編號：99001
Experimental Study on Effect of
Compounds in Inhibiting HCT-116
Human Colon Cancer Cells: The
Preliminary Results
1
中文摘要
目的: 分析數種藥物對於HCT-116人類大腸癌細胞株生長抑制的影響。
材料及方法: 一些具有抗發炎或抗氧化或具有自由基清除作用之藥物被選用，包
括厚朴酚(honokiol),大黃素(emodin),硫辛酸(lipoic acid),黃連素(berberine),皂甘
(diosgenin),白藜蘆醇(resveratrol),呂宋揪莢粉素(rottlerin), pinolo, 薑黃素
(curcumin), 退黑激素(melatonin), 以及丁酸鈉(sodium butyrate)。把HCT-116細胞
培養在無血清環境之培養盤，分別加入上述藥物，分別以不同藥物濃度之條件
下，於百分之五的二氧化碳濃度處理二十四或四十八小時。接著，再以
3-[4,5,-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay 分析
HCT-116細胞生長受影響的情形。 結果:厚朴酚，大黃素，白藜蘆醇，呂宋揪莢粉素，薑黃素，這些藥物對於HCT-116 細胞生長有明顯的抑制效果。經藥物處理四十八小時後，百分之五十的生長壓制 劑量分別是厚朴酚為18.5μM，大黃素為17.3μM，白藜蘆醇為25.3μM，呂宋揪莢 粉素為6.9μM，薑黃素為22.3μM。 結論: 厚朴酚，大黃素，白藜蘆醇，呂宋揪莢粉素，薑黃素，這些藥物對於HCT-116 細胞生長有明顯的抑制效果。對於已篩選出的這些藥物，應當設計進一步的實驗 探討牽涉在其中的機轉。 關鍵字: 增生,大腸癌, MTT 分析 類別: 原著論文
ABSTRACT
OBJECTIVE: Several compounds were studied for their growth inhibitory effects on cultured HCT-116 human colon cancer cells.
MATERIALS AND METHODS: Compounds with anti-inflammation,
anti-oxidation, or free-radical scavenging ability were used, including honokiol,
emodin, lipoic acid, berberine, diosgenin, resveratrol, rottlerin, pinolo, curcumin,
melatonin, and sodium butyrate. Cultured cells were incubated in a serum-free
medium with various concentrations of different compounds for 24 and 48 hours in a
5% CO2 incubator, after which the proliferation of HCT-116 cells was assessed by
3-[4,5,-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the
significance of differences was analyzed by Student's t test.
RESULTS: Honokiol, emodin, resveratrol, rottlerin, and curcumin used in this study were more effective than lipoic acid, berberine, diosgenin, pinolo, melatonin, and
sodium butyrate. The 50% suppression doses after 48-hour exposure were 18.5 μM
for honokiol, 17.3 μM for emodin, 25.3 μM for resveratrol, 6.9 μM for rottlerin, and
22.3 μM for curcumin respectively.
CONCLUSION: Further investigations should be conducted to elucidate the mechanisms modulating anti-tumor effects on HCT-116 cells for honokiol, emodin,
3
KEYWORDS: proliferation, colon cancer, MTT assay
INTRODUCTION
Colon cancer is the second leading cause of death for cancer worldwide. In Taiwan,
the Bureau of Health Promotion, Department of Health has proclaimed that about 19.5
people per 100 thousand die per year of colorectal cancer. Even though surgical
resection is curative for early stage diseases, currently used chemotherapeutic agents
for advanced stage colon cancer are palliative. Much research has been undertaken in
the battle against colon cancer over the past few decades. However, limited advances
have been obtained in spite of a substantial body of new discoveries about the
molecular biology of cancer cells.1 In addition, side effects of drugs are also potential
obstacles to successful chemotherapies. Compounds with anti-inflammation,
anti-oxidation, or free-radical scavenging ability have also been demonstrated to
possess varied degrees of anti-tumor activity in the literature. One promising approach
involves the administration of dietary phytochemicals that possess
cancer-preventative activity but with greater safety, better availability, and minimal
toxicity. Here we selected several candidate compounds with one or more of the
aforementioned properties (anti-inflammation, anti-oxidation, or free-radical
scavenging ability) for investigating the in-vitro anti-proliferative activity against the
HCT-116 cancer cell line in culture by
5
Berberine is found in plants such as Berberis, Hydrastis canadensis, and Coptis
chinensis, usually in the roots, rhizomes, stems, and bark. The traditional clinical applications of berberine include anti-infection2 and diabetic control.3 In addition,
berberine has been shown to suppress the growth of a wide variety of tumor cells
including prostate cancers4 and colon cancers.5
Diosgenin, a steroid sapogenin, is extracted from the tubers of Dioscorea wild yam.
