mL). Cell viability was measured using ELISA microplate reader at 405 nm. Values are expressed as mean ± SEM from three independent experiments (n = 3). * Compared with untreated HT-29 cells (p < 0.05), # compared with untreated SW 837 cells (p < 0.05).
Figure 3.
Combined treatment, 6G+γ-T3 after 24 h had no effect on human hepatic cells, WRL-68. No significance difference was observed between treated and untreated cells. Values are expressed as mean ± SEM from three independent experiments (n = 3).
Figure 3.
Combined treatment, 6G+γ-T3 after 24 h had no effect on human hepatic cells, WRL-68. No significance difference was observed between treated and untreated cells. Values are expressed as mean ± SEM from three independent experiments (n = 3).
2.4. Effects of Combined 6G+γ-T3 on Apoptosis of HT-29 and SW837 Cells
The IC
50 values of combined 6G+γ-T3 treatment of cells were used to determine the mode of cell death which was assayed by flow cytometry using annexin V FITC and active caspase-3 staining. Cytogram analysis suggested that the cells undergo apoptosis as the cells died in early and late apoptosis (
Figure 6). No significant difference was observed between control and sub-lethal concentration of individual 6G and γ-T3 treatment but combined treatment of 6G+γT3 increased cell death (
p < 0.05) (
Figure 7). Active caspase-3 is a key protease activated during early apoptosis, both in intrinsic and extrinsic pathway. In this study, active caspase-3 was not increased in all treatments after 24 h for both cells (
Figure 8).
Interest has increased in bioactive compounds present in plants which can modulate the development and progression of cancer. Accumulating evidence has suggested that many cancers are preventable, an effect attributed to dietary constituents, particularly phytochemicals which can modulate multiple stages carcinogenesis as well as reduce cancer risks [
17]. Promising anti-cancer agents including bioactive compounds such as curcumin in turmeric [
18] and epigallocatechin gallate (EGCG) found in green tea [
19] have reached clinical trials and shown promising results that can be further investigated for use as therapeutic agents. Currently, hundreds of bioactive compounds are being studied experimentally and clinically as chemopreventive agents.
Figure 6.
Effects of 6G, γ-T3 and 6G+γ-T3 on both HT 29 and SW 837 cells. Apoptosis was quantified using flow cytometry. Q1 represents dead cells/necrosis, An−/PI+. Q2 late apoptosis, An+/PI+, Q3 viable cells An−/PI−, and Q4 early apoptosis, An+/PI−.
Figure 6.
Effects of 6G, γ-T3 and 6G+γ-T3 on both HT 29 and SW 837 cells. Apoptosis was quantified using flow cytometry. Q1 represents dead cells/necrosis, An−/PI+. Q2 late apoptosis, An+/PI+, Q3 viable cells An−/PI−, and Q4 early apoptosis, An+/PI−.
Figure 7.
Annexin V FITC analysis on control, sub-lethal concentration of individual 6G and γ-T3, and combined 6G+γ-T3 treatment on both (A) HT-29 and (B) SW837 cells. The number of cells decreased (p < 0.05) in 6G+γ-T3 treatment as compared to control, both in HT-29 and SW837 cells. No significant difference was observed between individual treatment of 6G and γ-T3 when compared to control cells. Percentage of viable, early and late apoptotic cells. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 vs. control.
Figure 7.
Annexin V FITC analysis on control, sub-lethal concentration of individual 6G and γ-T3, and combined 6G+γ-T3 treatment on both (A) HT-29 and (B) SW837 cells. The number of cells decreased (p < 0.05) in 6G+γ-T3 treatment as compared to control, both in HT-29 and SW837 cells. No significant difference was observed between individual treatment of 6G and γ-T3 when compared to control cells. Percentage of viable, early and late apoptotic cells. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 vs. control.
Figure 8.
Combined of 6G+γ-T3 treatment did not increase active caspase-3 in both HT-29 and SW837. There was also no significant difference of active caspase-3 determined in sub-lethal concentrations of individual 6G and γ-T3 treatments. Data are presented as mean ± SEM of three independent experiments.
Figure 8.
Combined of 6G+γ-T3 treatment did not increase active caspase-3 in both HT-29 and SW837. There was also no significant difference of active caspase-3 determined in sub-lethal concentrations of individual 6G and γ-T3 treatments. Data are presented as mean ± SEM of three independent experiments.