Diosgenin is a well-known precursor of various synthetic steroidal drugs.6 Over the
past decade, much research has been conducted to understand the role of diosgenin on
human cancers and diosgenin has been found to have a role in multi-target based
chemopreventive or therapeutic properties.7
Lipoic acid is a naturally-occurring co-factor present in many enzyme complexes
regulating human metabolism. Lipoic acid has been demonstrated to have properties
of anti-oxidant activity8 and diabetic control.9 In addition, because of its free-radical
scavenging ability, lipoic acid has the potential to interfere with processes within
malignant cells.10
Pinolo is a nonselective beta-adrenergic blocker, possessing partial beta-adrenergic
receptor agonist activity. It also has membrane-stabilizing effects. Clinically, pinolo
has been used in angina pectoris, hypertension, arrhythmias, and prophylasix of acute
growth.11
Melatonin is a natural human hormone, produced by the pineal gland. It is essential
in the regulation of the circadian rhythms of several biological functions. Melatonin’s
biological effects are produced through activation of melatonin receptors or through
its powerful antioxidant activities.12 It has been demonstrated to have the properties of
antioxidant activities13 and prevention of ischemia brain damage.14 A systematic
review, involving 643 cancer patients, using melatonin found a reduced incidence of
death.15 Moreover, reduced melatonin level has been proposed as a likely
carcinogenic factor in night workers.16
Sodium butyrate, a short-chain volatile fatty acid in a non-toxic short-chain fatty
acid, is the product of large bowel microbial fermentation of dietary fiber in the colon.
Numerous studies have demonstrated the anti-proliferative effect of sodium butyrate
treatments in breast17, prostate18, and colon cancers.19
Curcumin is the chief ingredient in both traditional Chinese and Indian medicine
and in Indian turmeric spice, which is a member of the ginger family (Zingiberaceae).
Curcumin has been reported to have anti-inflammatory20 and anti-oxidant 21 activities.
It has been used for thousands of years by Asians in various clinical applications
including cancer treatment.22
7
other food sources. It has been demonstrated to have anti-inflammatory 23activities
and cancer chemopreventive properties.24
Honokiol is a pure biphenolic compound, present in cones, barks, and leaves of
Magnolia officinalis extracts, which is used in traditional Chinese medicine. Recent
research demonstrated that honokiol has variable biological activities including
anti-inflammatory25 and anti-oxidant effects.26
Emodin (1,3,8-trihydroxy-6-methylanthaquinone) is the main component in the
rhizome of Rheum palmatum L. (Polygonaceae). Emodin has been demonstrated to
have anti-bacterial27 and anti-tumor activities.28
Rottlerin, a compound from Indian tree, is a selective inhibitor of protein kinase
C-delta (PKC-delta). The PKC family is a major group of intracellular
phosphorylating enzymes which play a role in proliferation, differentiation, as well as
MATERIALS AND METHODS
Materials
The compounds (purity > 99%) were obtained from Alexis Biocompounds (San
Diego, CA). Culture medium RPMI-1640, fetal bovine serum (FBS) and
trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Invitrogen
(Carlsbad, CA).