Gingerol, a major constituent of ginger was found to have antioxidant [
20], anti-inflammatory [
21], anti-genotoxic [
22], anti-arthritic [
23] and anti-cancer effects [
5,
24,
25]. In this study, 6G alone was found to be effective in inhibiting growth of HT-29 and SW837 cells in a concentration dependent manner. This finding was consistent with a previous study that showed anti-proliferative effect of 6G in HPAC and BxPC-3 pancreatic cells [
26], HeLa cells [
5] and PC-3 prostate cancer cells [
27].
In our study, the IC
50 values of 6G on HT-29 and SW837 were 254 µg/mL and 158 µg/mL, respectively, after 24 h treatment. The range of concentrations was consistent with the previous study using HeLa cells where the IC
50 of 6G after 24 h treatment was 126 µg/mL [
6]. Other recent studies reported 6G reduced 50% of SW480 colon cancer cells growth at a concentration of 60 µg/mL or 205 ± 5 µM [
28] and 28% of HT-29 and HCT116 at concentration of 200 µM after 72 h treatment [
6]. The lower concentration of 6G needed could be due to the longer treatment time and a different method used in determining cell cytotoxicity. We believe that the concentration of 6G used in this study is achievable as daily human consumption of approximately 250 mg–1 g of ginger contains up to 1.0%–3.0% of 6G [
29]. In addition, 6-gingerol present in ginger has a high distribution in tissues of the gastrointestinal tract due to its lipophilicity [
30] with a short half-life of 75 to 120 min in human plasma [
31]. The other compound used in this study which was γ-T3 which is an isoform of vitamin E that has been suggested to have antioxidant properties and anti-cancer effects on a wide range of cancer cells including human colon carcinoma [
32,
33,
34], prostate [
35,
36], mammary [
37,
38], stomach [
11,
15], liver [
39] and skin cancer [
40]. In our study, γ-T3 significantly inhibited cell growth of both HT-29 and SW837 cells after 24 h treatment, in a concentration dependent fashion. The IC
50 of γ-T3 for HT-29 and SW837 was 138 µg/mL (equivalent to 336 µM) and 58 µg/mL (141 µM). This concentration was higher than that reported in other studies using DU145 prostate and MGH-U1 bladder cancer cells with IC
50 values of 45 to 60 µg/mL [
36], 31.4 ± 1.51 µM using SW 620 colon cancer cells [
34] and less than 50 µM using MCF-7 and MDA-MB-231 human mammary cells [
38]. The differences observed could be due to different number of cells and different incubation times used.
The inhibition of cell growth by 6G and γ-T3 alone on various cancer cells was reported in previous studies suggesting that anti-proliferative effect of these compounds on cancer cells is consistent. According to Lee
et al. [
6], 6G inhibits cell growth of HCT116, HT-29, SW480, LoVo and Caco-2 colorectal cancer cells by multiple mechanisms, such as by inhibiting transcription of cyclin D1 through the suppression of β-catenin, down-regulating cyclin D1 resulting in cell growth arrest and up-regulating NAG-1 expression through the GSK-3β and PKCε pathways. Both cell lines used were
APC-mutant cells. It has been known that colon cancer with a mutant
APC gene contained high levels of free β-catenin [
41]. 6G has been reported to suppress β-catenin in colon cancer cells through β-catenin/TCF-dependent gene transcription to induce anti-tumorigenesis effect shown by the reduction of cyclin D1 resulting in G1/S phase arrest as well as decreased β-catenin localization in cells [
6]. The similarities between HT-29 and SW837 cell lines were not only in
APC mutation, but also in the
p53 mutated gene. 6G is reported to modulate
p53 levels in HCT-116 and LoVo cells which are
p53-wild type cells, but not in SW-480, a
p53-mutant cell [
6,
42]. The results observed are consistent with the results for BxPC-3 mutant
p53 pancreatic cancer cells where 6G treatment (400 µM) showed a decline in p53 level but overexpression of p21 suggesting that cell death might be caused by
p53-independent events [
26]. It has been reported that inactivation of
p53 results in chemoresistance by chemopreventive agents [
6]. Mutation in
p53 decreased sensitivity to G1/S interface of cell cycle in prostate cancer cells [
43]. Thus, it is possible that 6G effects on cell cycle may depend on
p53 status.