Cell culture and treatment
HCT-116 cells are derived from a colon carcinoma [American Type Culture
Collection (ATCC), Rockville, MD; ATCC # CCL247] and serve as a useful model
for study. The culture medium used was RPMI 1640, containing 10% fetal bovine
serum, 20 mmol/L HEPES buffer, and 100 μg/mL gentamicin. Cells were incubated
at 37°C in a humidified atmosphere of 5% CO2 in air. The candidate compounds were
dissolved in dimethyl sulfoxide (DMSO) as a stock solution at 100 mmol/L and
diluted with an FBS-free medium to achieve the designated concentrations. The same
concentration of DMSO without any compounds added was used as a control.
MTT viability assay
Cell viability was assessed by MTT assay. HCT-116 cells cultured onto 24-well
plates were treated with various compounds. After 24 hours or 48 hours incubation,
9
saline (PBS), and MTT (100 μg/ 0.1 mL of PBS) was added to each well. The cells
were incubated at 37°C for 4 hours. Culture medium was then replaced with an equal
volume of DMSO to dissolve formazan crystals. The absorbance was measured at 550
nm by microplate reader (Bio-Tek, Winooski, VT). The cell proliferation inhibition
rate was calculated as 1 - (average OD value of wells with administered drug/average
OD value of control wells) × 100. All experiments were performed a minimum of 3
times and data was presented as the average value ± the standard error of the mean
(SEM).
Statistical analysis
The values given are means ± S.E.M. The significance of difference between the
experimental group and control was assessed by Student’s t test. The difference is
RESULT
To determine the optimal conditions for cytotoxicity in cultures of HCT-116 colon
cancer cells, a variety of compounds were used in different concentrations for the
indicated time periods. These compounds shown in Figure 1 include honokiol,
emodin, lipoic acid, berberine, diosgenin, resveratrol, rottlerin, pinolo, curcumin,
melatonin, and sodium butyrate. Cell viability at each time point was then assessed by
MTT assay.
The HCT-116 human colon cancer cells were treated with various compounds at
different concentrations in 10% FBS for 48 hours. Incubation with lipoic acid,
berberine, diosgenin, pinolo, melatonin, and sodium butyrate had no significant
anti-tumor effect on HCT-116 cells, while honokiol, emodin, resveratrol, rotterlin,
and curcumin caused a significant (*P < 0.05) reduction in total cell numbers (Figure
1). Subsequently, HCT-116 cells were treated with 3, 10, and 30 μM of the five
potentially cytotoxic compounds (honokiol, emodin, resveratrol, rotterlin, and
curcumin) for 24 and 48 hours in serum-free medium (Figure 2 and 3). The results
showed that the inhibitory effect of honokiol, emodin, resveratrol, rottlerin, and
curcumin on HCT-116 cell proliferation was both time-dependent and
concentration-dependent (Figure 3).
11
of a compound in inhibiting biological or biochemical function. According to the
criteria of the American National Cancer Institute, values of IC50 lower than
30μg/mL suggest a compound worth further purification.29 After 24 hour treatment,
the IC50 values were 18.0 μM for honokiol, 21.5 μM for emodin, infinity for
resveratrol, 13.1 μM for rottlerin, and 20.8 μM for curcumin (Table 1). However, only
the IC50 of rottlerin significantly decreased after 48 hour treatment, while the IC50
values of honokiol, emodin, resveratrol, and curcumin had no significant changes
DISCUSSION
In Cragg’s study 30, over 50% of the drugs in clinical trials for anticancer activity
were isolated from natural sources or were related to them. Recently the search for
natural compounds with active anti-tumor properties has been a popular research topic.
In this study, the cytotoxicities of the ten candidate compounds were tested against the
human colon carcinoma (HCT-116) cell line, using the thiazolyl blue test (MTT)
assay. Honokiol, emodin, resveratrol, rottlerin, and curcumin significantly inhibited
the growth of the HCT-116 cell line in a concentration- and time-dependent manner.
However, the anti-tumor activity of lipoic acid, berberine, diosgenin, pinolo,
melatonin or sodium butyrate on HCT-116 cells was not significant in the present
study.