Previous studies have shown that γ-T3 suppresses cell growth by reducing cyclin D1, CDK4, CDK6, and CDK2 levels in mammary cancer cells [
44] and SW620 human colon carcinoma [
34]. Cyclin D1, a protein that forms a complex with CDK4/6, mediates a growth factor involved in G1 phase progression. Thus, a decrease in cyclin D1 results in G0/G1 phase arrest [
9,
26]. Like 6-gingerol, γ-T3 also suppressed transcriptional activity of β-catenin/Tcf signalling in both
APC-mutant cells of HT-29 cells [
33], SW620 human colon carcinoma [
34] and in
APC-wild type prostate cancer stem cells [
36]. It is also reported γ-T3 is associated with the inhibition of the NF-kappa B (NF-κB) signalling pathway, a cell survival regulator which is frequently up-regulated in cancer. Inactivation of NF-κB by γT3 was reported in PCa prostate cancer cells [
35], Taken together, both 6G and γ-T3 may exert their anti-proliferative effect in HT-29 and SW837 which are
APC and
p53-mutant cells by modulating cyclin D1 in β-catenin/Tcf signalling pathway which later causes cell cycle arrest.
The idea of combining two or more bioactives as treatment may be helpful in reducing the toxicity and adverse effects of cancer treatment. Synergistic interactions have favourable outcomes such as enhanced efficacy, the need for lower concentrations or dosages at equal or increased level of efficacy, and simultaneous enhancement of therapeutic actions as well as reduction of unwanted actions during treatment [
45,
46,
47]. It is noted that anti-cancer therapy is more effective when multiple drugs with complimentary mechanisms and molecular targets are given to optimize the synergistic therapeutic response [
7]. Synergistic interactions were identified between combinations of indole-3-carbinol and genistein to reduce the growth and induce apoptosis of human colon cancer HT-29 cells [
48], while concurrent treatment of sulforaphane and eugenol synergistically induces cell cytotoxicity and apoptosis [
49] and co-treatment of sesamin with γ-tocotrienol inhibited the proliferation and caused cytostatic effects in mouse and human mammary cancer cells [
50].
In this study, we used sub-half maximal inhibitory concentrations of 6G alone, 105 µg/mL and 70 µg/mL on HT-29 and SW 837 cells with different γ-T3 concentrations. The inhibitory effect of 6G+γ-T3 in both HT-29 and SW837 cells occurred in a concentration dependent manner. The lower IC
50 of the combined treatment obtained for HT-29 and SW837 suggested a synergistic interaction based on the isobologram index (CI < 1, 0.89 for HT-29 and 0.79 for SW837). The respective concentrations (IC
50) had no effect on WRL-68 human hepatic cells after 24 h of treatment, consistent with a previous study that reported that γ-T3 showed no cytotoxicity on normal human peripheral blood mononuclear cells [
51]. Normal intestinal epithelial cells were unaffected when treated with high concentrations of 6G (500 µM) [
28] and human fibroblast cells were only inhibited at 500 µM [
52].
A number of studies have shown that combined treatment of γ-T3 with other chemotherapeutic agents induced synergistic interaction to enhance cytotoxic or cytostatic effects on tumor cells. γ-T3 in combination with resveratrol has been reported to enhance the apoptosis effects in estrogen receptor positive MCF-7 breast cancer cells [
53]. Combined treatment of sesamin and γ-T3 also showed an synergistic interaction and inhibited the growth of murine +SA, human MCF-7 and human MDA-MB231 mammary tumor cells [
50]. Other studies of γ-T3 combined with specific drugs such as statin [
54], celecoxib [
55], and erlonitib/gefitinib [
56] displayed significant chemopreventive effects against various cancers, as compared to individual treatments alone. In other studies, it was reported that treatment with γ-T3 alone significantly reduced quinone reductase, NQO1 while EGCG markedly increased the enzyme but when both compounds worked concurrently, they elicit synergism by boosting the NQO1 production in MCF-7 breast cancer cells [
53]. Since both 6G and γ-T3 have been reported to produce a variety of biological effects, we hypothesized that multiple pathways were probably involved. 6G exerts its anti-inflammatory properties by affecting various pro-inflammatory factors such as TNF, COX2 and PGE
2 [
57,
58] while γ-T3 alone may act as an angiogenic inhibitor by modulating the PI3K/PDK/Akt signalling pathway [
59] and down-regulating VEGF and VEGF receptors in the ERK-signaling pathway in SGC-7901 gastric adenocarcinoma cells [
10]. Perhaps, the different molecular targets affected by 6G and γ-T3 accelerate the cytotoxic and cytostatic effects. Furthermore, a previous study on different prostate cancer cell lines, namely DU145, LNCaP, and PC3 showed that γ-T3 exerts its anti-cancer action by modulating different mechanism routes as it depends on the existence of androgen receptors on cells [
60]. In the present study, as HT-29 carried
BRAF,
SMAD4 and
PI3KCA-mutated genes while SW837 is a
KRAS mutated cell, hence different mechanisms may contribute to increased cell death.