Extensive research over previous decades demonstrated that curcumin has potent
cancer-killing activity in vitro against various types of cancers including colon,
prostate, and breast.31 Mahmoud et al proved that dietary curcumin could suppress
intestinal carcinogenesis in a mouse model of familial adenomatous polyposis.32
Ushida et al confirmed that curcumin could prevent esophageal carcinogenesis in
mice.33 Huang et al demonstrated that curcumin could reduce the incidence
lymphomas and leukemias in rats.34 Our results demonstrated that proliferation of
13
time-dependent manner. On the other hand, we also demonstrated that 24 hour
treatment was optimal for curcumin-induced cytotoxicity in cultures of HCT-116
colon cancer cells, because the IC50 value had no significant change after 48 hour
treatment. Our results are consistent with other research that curcumin is cytotoxic for
HCT-116 human colon cancer cell line.35, 36 Moreover, curcumin is also cytotoxic
against many other types of cancer cells.37-39 Taken together, this suggests that
curcumin may have broad applications in cancer chemoprevention, although we did
not determine whether curcumin was selectively cytotoxic for neoplastic cells,
Watson et al demonstrated that the viability of normal human dermal fibroblasts was
not altered following 72 hour exposure to lower concentrations (10 and 20 μM) of
curcumin.40 This result is also consistent with Chen’s research.41
Resveratrol has been shown to have growth-inhibitory activity in several human
cancer cell lines including glioma 42, colorectal cancer 43, and epidermoid carcinoma.
44 In this study, we found that a 24 hour resveratrol exposure did not significantly alter
the number of viable cells, suggesting that the anti-proliferative effect was not
associated with obvious cell death at this exposure time. However, a 48 hour exposure
to 30 μM resveratrol resulted in a significant (**P < 0.01) decrease in cell viability.
This result might be indicative for delayed apoptotic death in HCT-116 cells exposure
cancer cell death. However, in Wolter’s study45 about resveratrol-induced Caco-2
human colon cancer cell death, the growth inhibitory results were similar to ours.
Honokiol also exhibited apoptosis induction and growth inhibition in some studies
including lung46, prostate47, and colon cancer cells.48 In this study, we demonstrated
that proliferation of HCT-116 cells in the presence of honokiol was inhibited in a
concentration- and time-dependent manner with an IC50 value of 18.5 μM (48-hour
treatment) and 18.0 μM (24-hour treatment) in serum-free condition. The sole
research investigating the anti-tumor effect of honokiol on human colon cancer cell
(HCT-116) also demonstrated similar results with an IC50 value of 23μM in 10%
FBS condition.48 The different IC50 values may be due to the presence or absence of
serum in medium. Recent study 49 also elucidated that the underlying mechanism is
through blocking of Nuclear Factor-kappa B.
A number of research has demonstrated the growth inhibitory effect of emodin on
cancer cells such as ovarian cancers,50 colon cancers,51 and breast cancers.52 Here,
proliferation of HCT-116 cells in the presence of emodin was inhibited in a
concentration- and time-dependent manner with an IC50 value of 17.3 μM (48 hour
treatment) and 21.5 μM (24 hour treatment) in serum-free condition. No previous
investigations reported any emodin-induced HCT-116 cancer cell deaths. However,
15
cells other than HCT-116.51, 53
Some research has already demonstrated the inhibitory effects of rottlerin on cancer
cells.54, 55 In the present study, we have shown that rottlerin inhibits HCT-116 cell
proliferation in a concentration-dependent and time-dependent manner both in 24
hour and 48 hour exposures. Although there has been no research about the anti-tumor
effects of rottlerin on the HCT-116 cell line, a few articles also demonstrated the
significant growth inhibition of rottlerin on various human colon cancer cell lines.56, 57
Progression of colon cancer is associated with activation of multiple signaling
pathways. Cell death is an essential event in normal life and in the pathophysiological
processes which lead to diseases. A pattern of cell death has emerged where each of
several distinct organelles (plasma membrane, mitochondrion, nucleus, endoplasmic
reticulum, lysosome) give rise to signals which induce cell death. Endoplasmic
reticulum (ER) is a central organelle engaged in protein production, folding and
maturation. Various toxic insults can perturb ER function and result in ER stress.58
There is increasing evidence that ER stress plays an important role in the regulation of
cell death.59 Until now, only one study reported about ER stress-mediated HCT-116
cell death in the literature.60 In the future, treatment of HCT-116 human colon
carcinoma cells with these five potential compounds (honokiol, emodin, resveratrol,
stress markers: phosphorylation of eukaryotic initiation factor-2α(eIF-2α),
up-regulation of glucose-regulated protein (GRP)-78, and up-regulation of CCAAT /
17
CONCLUSION
Here, we only presented the preliminary anti-tumor results of selected candidate
compounds on HCT-116 human colon cancer cells. Further investigations should be
conducted to elucidate the underlying mechanisms modulating anti-tumor effects on
FIGURES AND TABLES Figure 1
HCT-116 cells were treated with various concentrations (3, 10, 30μM) of different
compounds, in complete medium with 10% fetal bovine serum (FBS), for 48 hours as
described in the text. The cell viability of cells was counted by MTT assay. The
results represented the mean ± S.D. of three independent experiments and the
significant difference was established at *p<0.05, **p<0.01, ***p<0.001 compared
with the control group. DMSO served as the solvent control. Each column showed
19
Figure 2
HCT-116 cells were treated with various concentrations (3, 10, 30μM) of different
compounds (honokiol, emodin, resveratrol, rottlerin, and curcumin), in serum-free
medium, for 24 and 48 hours respectively, depicted in the linear regression mode. A.
Viable cells were detected by proliferation assay using MTT assay after 24-hour
exposure. B. Viable cells were detected by proliferation assay using MTT assay after
48-hour exposure.
21
Figure 3
Merged from Figures 2A and 2B. HCT-116 cells were treated with various
concentrations (3, 10, 30μM) of different compounds (honokiol, emodin, resveratrol,
rottlerin, and curcumin) in serum-free medium, for 24 (black columns) and 48 hours
(white columns) respectively, measured by MTT viability assay. Each column showed
growth inhibition after normalizing untreated cells to100%. The results represented
the mean ± S.D. of thouree independent experiments and the significant difference
was established at *p<0.05, **p<0.01, ***p<0.001 compared with the control group
Table 1
Effective doses (μM) of various compounds (honokiol, emodin, resveratrol, rottlerin,
and curcumin) that inhibited cell growth to 50% of control (IC50) in different
23
LEGENDS Figure 1
HCT-116 cells were treated with various concentrations (3, 10, 30μM) of different
compounds, in complete medium with 10% fetal bovine serum (FBS), for 48 hours as
described in the text. The cell viability of cells was counted by MTT assay. The
results represented the mean ± S.D. of three independent experiments and the
significant difference was established at *p<0.05, **p<0.01, ***p<0.001 compared
with the control group. DMSO served as the solvent control. Each column showed
growth inhibition after normalizing untreated cells to100%.
Figure 2
HCT-116 cells were treated with various concentrations (3, 10, 30μM) of different
compounds (honokiol, emodin, resveratrol, rottlerin, and curcumin), in serum-free
medium, for 24 and 48 hours respectively, depicted in the linear regression mode. A.
Viable cells were detected by proliferation assay using MTT assay after 24-hour
exposure. B. Viable cells were detected by proliferation assay using MTT assay after
Figure 3
Merged from Figures 2A and 2B. HCT-116 cells were treated with various
concentrations (3, 10, 30μM) of different compounds (honokiol, emodin, resveratrol,
rottlerin, and curcumin) in serum-free medium, for 24 (black columns) and 48 hours
(white columns) respectively, measured by MTT viability assay. Each column showed
growth inhibition after normalizing untreated cells to100%. The results represented
the mean ± S.D. of thouree independent experiments and the significant difference
was established at *p<0.05, **p<0.01, ***p<0.001 compared with the control group
for the indicated time. DMSO served as the solvent control.
Table 1
Effective doses (μM) of various compounds (honokiol, emodin, resveratrol, rottlerin,
and curcumin) that inhibited cell growth to 50% of control (IC50) in different
25
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