The inhibitory effect of the combined treatment is accompanied by apoptosis, shown by morphological changes of the cells. We observed significant characteristics of apoptotic cells in both 6G+γT3 treated cells, such as cell shrinkage, pyknosis (chromatin condensation), rounded and distorted cells, reduced adherence to adjacent cells, and blebbing of plasma membrane (
Figure 5). Furthermore, the apoptosis effect of 6G+γ-T3 is further supported by significant externalization of phosphotidylserine suggesting early apoptosis and existence of apoptotic bodies in late apoptosis (
Figure 6). Later, cells disintegrate into secondary necrosis, another programmed cell death form which is distinguished from necrosis without scavengers such as macrophages to remove the apoptotic bodies before proceeding to autolytic necrotic outcomes [
61,
62,
63]. As both HT-29 and SW837 cells died following the apoptosis sequence, it can be suggested that the cells died through the apoptotic pathway rather than due to necrosis. We also observed a difference in sensitivity of the two types of cells towards 6G+γ-T3 treatment, as HT-29 cells were more resistant compared to SW837 in cell viability and apoptosis. A previous study showed that SW-620 clones expressing
SMAD4 were three fold more sensitive to 5-FU treatment when compared with control based on the IC
50 values. Besides, treatment on SW-620 cells that were
SMAD4-deficient with 5-FU increased apoptotic cells by 26.3% ± 3.4% compared to 47.3% ± 6.2% in
SMAD4 expressing clone cells [
64]. HT-29 carries the
SMAD4 mutation, a tumor suppressor gene located at 18q21 chromosome. It has been reported that deficiency of
SMAD4 exhibits tumor progression, up-regulates VEGF expression related to vascular density progression, and chemoresistance to 5′-fluorouracil, anti-cancer drug-mediated apoptosis [
65,
66] and patients with high levels of
SMAD4 have better survival rates than patients with low
SMAD4 levels in CRC [
67,
68]. Moreover, loss of
SMAD4 in CRC plays an important role in chemoresistance to 5-FU by activating the PI3K/Akt pathway and regulating cell cycle and apoptosis-related proteins [
68]. In CRC that are
SMAD4 deficient, the changes of c-Myc and cyclin D1 increased levels and down-regulation of p21 and p27 are associated with a decrease of phosphorylation of Rb which later results in cell proliferation. Following the inhibition of
SMAD4, the Akt pathway is activated and the level of anti-apoptotic proteins such as Bcl-2 and survivin increased, and down-regulates pro-apoptotic keys, Bad and Bim, and finally suppressed apoptotic effects in cancer cells [
68,
69]. As both 6G and γ-T3 were reported to have effects on the PI3K/Akt signalling pathways, we hypothesized that the resistance of HT-29 towards 6G+γ-T3 treatment could be due to dysregulation of
SMAD4 through the PI3K/Akt signalling pathway.
By using morphological changes and membrane alteration evidence, we further tested the apoptotic effect of 6G+γ-T3 by activation of caspases via detection of active caspase-3. After 24 h of 6G+γ-T3 treatment, no significant production of active caspase-3 was observed in treated cells. Paradoxically, both 6G and γ-T3 alone were reported to mediate apoptosis in various cancer cell lines via the caspase-3 cascade, regardless by intrinsic or extrinsic pathways [
6,
28,
36,
56,
57]. In this study, neither compound alone produced significant active caspase-3 after 24 h. A study done by Kim
et al. [
60] reported that production of caspase-3 after 100–300 µM of 6G treatment of LNCaP prostate cancer cells was not evident at 24 h, but significant expression was observed after 48 h. In another study, γ-T3 and δT3 did not show significant activation of caspase-8 and caspase-3 in DU145 and PC3 prostate carcinoma cells at IC
50 concentrations, suggesting that tocotrienols could also mediate apoptosis by caspase-independent programmed cell death [
70]. The findings were similar to a study done by Takashi and Loo [
71] who reported that PARP, an enzyme involved in DNA repair which can be cleaved by caspase-3 during apoptosis was undetectable in MDA-MB-231 human breast cancer cells after treatment with γ-T3.
It is interesting to note that our findings showed apoptosis was increased without caspase-3 activation in 6G and γ-T3 alone as well as in combination treatment in both cell lines. The other possible explanation of this circumstance is that 6G+γ-T3 induced apoptosis synergistically by activating caspase-independent pathways as well. It has been reported that more than one type of programmed cell death may be activated at the same time [
72]. Therefore further studies are required to elucidate the mode of action of these bioactives in inducing apoptosis.